terminal differentiation in epithelia: the role of integrins in hensin polymerization

PH73CH18-Al-Awqati ARI 3 January 2011 14:40

Terminal Differentiationin Epithelia: The Roleof Integrins in HensinPolymerizationQais Al-AwqatiDepartment of Medicine and Department of Physiology & Cellular Biophysics,College of Physicians & Surgeons, Columbia University, New York, NY 10032;email: [email protected]

Annu. Rev. Physiol. 2011. 73:401–12

First published online as a Review in Advance onOctober 4, 2010

The Annual Review of Physiology is online atphysiol.annualreviews.org

This article’s doi:10.1146/annurev-physiol-012110-142253

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4278/11/0315-0401$20.00

Keywords

DMBT1, intercalated cells, acid-base

Abstract

Epithelia, the most abundant cell type, differentiate to protoepitheliafrom stem cells by developing apical and basolateral membrane do-mains and form sheets of cells connected by junctions. Following thisdifferentiation step, the cells undergo a second step (terminal differenti-ation), during which they acquire a mature phenotype, which unlike theprotoepithelial one is tissue and organ specific. An extracellular matrix(ECM) protein termed hensin (DMBT1) mediates this differentiationstep in the kidney intercalated cells. Although hensin is secreted as a sol-uble monomer, it requires polymerization and deposition in the ECMto become active. The polymerization step is mediated by the activationof inside-out signaling by integrins and by the secretion of two proteins:cypA (a cis-trans prolyl isomerase) and galectin 3.

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INTRODUCTION

From sponges to humans, each metazoanorganism contains an astonishing variety ofepithelial cell types. In higher organisms, ep-ithelia exist in two broad classes: single layered,e.g., columnar or squamous, or multilayered,e.g., transitional (ureter) or stratified (skin).Remarkably, embryonic epithelia, whethertheir fate is to become intestine or skin, beginas single-layered tissues. They exhibit all thedefining characteristics of epithelia: differenttypes of junctions (tight and adherent), apicaland basal polarity, transepithelial transport,and characteristic extracellular matrix (ECM)proteins. We term these phenotypes pro-toepithelia. Only later in the developmentof each tissue do differentiated character-istics appear. Differentiated characteristicsinclude many layers, specific cell shapes, apicalspecializations such as flagella or microvilli,secretory granules, and apical functionssuch as apical endocytosis and exocytosis(1).

What is the mechanism underlying suchterminal differentiation? Two questions arise inthis context. First, is there is a single mechanismthat applies to all epithelial differentiation? Ageneral mechanism that applies to squamousepithelia and columnar epithelia is plausible.However, of the many general mechanismsinvolved in differentiation, the appropriatemechanism is modified by each tissue to suitits particular context.

Even more difficult, is differentiationterminal? Tissue fibrosis, which is at the centerof many if not most diseases, displays trans-differentiation, during which epithelia convertto mesenchyme, a process well known duringembryonic development. Hence we use theterm terminal differentiation without implyingthat it is in fact terminal so as to be somewhatmore specific than using simply the worddifferentiation. Here terminal differentiationreflects the state of the epithelium in the adultmature organ.

KIDNEY INTERCALATED CELLS

Acid production by oxidation of dietary food-stuff would be lethal were it not for the kid-ney, which restores the blood’s buffering powerby producing new HCO3

−. H+ secretion bythe epithelial cells of the proximal tubule andthe cortical collecting duct (CCD) serves twofunctions: The proximal tubule reabsorbs thefiltered HCO3

−, and the distal nephron pro-duces new HCO3

−. Both processes are neededto titrate the generated metabolic acid. Twonephron segments mediate this process: theproximal convoluted tubule, where a Na+:H+

exchanger (NHE3) and a proton-translocatingATPase (V-ATPase) reabsorb filtered HCO3

−,and the collecting tubule, where there is onlythe V-ATPase.

The CCD of the kidney has two cell types:principal cells, which transport Na+, water, andK+, and intercalated cells, which mediate acid-base transport. Two functionally distinct sub-types of intercalated cells have been identifiedin the CCD: The β type secretes HCO3

−,whereas the α form secretes H+ (1). We firstshowed that the β intercalated cells mediateHCO3

− secretion by an apical Cl−:HCO3− ex-

changer that was later identified to be pendrin(2). These cells are present only in the cortex, inthe connecting and cortical collecting tubules.The α intercalated cell secretes H+ by an apicalH+-ATPase and by a basolateral Cl−:HCO3

−

exchanger, which is an alternately spliced formof the band 3 protein AE1. We found that feed-ing the animals an acid diet led to a conversionof the β intercalated cells to α intercalated cells(2). This phenotype change began within a fewhours of acid exposure and continued so that bya few days later most of the cells in the corti-cal collecting tubules turned into α intercalatedcells.

Intercalated cells were identified in the earlydays of microscopic anatomy. They are en-riched in mitochondria and exist in many ep-ithelia such as the skin of amphibia and somereptiles as well as in the gills of some fish (3).

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Their presence in all these epithelia forms aninteresting model of cell fate determinationwherein a single tissue has an apparently ran-dom distribution of a second cell type amongthe majority of cells. This salt-and-pepper ap-pearance has recently attracted much attentionand is thought to be mediated by the notch sig-naling pathway through the process of lateralinhibition (4, 5).

The response of the collecting tubule toacidosis can be thought of as representingtwo fundamentally different processes. Inthe α intercalated cell, an acute stimulussuch as an increase in the ambient pCO2

leads to intracellular acidosis, which in turncauses stimulation of H+ secretion due tothe insertion of H+-ATPases into the apicalmembrane. This effect is rapid and occurswithin minutes of stimulus exposure. Wepreviously showed that a rapid decline in cellpH causes an increase in cellular Ca2+ levelsthat is the proximate cause of the exocytoticinsertion of the V-ATPase vesicles (6). In the β

intercalated cell, basolateral acidification leadsto a decrease in apical Cl−:HCO3

− exchangewithin 3 h. Unfortunately, there are no studiesof shorter duration. Although the cell pHdeclined, this decrease showed no calcium de-pendency. Numerous other studies have shownthat chronic metabolic acidosis causes a severedecline in the abundance of apical Cl−:HCO3

−

exchange and/or pendrin expression.Metabolic acidosis induces a change in the

phenotype of the intercalated cell of the col-lecting tubule from a HCO3

−-secreting typeto one that absorbs HCO3

− by H+ secretion.This change is associated with a reversal ofthe polarized distribution of the H+-ATPaseand Cl−:HCO3

− exchange. Thus, this phe-nomenon attracted much attention among cellbiologists because it contradicted the deter-ministic idea (popular in the mid-1980s) thatpolarized protein sorting in epithelia was dueto targeting sequences in proteins and hencewas immutable. We first developed an immor-talized intercalated cell line that showed this

reversal of polarity (7). Our work has led us tothe finding that a new ECM protein we termedhensin causes this reversal of polarity. Thestudy of hensin biology has uncovered a criticalaspect of the origins of epithelial differentiation.It appears now that the reversal of polarity, al-though interesting, is only the local expressionin intercalated cells of a general differentiationprogram.

Since these initial studies, the methods foridentification and classification of these two in-tercalated cell types have become more sophis-ticated with the appearance of new molecularmarkers. Although the nomenclature remainssomewhat disparate, there is no doubt about thepresence of an acid-secreting canonical α celltype with an apical V-ATPase and a basolateralAE1. Similarly, there is agreement about thepresence of a canonical β cell type with an api-cal pendrin and a basolateral V-ATPase. Thepresence of intermediate cells raises many ques-tions about the origin of the diversity of thesecell types, and some AE1-negative intercalatedcells display bipolar and/or a diffuse cyto-plasmic distribution of the V-ATPase (8, 9).Additionally, there is good evidence that in thecortical collecting and connecting tubules, anintercalated cell type expresses pendrin and theV-ATPase together on the apical surface, theso-called non-A, non-B type (10–12). Induc-tion of metabolic acidosis or alkalosis induces aprofound change in the population distributionof these different cell types. Acidosis shifts allcells with basolateral, bipolar, or cytoplasmicATPase toward a cell type with apical ATPase,whereas alkalosis increases the number ofcanonical β cells at the expense of α cells (13,14). We provided evidence supporting the ideathat an individual β intercalated cell convertsto an α intercalated cell when we isolated andperfused cortical collecting tubules and foundthat, as a result of exposure to a basolaterallow-pH medium for 3 h, a significant fractionof identified intercalated cells that had an apicalCl−:HCO3

− exchanger converted to cells withbasolateral Cl−:HCO3

− exchangers (15).

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HENSIN MEDIATESPHENOTYPE CONVERSION

We began our molecular analysis by generat-ing an immortalized intercalated cell line thatshowed this reversal of polarity (7). When thesecells were seeded at subconfluent density andwere allowed to reach confluence, they resem-bled β intercalated cells. But when seeded atsuperconfluent density, this clonal cell line be-came identical to α intercalated cells (16). Thisresult was not simply a matter of density be-cause when the cells that were seeded at lowdensity became confluent, they had essentiallythe same cell number, yet they remained β in-tercalated cells. High-density seeding acted asa developmental switch that changed the fateof these cells from one cell type to another.To identify the mechanism of this fate change,we first searched for a secreted factor, to noavail. We then tested whether the ECM is thesource of this factor by seeding cells at highdensity on filters and then solubilizing themafter they achieved their final phenotype. Weseeded fresh cells at low density on these con-ditioned filters and found that they convertedto high-density-phenotype cells with columnarshape, apical microvilli, acid secretion, and api-cal endocytosis. To purify this factor, we useda miniaturized assay for apical endocytosis andidentified a single protein from the ECM of1,000 wells that could mediate this change byitself. We termed this protein hensin, which inJapanese means change in shape. Hensin causedlow-density cells to assume a columnar epithe-lial phenotype with all the differentiated prop-erties of columnar cells such as apical microvilliand apical endocytosis (16, 17). These resultsconclusively demonstrate that it was not den-sity per se but rather density in seeding thatproduced the change in phenotype.

Cloning of the hensin cDNA showed that itis composed of eight SRCR (scavenger recep-tor cysteine-rich) domains, two CUB (comple-ment C1r/C1s, Uegf, Bmp) domains, and onezona pellucida (Zp) domain. Five other proteinshave now been sequenced; all of them are com-posed of these three domains, but in different

combinations. We recently showed that allthese transcripts are derived from a single geneby alternative splicing (18). Hensin is expressedin most epithelia, and its expression begins inthe earliest epithelia, such as the trophoecto-derm, as well as in the primitive endoderm atapproximately day 3.5 of embryonic life.

The mechanism of the change in phenotypeis very complex. The apical microvilli are pro-duced by a mat of subapical actin, villin, andcytokeratin (17). Whereas actin is repositioned,villin and CK19 are induced, implying that atranscriptional event is induced. This action de-pends on DNA and protein synthesis but not onelevated intracellular Ca2+, implying that thisECM protein induces a transcriptional event.

Hensin is secreted as a monomer but un-dergoes a complex polymerization that leads tothe formation of insoluble ECM hensin; onlythe insoluble fibrillar hensin is capable of in-ducing columnarization (19). As is shown be-low, polymerization of hensin requires at leastthree other proteins: integrins, the cis-trans pro-lyl isomerase activity of cyclophilin A (cypA),and galectin 3 (20–22). These last two proteinsdo not have signal sequences and are secretedin high-density cells, presumably by activationof the nonclassical protein transport pathway(i.e., this pathway is not mediated by trafficfrom the endoplasmic reticulum and Golgi tothe outside). Activated cells generally secretesuch leaderless peptides following their overex-pression. In summary, the program induced byhensin includes changes in cell shape, the devel-opment of apical endocytosis, the redistributionof actin, the induction of new proteins, and thesecretion of cytoplasmic proteins.

SECRETED GALECTIN 3 ANDCYCLOPHILIN A ARE REQUIREDFOR HENSIN POLYMERIZATION

Hensin is a high-molecular-weight (approxi-mately 240-kDa) protein. Hence, when we pu-rified it, we used the appropriate gels to estimateits Mr and the degree of purity of the prepa-ration. In retrospect, we missed the presenceof two smaller proteins that we now know are

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present in the highly purified preparation. Wefound that galectin 3 binds to hensin and thatthe extraction of galectin 3 from purified hensinleads to loss of hensin activity. Furthermore,when galectin 3 was added back to this fraction,it recovered its ability to induce columnariza-tion and apical endocytosis (20). Purified re-combinant galectin 3 was able to bind to hensinand to polymerize it in vitro. Seeding cells athigh density induced the secretion of galectin 3into the ECM, where galectin 3 bundled hensin.Knockout of galectin 3 did not reveal any acid-base phenotype (23), perhaps because the kid-ney expresses several other galectins that mayhave compensated for the loss of galectin 3.

Cyclosporine A, a widely used immunosup-pressant, causes distal renal tubular acidosis(24). Cyclosporine binds to cypA, a cytoplasmicpeptidyl cis-trans prolyl isomerase that binds tocalcineurin, blocking calcineurin’s phosphataseactivity and thereby producing immunosup-pression. Given that hensin is a proline-richprotein and that prolines are a critical determi-nant of protein tertiary structure, we tested thehypothesis (in collaboration with Dr. George J.Schwartz) that cyclosporine might block hensinpolymerization and deposition in the ECM(21). We extracted the ECM of high-densitycells and blotted it with hensin antibodies andfound that high-density cells’ ECM has anabundant band at 200 kDa that disappears fromthe ECM of cells seeded also at high density butin the presence of cyclosporine A. Similarly, thenewly derived specific inhibitor (25) of the pep-tidyl prolyl cis-trans isomerase (Cs9) blockedhensin deposition, but the specific calcineurinblocker LIE or FK-506 had no effect. We alsotested the effect of cyclosporine on the conver-sion of β intercalated cells to α intercalated cellsin response to extracellular acidosis. We foundthat cyclosporine prevented adaptive decreasesin HCO3

− secretion and apical Cl−:HCO3− ex-

change of identified β intercalated cells in iso-lated perfused rabbit cortical collecting tubules.[AD-Ser]CsA, a cyclosporine derivative, whichdoes not inhibit calcineurin but inhibits the pro-lyl isomerase activity of cyclophilin A, com-pletely blocked the effect of acid incubation on

apical Cl−:HCO3− exchange. Acid incubation

resulted in prominent clumpy staining of extra-cellular hensin, whereas cyclosporine and [AD-Ser]CsA prevented most hensin staining. Thesestudies demonstrate that hensin polymeriza-tion requires cis-trans prolyl isomerase activity.To identify the specific isomerase that causesthe polymerization, we attempted to distinguishamong the three cyclophilin forms: cypA is cy-toplasmic, cyclophilin B (cypB) is located inthe endoplasmic reticulum, and cyclophilin C(cypC) is secreted. We initially thought thatcypC is the form most likely responsible for ex-tracellular hensin polymerization, given that itis a secreted protein. Using antibodies to cypCand cypB, we found that, although the interca-lated cells express these proteins, neither typeis secreted by high-density cells. Surprisingly,cypA, the cytoplasmic form without a signal se-quence, is secreted into the basolateral medium(22). However, recent studies have shown thatcypA is secreted by vascular smooth muscle cellsin response to oxidative stress and that vesicu-lar transport mediates this secretion (26). Moreinterestingly, when we separated the mediumof high-density cells on sucrose density gradi-ents, we found that cypA, an 18-kDa protein,not only was present at the top of the gradient,as expected, but was spread out over the wholegradient, even in the pellet, in a manner simi-lar to the distribution of hensin. Furthermore,using pull-down studies, we found that agarosebeads loaded with cypA bind hensin in a specificmanner. These studies demonstrate that cypA issecreted and acts through its isomerase activityto mediate one critical aspect of hensin poly-merization. Structural studies will be neededto identify the regions of hensin where cypAbinds and exerts its isomerase effect. A simplecomputer model showed that, given that thesequences in between all the SRCR domainsare proline rich, if all the intervening prolinewere in a cis or a trans configuration, they woulddevelop a superhelix. Because our preliminarynegative-staining electron microscopy showedthat hensin is composed of fibrils, the superhe-lix may be a mechanism of winding one hensinchain around others.


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To confirm these studies performed inthe immortalized intercalated cell culture, weused the in situ–perfused collecting tubule andfound that in response to acidosis, β inter-calated cells began to deposit fibrillar hensinand galectin 3 (27) in their ECM. Simulta-neously, the intercalated cells lost their apicalCl−:HCO3

− exchange ability and acquired ba-solateral Cl−:HCO3

− exchange ability, provid-ing unequivocal evidence for conversion of thepolarity of β intercalated cells to that of α in-tercalated cells. Blocking antibodies to hensinprevented this change in polarity. Furthermore,prevention of hensin polymerization by an in-hibitor of the cis-trans prolyl isomerase also pre-vented the conversion of polarity and, in wholeanimals, produced distal renal tubular acidosis,the condition expected from lack of generationof α intercalated cells.

In summary, we propose that hensin is de-posited in the basement membrane followingan ordered scheme. First the integrins are acti-vated whereby the multimeric hensin is assem-bled by binding of the repeat domains (SRCR)of hensin to clustered integrins. This is followedor aided by the secretion of galectin 3 and cypA,each of which plays a critical role in assem-bling the hensin fiber. Only the fiber form ofhensin can mediate the conversion of β to α

α β

α βα β α β α β α β

Inactive

Priming Active Clustered

CypA, cis-trans prolyl isomerase

Galectin 3

Hensin

Figure 1Polymerization of hensin following inside-out integrin activation. A potentialmechanism of an ordered process whereby hensin first binds to αvβ integrin.This binding is followed by polymerization that is strengthened by the presenceof the cis-trans prolyl isomerase cyclophilin A (cypA) and galectin 3. The netresult is a hensin fiber that is insoluble and remains in the extracellular matrix.

intercalated cells and perhaps other cell types(see Figure 1).

ROLE OF αvβ1 INTEGRIN INHENSIN POLYMERIZATION

Many extracellular matrix proteins are de-posited in the basement membrane by poly-merization. To investigate the mechanism ofhensin’s polymerization, we reasoned that, asin the case of fibronectin, hensin might alsobind to a cell surface protein that then helpsit to polymerize. We cross-linked basolateralmembrane proteins from high-density and low-density cells, using the cleavable chemical cross-linker DTSSP, and subjected equal amountsof cell lysate proteins to immunoprecipitationwith antihensin antibodies. Performing proteinstains to identify these proteins was not possi-ble, given that the abundance of the surface pro-teins was low. Because we were studying termi-nal differentiation, we felt that perhaps the sur-face proteins might be phosphorylated as partof the activation process, so we used antibodiesto serine- or tyrosine-phosphorylated proteins.Although antiphosphoserine antibodies did notreveal any specifically phosphorylated proteins,we used antiphosphotyrosine antibodies andfound two tyrosine-phosphorylated bands ofmolecular masses of 110 kDa and 130 kDa.These bands appeared only in the high-densitylane and not in the low-density lane. These twoproteins bound to wheat-germ agglutinin, andwe developed a purification scheme using ionexchange chromatography, starting with mem-brane proteins from the equivalent of 800 wells.After tryptic digestion and microcleanup pro-cedures, analysis of the peptide pools by massspectrometry was carried out, revealing that thepeptides matched the chicken αv integrin andmouse β1 integrin (28).

We then began studying the integrins ex-pressed by the intercalated cell line. Using im-munocytochemistry and/or immunoprecipita-tion, we found that the cell line expressed onlyα1, αv, α6, and β1 integrins; i.e., these anti-bodies stained or immunoprecipitated proteinsof the appropriate Mr from these cells, whereas

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other antibodies did not. The caveat here is thatthe intercalated cell line is derived from rabbits,and hence there may be other integrins that arepresent in these cells but that are not recog-nized by commercial antibodies. Regardless, wewere able to find a sufficient number of func-tional antibodies to αvβ1 integrins to allow forprogress. Studies using blocking antibodies tothese integrins showed that only antibodies toαv and to β1 blocked the conversion of interca-lated cells from squamous epithelia to columnarepithelia. Cells seeded at high density alwayshad subapical actin, and the anti-αv and anti-β1 antibodies blocked actin’s subapical localiza-tion. Moreover, apical endocytosis, a genuinemarker of terminal differentiation, was blockedby these antibodies but not by other integrinantibodies such as α1. We performed crossim-munoprecipitation wherein we reversibly cross-linked the basolateral surfaces of high-densityand low-density cells and immunoprecipitatedwith either hensin or integrin antibodies fol-lowed by Western blotting with the hensin orintegrin antibodies.

We found that antihensin antibodies canimmunoprecipitate β1 integrin and vice versa,but only in high-density cells. Furthermore, ananti-β1 integrin brought down αv integrin butnot α1 integrin or α6 integrin. Similarly, hensinantibodies brought down αv but not α1 inte-grins. Anti-β1 antibodies also immunoprecipi-tated α6 integrins (discussed below). In thesestudies, we also found that the immunopre-cipitate of β1 integrin from high-density cellsshowed that this integrin is tyrosine phosphory-lated. Although previous studies had shown thattyrosine phosphorylation of β1 integrin is func-tionally significant, more recent studies showedthat replacement of tyrosines with phenylala-nine resulted in apparently normal mice (29,30), but replacement of tyrosine with alanineabolished integrin function. Whether the tyro-sine phosphorylation of β1 integrin of interca-lated cells led to its activation is unknown.

αβ integrin dimers exist in a bent inactivestate, but when they become activated, theypass through a series of conformational statesfrom primed to active to clustered (31). We first

found that αvβ1 is clustered in high-densitybut not in low-density cells. Immunocyto-chemistry of these integrins showed that theintegrins in low-density cells are present ina diffuse punctate manner typical of inactiveintegrins, whereas in high-density cells theintegrins are present in thick rope-like clusterslocated on the cells’ basal surfaces. Blockingantibodies to αvβ1 integrin prevented thedeposition of hensin in the ECM, as assayed bytwo methods. In one method we used stainingof unpermeabilized cells and found that high-density cells stained for extracellular hensin inthe ECM, whereas high-density cells treatedwith blocking antibodies to αvβ1 integrin didnot. In other studies we extracted the ECMof high-density cells and found that antibody-treated cells did not contain any ECM hensin.Finally, we treated low-density cells with anactivating antibody to β1 integrin and found—using our two assays, immunocytochemistryof unpermeabilized cells and ECM extractionfollowed by blotting for hensin—that hensinbecame deposited in the ECM.

In all these studies, we also examined the cellheight and the presence of apical endocytosis.When we blocked hensin deposition, the cellswere no longer columnar in shape; when we ac-tivated hensin deposition by β1-activating an-tibodies, the cells became columnar. The finalpiece of evidence was provided by seeding cellsat low density on purified ECM of high-densitycells; such ECM contains polymerized hensin.In control studies, low-density cells convertedto a high-density phenotype, with tall cells thatexhibited apical endocytosis. When we seededlow-density cells on ECM containing hensinin the presence of αvβ1-blocking antibodies,they also became tall and columnar and had api-cal endocytosis. These data conclusively showthat the role of αvβ1 integrin is to deposithensin in the ECM, which in turn converts thecells to a new phenotype. However, how theacid signal leads to β1 integrin phosphoryla-tion and clustering is unknown at present. Inmany systems, initiation of integrin phospho-rylation and activation begins when a recep-tor tyrosine kinase binds its cognate ligand and


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phosphorylates talin, which in turn mediatesthe effect on integrin (32). Perhaps a similarevent operates in the intercalated cell during itsresponse to acidosis.

We recently used conditional β1 integrinknockout mice to delete the integrin from theintercalated cells. The cells continued to ex-press hensin, and all cortical intercalated cellswere of the β phenotype. Moreover, the micedeveloped distal renal tubular acidosis, provid-ing compelling evidence for the role of integrinin hensin polymerization. Conditional deletionof β1 integrin in other epithelia suggested thatthis integrin is involved in differentiation. Forinstance, its deletion in intestinal epitheliumled to the inhibition of epithelial differentia-tion and to increased proliferation, leading topostnatal lethality (33), and deletion from thebreast epithelium resulted in aberrant differen-tiation (34). There were anomalies in acinar for-mation, and although the β1 integrin–null cellsin vitro remained associated with each other,they did not attach to the ECM and failed tosynthesize milk proteins adequately in responseto prolactin.

THE RECEPTOR FOR ECMHENSIN IS α6 INTEGRIN

As mentioned above, seeding cells at lowdensity on hensin-containing ECM causedconversion of low-density cells to a high-density phenotype, even in the presence ofblocking antibodies to αvβ integrin or to α1integrin. However, when the cells were seededin the presence of blocking antibodies to α6integrin, the cells failed to columnarize or to de-velop apical endocytosis, even though they wereseeded on top of pylerized hensin. These stud-ies suggested that α6 might be a receptor forECM hensin. We then performed immunopre-cipitation studies and found that in cross-linkedhigh-density cells, hensin antibodies immuno-precipitated α6 integrin but not α1 integrin.Furthermore, seeding low-density cells onhensin-containing ECM caused clustering ofα6 integrin. Although these results show thatpolymerized hensin binds to α6 integrin and

that this binding is critical for its function, theresults do not prove that this integrin actuallysignals to the cell interior to cause phenotypeconversion. The alternative mechanism is thata specific factor binds to polymerized hensin,which changes the conformation of hensinand brings it close to its cognate receptor.Here α6 simply reduces the distance betweenthe hensin:growth factor complex. Similarscenarios have been described for a numberof fibroblast growth factors, wherein thefactor binds to an ECM protein such as aheparan sulfate proteoglycan, which changesits conformation and allows it to bind to thefibroblast growth factor receptor (35).

PATHWAYS OF HENSINSIGNALING

Hensin deposition in the ECM allowed us todevise an assay that divided the hensin signal-ing pathway into an upstream arm, which causeshensin polymerization (and hence ECM depo-sition), and a downstream arm, in which poly-merized hensin binds to a receptor that activatesa signaling cascade that in turn leads to colum-narization. As this idea suggests, hensin is in themiddle of a pathway of differentiation, raisingthe questions of how the process starts and whathensin does. The process in the intercalatedcells starts in vivo by an acid stimulus. How doesacidosis activate integrin signaling secretion ofgalectin 3 and cypA? The mechanism by whichlow pH signals to the cell remains a mystery.Studies by Preisig, Alpern, and their cowork-ers suggest that, at least in the proximal tubule,there is activation of the src-type tyrosine ki-nases sec and pyk2 (36). Whether this mech-anism applies to the intercalated cells will re-quire further studies. Regardless, we found thata nonspecific tyrosine kinase inhibitor blockedthe conversion of low-density cells to a high-density phenotype in culture and furthermorethat β1 integrins were indeed tyrosine phos-phorylated. All these studies indirectly suggestthat tyrosine phosphorylation is critically in-volved in the mechanism of induction of thisprocess, but more work is required to document

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Differentiation

Acid

PI3K/Akt/mTOR

β1 integrin

Secreted cyclophilin and galectin 3

Polymerized hensin

Progenitor cell activator

Figure 2Pathway of the mechanism of terminaldifferentiation of the intercalated cell. Recentstudies have shown that blockers ofphosphoinositide 3 kinase (PI3K), Akt, andmammalian target of rapamycin (mTOR) preventhensin polymerization. Because these kinases havebeen implicated in integrin activation, we proposethe pathway shown here. We also propose thatpolymerized hensin binds and sequesters anantidifferentiation factor that tonically blocksdifferentiation, allowing differentiation to proceed.

the proximate and intermediate steps betweenacidosis and β1 integrin activation.

How does polymerized hensin produce itseffect? Does it act as a ligand to a signalingreceptor? We demonstrated that α6 integrinbinds to hensin, but whether α6 integrin is thesignaling receptor remains to be demonstrated.Alternatively, hensin may bind one ligand topresent it to its receptor or may bind a ligandand, by sequestering it, prevent it from bindingto its cognate receptor. Sequestration of ligandsby ECM proteins is a very attractive possibilitybecause a common theme in developmental bi-ology is that progenitor cells are under a tonicsignal that prevents them from differentiation;removal of the signal that favors the progenitor(less-differentiated) phenotype is required forthe differentiation program to commence (seeFigure 2).

EPITHELIAL DIFFERENTIATIONAND HENSIN

One of the most surprising outcomes of ourwork was the discovery that the change in

intercalated cell phenotype is identical to termi-nal differentiation of epithelial cells. The phe-notype change recapitulated all the character-istics of terminal differentiation. For example,β cells are flat, whereas α cells jut into the lu-men and are taller; i.e., they are more colum-nar. β cells have no apical microvilli, whereas α

cells have exuberant apical structures (microvilliand ruffles). β cells have no apical endocytosis,whereas α cells endocytose and secrete materi-als in a regulated manner from the apical space.All these are characteristic features of terminaldifferentiation of columnar epithelia. Most ofthese characteristics are those of the apical com-partment. We speculate that the mechanism ofterminal differentiation induces a program ofapical proteins that mediate these functions. Itis as if a protoepithelium has a basic apical com-partment that allows only for polarized sort-ing of proteins and lipids essential for polaritybut nothing else. Differentiation causes the ap-pearance of a mature apical compartment thatpermits the development of surface organelles,more specific apical sorting machinery such asdense core vesicles (e.g., pancreas acinar cells),H+-ATPase vesicles in intercalated cells, ciliaor microvilli, etc. Identification of such a tran-scriptional program will be a major discovery inthe cell biology of epithelia.

Hensin is expressed not only in mostsimple epithelia such as the collecting duct, in-testine, pancreas, and lung but also in all multi-layered epithelia such as stratified (skin, esoph-agus), pseudostratified (prostate, bronchi), andtransitional (ureter and urinary bladder) epithe-lia. These studies raise the question of whetherhensin mediates a general mechanism of dif-ferentiation in these epithelia. Indeed, globaldeletion of hensin was lethal to embryos atthe time of first appearance of a columnar ep-ithelium, the visceral endoderm. Seeding em-bryonic stem cells on polymerized hensin ledto the development of terminally differentiatedcolumnar cells expressing many of the changesspecific to the visceral endoderm (37). Thesestudies suggest that the hensin signaling path-way may facilitate a general process of differ-entiation of epithelia. In epithelia, hensin is


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present as an ECM protein, implying (by ex-tension of our previous results) that it plays acritical role in terminal differentiation of ep-ithelia. Using our newly developed conditionalhensin/loxP mice, we should be able to test thishypothesis in vivo. If this indeed turns out tobe the case, a novel hypothesis will emerge re-garding the general differentiation of epithe-lial organs wherein epithelia develop in the fol-lowing defined stages: Differentiation beginsby conversion of stem cells to a protoepithe-lial phenotype. However, these protoepithe-lia are organ specific; that is, their identityhas already been specified to become simple

epithelia or multilayered and to be determinedto be, for example, columnar or transitional.This picture is consonant with the current viewsin developmental biology in which the deter-mination of cells is largely organ and tissuespecific.

Many cancer biologists believe that blockadeof terminal differentiation is a critical determi-nant of oncogenesis, and the human orthologof hensin [deleted in malignant brain tumors 1(DMBT1)] is deleted in a vast number of ep-ithelial cancers (38). These studies are compat-ible with the idea that hensin mediates terminaldifferentiation.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Most of the work described here has been funded by the NIH (DK20999) and by a grant-in-aidfrom the American Heart Association. I am grateful to Dr. George J. Schwartz for our long andcontinuing collaboration on many aspects of this project. Members of my lab who performed thesestudies deserve any credit garnered by this work.

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terminal differentiation in epithelia: the role of integrins in hensin polymerization

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