Regulation of paracellular permeability: factors and mechanisms

Yan-Jun Hu • Yi-Dong Wang • Fu-Qing Tan •

Wan-Xi Yang

Received: 8 December 2012 / Accepted: 14 September 2013 / Published online: 24 September 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Epithelial permeability is composed of trans-

cellular permeability and paracellular permeability. Para-

cellular permeability is controlled by tight junctions (TJs).

Claudins and occludin are two major transmembrane pro-

teins in TJs, which directly determine the paracellular

permeability to different ions or large molecules. Intra-

cellular signaling pathways including Rho/Rho-associated

protein kinase, protein kinase Cs, and mitogen-activated

protein kinase, modulate the TJ proteins to affect paracel-

lular permeability in response for diverse stimuli. Cyto-

kines, growth factors and hormones in organism can

regulate the paracellular permeability via signaling path-

way. The transcellular transporters such as Na-K-ATPase,

Na?-coupled transporters and chloride channels, can

interact with paracellular transport and regulate the TJs. In

this review, we summarized the factors affecting paracellular

permeability and new progressions of the related mecha-

nism in recent studies, and pointed out further research

areas.

Keywords Tight junctions � Paracellular

permeability � Occludin � Claudins � Signal pathway �Mechanism

Abbreviations:

BBB Blood brain barrier

BTB Blood testis barrier

CFTR Cystic fibrosis transmembrane conductance

regulator

ECL Extracellular loops

EGF Epidermal growth factor

ERK Extracellular signal-related kinases

GAPs GTPase activating proteins

GEFs Guanine nucleotide exchange factors

GUK Guanylate kinase

JAM Junctional adhesion molecule

JNK c-Jun amino-terminal kinases

MAGUK Membrane-associated guanylate kinases

MARVEL MAL and related proteins for vesicle

trafficking and membrane link

MAPK Mitogen-activated protein kinase

MLC Myosin light chain

MLCK Myosin light chain kinase

NHE Sodium–hydrogen exchanger

PDGF Platelet-derived growth factor

PKCs Protein kinase Cs

ROCK Rho/Rho-associated protein kinase

SGLT Sodium–glucose transporters

TER Transepithelial electrical resistance

TGF Transforming growth factor

TJs Tight junctions

Yan-Jun Hu and Yi-Dong Wang contributed equally to this study.

Y.-J. Hu � Y.-D. Wang � W.-X. Yang

Department of Reproductive Endocrinology, Women’s Hospital,

School of Medicine, Zhejiang University, Hangzhou 310006,

People’s Republic of China

Y.-D. Wang � W.-X. Yang (&)

The Sperm Laboratory, Institute of Cell and Developmental

Biology, College of Life Sciences, Zhejiang University, 866 Yu

Hang Tang Road, Hangzhou 310058, People’s Republic of

China

e-mail: wxyang@spermlab.org

F.-Q. Tan

The First Affiliated Hospital, College of Medicine, Zhejiang

University, Hangzhou 310003, People’s Republic of China

123

Mol Biol Rep (2013) 40:6123–6142

DOI 10.1007/s11033-013-2724-y

VEGF Vascular endothelial growth factor

ZO Zonula occludens

Introduction

Tight junctions (TJs) are a protein complex which connect

the adjacent cells in the epithelial or endothelial cells, and

was first described by Farquhar and Palade in 1963 [1].

Working together with other junctions, TJs integrate the

tissues and form the barriers to resist alien invader.

Besides, they also work as filters with selective paracellular

passage to ions, water, larger molecules and even cells,

which contribute to maintain homeostasis in body [2]. Due

to the wide distribution in the epithelium and the impor-

tance for homeostasis, dysfunction of this barrier results in

numerous diseases including diarrhea, jaundice, asthma,

pulmonary edema, inflammatory bowel disease and rheu-

matoid arthritis, which finally deteriorate the organism [3,

4]. Over the years, further studies indicate additional

functions of TJs in cell differentiation, proliferation, signal

transduction and gene expression, underlining the diverse

roles of TJs in cells [5–8].

TJ proteins are dynamic, which can be roughly divided

into three groups: transmembrane proteins, cytoplasmic

scaffolding proteins and cytoskeletal proteins (Fig. 1a).

The transmembrane proteins are composed of claudins,

occludin and junctional adhesion molecule (JAM). Clau-

dins and occludin are two principal proteins, whose

extracellular strands are thought to directly control the

intercellular space flux, and select the diffusion of different

kinds of ions and molecules (Fig. 1b, c). Tricellulin, first

reported in 2005, is another transmembrane protein local-

izing among three adjacent cell corners, which is also

involvement in the regulation of paracellular permeability

[9–11]. Zonula occludens-1,-2,-3 (ZO-1,2,3) are scaffold-

ing proteins belonging to the MAGUK family, and contain

three PDZ domains, a SH3 domain and a guanylate kinase

(GUK) domain in the amino-terminal region [12, 13],

interacting with claudins and occludin. Their carboxy-ter-

minal region directly link to the cytoskeletal proteins, actin

and myosin, which constitute a belt ring surrounding the

intracellular surface. The ZO proteins can recruit a large

amount of proteins to the site of TJs and play a crucial role

in the assembly of TJs [12].

Although it is discovered that this complex can perform

lots of missions, the primary functions of TJs are the barrier

and fence function. TJs are regarded as a fence, which

restrict the movement of proteins in the membrane in order

to maintain the cell polarity. However, their barrier function

is the ability of limiting the transport of ions and nonelec-

trolytes via the intercellular space, which directly affect the

paracellular permeability. It is known that the epithelial

permeability is composed of two parts, the transcellular

permeability and paracellular permeability [14]. The trans-

cellular permeability is the movement of ions or molecules

through apical and basolateral transporters or channels

across the membrane, while the paracellular permeability is

the diffusion of ions or molecules via the intercellular space,

driven by the transepithelial electrochemical gradient.

Paracellular permeability is featured as size and charge

selectivity and the physiological function is various, to a

great extent, dependent on the cell type.

Traditionally, the paracellular permeability can be eval-

uated by two methods [15–17]. Transepithelial electrical

resistance (TER) is the measurement of the flux of ions

across the epithelium immediately. Unfortunately, this

parameter is unable to reflect the characteristic of size or

charge selectivity. TJ assembly is often accompanied by the

increase of TER and decrease in paracellular permeability,

and vice versa. It is the best choice when the ion flux across

the TJ is far greater than the total flux across membrane [16].

The other method is the measurement of paracellular trace

flux. Applying the radioactive or fluorescently connected

tracers including mannitol or dextran, the permeability of the

paracellular tracers can be measured over a period of time

when the tracers transit a monolayer. This method can

reflect the size selectivity but vesicular transport will be an

influencing factor. In sum, these two methods are comple-

mentary to each other and the combined application mirror

the paracellular permeability objectively.

In the past several decades, an ocean of researches have

unfolded that the barrier properties of TJs can be regulated

in either physiological or pathological conditions, and then,

affect the paracellular permeability. These factors includ-

ing the cytokines, growth factors, and hormones modulate

the TJs, and the apical transporters are also involved. The

mechanism of the regulation is multiple and several sig-

naling pathways, such as, Rho/ROCK, PKCs and MAPK,

participate in the regulation. Now, it is essential to give a

updated conclusion including recent progressions of TJs,

considering universal distribution and diverse roles TJs

play in living organism. Here, claudins and occludin will

be discussion in the control of paracellular flux, and

modulators of the TJ proteins and mechanism will be

concluded in this review.

Claudins and occludin: two guards in intercellular

space

Claudins

The family of claudins were first reported by Furuse [18],

and until now, 27 members are identified in mammalian

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Fig. 1 Schematic of the tight

junctions. a The three groups of

proteins and their interaction are

showing in this schematic.

Scaffolding proteins, ZO

proteins, mediate the interaction

between transmembrane

proteins, mainly claudin and

occludin, and cytoskeletal

proteins actin and myosin.

b The claudins contain four

transmembrane domains, two

extracellular loops, amino- and

carboxy-terminal cytoplasmic

regions. The carboxy-termini

can be phosphorylated and the

PDZ domain binding motif can

bind PDZ domain of ZO

proteins. c The structure of

occludin is roughly similar to

claudin, but weigh more than

claudin. The carboxy-terminus

is rich in serine, thereonine and

tyrosine residues for

phosphorylation and coiled-coil

domain interacts with ZO

proteins

Mol Biol Rep (2013) 40:6123–6142 6125

123

[19], with a molecular weight ranging from 20 to 34 kDa

[20]. Claudins are four transmembrane proteins, which

contain four transmembrane domains, two extracellular

loops, amino-and carboxy-terminal cytoplasmic regions

(Fig. 1b). The carboxy-terminal regions of claudins have

PDZ binding motifs which directly interact with the cyto-

plasmic scaffolding proteins ZO-1,-2,-3, MUPP1 and PATJ

[20, 21]. Ser or Thr residues in this region can also be

phosphorylated in regulation by kinases. The distribution

of the isoforms of claudins is tissue-specific, largely

dependent on the tissue or organ characteristics. For

example, claudin-1, is universally expressed, while other

isoforms are limited in some certain cell types or stage of

development [22]. In placental endothelial vessel, claudin-

1,-3,-5 usually express [23, 24]. Claudin-2,-10 and -11

express in proximal tubules, while claudin-3,-10,-11 and -

16 express in the thick ascending limb of the Loop of

Henle, indicating the variation in the expression of human

claudins, which probably the reason for different paracel-

lular permeability in kidney [25].

Claudins are vital for the establishment of TJs. In rat

lung endothelial cell line which barely form TJs, the

transfection of claudin-1 resulted in the formation of

functional TJs, assessed by the TER and paracellular trace

flux [3]. Instead, claudin-1-deficient mice born with wrin-

kled skin died within 1 day of birth, and claudin-6-deficient

mice died within 10 h of birth, with the size-selective

loosening in the blood–brain barrier [26, 27]. In many

cases, TJ strands are generally the mixture of different

isoforms of claudins, and the appropriate combination of

claudins is significant for the function of TJs, which

determine the barrier properties in a specific tissue [28, 29].

The family of claudins seems to stand in two rows,

according to their dual effects on the paracellular perme-

ability. Several claudins, when overexpressing in cells,

have been revealed to increase the paracellular perme-

ability to ions or water, which are regarded as the pore-

forming claudins, e.g. claudin-2, claudin-16 and claudin-

19, while some others, when overexpressing, paracellular

permeability to ions or water decrease, which are regarded

as the seal-forming claudins, e.g., claudin-3, claudin-4 and

claudin-8. After transfecting claudin-8 to MDCK II cells,

the paracellular permeability to cations reduced [30], and

the later research revealed that claudin-8 also decreased the

flux to protons, ammonium and bicarbonate [31]. Claudin-

4, when inhibited by small interfering RNA (siRNA),

decreased TER but the macromolecule permeability didn’t

change, and claudin-4 expression in uterine LE and GE

cells increase the tightness [32, 33]. Claudin-3 was repor-

ted to act as a seal against cations, anions and larger

molecules, but didn’t affect water permeability [34].

The pore-forming claudins are major participant of the

permeability to ions. Claudin-2 has been reported to

constitute the cation (Na?) selective paracellular channels

in mouse proximal tubules and in intestinal Caco-2 cells,

working as a pore for cation [35, 36], and calcium’s

competition sodium for binding Asp-65 resulted in inhib-

iting sodium reabsorption in the proximal tubule of kidney

[37]. In the SRL- and CsA-induced barrier alteration,

decrease the permeability to Na? ions accompanied by the

increase of claudin-1 and decrease of claudin-2 [38]. In

addition to be considered as a cation pore, recent advance

also showed that claudin-2 act as a water channel and

mediate the water flux in leaky epithelia [39], suggesting

the water transport of TJs partially relies on the claudins.

Claudin-16 mutation in human results in magnesium

wasting [40], and Hou’s work [41, 42] demonstrated that

the heteromeric claudin-16 and claudin-19 interaction

generated cation-selective pores, indicating claudin-16 and

claudin-19 have coordinated and affect paracellular mag-

nesium permeability. Interestingly, the so-called seal-

forming claudins—claudin-4 and claudin-8, are well

cooperated and form Cl- channel. Claudin-4 works as a

Cl- channel and the lysine residue at 65 is critical for its

channel. Yet without claudin-8, claudin-4 can’t recruit to

the TJs [43]. Recent reports showed claudin-17, abundant

expression in proximal tubules, forms anion channels

which can paracellularly reabsorb chloride and bicarbon-

ate, and the anion selectivity depend on positive amino acid

at position 65 [44]. From the above facts, it can be con-

cluded that claudins are the main components to constitute

pore, and are responsible for the charge and size selective

flux.

Claudins have two extracellular loops (ECL), and the

amino acid residues with different types determine per-

meability to ions or solute (Fig. 1b). Using site-directed

mutagenesis, replacement of negative by positive charge at

the position in claudin-4 ECL1 increased paracellular Na?

permeability, while replacement of positive by negative in

claudin-15 reversed the phenomenon [45]. For claudin-7,

the mutation of ECL1, which replace negative charged

with positive charged amino acids, increase the Cl- per-

meability, but the mutation of ECL2 have little effect on

the paracellular permeability [46]. Two splice variants of

claudin-10, claudin-10a and claudin-10b, show a typical

example [47]. The two splice variants have different

domains in ECL1. Claudin-10a, which contain more posi-

tively charged residues, more permeable to anions than

claudin-10b. The study also found that not all the residues

in the ECL1 were responsible for the selectivity and dif-

ferent charged residues exert different effects on the

selectivity [47].

Until now, the behavior of claudins in molecule level

has not been well described, which may find out the rea-

sonable explanation for abovementioned roles claudins

play in paracellular permeability, such as the different

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actions of claudin-4. The increasing data of the role of

particular sites help to recognize molecular mechanism of

pore or seal forming. It is reported that there exist four

patterns of interaction between claudins: homophilic cis-

interaction, homophilic trans-interaction, heterophilic cis-

interaction and heterophilic trans-interaction [29]. The

homophilic interaction means the one claudin binding to

each other while heterophillic interaction means binding

other claudin members. The cis-interaction occurs when

the molecules interact in the same side of cell while trans-

interaction in the opposing cells. How the extracellular

loops and interaction between same or different claudins

organize the structure and contribute to the selectivity

should be investigated in the future studies. What’s more,

whether the intracellular part of claudins is also involved in

the interaction, such as cis-interaction is unknown.

Occludin

Occludin, first identified in 1993 [48], is an approximately

65 kD protein containing four transmembrane domains,

two extracellular loops, and amino- and carboxy-terminal

cytoplasmic regions (Fig. 1c). The coiled-coil domain of

carboxy-terminal region of occludin interacts with ZO-1,

ZO-2 and ZO-3, which is important for the regulation of

TJs [2, 49]. Occludin also has a MARVEL domain, a four

transmembrane-helix structure whose function related to

membrane apposition events [50].

In vivo and in vitro studies manifest that occludin is not

essential for the formation of TJ strands, since the mor-

phology of TJ strands seems not to be affected [51–53].

However, abnormalities of occludin-deficiency mice have

been found, including the chronic inflammation, brain

calcification and testicular atrophy, while the gastric epi-

thelial permeability measured by TER and mannitol flux

were normal. This phenomenon may indicate that occludin

gene is indispensable during the development of organ-

isms. In addition, numerous investigations also explore the

role of occludin in the paracellular permeability. Overex-

pression of occludin in MDCK cells and brain–blood bar-

rier decreased the permeability [54, 55], while

down regulation and alteration of localization of occludin

increased paracellular permeability in a murine model of

GvHD in small intestine [56]. Similar to claudin, extra-

cellular loops of occludin decide the TJ’s permeability,

which can be perturbed by the synthetic peptide [57, 58].

Except the seal function, studies also demonstrate selective

function of occludin. Balda’s [59] research indicated that

occludin may work together with claudin-4, increase the

permeability to hydrophilic molecules. siRNA-induced

knockdown of occludin in Caco-2 cells and mouse intestine

increase the transepithelial flux of large molecules such as

dextran, in the meantime, TER was not affected [60].

However, the selective increase the macromolecule flux

accompanied by the increase expression of claudin-2,

which is regarded as cation channel. Although the possi-

bility of the role of claudin-2 expression in the increase in

macromolecule flux is excluded, it seems that claudins

always engage in the selective function of occludin. How

the occludin block the macromolecule flux and whether

claudins exert effects on the function of occludin is

unknown. The rational role of occludin in paracellular

permeability should be completely defined in the coming

researches.

The prominent feature of occludin is the various post-

translational modifications, such as phosphorylation, pro-

teolysis and ubiquitination [2]. Phosphorylation is a far-

ranging regulation and detailed discussed in recent reviews

[2, 61–63]. The carboxy-terminal region of occludin is rich

in serine, threonine and tyrosine residues, the substrates for

quantities of kinases and phosphatases in various condi-

tions. It is concluded that the effect of phosphorylation is

paradoxical, since phosphorylation of serine, threonine and

tyrosine can both enhance or reduce the barrier function

[2]. Abundant phosphosites in occludin may largely con-

tribute to the precise effect on paracellular permeability,

but the molecule mechanism how the phosphorylation

affects the permeability remains unknown. What’s more,

phosphorylation can also be activated by distinct modula-

tors in specific tissues, such as PKCs that will be mentioned

below, which is another possible influence factors. How-

ever, a definitive explanation for the paradoxical result

needs to be established.

Intracellular signaling pathway to modulate TJ

Enormous evidences indicate that the assembly, mainte-

nance, and disassembly of TJ can be regulated by the

signaling pathway, affecting the permeability to solute.

Past pharmacological and molecular researches have

showed that three signaling pathways, Rho/Rho-associated

protein kinase (ROCK), protein kinase Cs (PKCs), and

mitogen-activated protein kinase (MAPK), deeply modify

the phosphorylation or expression of TJ proteins [5, 64].

These signaling pathways usually work as an intermediate

to execute the intracellular process in the cytokines, nox-

ious agents or other chemicals-induced regulation of TJs.

The alterations of TJs always reflect the combination of the

crosstalk of different pathways, which may have syner-

gistic or antagonistic action.

Rho/Rho-associated protein kinase

The small GTPases are important molecules in the intra-

cellular process, for they acts as molecular switches that

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cycle between GTP-bound(active) and GDP-bound(inac-

tive) states [5, 6, 65] (Fig. 2). The activated state of

GTPases change the conformation and initiate the down-

stream reactions. The transformation of the active and

inactive states can be catalyzed by guanine nucleotide

exchange factors (GEFs) and GTPase activating proteins

(GAPs). Rho GTPase is one family of the small GTPase,

and plays prominent roles in the regulation of TJs and

paracellular permeability, especially in the vascular endo-

thelium [66, 67]. Three members of Rho family, RhoA,

Rac and Cdc42 are all reported to exert effects on the TJs

[68]. In this part, the Rho, mainly RhoA, the downstream

molecular ROCK and related signaling molecules will be

detailed discussed.

ROCK, the major target of Rho, is Ser/Thr kinase that

regulates the TJ permeability. ROCK have two isoforms,

ROCK I ubiquitously expressed except in the brain and

muscle, and ROCK II abundantly in the brain, muscle,

heart, lung and placenta [69]. As a kinase, ROCK can

directly phosphorylate occludin (T382 and S507) and

claudin-5(T207) in brain endothelial cells after activation

in mouse BMVECs [70]. The phosphorylation of occludin

and claudin correlate with the decrease barrier function and

enhance monocyte migration, which can be prevented by

ROCK inhibitor.

The cytoskeletal proteins of TJs, myosin and actin, are

also the downstream effectors of ROCK, which play a

central role in the regulation of permeability (Fig. 2). The

one kind of myosin isoform, myosin II, bind to the actin

cytoskeleton and the phosphorylation of myosin light chain

(MLC) contract actin belt, resulting in the actin rear-

rangements [71]. In addition to the direct phosphorylation

of MLC, ROCK can also inactive the MLC phosphatase,

thereby maintain the phosphorylation state of MLC [14,

72]. It is also found that cofilin, one actin-remodeling

protein, can be direct phosphorylated by ROCK, or via

LIM kinase indirectly [73–75]. Phosphorylation of cofilin

will stabilize actin filament and enhance barrier function,

while dephosphorylation speed up the depolymerization of

actin and disruption of TJ by endocytosis mediated by

caveolin-1 [76].

Recently, it is reported that the epithelial iNOS causes

the Rho/ROCK signals to MLC phosphorylation in en-

terocytes, which leads to gut barrier damage [77]. Rho/

ROCK/MLC phosphorylation also mediated the thrombin-

induced disruption of blood–retina barrier [78]. When treat

rat brain microvascular endothelial cells (RBMECs) with

bradykinin (BK), TJ disassembly and the blood–tumor

barrier increase permeability, accompanied by the phos-

phorylation of MLC and cofilin, stress fiber formation and

relocation of occludin and claudin-5 [79]. Their other

research also conform the observation [80]. However, the

cofilin phosphorylation will stabilize TJ barriers, which

seems to contradictory [81]. The increase permeability of

blood–tumor barrier is the combined effect of the changes

of myosin and actin, but the individual contribution of

myosin and actin to the increase of permeability is not

calculated. A quantitative analysis, if possible, can be used

so as to give us a deep understanding of the effect of

cytoskeletal proteins on TJs.

Myosin can also be phosphorylated by myosin light

chain kinase (MLCK) which activated by binding of Ca?–

calmodulin [82]. MLCK mediate the thrombin, ethanol and

histamine-induced damage of barrier function [83–85],

indicating it is a common way to regulate the TJs. Heli-

cobacter pylori is able to active MLCK and disrupt the

gastric epithelial TJs [86, 87], paralleled to the occludin

internalization and disruption of claudin-4 and claudin-5.

Excluding other factors, Shen’s works [88] suggest that the

MLCK alone is enough to regulate the TJs without any

upstream stimuli, and occludin and ZO-1 are relocated.

The GEF, the transformer of the inactive state to active

state, are involved in the TJ regulation (Fig. 2). GEF-H1 not

only can increase the paracellular permeability in MDCK

cells [89, 90], but also mediate the agonist-or ventilator-

induced lung vascular endothelial barrier disruption [91, 92].

p115RhoGEF played a role in LPS signaling to activate

RhoA and increase paracellular permeability bEnd.3 cells,

accompanied by the change of actin and degradation of TJ

proteins [93]. However, p115RhoGEF depletion partially

Fig. 2 The Rho signaling pathway has been revealed in the

regulation of tight junction and paracellular permeability. GEFs can

activate Rho and initiate the signaling pathway. GEF-11 and

p115RhoGEF can induce the disruption while p114RhoGEF and

ARHGEF11 activate Rho at junctions and facilitate the formation of

tight junctions. ROCK is the downstream molecules activated by Rho,

which can directly phosphorylate claudin, occludin, myosin light

chain (MLC) and cofilin. In addition, cofilin can also indirectly

phosphorylated by ROCK through LIM kinase (LIMK). MLC kinase

(MLCK) is another member engaging in the regulation, also showing

in the schematic

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prevented the TER decrease and TJ proteins degradation,

suggesting that other pathways may exist in the LPS-induced

TJ disruption. Since the GEF family for Rho is large, other

GEFs which may mediate the signaling pathway can be

excluded and need to explore further. In contrast to mediate

disruption of TJ, p114RhoGEF was reported that spatially

restricted activate the RhoA at junctions can drive TJ

assembly and regulate actomyosin contractility, suggesting

the GEF and RhoA in distinct subcellular regions exert dif-

ferent effects on TJs at the stage of TJ assembly [94]. Recent

Itoh’s research [95] showed that knockdown of ARHGEF11,

which directly bind to ZO-1, decelerated junction assembly

and barrier maturation, indicating the ARHGEF11 also

involve in the formation of TJs. They supposed that proper

contraction of actomyosin is necessary, while overmuch or

reduced phosphorylation of MLC result in the inadequate

contraction, which lead to the TJ disruption. The study gives

a new insight in the role of Rho in the TJ regulation, but

whether the assumption is reasonable should be ensured by

more facts.

Protein kinase Cs

Protein kinase Cs (PKCs) is a family of serine/threonine

kinases, and until now, at least ten different isoforms of

PKCs have been discovered [96]. The isoforms can be

divided into three groups: (1) the classical PKC(cPKC): a,

b1, b2 and c. (2) The novel (nPKC): d, e, h, g and l. (3)

Atypical PKC (aPKC): k, s and f. Diacylglycerol (DAG) and

calcium can activate cPKCs, while nPKCs don’t need cal-

cium. The aPKCs are both calcium and DAG insensitive.

DAG analogs, such as phorbol esters and 1,2-dioctanoyl-

glycerol(diC8), are always used in the experiments [96].

However, conflicting observations have been reported that

PKCs increase or decrease paracellular permeability of TJs,

suggesting the various roles of PKCs in the regulation of TJs.

PKC’s role in TJs’s assembly and maintenance

The early studies showed the DAG analogue, diC8,

enhanced the junction assembly [97]. The subsequent

studies showed the PKC’s ability of enhancement of bar-

rier, accompanied by the translocation of claudin, occludin

and ZO proteins. In T84 epithelia, PKC-e involved the

bryostatin-1-induce enhanced barrier function, and the

signaling pathway cause the recruitment of claudin-1 and

ZO-2 [98]. nPKC d and aPKC, not cPKC, are thought to

stimulate TJ formation in isolated inner cell masses, in line

with the assembly of ZO-1a? and ZO-2 [99]. PKC-h, one

of the nPKC isoform, is required for the formation of TJs

[100]. Claudin-1 and claudin-4 are phosphorylated and

organized into the TJs in the regulation of barrier function

in the intestinal epithelium.

Occludin, rich in serine, threonine and tyrosine residues

in carboxy-terminal region, is the ideal substrate for PKCs.

Addition of phorbol 12-myristate 13-acetate (PMA) and

diC8, two PKC agonist, a rapid phosphorylation of occlu-

din is induced and occludin is redistribution to the cell

borders, resulting in the assembly of TJs [101]. Recent

studies showed that PKC-g plays a critical role in the

formation of TJ. PKC-g directly phosphorylate the occlu-

din on threonine residues T403 and T404, two sites seems

to require for the assembly of TJs [102]. Inhibition and

knockdown PKC-g induce the dephosphorylate occludin

on threonine. PKC-g also mediate the different isoforms of

apolipoprotein E induced regulation of BBB, via phos-

phorylation of occludin on threonine residues [103].

According to the abovementioned facts, subfamily of

nPKC is plays a key role in the assembly and maintenance

of TJ, which is supported by Anna Y. Andreeva’s work

[104].

aPKC involve the formation of TJ in a distinctive way,

by binding with Par3 and Par6. The complexes at TJ is

important for the TJ formation and epithelial polarization

[5]. When overexpression of a mutant aPKC in MDCK II

cells, PAR-3 is mislocalization and the TJ is seriously

affected by the measurement of ions and solutes [105]. In

mouse epidermis, aPKC-i/k are observed rich in the TJs,

implying the role in the TJ function [106]. And in vitro

inhibition of aPKC in keratinocytes results in lower TER

compared to the control keratinocytes. Overexpression of

aPKC increases the TER, while overexpression of mutant

aPKC abrogated the increase. Although the aPKC–PAR3–

PAR6 complex is important for the assembly of TJs, the

molecule mechanism of regulation is still unknown. Except

the formation of complex, PKC-f, one isotype of aPKC,

directly interacts with occludin and phosphorylate the

occludin on Thr438, Thr403, Thr404 and Thr424 in Caco-2

and MDCK cells [107].

PKC’s role in TJs’s disassembly

The participation of PKCs in TJ disassembly has been

studied by diverse experiments. Treatment chorioid plexus

epithelium with PMA, the TER decreased and the decrease

can be block by Rottlerin, a specific inhibitor of PKC-d,

indicating the participation of PKC-d [108]. In the oxidant-

induced disruption of TJs, PKC-d mediates a portion

(nearly 50 %) of the disruption [109]. Later, PKC-kmediate another 50 % of the disruption, suggesting that the

coordination of PKC-d and PKC-k accounts for the oxi-

dant-induced disruption in intestinal epithelium [110].

PKC-d activation and the its subcellular translocation from

cytosol to membrane is tightly associate with the vascular

permeability in diabetic retinopathy, which can be pre-

vented by treatment of rottlerin, transfection of PKC-d-DN,

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123

or siRNA [111]. The increase permeability also accompany

the decrease proteins ZO-1,-2 in AGE-treated HRMECs.

The decrease expression of ZO-1 and ZO-2 may be the

direct cause of blood–retinal barrier break in diabetic ret-

ina, but the mechanism why the loss of ZO-1 and ZO-2,

and the possible relationship between the PKC-d should be

elucidated.

MLC, the phosphorylation substrate of ROCK and

MLCK, are also involved in the regulation of PKCs. In the

HIV-1 envelope glycoprotein gp120-induced dysfunction

of BBB, the gp120 evokes calcium release and activate

PKC-f/k, PKC-a/b II and PKC(pan)-bII, resulting in the

MLCK activation and decrease TER [112]. PKC-f and

Rho/ROCK, well collaboration, participate in the throm-

bin-induced damage of blood–retina barrier and iNOS-

induced TJ disruption in enterocytes [77, 78], paralleled to

the MLC phosphorylation. But their individual precise

contribution to the phosphorylation is not known. In con-

trast, in ischemia–reperfusion rat, the BBB permeability is

remarkable decrease after Ang-1 injunction, and in the

meantime, PKC-a and phosphorylated MLC decrease [55].

The possible reasons for the diverse effects

The effects of PKCs on the regulation of paracellular

permeability are various, some even contradictory. The

possible hypothesis for the diverse functions of PKCs may

be that the PKC family have at least ten isoforms, which

have different structures, substrates, subcellular location

and cell distribution, each one important for the regulation.

The domains every isoform has are different, and some

isoforms are wide spread, while others are restrict to some

specific cells [96]. In the study of aglycemic hypoxia-

induced BBB hyperpermeability, PKC-b II and PKC-d act

oppositely [113]. PKC-b II increases the BBB permeability

while PKC-d attenuates the hyperpermeability in one

in vitro cell model. In PMA-induce activation of PKC on

the HT29 and MDCK I cells, barrier function increase in

HT29 and decrease in MDCK I [114]. Later study showed

that the alteration of several claudins, especially claudin-2,

account for the results [22]. It is suggested that different

cell types have distinct mechanisms in the regulation of

barrier function when give the same treatment. However,

the definitive role for specific PKC isoform in modulation

of permeability and their mechanisms have largely

unknown, which is an area need to elaborate well.

MAPK pathway

The mitogen-activated protein kinases (MAPK) family are

serine-threonine kinases, which have four primary mem-

bers: extracellular signal-related kinases (ERK1/2), ERK5,

c-Jun amino-terminal kinases (JNK) and p38 kinases. The

upstream of MAPK pathway includes many receptors,

which react to extracellular stimuli such as stress and

growth factors, and downstream MAPK pathway has a lot

of transcription factors, which can regulate gene expres-

sion, proliferation, differentiation and apoptosis [64, 115,

116]. MAPK signaling pathway has subsequent activation

by kinase. MAPKKK is first activated and stimulate its

downstream MAPKK. Then MAPKK activates MAPK by

phosphorylation. MAPK can regulate intracellular process

by direct phosphorylation or activation transcription factors

to regulate gene expression.

Just like other kinases such as ROCK and PKCs,

MAPKs can direct phosphorylate transmembrane proteins

and affect paracellular permeability. In rat lung endothelial

cell line RLE, MAPK may phosphorylate the claudin-1 at

Thr203 and enhances the barrier function [3]. However, in

more cases, regulation of gene expression is another way to

influence TJ permeability. When addition of bile to IEC-6

enterocytic monolayers, the expression of ZO-1 and

occludin increase detected by western blots and the per-

meability decrease, accompanied by the enhanced phos-

phorylation of ERK1/2 [117]. The two immunosuppressant

drug, CsA and SRL, treatment alone or in combination

increase TER in LLC-PK1 monolayers, and the expression

of claudin-1 increase [38]. Inhibition of activation of

ERK1/2 can prevent the change, suggesting the ERK1/2

are involved in the regulation. In rat alveolar epithelial

cells, ERK, JNK, and p38 kinase mediate the cyclic stretch

induce dysfunction of barrier [118]. Using specific inhibi-

tor U0126 and SP600125, it is found that JNK and ERK is

responsible for the loss of barrier function, and JNK, is

associated with the reduction of occludin expression.

What’s more, it is reported that p38 kinase mediate the

burn-induced intestinal barrier dysfunction by overexpres-

sion of MLCK [119]. It is seems that the effect of para-

cellular permeability largely depend on the characteristics

of overexpression proteins, and phosphorylation and gene

expression are two major ways to modulate TJs. Since

several growth factors receptors exist in the plasma mem-

brane which can initiate the MAPK pathway, the role of

MAPK in growth factor-induced permeability increase or

decrease will be detailed talked later.

Cytokines, growth factors and hormones

The studies over the years have revealed that cytokines,

growth factors and hormones are capable of affecting TJ

proteins and paracellular permeability. As is well known,

cytokines such as IFN-c and TNF-a accelerate pathogen-

esis of inflammation, and sex hormones tightly associate

with human’s reproduction. The understanding of the

mechanisms of the physiological or pathologic procedures

6130 Mol Biol Rep (2013) 40:6123–6142

123

has a broad clinical and pharmacological application to

develop new therapies. However, until now, our knowledge

of molecular mechanisms that mediate the procedures

raised by cytokines, growth factors and hormones is limited

and many entire pathways have not been described. This

section concludes the recent observations and discusses the

putative connections among the isolated results.

Cytokines: IFN-c, TNF-a and interleukin family

IFN-c and TNF-a are two proinflammatory cytokines

which play a role in the inflammation. The modulation of

TJs and epithelial barrier function is one step in the path-

ogenesis. The past studies showed that IFN-c can impair

the barrier function in T84 human intestinal epithelial cells

and thyroid epithelial cells, with the downregulation of ZO-

1 and claudin-1, respectively [120–122]. A series of

researches gradually uncover the mechanism of regulation

the permeability. It is demonstrated that IFN-c impair the

barrier function in T84 cells by the internalization of

transmembrane proteins, including occludin, claudin-1,

claudin-4 and JAM1, visualized by immunofluorescence

microscopy [123]. The transmembrane proteins are

resembled into vacuolar apical compartment (VACs) after

treatment of IFN-c by macropinocytosis, which require the

myosin-based contraction activated by Rho/ROCK signal-

ing pathway [124, 125] (Fig. 3a). They also observed that

IFN-c increase the expression of ROCK protein and

activity of Rho. Recent study indicates that PI3-K/Akt and

NF-jB are involved in the IFN-c-induced permeability

increase in T84 cells [126] (Fig. 3a). IFN-c can activate

PI3K/Akt signaling pathway, leading to the decrease

occludin protein, and NF-jB activation depend on the

activation of PI3K. Whether PI3K/Akt pathway is a part of

pathway to endocytosis or just independently regulates the

TJ proteins is a question. And which molecule does acti-

vate NF-jB and NF-jB’s role in TJ regulation is still

remain unknown.

TNF-a is reported to increase permeability, accompanied

the change of occludin, claudin and ZO proteins [127–130].

NF-jB plays a central role in the TNF-a induced perme-

ability increase [131] (Fig. 3a). NF-jB decrease the

expression of claudin-5 and ZO-1 and increase permeability

[132]. In the process, PKC-f is essential for the TNF-a-

induced permeability, since PKC-f can regulate the NF-jB

activity. Another study showed that NF-jB mediate the

downregulation of claudin-5 in BBB, and mechanism is that

the subunit p65 inactivate claudin-5 promoter activity [133].

NF-jB can also increase the expression of MLCK in TNF-a-

induced increase permeability in Caco-2 cells, leading to the

rearrangement of actin [134–136]. They showed that MLCK

promoter contained sites for the binding of NF-jB and

increased the transcription of MLCK. The occludin

endocytosis was identified in the MLCK-dependent barrier

loss, and the process was mediated by caveolin-1 [137, 138].

Several studies indicate that the ROCK are also involved in

the regulation, and the ERK/GEF-H1/Rho/ROCK/p-MLC

pathway is the major route [139, 140] (Fig. 3a). These facts

indicate that the endocytosis of transmembrane proteins is a

important way in the regulation of TJ and paracellular per-

meability, which plays a role in cytokine-induced disruption

TJs [141]. The phosphorylation of MLC by ROCK or MLCK

can both result in the endocytosis of transmembrane proteins.

IFN-c induce macropinocytosis activated by ROCK while

TNF-a induce caveolin-1 mediated endocytosis activated by

MLCK. How the two kinases lead to different kinds of

endocytosis is waited for solving, are there other molecules

involved in the process?

Except the dissection researches of IFN-c and TNF-a,

their effects and mechanisms are usually studied together,

since the proinflammatory cytokines coexist in pathogenesis

in inflammation. The studies of Caco-2, MDCK, T84 and

Par-C10 cells, synergistical effects are observed [142–145],

while in recent paper, the synergistical effects are not

observed in hfRPE cells [146]. The study in Caco-2 cells

showed that treatment of IFN-c and TNF-a induced up-

regulation of MLCK and increase MLC phosphorylation,

which was not mediated by NF-jB [142]. Interestingly,

sequential treatment with IFN-c and TNF-a decrease TER

more than those treated simultaneously, suggesting that IFN-

c seems to prime cells for the coming of TNF-a. Their later

work interpreted that IFN-c increase the expression of TNF

receptor 2, which is responsible for the TJ disruption [147].

In the co-administration of IFN-c and TNF-a, TNF-a may

exert major effects on the cells. Additionally, in the study of

MDCK cells, the exposure of IFN-c and TNF-a activate

MEK1 and p38 MAPK pathway, accompanied by the alter-

ation of expression and location of claudin-1, claudin-2 and

occluding [143]. The activation of MAPK pathway has also

been talked in the individual TNF-a-induced disruption of

TJs. From these studies, TNF-a is thought to be the primary

function while IFN-c is accessory. Whether the MAPK

pathway is a proper candidate mediating the up-regulation of

MLCK has not been proved by facts.

Interleukins (IL) are a large groups of cytokines, and

until now, IL-1, 2, 4, 6, 10, 13, 15,17 and 18 have been

showed for the regulation of paracellular permeability in

various ways. IL-1b has been showed to increase the per-

meability and the mechanism is similar to TNF-a: activate

NF-jB and increase the transcription of MLCK gene [132,

148, 149]. Later study elucidated that how the IL-1b acti-

vate NF-jB, and the MAP3 Kinase, MEKK-1 play a cen-

tral role in the activation [150] (Fig. 3a). The activation of

MEKK-1 result in the activation of IKK catalytic subunits,

and the catalytic subunits phosphorylate and degrade IjB,

leading to the activation of NF-jB. Interestingly, recent

Mol Biol Rep (2013) 40:6123–6142 6131

123

study show that PKC-h and phosphorylation of ZO-1 is

another intracellular process in the IL-1b induced TER

decrease in BBB, which can be prevent by shRNA silence

or inhibitor Go6976 [151] (Fig. 3a). Although it can be

observed that the decrease TER is accompanied by the

phosphorylation of MLC, the effects can’t be prevented by

Go6976, suggesting phosphorylation of MLC has little

relevance to the decrease TER. How the phosphorylation of

ZO-1 affects paracellular permeability and the specific sites

which involve in the regulation should be explained [151].

In vitro and in vivo studies showed IL-6 increased the

permeability, accompanied by the redistribution of ZO

proteins [152, 153], and the PKCs plays a partial role in the

alteration. However, IL-6 can also increase the perme-

ability to cation by overexpression of claudin-2 [36]. The

transcriptional factor, caudal-related homeobox (Cdx) 2,

activated by the MEK/ERK and PI3K pathway, bind the

claudin-2 promoter sequence and increase the expression

of claudin-2. The function of IL-13 also correlates with the

increase claudin-2 levels, and PI3K and STAT6 pathway

are responsible for the effects [127, 154]. IL-15 activates

an NF-jB luciferase reporter, and regulates the transcrip-

tion of p65, which phosphorylate and degrade IjB,

resulting in activation of NF-jB [155]. IL-17A induced

disruption of the BBB involves the generation of ROS by

NAD(P)H oxidase and xanthine oxidase, and MLCK is the

downstream molecular [156]. IL-17F lead to the hyper-

permeability and actin rearrangement in RPMVECs mon-

olayers via a PKC-dependent way [157]. In the condition of

EtOH intoxication together with burn injury, IL-18 directly

increase paracellular permeability in the intestinal mucos,

measured by the FITC-dextran [158]. Occludin and

Fig. 3 Brief conclusions of

signaling pathway induced by

cytokines, growth factors and

hormones. a IFN-c can activate

Rho signaling pathway and

PI3K. Rho signaling provokes

macropinocytosis of

transmembrane proteins, and

PI3K decreases the expression

of occludin. NF-jB increases

the expression of MLCK in

TNF-a and IL-1b treatment,

however, decreases the

expression of claudin and ZO in

TNF-a treatment. The

overexpression of MLCK in

TNF-a treatment instigates

occludin endocytosis mediated

by caveolin-1. TNF-a can also

activate ERK, subsequent, Rho

signaling pathway to disrupt

tight junctions. Besides the

activation of NF-jB, activation

of PKC-h and phosphorylation

of ZO-1 is another way to affect

paracellular permeability. b The

effect of TGF-b relies on

activation of Smad and ERK

pathway, which both increases

the expression of claudin. Smad

can also activate p38 MAPK

and then Rho signaling

pathway. Two hormones,

glucocorticoid and

progesterone, both bind to the

receptors in cells and regulate

the expression of

transmembrane proteins

6132 Mol Biol Rep (2013) 40:6123–6142

123

claudin-1 are observed dephosphorylation. But the mech-

anism how IL-18 regulate transmembrane proteins remains

unknown. One hypothesis is that MyD88/IRAK and the

downstream molecules such as MAPK and PKC, are

involved in the IL-18-induced regulation.

Growth factors

Growth factors are a family of proteins or steroid hor-

mones, which can stimulate cell growth, proliferation and

differentiation. Several growth factors have been found to

modulate the barrier function, and the effects are divided,

dependent on growth factor and tissue specific. Hepatocyte

growth factor (HGF) is showed to be secreted by stromal

cells and increase the TER of uterine epithelial cells, with

the decrease of TNF-a [159]. It is reported that HGF can

maintain the TJs and against the disruption of BBB, while

in MDCK cells and blood–testis barrier, HGF correlates

with the disassembly of TJs [160–162]. Another research

showed that HGF inhibits the expression of claudin-2 by

the activation of ERK1/2, and increase TER, which can be

prevented by ERK 1/2 inhibitor U0126 [163]. MAPK

pathway also mediate epidermal growth factor (EGF)-

induced protection of TJs. MAPK interacts with occludin

and mediates EGF-induced prevention of TJ disruption by

hydrogen peroxide [164], while EGF also accelerates

clathrin-dependent endocytosis of claudin-2 by activation

MEK/ERK pathway [165]. PKCs are involved in the EGF-

induced translocation of ZO-1 and occludin from cyto-

plasm to cell borders, facilitating the assembly of TJ in

poorly differentiated gastric cancer cell line [166]. Previous

study showed platelet-derived growth factor (PDGF)

increase the permeability to dextran, with the redistribution

of occludin and ZO-1 from cell borders to cytoplasm [167].

Recent two studies indicates that PDGF mediate the mor-

phine and cocaine-induced vascular permeability increase

in BBB [168, 169]. Vascular endothelial growth factor

(VEGF) can increase the vascular permeability and asso-

ciate with diabetic retinopathy and brain tumors, and its

effect mediate by the activation of PKC and phosphoryla-

tion of occluding [170]. Phosphorylation of occludin at

Ser-490 causes occludin ubiquitination and trafficking,

leading to increase permeability [171].

Transforming growth factor (TGF)-b is a multifunction

growth factors which has roles in the cell proliferation and

differentiation. The family has three members: TGF-b1,

TGF-b2 and TGF-b3 [172, 173]. TGF-b displays exert

opposite effects on TJs. In studies of uterine, vas deferens,

and vascular endothelial cell, TGF-b are showed to

increase permeability [130, 174–176], while in response for

extracellular stimuli such as cyclosporine and enterohem-

orrhagic Escherichia coli, TGF-b tend to increase barrier

function [177, 178]. The above mentioned Rho/ROCK and

MAPK pathway are still mediate the intracellular process.

Rho/ROCK signaling pathway mediate the TGF-b induced

increase permeability, with the MLC phosphorylation and

cytoskeletal reorganization [174, 175] (Fig. 3b). TGF-b1

also activate ERK1/2, increase the expression of claudin-1

and ZO-2, then increase TER when treat MDCK cells with

cyclosporine [177]. The canonical Smad dependent sig-

naling pathway of TGF can’t be overlooked during the TJ

regulation. In response to the invasion of enterohemor-

rhagic Escherichia coli (EHEC), TGF-b increase expres-

sion of claudin-1 and increase TER via activation the ERK

MAPK and Smad pathways, which can be prevented by

inhibitor PD98059 and Ad-SMAD7, respectively [178].

Another mechanism found in lung endothelial cells is

Smad2, activated by TGF-b1, subsequent activate p38

MAPK and RhoA signaling pathway, leading to the dis-

ruption of TJs [179] (Fig. 3b). These facts suggest the

different signal pathways are crosstalk to influence the TJ

proteins and paracellular permeability. A novel mechanism

reported that TGF-b3 perturb the BTB by clathrin-medi-

ated endocytosis of occludin and JAM-A, which can be

specific prevented by knockdown of TGF- b receptor I (T

bR1) [180]. When adding TGF-b3 to oral epithelial cells,

the permeability first decreases, and then increases,

accompanied by the change of claudin and ZO proteins

[181]. It seems that the members of TGF-b family acts in

different mechanism, so it is better to clarify function of

one member in the research, which will be more precise.

Hormones

Hormones are the important chemicals regulating the

development and metabolism of organism, released by

specific cells or tissues and brought by bloodstream. They

also regulate the paracellular permeability. Mineralocorti-

coid and glucocorticoid, released by adrenal gland, are

function as regulation of TJs. Aldosterone, the mineralo-

corticoid, can phosphorylate endogenous claudin-4 and

increase permeability to anion, while the Na? passage is

unaffected in collecting duct cells [182]. Glucocorticoid

has been detailed researched in past years. Glucocorticoid

can increase the expression of occludin and claudin-5 and

stabilize the brain–blood barrier [183, 184]. Dexametha-

sone exposure in utero improve fetal blood–brain barrier

function by increase claduin-1, claudin-5, but induce the TJ

disassembly in human amniotic epithelium [185, 186].

Glucocorticoid receptor (GR) is responsible for the over-

expression of proteins [187–190] (Fig. 3b). GR directly

bind promoter regions in occludin and claudin so as to

increase the transcriptional activity to preserve the barrier

function, and can also inhibit the MLCK promoter activity

in TNF-a-induced condition, although precise mechanism

should be elucidated in future [191]. Recent study on the

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123

airway epithelial cell monolayers showed that permeability

measured by TER and dextran are increased when treating

dexamethasone, fluticasone propionate or budesonide

[192]. The enhanced barrier function is accompanied by

redistribution of ZO-1 and claudin, which can be prevented

by the knockdown of EGFR, suggesting EGFR signaling

pathway mediate the TJs assembly. It is hypothesis that

glucocorticoid increase the binding between EGF and

EGFR, but does not prove with facts.

Sex hormones play an important role in the growth and

function of reproductive system, the development of sec-

ondary sex characteristics and estrous cycle, which contain

androgen, estrogen, and progesterone. Testosterone, one

member of androgens, have been indicated down-regula-

tion the expression of claudin-3 and increase the perme-

ability of BTB to biotin [193]. Recent study showed that

testosterone internalized occludin mediated by clathrin,

similar to TGF-b3’s action [194]. Interestingly, testoster-

one promotes BTB barrier function, in contrast to TGF-

b3’s role discussed above. It is found that testosterone

endocytose the proteins and then still transcytose and

recycle to the Sertoli cell surface, while TGF-b3 internalize

proteins and the protein will be degradated in cell. The

coordinated regulation of TGF-b3 and testosterone help

preleptotene spermatocytes transit while immunological

barrier is not disruption, which is important for spermato-

genesis. However, how the vesicles are determined and

how much the proteins are endocytosis to recycle or deg-

radation is not answered. In female, junction proteins are

change when in the period of menstrual cycle and preg-

nancy, and progesterone are involved in the regulation [33,

195–197]. In the rat early pregnancy, progesterone increase

occludin and claudin-4 and improve the barrier function of

glandular epithelium [33]. It is also reported that proges-

terone increase claudin-3 and claudin-4 in a dose-depen-

dent manner organ-cultured amniotic membranes, and

in vivo study showed that with the treatment of proges-

terone receptor antagonist, RU-486, paracellular perme-

ability increase accompanied by the internalization and

decrease expression of claudin-3 and claudin-4 [198]. It is

indicated that progesterone/PR signaling pathway mediate

the increase barrier function of TJs (Fig. 3b).

Interaction of transport processes and paracellular

permeability

It is mentioned above that epithelial permeability are the

sum of transcellular transport and paracellular transport.

The transcellular transport largely depends on the specific

channels, primary and secondary active or passive. The

past researches indicate the transporters and the transport

process affect the paracellular permeability. It seems that

the modifications of paracellular permeability often initiate

in two ways: the change of driving force between intra-

membrane and extramembrane cause the paracellular per-

meability, or directly induces the signaling pathway to

regulate the paracellular permeability[14].

Na,K-ATPase

Na,K-ATPase, one member of the P-type subfamily, is a

heterodimer composed of a subunit and b subunit, and the

c subunit is optional. The major function of Na,K-ATPase

is the carrier of Na? and K? ions across the plasma

membranes and maintain Na? and K? gradients. The

energy is from the hydrolysis of ATP, and the a subunit is

responsible for the hydrolysis [199, 200]. In addition to the

ion transport, many studies imply Na,K-ATPase can also

modulate TJs and paracellular permeability, and often

works as a signal transducer, independent on ion channel

function. Interestingly, high concentration of ouabain can

block the Na,K-ATPase, while low concentration of oua-

bain seems to initiate the signaling pathway [201, 202].

Na,K-ATPase is the putative receptor of the both high and

low concentrations, and the effect of ouabain is dose-

dependent [203]. For example, when treatment of mouse

embryos with high concentration of ouabain in preim-

plantation development, the permeability to FITC-dextran

increase, accompanied by the redistribution of occludin and

ZO-1, while the low concentration of ouabain seems have

no effect on mouse embryos [204]. Their later study

showed that low concentration of ouabain can phosphory-

late and activate SRC family kinase (SFK) members, Src

and Yes, which are required for the TJ formation and

permeability decrease in trophectoderm of blastocyst [201].

Low concentrations of ouabain is also showed to increase

expression of claudin-1, claudin-2 and claudin-4 so as to

increase TER, the increase level of claudin-1 through c-Src

and ERK1/2 activation, while claudin-4 only depend on

ERK1/2 [202]. The molecular mechanism of ouabain’s

dose-dependent manner have been uncovered in recent

papers. Low concentration of ouabain stimulates the

association of a1 subunit and NHE-1 and the inhibition

function resulted from redistribution of the a1 subunit and

NHE3 [205, 206]. It is indicated that a1 subunit, play an

important roles in the ouabain-induced regulation.

The past evidences have showed that, a subunit and bsubunit, can individually interact with other proteins, may

have a further understanding the role of Na,K-ATPase in

the regulation. a-Subunit have been showed to interact with

Src, PLC-c1, PP2A, PI3K, PKC-g [207–212] (Fig. 4). In

the blood–labyrinth barrier in CBA/CaJ mouse cochlea,

Na,K-ATPase a1 is abundant in the barrier, interaction

with PKC-g, occludin phosphorylation and vascular per-

meability increase when noise induce condition [211]. b-

6134 Mol Biol Rep (2013) 40:6123–6142

123

Subunit can interact with annexin II, PP2A [210, 212]

(Fig. 4). In human pancreatic epithelial cell line HPAF-II,

inhibition Na,K-ATPase by ouabain and K? depletion

reduced the activity of PP2A, increased the phosphoryla-

tion of occludin and increased the permeability [210].

PP2A is observed associates with the b subunit and interact

with occludin, suggesting PP2A, occludin and Na,K-

ATPase may form a complex to regulate the TJ perme-

ability. For the formation of TJs of trophectoderm just

talked abovementioned, b1 subunit is required for the

blastocyst formation, which inhibited by specific knock-

down b1 subunit in mouse one-cell zygotes using siRNA

[213]. In light of these studies, Na,K-ATPase function as a

scaffolding protein and the subunit interactions with dif-

ferent proteins in specific tissues to regulate the paracel-

lular permeability. How the subunits provoke the

downstream signaling pathways remains to be determined.

Hydrolysis of a subunit may involve, but the interaction of

b subunit and other molecules and the possible binding

domains is waited for defined.

Na?-coupled transporters: SGLT and NHE

Sodium-glucose transporters (SGLT) and sodium–hydro-

gen exchanger (NHE) are two secondary active transport-

ers, which depend on sodium gradient across the cell

membrane. The SGLT family has 12 members, which play

a important role in glucose absorption [214]. SGLT can

transport sodium and glucose in the same direction, known

as symporters, while NHE transport sodium and hydrogen

in opposite direction, known as antiporters, which maintain

cytoplasmic pH and sodium reabsorption [215, 216]. The

activation of SGLT have been described to increase the

paracellular permeability, both in the Caco-2 cells after

transfection and in human beings in vivo, suggesting a

coordinating work of transcellular and paracellular

absorption [217, 218]. In intestinal epithelial cell, MLCK

and MLC phosphorylation mediate the SGLT1 induce

permeability increase, and the isoform of MLCK1 strongly

associate with the regulation [217, 219, 220]. Later, a rel-

atively complete signaling pathway emerged after a series

of studies, explain how the SGLT1 activate MLCK and the

NHE are also involved in the regulation of TJ (Fig. 4).

Activation of NHE3, one isoform of NHE familiy, lead to

the cytoplasmic alkalinization, and activate MLCK activ-

ity, resulting in TER decrease [221]. This effect can be

inhibited by amiloride, the NHE inhibitor. However, the

inhibition does not work when SGLT1 is inhibited, sug-

gesting activation of NHE3 is downstream action of

SGLT1. SGLT1 is showed to activate p38 MAPK, and the

kinase subsequent phosphorylate downstream moleculers,

MAPK-activated proteins kinase-2 (MAPKAPK-2) [222,

223]. Akt2 can be directly phosphorylated by MAPKAPK-

2 at serine 473, and phosphorylate threonine 567 of ezrin,

which is thought as a critical intermediate in the recruit-

ment and increase of NHE3 [224, 225]. Ezrin can bind to

the NHE regulatory factors NHERF-1 and -2, and link the

NHERF–NHE complex to actin. Although the signal

pathway of SGLT1 induced permeability increase is seems

perfect, the mechanism how SGLT activate p38 MAPK

and cytoplasmic alkalinization activate MLCK is unknown.

Waheed’s group showed that membrane depolarization can

regulate TJs by ERK/GEF-H1/Rho/ROCK/MLC pathway

[90]. These facts illustrate the osmotic pressure change

could be a central process in the activation of p38 MAPK,

ERK and MLCK, and well understanding the mechanism

of osmosensing can give a deep insight into the regulation

process in the future.

Since NHE involves in SGLT-induced regulation of TJs,

NHE individually can regulate the paracellular permeability.

Fig. 4 The left is Na-K-ATPase, composed of a subunit and bsubunit. The a subunit interacts with Src, PP2A, PI3K, PKC-g, while

b subunit interacts with Src and PP2A. Activation of Na-K-ATPase

initiates downstream pathways to affect tight junctions. The right is

the couple of sodium–glucose transporters (SGLT) and sodium–

hydrogen exchanger (NHE) involved in the regulation of paracellular

permeability. SGLT activate p38 MAPK, subsequent, MAPKAPK-2,

AKT2 and Ezrin, and then recruitment NHE3 to apical membrane.

The alkalinization raised by NHE3 activates MLCK and results in

permeability increase

Mol Biol Rep (2013) 40:6123–6142 6135

123

Sabiporide, a specific NHE-1 inhibitor, can attenuate the

BBB permeability increase and disruption of occludin and

ZO-1 in vivo and in vitro when confront ischemia/hypoxia

[226]. It is hypothesis that in ischemia/hypoxia, NHE-1 is

activated by intracellular acidosis, leading to the increase

Na? in cell. The excessive Na? in cell stimulate Na?/Ca?

exchanger, which result in the Ca2? overload and negatively

affect the TJs [226, 227]. Next step is to prove this mech-

anism of the protective function of NHE-1 inhibition on

BBB, for example, Ca?/- chelation, can be used. In the

study of porcine ileal mucosa, inhibition NHE2 remarkably

increase the TER during the recovery after ischemia [228].

In contrast, NHE2 knockdown mice have greater paracel-

lular permeability during the recovery, accompanied by the

dephosphorylation of occludin and claudin-1 [229]. It seems

that NHE2 have different functions in different species, and

the mechanism of NHE2’s effect on TJs can be studied in

the future. Unlike the NHE3, which can activate MLCK, the

dephosphorylation of occludin indicates that NHE2 may

activate ROCK or PKC or inhibit phosphatase.

Chloride channels: CFTR and ClC-2

The cystic fibrosis transmembrane conductance regulator

(CFTR) is a cAMP-activated glycoprotein which functions

as an chloride channel. The protein has a wide range dis-

tribution, including intestine, airways, secretory glands,

bile ducts, and epididymis, and the malfunction of CFTR

lead to cystic fibrosis [230]. The recent studies in airway

cell lines showed different attitudes towards CFTR and

TER change. Pierre LeSimple’s works show that CFTR

trafficking and apical membrane localization increase TER

and decrease the paracellular permeability, which can’t be

affected by CFTR inhibitor or low-chloride medium, sug-

gesting the ion channel function is not required for the

maintenance of TJs [231]. Treatment of enistein, the

tyrosine kinase inhibitor, reduced CFTR’s effect on barrier

function, indicating tyrosine kinases are involved in the

regulation. However, it is observed that CFTR-defective

cell line have higher TER than its corrected counterpart,

and inhibition of CFTR of normal cell or corrected cell line

increase TER, while activation of CFTR by IBMX and

forskolin decrease TER [232]. The inhibition of CFTR is

accompanied by the alteration of actin, suggesting CFTR

may interact with cytoskeleton to modulate the TJ. Whe-

ther the interaction is direct or indirect is unknown. In

consistent with the result, Nelly Weiser’s work indicates

activation of CFTR in normal cell line by cAMP increase

the paracellular permeability [233]. This effect can be

blocked by MLCK inhibitor ML-7, implying the MLCK is

involved. The study also demonstrates that the ion channel

function is required for the regulation, in contrast to the

early study, and the different cell culture method and the

way of treatment may be responsible for the paradoxical

conclusion [233]. It is assumed that activation of CFTR and

depolarization may induce the TJ regulation, similar to

Waheed’s work. Whether CFTR individually interact with

TJ proteins just like Na-K-ATPase or depolarization is the

central mechanism remains unknown. More facts need to

accumulate to reveal the effect and mechanism of CFTR in

TJ regulation.

Another chloride channel, ClC-2, one member of ClC

family, is also involved in the regulation. Adding the lubi-

prostone, ClC-2 agonist, to porcine ileum and ascending

colon after ischemia, TER increases and the permeability to

mannitol decreases, accompanied by the occludin exclusively

localized to TJs [234]. However, the later study in murine

intestinal mucosa is opposite. The knockdown mice signifi-

cantly increase the TER and decrease permeability to man-

nitol compared to normal mice, suggesting ClC-2 improve the

barrier function [235]. Inhibition MLCK in normal mice

mucosa intestine increase TER, but the relationship between

ClC-2 and MLCK is not clear. And the contradictory results

of two studies need to be elucidated in the future.

Conclusion and perspective

TJs are intercellular protein complexes which determines

the paracellular passage to ions or solutes. Until now, the

protein composition of TJs is well unravelled. However,

our understanding of protein interactions and modifications

in molecular levels is so limited. The extracellular loops of

claudin and occludin and their interactions organize pores

to select ions by charges and macromolecules by size, and

these selectivity and molecular basis is a field that could be

explored. The modifications of occludin and claudin, such

as phosphorylation, and their effects on TJs are still

unpredictable. The signaling pathways, mainly ROCK,

PKCs and MAPK pathway, phosphorylate, relocate or

affect the expression of TJ proteins and the paracellular

permeability is alteration. The signaling pathway also

mediate the cytokines, growth factors and hormone’s reg-

ulation of TJs, and the interaction between transcellular

transporters and TJ proteins. However, in many cases the

complete signaling pathway to regulate TJ is still unclear,

which may represent a novel therapeutic target for

inflammation or burn or other stimulus-induced epithelium

disruption. The signaling pathway is always intricate, and

most of it is crosstalk, which makes it difficult to give a

conclusive illumination. The paracellular passage, together

with transcellular process, affects the absorption and

secretion in epithelium. However, the precise molecular

mechanisms of interaction between paracellular and

transcellular passage are waiting for elucidated.

6136 Mol Biol Rep (2013) 40:6123–6142

123

Acknowledgments We are indebted to all members of the Sperm

Laboratory at Zhejiang University for their enlightening discussion.

This project was supported in part by Zhejiang Provincial Natural

Science Foundation of China (Grant No. Y2080362), the National

Natural Science Foundation of China (Nos. 81100393 and 41276151),

and Zhejiang Provincial Natural Science Foundation of China (Grant

No. Y2100296).

References

1. Farquhar MG, Palade GE (1963) Junctional complexes in vari-

ous epithelia. J Cell Biol 17:375–412

2. Cummins PM (2012) Occludin: one protein, many forms. Mol

Cell Biol 32(2):242–250

3. Fujibe M (2004) Thr203 of claudin-1, a putative phosphoryla-

tion site for MAP kinase, is required to promote the barrier

function of tight junctions. Exp Cell Res 295(1):36–47

4. Harhaj NS, Antonetti DA (2004) Regulation of tight junctions

and loss of barrier function in pathophysiology. Int J Biochem

Cell Biol 36(7):1206–1237

5. Matter K, Balda MS (2003) Signalling to and from tight junc-

tions. Nat Rev Mol Cell Biol 4(3):225–236

6. Terry S (2010) Rho signaling and tight junction functions.

Physiology 25(1):16–26

7. Balda MS, Matter K (2009) Tight junctions and the regulation of

gene expression. Biochim Biophys Acta 1788(4):761–767

8. Farkas AE, Capaldo CT, Nusrat A (2012) Regulation of epi-

thelial proliferation by tight junction proteins. Ann NY Acad Sci

1258(1):115–124

9. Ikenouchi J (2005) Tricellulin constitutes a novel barrier at tri-

cellular contacts of epithelial cells. J Cell Biol 171(6):939–945

10. Krug SM (2009) Tricellulin forms a barrier to macromolecules

in tricellular tight junctions without affecting ion permeability.

Mol Biol Cell 20(16):3713–3724

11. Mariano C (2011) A look at tricellulin and its role in tight

junction formation and maintenance. Eur J Cell Biol

90(10):787–796

12. Guillemot L (2008) The cytoplasmic plaque of tight junctions: a

scaffolding and signalling center. Biochim Biophys Acta

1778(3):601–613

13. Bauer H (2010) The dual role of zonula occludens (ZO) pro-

teins. J Biomed Biotechnol 2010:402593

14. Kapus A, Szaszi K (2006) Coupling between apical and para-

cellular transport processes. Biochem Cell Biol 84(6):870–880

15. Buchert M, Turksen K, Hollande F (2011) Methods to examine

tight junction physiology in cancer stem cells: TEER, paracel-

lular permeability, and dilution potential measurements. Stem

Cell Rev 8(3):1030–1034

16. Shen L (2011) Tight junction pore and leak pathways: a

dynamic duo. Annu Rev Physiol 73:283–309

17. Rajasekaran SA, Beyenbach KW, Rajasekaran AK (2008)

Interactions of tight junctions with membrane channels and

transporters. Biochim Biophys Acta 1778(3):757–769

18. Furuse M (1998) Claudin-1 and -2: novel integral membrane

proteins localizing at tight junctions with no sequence similarity

to occludin. J Cell Biol 141(7):1539–1550

19. Mineta K (2011) Predicted expansion of the claudin multigene

family. FEBS Lett 585(4):606–612

20. Lal-Nag M, Morin PJ (2009) The claudins. Genome Biol

10(8):235

21. Findley MK, Koval M (2009) Regulation and roles for claudin-

family tight junction proteins. IUBMB Life 61(4):431–437

22. Sjo A, Magnusson KE, Peterson KH (2010) Protein kinase C

activation has distinct effects on the localization, phosphorylation

and detergent solubility of the claudin protein family in tight and

leaky epithelial cells. J Membr Biol 236(2):181–189

23. Leach L (2002) Vasculogenesis, angiogenesis and the molecular

organisation of endothelial junctions in the early human pla-

centa. J Vasc Res 39(3):246–259

24. Lievano S (2006) Endothelia of term human placentae display

diminished expression of tight junction proteins during pre-

eclampsia. Cell Tissue Res 324(3):433–448

25. Kirk A (2010) Differential expression of claudin tight junction

proteins in the human cortical nephron. Nephrol Dial Transplant

25(7):2107–2119

26. Furuse M (2002) Claudin-based tight junctions are crucial for

the mammalian epidermal barrier: a lesson from claudin-1-

deficient mice. J Cell Biol 156(6):1099–1111

27. Nitta T (2003) Size-selective loosening of the blood–brain

barrier in claudin-5-deficient mice. J Cell Biol 161(3):653–660

28. Furuse M (2010) Molecular basis of the core structure of tight

junctions. Cold Spring Harb Perspect Biol 2(1):a002907

29. Krause G (2008) Structure and function of claudins. Biochim

Biophys Acta 1778(3):631–645

30. Yu AS (2003) Claudin-8 expression in Madin–Darby canine

kidney cells augments the paracellular barrier to cation perme-

ation. J Biol Chem 278(19):17350–17359

31. Angelow S, Kim KJ, Yu AS (2006) Claudin-8 modulates

paracellular permeability to acidic and basic ions in MDCK II

cells. J Physiol 571(Pt 1):15–26

32. Wray C (2009) Claudin-4 augments alveolar epithelial barrier

function and is induced in acute lung injury. Am J Physiol Lung

Cell Mol Physiol 297(2):L219–L227

33. Nicholson MD, Lindsay LA, Murphy CR (2010) Ovarian hor-

mones control the changing expression of claudins and occludin

in rat uterine epithelial cells during early pregnancy. Acta His-

tochem 112(1):42–52

34. Milatz S (2010) Claudin-3 acts as a sealing component of the

tight junction for ions of either charge and uncharged solutes.

Biochim Biophys Acta 1798(11):2048–2057

35. Muto S (2010) Claudin-2-deficient mice are defective in the

leaky and cation-selective paracellular permeability properties

of renal proximal tubules. Proc Natl Acad Sci USA

107(17):8011–8016

36. Suzuki T, Yoshinaga N, Tanabe S (2011) Interleukin-6 (IL-6)

regulates claudin-2 expression and tight junction permeability in

intestinal epithelium. J Biol Chem 286(36):31263–31271

37. Yu AS, Cheng MH, Coalson RD (2010) Calcium inhibits

paracellular sodium conductance through claudin-2 by compet-

itive binding. J Biol Chem 285(47):37060–37069

38. Martin-Martin N (2010) Sirolimus and cyclosporine A alter

barrier function in renal proximal tubular cells through stimu-

lation of ERK1/2 signaling and claudin-1 expression. Am J

Physiol Renal Physiol 298(3):F672–F682

39. Rosenthal R (2010) Claudin-2, a component of the tight junc-

tion, forms a paracellular water channel. J Cell Sci 123(Pt

11):1913–1921

40. Simon DB (1999) Paracellin-1, a renal tight junction protein

required for paracellular Mg2? resorption. Science 285(5424):

103–106

41. Hou J (2008) Claudin-16 and claudin-19 interact and form a

cation-selective tight junction complex. J Clin Invest

118(2):619–628

42. Hou J (2009) Claudin-16 and claudin-19 interaction is required

for their assembly into tight junctions and for renal reabsorp-

tion of magnesium. Proc Natl Acad Sci USA 106(36):

15350–15355

43. Hou J (2010) Claudin-4 forms paracellular chloride channel in

the kidney and requires claudin-8 for tight junction localization.

Proc Natl Acad Sci USA 107(42):18010–18015

Mol Biol Rep (2013) 40:6123–6142 6137

123

44. Krug SM (2012) Claudin-17 forms tight junction channels with

distinct anion selectivity. Cell Mol Life Sci 69(16):2765–2778

45. Colegio OR (2002) Claudins create charge-selective channels in

the paracellular pathway between epithelial cells. Am J Physiol

Cell Physiol 283(1):C142–C147

46. Alexandre MD (2007) The first extracellular domain of claudin-

7 affects paracellular Cl-permeability. Biochem Biophys Res

Commun 357(1):87–91

47. Van Itallie CM (2006) Two splice variants of claudin-10 in the

kidney create paracellular pores with different ion selectivities.

Am J Physiol Renal Physiol 291(6):F1288–F1299

48. Furuse M (1993) Occludin: a novel integral membrane protein

localizing at tight junctions. J Cell Biol 123(6 Pt 2):1777–1788

49. Feldman GJ, Mullin JM, Ryan MP (2005) Occludin: structure,

function and regulation. Adv Drug Deliv Rev 57(6):883–917

50. Sanchez-Pulido L (2002) MARVEL: a conserved domain

involved in membrane apposition events. Trends Biochem Sci

27(12):599–601

51. Saitou M (1998) Occludin-deficient embryonic stem cells can

differentiate into polarized epithelial cells bearing tight junc-

tions. J Cell Biol 141(2):397–408

52. Saitou M (2000) Complex phenotype of mice lacking occludin, a

component of tight junction strands. Mol Biol Cell

11(12):4131–4142

53. Schulzke JD (2005) Epithelial transport and barrier function in

occludin-deficient mice. Biochim Biophys Acta 1669(1):34–42

54. McCarthy KM (1996) Occludin is a functional component of the

tight junction. J Cell Sci 109(Pt 9):2287–2298

55. Yu H (2012) Recombinant human angiopoietin-1 ameliorates

the expressions of ZO-1, occludin, VE-cadherin, and PKCalpha

signaling after focal cerebral ischemia/reperfusion in rats. J Mol

Neurosci 46(1):236–247

56. Noth R (2011) Increased intestinal permeability and tight

junction disruption by altered expression and localization of

occludin in a murine graft versus host disease model. BMC

Gastroenterol 11:109

57. Wong V, Gumbiner BM (1997) A synthetic peptide corre-

sponding to the extracellular domain of occludin perturbs the

tight junction permeability barrier. J Cell Biol 136(2):399–409

58. Lacaz-Vieira F (1999) Small synthetic peptides homologous to

segments of the first external loop of occludin impair tight

junction resealing. J Membr Biol 168(3):289–297

59. Balda MS (2000) Multiple domains of occludin are involved in

the regulation of paracellular permeability. J Cell Biochem

78(1):85–96

60. Al-Sadi R (2011) Occludin regulates macromolecule flux across

the intestinal epithelial tight junction barrier. Am J Physiol

Gastrointest Liver Physiol 300(6):G1054–G1064

61. Rao R (2009) Occludin phosphorylation in regulation of epi-

thelial tight junctions. Ann NY Acad Sci 1165:62–68

62. Dorfel MJ, Huber O (2012) Modulation of tight junction

structure and function by kinases and phosphatases targeting

occludin. J Biomed Biotechnol 2012:807356

63. Dorfel MJ, Huber O (2012) A phosphorylation hotspot within the

occludin C-terminal domain. Ann NY Acad Sci 1257(1):38–44

64. Gonzalez-Mariscal L, Tapia R, Chamorro D (2008) Crosstalk of

tight junction components with signaling pathways. Biochim

Biophys Acta 1778(3):729–756

65. Etienne-Manneville S, Hall A (2002) Rho GTPases in cell

biology. Nature 420(6916):629–635

66. Beckers CM, van Hinsbergh VW, van Nieuw AG (2010)

Driving Rho GTPase activity in endothelial cells regulates

barrier integrity. Thromb Haemost 103(1):40–55

67. Spindler V, Schlegel N, Waschke J (2010) Role of GTPases in

control of microvascular permeability. Cardiovasc Res

87(2):243–253

68. Bruewer M (2004) RhoA, Rac1, and Cdc42 exert distinct effects

on epithelial barrier via selective structural and biochemical

modulation of junctional proteins and F-actin. Am J Physiol Cell

Physiol 287(2):C327–C335

69. Nakagawa O (1996) ROCK-I and ROCK-II, two isoforms of

Rho-associated coiled-coil forming protein serine/threonine

kinase in mice. FEBS Lett 392(2):189–193

70. Yamamoto M (2008) Phosphorylation of claudin-5 and occludin

by rho kinase in brain endothelial cells. Am J Pathol

172(2):521–533

71. Ivanov AI (2004) Role for actin filament turnover and a myosin

II motor in cytoskeleton-driven disassembly of the epithelial

apical junctional complex. Mol Biol Cell 15(6):2639–2651

72. Hirano K (2003) Protein kinase network in the regulation of

phosphorylation and dephosphorylation of smooth muscle

myosin light chain. Mol Cell Biochem 248(1–2):105–114

73. Maciver SK, Hussey PJ (2002) The ADF/cofilin family: actin-

remodeling proteins. Genome Biol 3(5):reviews3007

74. Ishibashi F (2008) High glucose increases phosphocofilin via

phosphorylation of LIM kinase due to Rho/Rho kinase activa-

tion in cultured pig proximal tubular epithelial cells. Diabetes

Res Clin Pract 80(1):24–33

75. Thirone AC (2009) Hyperosmotic stress induces Rho/Rho

kinase/LIM kinase-mediated cofilin phosphorylation in tubular

cells: key role in the osmotically triggered F-actin response. Am

J Physiol Cell Physiol 296(3):C463–C475

76. Shen L, Turner JR (2005) Actin depolymerization disrupts tight

junctions via caveolae-mediated endocytosis. Mol Biol Cell

16(9):3919–3936

77. Wu LL (2011) Epithelial inducible nitric oxide synthase causes

bacterial translocation by impairment of enterocytic tight junc-

tions via intracellular signals of Rho-associated kinase and

protein kinase C zeta. Crit Care Med 39(9):2087–2098

78. Ruiz-Loredo AY, Lopez E, Lopez-Colome AM (2011) Throm-

bin promotes actin stress fiber formation in RPE through Rho/

ROCK-mediated MLC phosphorylation. J Cell Physiol

226(2):414–423

79. Ma T (2012) Evidence for involvement of ROCK signaling in

bradykinin-induced increase in murine blood–tumor barrier

permeability. J Neurooncol 106(2):291–301

80. Xie H (2012) Role of RhoA/ROCK signaling in endothelial-

monocyte-activating polypeptide II opening of the blood–tumor

barrier: role of RhoA/ROCK signaling in EMAP II opening of

the BTB. J Mol Neurosci 46(3):666–676

81. Nagumo Y (2008) Cofilin mediates tight-junction opening by

redistributing actin and tight-junction proteins. Biochem Bio-

phys Res Commun 377(3):921–925

82. Hong F (2011) Biochemistry of smooth muscle myosin light

chain kinase. Arch Biochem Biophys 510(2):135–14683. Satpathy M (2004) Thrombin-induced phosphorylation of the

regulatory light chain of myosin II in cultured bovine corneal

endothelial cells. Exp Eye Res 79(4):477–486

84. Haorah J (2005) Ethanol-induced activation of myosin light

chain kinase leads to dysfunction of tight junctions and blood–

brain barrier compromise. Alcohol Clin Exp Res

29(6):999–1009

85. Srinivas SP (2006) Histamine-induced phosphorylation of the

regulatory light chain of myosin II disrupts the barrier integrity

of corneal endothelial cells. Invest Ophthalmol Vis Sci

47(9):4011–4018

86. Fedwick JP (2005) Helicobacter pylori activates myosin light-

chain kinase to disrupt claudin-4 and claudin-5 and increase

epithelial permeability. Infect Immun 73(12):7844–7852

87. Wroblewski LE (2009) Helicobacter pylori dysregulation of

gastric epithelial tight junctions by urease-mediated myosin II

activation. Gastroenterology 136(1):236–246

6138 Mol Biol Rep (2013) 40:6123–6142

123

88. Shen L (2006) Myosin light chain phosphorylation regulates

barrier function by remodeling tight junction structure. J Cell Sci

119(Pt 10):2095–2106

89. Benais-Pont G (2003) Identification of a tight junction-associ-

ated guanine nucleotide exchange factor that activates Rho and

regulates paracellular permeability. J Cell Biol 160(5):729–740

90. Waheed F (2010) Extracellular signal-regulated kinase and

GEF-H1 mediate depolarization-induced Rho activation and

paracellular permeability increase. Am J Physiol Cell Physiol

298(6):C1376–C1387

91. Birukova AA (2006) GEF-H1 is involved in agonist-induced

human pulmonary endothelial barrier dysfunction. Am J Physiol

Lung Cell Mol Physiol 290(3):L540–L548

92. Birukova AA (2010) Mechanotransduction by GEF-H1 as a

novel mechanism of ventilator-induced vascular endothelial

permeability. Am J Physiol Lung Cell Mol Physiol

298(6):L837–L848

93. Xiaolu D (2011) Role of p115RhoGEF in lipopolysaccharide-

induced mouse brain microvascular endothelial barrier dys-

function. Brain Res 1387:1–7

94. Terry SJ (2011) Spatially restricted activation of RhoA signal-

ling at epithelial junctions by p114RhoGEF drives junction

formation and morphogenesis. Nat Cell Biol 13(2):159–166

95. Itoh M (2012) Rho GTP exchange factor ARHGEF11 regulates

the integrity of epithelial junctions by connecting ZO-1 and

RhoA-Myosin II signaling. Proc Natl Acad Sci USA

109(25):9905–9910

96. Zeng L, Webster SV, Newton PM (2012) The biology of protein

kinase C. Adv Exp Med Biol 740:639–661

97. Balda MS (1993) Assembly of the tight junction: the role of

diacylglycerol. J Cell Biol 123(2):293–302

98. Yoo J (2003) Bryostatin-1 enhances barrier function in T84

epithelia through PKC-dependent regulation of tight junction

proteins. Am J Physiol Cell Physiol 285(2):C300–C309

99. Eckert JJ (2004) PKC signalling regulates tight junction mem-

brane assembly in the pre-implantation mouse embryo. Repro-

duction 127(6):653–667

100. Banan A (2005) theta Isoform of protein kinase C alters barrier

function in intestinal epithelium through modulation of distinct

claudin isotypes: a novel mechanism for regulation of perme-

ability. J Pharmacol Exp Ther 313(3):962–982

101. Andreeva AY (2001) Protein kinase C regulates the phosphor-

ylation and cellular localization of occludin. J Biol Chem

276(42):38480–38486

102. Suzuki T (2009) PKC eta regulates occludin phosphorylation

and epithelial tight junction integrity. Proc Natl Acad Sci USA

106(1):61–66

103. Nishitsuji K (2011) Apolipoprotein E regulates the integrity of

tight junctions in an isoform-dependent manner in an in vitro

blood–brain barrier model. J Biol Chem 286(20):17536–17542

104. Andreeva AY (2006) Assembly of tight junction is regulated by

the antagonism of conventional and novel protein kinase C

isoforms. Int J Biochem Cell Biol 38(2):222–233

105. Suzuki A (2001) Atypical protein kinase C is involved in the

evolutionarily conserved par protein complex and plays a criti-

cal role in establishing epithelia-specific junctional structures.

J Cell Biol 152(6):1183–1196

106. Helfrich I (2007) Role of aPKC isoforms and their binding

partners Par3 and Par6 in epidermal barrier formation. J Invest

Dermatol 127(4):782–791

107. Jain S (2011) Protein kinase Czeta phosphorylates occludin and

promotes assembly of epithelial tight junctions. Biochem J

437(2):289–299

108. Angelow S (2005) Phorbol ester induced short- and long-term

permeabilization of the blood–CSF barrier in vitro. Brain Res

1063(2):168–179

109. Banan A (2002) Activation of delta-isoform of protein kinase C

is required for oxidant-induced disruption of both the microtu-

bule cytoskeleton and permeability barrier of intestinal epithelia.

J Pharmacol Exp Ther 303(1):17–28

110. Banan A (2005) Critical role of the atypical lambda isoform of

protein kinase C (PKC-{lambda}) in oxidant-induced disruption

of the microtubule cytoskeleton and barrier function of intestinal

epithelium. J Pharmacol Exp Ther 312(2):458–471

111. Kim JH (2010) Inhibition of protein kinase C delta attenuates

blood–retinal barrier breakdown in diabetic retinopathy. Am J

Pathol 176(3):1517–1524

112. Kanmogne GD (2007) HIV-1 gp120 compromises blood–brain

barrier integrity and enhances monocyte migration across

blood–brain barrier: implication for viral neuropathogenesis.

J Cereb Blood Flow Metab 27(1):123–134

113. Kim YA (2010) Role of PKCbetaII and PKCdelta in blood–

brain barrier permeability during aglycemic hypoxia. Neurosci

Lett 468(3):254–258

114. Sjo A, Magnusson KE, Peterson KH (2003) Distinct effects of

protein kinase C on the barrier function at different develop-

mental stages. Biosci Rep 23(2–3):87–102

115. Dhillon AS (2007) MAP kinase signalling pathways in cancer.

Oncogene 26(22):3279–3290

116. Gehart H (2010) MAPK signalling in cellular metabolism: stress

or wellness? EMBO Rep 11(11):834–840

117. Yang R (2005) Bile modulates intestinal epithelial barrier

function via an extracellular signal related kinase 1/2 dependent

mechanism. Intensive Care Med 31(5):709–717

118. Cohen TS (2010) MAPK activation modulates permeability of

isolated rat alveolar epithelial cell monolayers following cyclic

stretch. PLoS One 5(4):e10385

119. Costantini TW (2009) Role of p38 MAPK in burn-induced

intestinal barrier breakdown. J Surg Res 156(1):64–69

120. Madara JL, Stafford J (1989) Interferon-gamma directly affects

barrier function of cultured intestinal epithelial monolayers.

J Clin Invest 83(2):724–727

121. Youakim A, Ahdieh M (1999) Interferon-gamma decreases bar-

rier function in T84 cells by reducing ZO-1 levels and disrupting

apical actin. Am J Physiol 276(5 Pt 1):G1279–G1288

122. Tedelind S (2003) Interferon-gamma down-regulates claudin-1

and impairs the epithelial barrier function in primary cultured

human thyrocytes. Eur J Endocrinol 149(3):215–221

123. Bruewer M (2003) Proinflammatory cytokines disrupt epithelial

barrier function by apoptosis-independent mechanisms.

J Immunol 171(11):6164–6172

124. Bruewer M (2005) Interferon-gamma induces internalization of

epithelial tight junction proteins via a macropinocytosis-like

process. FASEB J 19(8):923–933

125. Utech M (2005) Mechanism of IFN-gamma-induced endocytosis

of tight junction proteins: myosin II-dependent vacuolarization of

the apical plasma membrane. Mol Biol Cell 16(10):5040–5052

126. Boivin MA (2009) Mechanism of interferon-gamma-induced

increase in T84 intestinal epithelial tight junction. J Interferon

Cytokine Res 29(1):45–54

127. Prasad S (2005) Inflammatory processes have differential effects

on claudins 2, 3 and 4 in colonic epithelial cells. Lab Invest

85(9):1139–1162

128. Cui W (2010) Tumor necrosis factor alpha increases epithelial

barrier permeability by disrupting tight junctions in Caco-2

cells. Braz J Med Biol Res 43(4):330–337

129. Ewert P (2010) Disruption of tight junction structure in salivary

glands from Sjogren’s syndrome patients is linked to proinflam-

matory cytokine exposure. Arthritis Rheum 62(5):1280–1289

130. Grant-Tschudy KS, Wira CR (2005) Paracrine mediators of

mouse uterine epithelial cell transepithelial resistance in culture.

J Reprod Immunol 67(1–2):1–12

Mol Biol Rep (2013) 40:6123–6142 6139

123

131. Ma TY (2004) TNF-alpha-induced increase in intestinal epithelial

tight junction permeability requires NF-kappa B activation. Am J

Physiol Gastrointest Liver Physiol 286(3):G367–G376

132. Aveleira CA (2010) TNF-alpha signals through PKCzeta/NF-

kappaB to alter the tight junction complex and increase retinal

endothelial cell permeability. Diabetes 59(11):2872–2882

133. Aslam M (2012) TNF-alpha induced NFkappaB signaling and

p65 (RelA) overexpression repress Cldn5 promoter in mouse

brain endothelial cells. Cytokine 57(2):269–275

134. Ma TY (2005) Mechanism of TNF-{alpha} modulation of Caco-

2 intestinal epithelial tight junction barrier: role of myosin light-

chain kinase protein expression. Am J Physiol Gastrointest Liver

Physiol 288(3):G422–G430

135. Ye D, Ma I, Ma TY (2006) Molecular mechanism of tumor

necrosis factor-alpha modulation of intestinal epithelial tight

junction barrier. Am J Physiol Gastrointest Liver Physiol

290(3):G496–G504

136. He F (2012) Mechanisms of tumor necrosis factor-alpha-

induced leaks in intestine epithelial barrier. Cytokine

59(2):264–272

137. Marchiando AM (2010) Caveolin-1-dependent occludin endo-

cytosis is required for TNF-induced tight junction regulation

in vivo. J Cell Biol 189(1):111–126

138. Van Itallie CM (2010) Occludin is required for cytokine-induced

regulation of tight junction barriers. J Cell Sci 123(Pt

16):2844–2852

139. McKenzie JA, Ridley AJ (2007) Roles of Rho/ROCK and

MLCK in TNF-alpha-induced changes in endothelial morphol-

ogy and permeability. J Cell Physiol 213(1):221–228

140. Kakiashvili E (2009) GEF-H1 mediates tumor necrosis factor-

alpha-induced Rho activation and myosin phosphorylation: role

in the regulation of tubular paracellular permeability. J Biol

Chem 284(17):11454–11466

141. Utech M, Mennigen R, Bruewer M (2010) Endocytosis and

recycling of tight junction proteins in inflammation. J Biomed

Biotechnol 2010:484987

142. Wang F (2005) Interferon-gamma and tumor necrosis factor-

alpha synergize to induce intestinal epithelial barrier dysfunc-

tion by up-regulating myosin light chain kinase expression. Am

J Pathol 166(2):409–419

143. Patrick DM (2006) Proinflammatory cytokines tumor necrosis

factor-alpha and interferon-gamma modulate epithelial barrier

function in Madin–Darby canine kidney cells through mitogen

activated protein kinase signaling. BMC Physiol 6:2

144. Li Q (2008) Interferon-gamma and tumor necrosis factor-alpha

disrupt epithelial barrier function by altering lipid composition

in membrane microdomains of tight junction. Clin Immunol

126(1):67–80

145. Baker OJ (2008) Proinflammatory cytokines tumor necrosis

factor-alpha and interferon-gamma alter tight junction structure

and function in the rat parotid gland Par-C10 cell line. Am J

Physiol Cell Physiol 295(5):C1191–C1201

146. Peng S (2012) Effects of proinflammatory cytokines on the

claudin-19 rich tight junctions of human retinal pigment epi-

thelium (RPE). Invest Ophthalmol Vis Sci 53(8):5016–5028

147. Wang F (2006) IFN-gamma-induced TNFR2 expression is

required for TNF-dependent intestinal epithelial barrier dys-

function. Gastroenterology 131(4):1153–1163

148. Al-Sadi RM, Ma TY (2007) IL-1beta causes an increase in

intestinal epithelial tight junction permeability. J Immunol

178(7):4641–4649

149. Al-Sadi R (2008) Mechanism of IL-1beta-induced increase in

intestinal epithelial tight junction permeability. J Immunol

180(8):5653–5661

150. Al-Sadi R (2010) IL-1beta-induced increase in intestinal epi-

thelial tight junction permeability is mediated by MEKK-1

activation of canonical NF-kappaB pathway. Am J Pathol

177(5):2310–2322

151. Rigor RR (2012) Interleukin-1beta-induced barrier dysfunction

is signaled through PKC-theta in human brain microvascular

endothelium. Am J Physiol Cell Physiol 302(10):C1513–C1522

152. Desai TR (2002) Interleukin-6 causes endothelial barrier dys-

function via the protein kinase C pathway. J Surg Res

104(2):118–123

153. Yang R (2003) IL-6 is essential for development of gut barrier

dysfunction after hemorrhagic shock and resuscitation in mice.

Am J Physiol Gastrointest Liver Physiol 285(3):G621–G629

154. Weber CR (2010) Epithelial myosin light chain kinase activa-

tion induces mucosal interleukin-13 expression to alter tight

junction ion selectivity. J Biol Chem 285(16):12037–12046

155. Stone KP, Kastin AJ, Pan W (2011) NFkB is an unexpected

major mediator of interleukin-15 signaling in cerebral endo-

thelia. Cell Physiol Biochem 28(1):115–124

156. Huppert J (2010) Cellular mechanisms of IL-17-induced blood–

brain barrier disruption. FASEB J 24(4):1023–1034

157. You QH (2010) Interleukin-17F-induced pulmonary microvas-

cular endothelial monolayer hyperpermeability via the protein

kinase C pathway. J Surg Res 162(1):110–121

158. Li X, Akhtar S, Choudhry MA (1822) Alteration in intestine

tight junction protein phosphorylation and apoptosis is associ-

ated with increase in IL-18 levels following alcohol intoxication

and burn injury. Biochim Biophys Acta 2:196–203

159. Grant-Tschudy KS, Wira CR (2005) Hepatocyte growth factor

regulation of uterine epithelial cell transepithelial resistance and

tumor necrosis factor alpha release in culture. Biol Reprod

72(4):814–821

160. Date I (2006) Hepatocyte growth factor attenuates cerebral

ischemia-induced increase in permeability of the blood–brain

barrier and decreases in expression of tight junctional proteins in

cerebral vessels. Neurosci Lett 407(2):141–145

161. Togawa A (2010) Hepatocyte Growth Factor stimulated cell

scattering requires ERK and Cdc42-dependent tight junction

disassembly. Biochem Biophys Res Commun 400(2):271–277

162. Catizone A (2012) Hepatocyte growth factor (HGF) regulates

blood–testis barrier (BTB) in adult rats. Mol Cell Endocrinol

348(1):135–146

163. Lipschutz JH (2005) Extracellular signal-regulated kinases 1/2

control claudin-2 expression in Madin–Darby canine kidney

strain I and II cells. J Biol Chem 280(5):3780–3788

164. Samak G, Aggarwal S, Rao RK (2011) ERK is involved in EGF-

mediated protection of tight junctions, but not adherens junc-

tions, in acetaldehyde-treated Caco-2 cell monolayers. Am J

Physiol Gastrointest Liver Physiol 301(1):G50–G59

165. Ikari A (2011) Epidermal growth factor increases clathrin-

dependent endocytosis and degradation of claudin-2 protein in

MDCK II cells. J Cell Physiol 226(9):2448–2456

166. Yoshida K (2005) EGF rapidly translocates tight junction pro-

teins from the cytoplasm to the cell–cell contact via protein

kinase C activation in TMK-1 gastric cancer cells. Exp Cell Res

309(2):397–409

167. Harhaj NS, Barber AJ, Antonetti DA (2002) Platelet-derived

growth factor mediates tight junction redistribution and increases

permeability in MDCK cells. J Cell Physiol 193(3):349–364

168. Wen H (2011) Morphine induces expression of platelet-derived

growth factor in human brain microvascular endothelial cells:

implication for vascular permeability. PLoS One 6(6):e21707

169. Yao H, Duan M, Buch S (2011) Cocaine-mediated induction of

platelet-derived growth factor: implication for increased vascu-

lar permeability. Blood 117(8):2538–2547

170. Harhaj NS (2006) VEGF activation of protein kinase C stimu-

lates occludin phosphorylation and contributes to endothelial

permeability. Invest Ophthalmol Vis Sci 47(11):5106–5115

6140 Mol Biol Rep (2013) 40:6123–6142

123

171. Murakami T, Felinski EA, Antonetti DA (2009) Occludin

phosphorylation and ubiquitination regulate tight junction traf-

ficking and vascular endothelial growth factor-induced perme-

ability. J Biol Chem 284(31):21036–21046

172. Goumans MJ, Liu Z, Ten DP (2009) TGF-beta signaling in

vascular biology and dysfunction. Cell Res 19(1):116–127

173. Drabsch Y, Ten DP (2011) TGF-beta signaling in breast cancer

cell invasion and bone metastasis. J Mammary Gland Biol

Neoplasia 16(2):97–108

174. Birukova AA (2005) Involvement of microtubules and Rho

pathway in TGF-beta1-induced lung vascular barrier dysfunc-

tion. J Cell Physiol 204(3):934–947

175. Clements RT (2005) RhoA and Rho-kinase dependent and

independent signals mediate TGF-beta-induced pulmonary

endothelial cytoskeletal reorganization and permeability. Am J

Physiol Lung Cell Mol Physiol 288(2):L294–L306

176. Pierucci-Alves F, Yi S, Schultz BD (2012) Transforming growth

factor Beta 1 induces tight junction disruptions and loss of

transepithelial resistance across porcine vas deferens epithelial

cells. Biol Reprod 86(2):36

177. Feldman G (2007) Role for TGF-beta in cyclosporine-induced

modulation of renal epithelial barrier function. J Am Soc

Nephrol 18(6):1662–1671

178. Howe KL (2005) Transforming growth factor-beta regulation of

epithelial tight junction proteins enhances barrier function and

blocks enterohemorrhagic Escherichia coli O157:H7-induced

increased permeability. Am J Pathol 167(6):1587–1597

179. Lu Q (2006) Transforming growth factor-beta1-induced endo-

thelial barrier dysfunction involves Smad2-dependent p38 acti-

vation and subsequent RhoA activation. J Appl Physiol

101(2):375–384

180. Xia W (2009) TGF-beta3 and TNFalpha perturb blood–testis

barrier (BTB) dynamics by accelerating the clathrin-mediated

endocytosis of integral membrane proteins: a new concept

of BTB regulation during spermatogenesis. Dev Biol

327(1):48–61

181. Ye P (2012) Modulation of epithelial tight junctions by TGF-

beta 3 in cultured oral epithelial cells. Aust Dent J 57(1):11–17

182. Le Moellic C (2005) Aldosterone and tight junctions: modula-

tion of claudin-4 phosphorylation in renal collecting duct cells.

Am J Physiol Cell Physiol 289(6):C1513–C1521

183. Forster C (2006) Glucocorticoid effects on mouse microvascular

endothelial barrier permeability are brain specific. J Physiol

573(Pt 2):413–425

184. Forster C (2008) Differential effects of hydrocortisone and

TNFalpha on tight junction proteins in an in vitro model of the

human blood–brain barrier. J Physiol 586(7):1937–1949

185. Kobayashi K (2010) Expression and distribution of tight junc-

tion proteins in human amnion during late pregnancy. Placenta

31(2):158–162

186. Sadowska GB, Malaeb SN, Stonestreet BS (2010) Maternal

glucocorticoid exposure alters tight junction protein expression

in the brain of fetal sheep. Am J Physiol Heart Circ Physiol

298(1):H179–H188

187. Forster C (2005) Occludin as direct target for glucocorticoid-

induced improvement of blood–brain barrier properties in a

murine in vitro system. J Physiol 565(Pt 2):475–486

188. Harke N (2008) Glucocorticoids regulate the human occludin

gene through a single imperfect palindromic glucocorticoid

response element. Mol Cell Endocrinol 295(1–2):39–47

189. Felinski EA (2008) Glucocorticoids induce transactivation of

tight junction genes occludin and claudin-5 in retinal endothelial

cells via a novel cis-element. Exp Eye Res 86(6):867–878

190. Kashiwamura Y (2011) Hydrocortisone enhances the function of

the blood–nerve barrier through the up-regulation of claudin-5.

Neurochem Res 36(5):849–855

191. Boivin MA (2007) Mechanism of glucocorticoid regulation of

the intestinal tight junction barrier. Am J Physiol Gastrointest

Liver Physiol 292(2):G590–G598

192. Sekiyama A (2012) Glucocorticoids enhance airway epithelial

barrier integrity. Int Immunopharmacol 12(2):350–357

193. Meng J (2005) Androgens regulate the permeability of the blood–

testis barrier. Proc Natl Acad Sci USA 102(46):16696–16700

194. Su L (2010) Differential effects of testosterone and TGF-beta3

on endocytic vesicle-mediated protein trafficking events at the

blood–testis barrier. Exp Cell Res 316(17):2945–2960

195. Mendoza-Rodriguez CA, Gonzalez-Mariscal L, Cerbon M

(2005) Changes in the distribution of ZO-1, occludin, and

claudins in the rat uterine epithelium during the estrous cycle.

Cell Tissue Res 319(2):315–330

196. Buck VU (2012) Redistribution of adhering junctions in human

endometrial epithelial cells during the implantation window of

the menstrual cycle. Histochem Cell Biol 137(6):777–790

197. Satterfield MC (2007) Tight and adherens junctions in the ovine

uterus: differential regulation by pregnancy and progesterone.

Endocrinology 148(8):3922–3931

198. Kobayashi K, Miwa H, Yasui M (2011) Progesterone maintains

amniotic tight junctions during midpregnancy in mice. Mol Cell

Endocrinol 337(1–2):36–42

199. Kaplan JH (2002) Biochemistry of Na,K-ATPase. Annu Rev

Biochem 71:511–535

200. Geering K (2008) Functional roles of Na,K-ATPase subunits.

Curr Opin Nephrol Hypertens 17(5):526–532

201. Giannatselis H, Calder M, Watson AJ (2011) Ouabain stimulates

a Na?/K?-ATPase-mediated SFK-activated signalling pathway

that regulates tight junction function in the mouse blastocyst.

PLoS One 6(8):e23704

202. Larre I (2010) Ouabain modulates epithelial cell tight junction.

Proc Natl Acad Sci USA 107(25):11387–11392

203. Larre I, Cereijido M (2010) Na,K-ATPase is the putative

membrane receptor of hormone ouabain. Commun Integr Biol

3(6):625–628

204. Violette MI, Madan P, Watson AJ (2006) Na?/K?-ATPase

regulates tight junction formation and function during mouse

preimplantation development. Dev Biol 289(2):406–419

205. Yan Y (2012) Ouabain-stimulated trafficking regulation of the

Na/K-ATPase and NHE3 in renal proximal tubule cells. Mol

Cell Biochem 367(1–2):175–183

206. Holthouser KA (2010) Ouabain stimulates Na-K-ATPase

through a sodium/hydrogen exchanger-1 (NHE-1)-dependent

mechanism in human kidney proximal tubule cells. Am J

Physiol Renal Physiol 299(1):F77–F90

207. Haas M (2002) Src-mediated inter-receptor cross-talk between

the Na?/K?-ATPase and the epidermal growth factor receptor

relays the signal from ouabain to mitogen-activated protein

kinases. J Biol Chem 277(21):18694–18702

208. Yuan Z (2005) Na/K-ATPase tethers phospholipase C and IP3

receptor into a calcium-regulatory complex. Mol Biol Cell

16(9):4034–4045

209. Lecuona E (2006) Na,K-ATPase alpha1-subunit dephosphoryl-

ation by protein phosphatase 2A is necessary for its recruitment

to the plasma membrane. FASEB J 20(14):2618–2620

210. Rajasekaran SA (2007) Na-K-ATPase regulates tight junction

permeability through occludin phosphorylation in pancreatic

epithelial cells. Am J Physiol Gastrointest Liver Physiol

292(1):G124–G133

211. Yang Y (2011) Na?/K?-ATPase alpha1 identified as an abun-

dant protein in the blood–labyrinth barrier that plays an essential

role in the barrier integrity. PLoS One 6(1):e16547

212. Barwe SP (2005) Novel role for Na,K-ATPase in phosphati-

dylinositol 3-kinase signaling and suppression of cell motility.

Mol Biol Cell 16(3):1082–1094

Mol Biol Rep (2013) 40:6123–6142 6141

123

213. Madan P, Rose K, Watson AJ (2007) Na/K-ATPase beta1

subunit expression is required for blastocyst formation and

normal assembly of trophectoderm tight junction-associated

proteins. J Biol Chem 282(16):12127–12134

214. Wright EM, Loo DD, Hirayama BA (2011) Biology of human

sodium glucose transporters. Physiol Rev 91(2):733–794

215. Avkiran M (2003) Basic biology and pharmacology of the car-

diac sarcolemmal sodium/hydrogen exchanger. J Card Surg

18(Suppl 1):3–12

216. De Vito P (2006) The sodium/hydrogen exchanger: a possible

mediator of immunity. Cell Immunol 240(2):69–85

217. Turner JR (1997) Physiological regulation of epithelial tight

junctions is associated with myosin light-chain phosphorylation.

Am J Physiol 273(4 Pt 1):C1378–C1385

218. Turner JR (2000) Noninvasive in vivo analysis of human small

intestinal paracellular absorption: regulation by Na?–glucose

cotransport. Dig Dis Sci 45(11):2122–2126

219. Berglund JJ (2001) Regulation of human jejunal transmucosal

resistance and MLC phosphorylation by Na(?)–glucose cotransport.

Am J Physiol Gastrointest Liver Physiol 281(6):G1487–G1493

220. Clayburgh DR (2004) A differentiation-dependent splice variant

of myosin light chain kinase, MLCK1, regulates epithelial tight

junction permeability. J Biol Chem 279(53):55506–55513

221. Turner JR (2000) Transepithelial resistance can be regulated by

the intestinal brush-border Na(?)/H(?) exchanger NHE3. Am J

Physiol Cell Physiol 279(6):C1918–C1924

222. Hu Z (2006) MAPKAPK-2 is a critical signaling intermediate in

NHE3 activation following Na?–glucose cotransport. J Biol

Chem 281(34):24247–24253

223. Turner JR, Black ED (2001) NHE3-dependent cytoplasmic alka-

linization is triggered by Na(?)-glucose cotransport in intestinal

epithelia. Am J Physiol Cell Physiol 281(5):C1533–C1541

224. Zhao H (2004) Ezrin regulates NHE3 translocation and activa-

tion after Na?–glucose cotransport. Proc Natl Acad Sci USA

101(25):9485–9490

225. Shiue H (2005) Akt2 phosphorylates ezrin to trigger NHE3

translocation and activation. J Biol Chem 280(2):1688–1695

226. Park SL (2010) The effect of Na(?)/H(?) exchanger-1 inhibi-

tion by sabiporide on blood–brain barrier dysfunction after

ischemia/hypoxia in vivo and in vitro. Brain Res 1366:189–196

227. Brown RC, Davis TP (2002) Calcium modulation of adherens

and tight junction function: a potential mechanism for blood–

brain barrier disruption after stroke. Stroke 33(6):1706–1711

228. Moeser AJ (2006) Prostaglandin-mediated inhibition of Na?/H?

exchanger isoform 2 stimulates recovery of barrier function in

ischemia-injured intestine. Am J Physiol Gastrointest Liver

Physiol 291(5):G885–G894

229. Moeser AJ (2008) Mice lacking the Na?/H? exchanger 2 have

impaired recovery of intestinal barrier function. Am J Physiol

Gastrointest Liver Physiol 295(4):G791–G797

230. Jentsch TJ (2002) Molecular structure and physiological func-

tion of chloride channels. Physiol Rev 82(2):503–568

231. LeSimple P (2010) Cystic fibrosis transmembrane conductance

regulator trafficking modulates the barrier function of airway

epithelial cell monolayers. J Physiol 588(Pt 8):1195–1209

232. Nilsson HE (2010) CFTR and tight junctions in cultured bron-

chial epithelial cells. Exp Mol Pathol 88(1):118–127

233. Weiser N (2011) Paracellular permeability of bronchial epithe-

lium is controlled by CFTR. Cell Physiol Biochem

28(2):289–296

234. Moeser AJ (2007) Recovery of mucosal barrier function in

ischemic porcine ileum and colon is stimulated by a novel

agonist of the ClC-2 chloride channel, lubiprostone. Am J

Physiol Gastrointest Liver Physiol 292(2):G647–G656

235. Nighot PK, Blikslager AT (2010) ClC-2 regulates mucosal

barrier function associated with structural changes to the villus

and epithelial tight junction. Am J Physiol Gastrointest Liver

Physiol 299(2):G449–G456

6142 Mol Biol Rep (2013) 40:6123–6142

123