selective changes inlaminin adhesion anda634integrin...

Vol. 7, 615-628, May 1996 Cell Growth & Differentiation 615

Selective Changes in Laminin Adhesion and a6�34 IntegrinRegulation Are Associated with the Initial Steps inKeratinocyte Maturation1

Tamar Tennenbaum,2 Luowei Li, Adam J. Belanger,Luigi M. De Luca, and Stuart H. Yuspa3

Laboratory of Cellular Carcmnogenesis and Tumor Promotion, NationalCancer Institute, National Institutes of Health, Bethesda, Maryland20892

AbstractIn skin, the distribution of integrins iscompartmentalized. Whereas the a�IJ� integrin complexis polarized to the basal portion of proliferating cells inthe basal layer juxtaposed to the basement membrane,

��3I�1 integrin receptors are localized on the cell surfacesurrounding basal and suprabasal cells, suggesting fi�integrins mediate both cell-matrix and cell-cellinteractions. As initiation of maturation in skin isassociated with the detachment of cells from thebasement membrane, the early loss of a�I3�, but nota3�1� integrin expression could be a determining factorin the transition from the proliferating to adifferentiating keratinocyte. We have studied theregulation of adhesion potential and integrinexpression during differentiation of mouse basalkeratinocytes cultured in 0.05 m�.i Ca2 medium andinduced to differentiate in 0.12 m�.i Ca2’ medium.Within 12-24 h after elevation of Ca2�, a selective lossof the a6134integrin complex is associated with theinduction of the spinous cell marker keratin I . Thisearly differentiation phenotype coincides with loss ofcell attachment mediated by a�fi� to laminins 1 and 5but not to fibronectin or collagen IV. Selective loss ofattachment to laminin is also detected in spinous cellsisolated from newborn epidermis in vivo. The loss ofcreIJ4 protein expression is a consequence oftranscriptional and posttranslational events, includingreduction in mRNA transcripts, reduced synthesis ofthe a� protein, and enhanced processing of the a� andp4 chains as determined by Western blots and pulse-chase experiments in metabolically labeledkeratinocytes. Selective processing of the I3�

Received 1 1/14/95; revised 2/12/96; accepted 2/26/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdi-cate this fact.1 This work was supported by a grant from Johnson & Johnson ConsumerProducts, Inc.2 Present address: Department of Life Sciences, Bar Ilan University,Aamat Gan, Israel.3 To whom all correspondence should be addressed, at Laboratory ofCellular Carcmnogenesis and Tumor Promotion, National Cancer Institute,Building 37, Room 3B25, 37 Convent Drive MSC 4255, Bethesda, MD20892-4255. Phone: 301 -496-2162; Fax: 301-496-8709.

intracellular domain is detected before loss of I3, fromthe cell surface in basal keretinocytes, and thisprocess is accelerated during differentiation. Whereasearly keratinocyte maturation is linked to the selectiveloss of the a6134complex, loss of both fi� and fi,�integrin mRNA and protein occurs as cells proceed tolater stages in the differentiation program as inducedby 0.5 mM Ca2� or suspension culture. Theseconditions are characterized by accelerated expressionof transglutaminase; reduced keratin 1 protein; loss ofadhesion to fibronectin, laminin I , laminin 5, andcollagen IV; and rapid cell death. Contributing to thedown-regulation of 13, integrins during terminaldifferentiation is a selective sensitivity of a3131 but nota6134 to down-regulation by transforming growthfactors � and 1�2’ factors that are also expresseddifferentially in normal skin. This study indicates thatdown-regulation of the a�J, but not � integrins occursduring the initial steps of keratinocyte differentiationand is associated with detachment from the Iamininmatrix. Such changes could contribute an importantsignal to initiate the process of terminal keratinocytedifferentiation.

Introduction

The integnins are heterodimenic glycoproteins composed of a

and f3 subunits expressed in a variety of cell types andinvolved in cell-matrix and cell-cell interactions (1). Specificcombinations of a and j3 heterodimers determine ligandspecificity (2-4). However, the same integnins can bind tomore than one ligand, and coexpressed integnins can medi-ate the interaction with the same ligand (3, 5). A specializedtissue distribution is associated with the a6 integnin subunit,

where a6 associates preferentially with the f3� subunit inepithelial and neuroepithelial cells, while mesenchyme-derived cells express a6f31 (6, 7).

The a6f34 integnn complex is a unique receptor among the

integnin family. The a6(34 ligand is thought to be a laminin orlaminin isoform (6044, 6043). Nevertheless, cells bind tolaminins via a6f34 with varying affinities (8-13). The �34 subunit

is unique among the p subunits because of its large cyto-plasmic domain composed of 1000 amino acids comparedwith 50 amino acids in cytoplasmic domains of other mem-

bers of the 13family (6, 14-1 6). Posttranslational modificationby proteolytic digestion of the �34 cytoplasmic domain has

been described (1 7). Several studies have identified uniqueintracellular associations of the a6f34 integnin. Whereas most

integnins are thought to localize to focal contacts and interact

with the actin cytoskeleton, a6�4 is concentrated at the

hemidesmosomes of keratinocytes, where it most likely in-teracts with the keratin intermediate filaments (18-22).

U)

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0zLU0U)LU

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Fig. 1 . Assay of primary mouse keratmnocyte attachment to matrix pro-teins. Primary mouse keratmnocytes were isolated and plated in EMEM,8% FCS, and 0.05 mM Ca2� as described in “Matedals and Methods.”After 5 days in culture, cells were trypsinized briefly, resuspended in 0.1%BSA in 0.05 mM Ca2� EMEM medium without FCS, and reattachedto Petridishes coated with increasing amounts of fibronectin (U), EHS laminin 1(A), and collagen type IV(#{149}).After 1 h incubation at 37#{176}C,unattached cellswere removed and wells rinsed twice with PBS. 4-Methylumbelliferylheptanoate (1 0 �tg/1 00 pi) was added to each well, and fluorescence wasmeasured after 30 mm using a microfluor reader as described in “Mate-rials and Methods.” For each point, results are presented in arbitraryfluorescence units released from six wells as means; bars, SD.

MATRIX PROTEIN i�ig/mll

4 The abbreviations used are: K1 , KS, K1 0, and K1 4, keratins 1 , 5, 10, and14, respectively; MiT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-hum bromide; TGF-�31, transforming growth factor �3�; EMEM, Eagle’sminimal essential medium without calcium; mAb, monoclonal antibody;ECL enhanced chemiluminescence; EHS, EH sarcoma.

616 Integrin Regulation in Keratmnocyte Differentiation

To understand the specific role of a6(34 as compared with

13, integnns, we have studied the differential regulation ofadhesion and integnin expression during differentiation ofmouse epidermis. In skin, the a6�4 integnin complex is re-stncted to the cell surface of the basal portion of the basalcell compartment, suggesting an interaction with basementmembrane components (6, 8, 10). In contrast, f3� integnins,including a3�1 , a2�1 , and a5131 are localized on the cellsurface surrounding basal and supnabasal cells mediatingcell-matrix as well as cell-cell interactions (23-25). Prolifera-tion of keratinocytes in normal epidermis is maintained in thebasal layer in cells expressing the a6�4 integnn. Because theinitiation of terminal differentiation is associated with detach-ment of cells from the basement membrane and suprabasal

migration, the loss of a5�34, but not j3� integnn expressiondunng the early stages of differentiation, could be a deter-mining factor in the transition from the proliferating to adifferentiating keratinocyte.

In vitro, the a3�1 integnin complex is redistributed to cell-

to-cell junctions when human keratinocytes are induced todifferentiate by Ca2� (23). In contrast, p� integnns are lostwhen human kenatinocytes are induced to differentiate insuspension culture (26). Because the loss of � integnin ex-pression was associated with reduced attachment to fi-bronectin (27), it has been proposed to represent a critical

early event related to detachment from the basement mem-brane of differentiating keratinocytes. However, fibronectin isnot a component of the epidermal basement membrane, andboth a6f34 and � integnins are distributed on the basal as-

pect of basal cells that contact the basement membrane (19).In vivo analysis of the distribution of a6f34 in normal skin andchanges in skin diseases, wound healing, psonasis, andcancer have been reported previously (28-31). The localiza-tion and ligand binding properties of a6�4 have been evalu-ated in vitro (7, 8, 19). However, a molecular and functionalanalysis of �L5�34 integnn complexes during kenatinocyte dif-ferentiation has not been explored.

In this report, we have analyzed the relationship of a6j34

and a3f31 expression and keratinocyte differentiation in cul-tuned mouse kenatinocytes. When mouse keratinocytes arecultured in medium with a reduced Ca2� concentration (0.05mM), the cells maintain a basal phenotype. Elevation of Ca2�above 0.1 mM induces terminal differentiation in a coondi-nated sequence of maturation steps that closely mimic the invivo process in epidermis (32, 33). These studies reveal thata6f34 and a3J31 integnins are differentially regulated during the

induction of terminal differentiation. These changes are as-sociated with specific alterations in attachment to extnacel-lulan matrix components and expression of stage-specificmarkers of terminal differentiation.

Results

Attachment of Keratinocytes to Laminin I and Laminin 5Is Selectively Reduced in Early Stages of Ca2�-inducedDifferentiation. To assess adhesion potential, primary ke-

natinocytes, maintained as basal cells in 0.05 m� Ca2’� me-

dium for 6 days, were replated onto collagen IV, fibronectin,laminin 1 , and laminin 5 matrices. Within 1 h, 60-70% of cellsadhered to fibronectin and collagen type IV, but only 30%

attached to laminin 1 (Fig. 1). While spreading of cells onfibronectin and collagen IV matrix occurred within 2 h afterthe initial plating, cells plated on laminin 1 initially remained

round. Attachment to another laminin isoform, laminin 5, wasrapid, with 80% of the kenatinocytes attaching to the cultureplate within 10 mm (Fig. 2B) and full spreading detected by 1h. However, spreading on all matrices was similar 24 h afterplating (not shown). When kenatinocytes were induced toundergo spinous cell differentiation by growth in 0.1 2 mt�iCa2’� medium for 24 h, proliferation stopped, and 20-30% ofcells expressed the spinous cell proteins � and K1 0 (32,34). After 24 h in 0.12 m� Ca2� medium, differentiatingkenatinocyte attachment to laminin 5 and 1 matrices de-

creased, but attachment to fibnonectin was maintained

(Fig.2). By 48 h in 0.12 m�i Ca2� medium, kenatinocyte at-tachment to all matrix proteins, including fibronectin, laminin1 , and laminin 5 (Fig. 2), as well as to collagens I and IV (notshown), was greatly reduced. At this time, these cells nepne-sent the granular cell stage as determined by the expressionof Ioncnin, filaggnin, and transglutaminase (32, 35).

Adhesion to Laminin I Is Reduced Selectively in Kera-tinocytes Expressing KI . To determine whether there is aspecific reduction in matrix adhesion of cells expressing Ki,kenatinocytes were trypsinized after 24 h in 0.12 m�i Ca2�

medium, and the single cell suspension reattached to Petnidishes coated with 20 �g fibnonectin, collagen IV, on laminin1 (Table 1). After 1 h, adherent cells were analyzed by im-munofluorescence for the expression of Ki . As summarized

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in Table 1 , a population of spinous keratinocytes expressing

Ki maintained their attachment to fibronectin and collagen

IV, but did not attach to laminin 1 . Cells maintained in 0.05

mM Ca2� medium expressing K14 but not expressing Ki

maintained their adherence to both fibronectin and laminin

matrices, as shown in Fig. 2. To confirm the in vitro findings,

keratinocytes were isolated from newborn epidermis and

analyzed for attachment to matrix-coated dishes. As seen in

Fig. 3, K1 -positive keratinocytes isolated from newborn ep-

idermis maintained their adhesion to fibronectin (Fig. 3A) but

did not attach to a laminin 1 matrix (Fig. 3C). Keratinocytes

expressing granular cell markers lonicnin (Fig. 3E) and filag-

grin (not shown) adhered poorly to both fibronectin and

laminin 1 as seen for cultured keratinocytes after 48 h in 0.12

mM Ca2 � medium (Fig. 2). Keratinocytes expressing the ba-

sal cell K14 adhered to both laminin 1 and fibronectin.

24 hr. 4Jhr.

Selective Down-Regulation of a6�4 and a3�1 Is Asso-ciated with Ca2�-lnduced Differentiation. Two integrin re-

ceptors, a6�4 and a3f31, are thought to mediate the attach-ment of keratinocytes to both laminins 1 and 5 (12, 1 3). The

differential expression of a6�4 and a3/31 in stratifying epider-

mis suggested these integnins may be regulated by the state

of differentiation, and in turn this may alter the adhesion of

cells to laminin matrix in the early stages of keratinocyte

differentiation. Membranal fractions of cultured basal kerati-

nocytes in 0.05 mM Ca2� contained abundant a6f34 and a3�1

complexes when assayed by immunoblotting (Figs. 4A and

B). Induction of spinous cell differentiation by 0.12 m� Ca2�

medium reduced immunodetection of a6 by up to 90% and

134 integnin subunit by 50% in 24 h, a time point coincidingwith reduced attachment to a laminin 1 and laminin 5 matrix

and increased expression of K1 and Ki 0. In contrast,

Cell Growth & Differentiation 617

Fig. 2. The effects of calcium-induced dif-ferentiation on the attachment potential ofprimary mouse keratinocytes. Primary kera-tinocytes were cultured for 5 days in 0.05 m�Ca2� EMEM as described in the legend toFig. 1 . At day 6, differentiation was inducedby elevating the Ca2 ‘ to 0.1 2 m� for anadditional 24 or 48 h. A, cells cultured in 0.05(#{149})and 0.12 (0) mM Ca2 � were trypsinizedbriefly, resuspended in 0.05 m� Ca2 ‘ . andallowed to reattach for 1 h to Petn dishescoated with increasing concentrations of fi-bronectin or laminin 1. Attachment was as-sayed as described in the legend to Fig 1.For each point, results are presented as ar-bitrary fluorescence units released from sixwells as means; bars, SD. Attachment as-says carried out in 0.12 m� Ca2 ‘ yieldedsimilar results. B, cells cultured in 0.05 m�iCa2� and differentiating cells in 0.12 m�Ca2 � were trypsinized briefly, resuspendedin 0.05 mM Ca2 ‘ , and replated for 10 mmonto 24-well Petri plates coated with 10jtg/ml laminin 5, as described in “Materialsand Methods.” Attachment to laminin 5 ma-trix is presented as mean percentage of dif-ferentiating keratinocytes (0.1 2 mr�i Ca2’)from three wells (bats, SD) relative to at-tached cells maintained in 0.05 m� Ca2’medium determined as 100%.

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618 lntegrin Regulation in Keratinocyte Differentiation

Table 1 Attachment of differentiating keratinocytes expressing keratin1 to matrix proteins

Keratinocytes cultured for 6 days in 0.05 m� Ca2� were induced todifferentiate by elevating the Ca2 ‘ concentration to 0.1 2 mt�i for 24 hours.Keratinocytes were then trypsinized briefly and resuspended in 0.05 m�Ca2� EMEM, plated on Petri dishes coated with 20 �g/ml of fibronectin,EHS laminin 1, or collagen type IV, and incubated for 1 h at 37#{176}C.Unattached cells were removed, and plates were rinsed twice with PBS.Thereafter, plates were fixed in 1 :1 methanol:acetone (v/v) for 5 mm atroom temperature and processed for double immunofluorescence asdescribed in “Materials and Methods.” Mouse Ki was visualized usingFITC-conjugated secondary antibody. Mouse K14 was visualized byTexas red. For six fields, each of 100 cells was counted on duplicatedishes.

Experiment no. Collagen IV Fibronectin Laminin 1

1 35#{176}35

50

60

0.050.05

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32

40

44

0.06

0.05

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3248

440.04

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a Results are presented as mean percentage of Ki -positive cells attached

to coated plates relative to total K14-positive cells, representing all kera-tinocytes. Results have been adjusted to reflect the percent of K1 -positivecells in the starting population after 24 h in 0.12 m� Ca2� , which rangedbetween 20 and 30% of total keratinocytes.

although there was a 50% reduction in the expression of the

13� subunit, the level of a3�1 complex as determined by a3protein was maintained for up to 60 h in 0.12 mr�i Ca2� (Fig.

4). When Ca2� was increased from 0.05 to 0.5 m�, down-

regulation of both a6�4 and a3�1 occurred more rapidly. In

fact, both a6 and �34 were reduced markedly by 6 h. As seen

for 0.12 mM Ca2�, a3 reduction occurred more slowly in 0.5

mM Ca2� but was obvious by 48 h. This Ca2� level acceler-

ates the terminal phase of keratinocyte maturation without

optimal induction of spinous cell markers (32, 35).

a6f34 Integrin Is a Major Receptor Mediating Attach-ment of Mouse Keratinocytes to Laminins I and 5. To

examine further the contribution of cell surface a6f34 integnin

to attachment of keratinocytes to laminins 1 and 5, we per-

formed attachment assays using a blocking antibody to the

a6 integnin, GoH3. As seen in Fig. 5, attachment of keratino-

cytes to both laminins 1 and 5 was efficiently blocked by the

addition of the antibody (75 and 67%, respectively). How-

ever, the attachment to fibronectin was not altered. These

results suggest that reduced a6�4 protein expression on the

cell surface of keratinocytes in the early stages of keratino-

cyte differentiation could reduce selectively the ability of

keratinocytes to attach to laminin 1 and 5 matrices.

Ca2’-induced Differentiation Is Associated with Selec-tive Regulation of Integrin mRNA. Protein data indicated a

rapid decrease in a6 and �34 proteins associated with spinous

cell differentiation. A similar change was noted in the level of

mRNA transcripts (Fig. 6). Within 24 h of raising Ca2� from

0.05 to 0.12 mM, transcript levels for both a6 and p4 were

substantially reduced. This coincides with an increase in Ki

transcripts as seen previously (32). The expression of trans-

glutaminase transcripts also increased gradually during this

time. In contrast, transcripts for the /3� integrin were main-

tamed at the 0.05 mM Ca2� level for 48 h. It is interesting that

there appears to be a transient increase in the level of f3�

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Fig. 3. Attachment of differentiating keratinocytes isolated from new-born epidermis to matrix proteins. Keratinocytes isolated from newbornskin were resuspended in 0.05 m� Ca2 EMEM and plated on Petri dishescoated with 20 �g/ml offibronectin (A, B, E, and F) or EHS laminin 1 (C and0) and allowed to attach for 1 h at 37CC. Unattached cells were removed,and plates were rinsed twice with PBS. Thereafter, plates were fixed in 1:1methanol:acetone (v/v) for 5 mm at room temperature and processed fordouble immunofluorescence. Mouse Ki (A and C) and mouse loricrin (E)are recognized by rabbit polyclonal antibodies and visualized using FITC-conjugated secondary antibody. Mouse K14 (B, D, and F) is recognized bya polyclonal guinea pig antibody and visualized by Texas red. Photo-graphs (x200) of the same fields are presented for the following pairs: A(Ki), and B (K14); C (Ki) and D (K14); and E (loricrin) and F (K14).

transcripts within the first 1 5 h of the Ca2� switch preced-

ing the increase in Ki transcription. Similar results were

obtained for a3 transcripts (not shown). Coinciding with

the protein data, elevation of Ca2 � to 0.5 m� caused

accelerated loss of transcripts for a6f34, but this was less

rapid than the protein loss (Fig. 6B). The rapid down-

regulation of 13� protein occurring within a few hours of

culture in 0.5 mM Ca2� (Fig. 4) was not associated with a

loss of 13� mRNA until 24 h in 0.5 mM Ca2� (Fig. 6B). This

implies that both transcriptional and posttranscniptional

changes are involved in the regulation of f3� and /34 integrin

expression in keratinocytes. The accelerated expression

of transglutaminase within 1 2 h and aborted expression of

Ki in 0.5 mM Ca2� medium are consistent with the rapid

terminal differentiation that occurs in this higher-Ca2

medium (35, 36).

Kinetics of a61J4 Synthesis and Turnover during Ca2�-induced Differentiation. The reduction in a6�4 expression on

the cell surface after Ca2 � -induced spinous differentiation

could result from modulation of preexisting receptors on the cell

surface or reduced synthesis of integrin subunits. To determine

the turnover kinetics of both preexisting receptors as well as

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Fig. 4. Western blots a6,f3�, J13, and (3� integrin subunits were analyzed in keratinocyte membranal lysates from cells induced to differentiate by elevationof Ca2 � concentrations. Primary keratinocytes were cultured in 0.05 m� Ca2� medium for 5 days as described in the legend to Fig. 1 . At day 6, the Ca2concentrations were elevated to 0.12 and 0.5 m� as indicated, and cell lysates were collected at the indicated time points and samples analyzed bySDS-PAGE run under nonreducing conditions. A, Western blots of the membranal fractions were visualized by ECL using polyclonal rabbit antibodiesagainst a6 and (13 and rat antibodies mAbs against j3� and /3� as described in “Materials and Methods.” B, densitometric quantitation of Western blot analysisof a6,L�4. �3’ and (3� integrin subunits in keratmnocyte lysates from cells induced to differentiate by elevation of Ca2 ‘ concentrations. Results were visualizedby ECL. The densitometric results were obtained from blots presented in A.

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620 Integrin Regulation in Keratmnocyte Differentiation

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Fig. 5. The effects of (16 blocking antibody (G0H3) on the attachment ofprimary keratmnocytes. Primary keratinocytes were cultured for 5 days in0.05 mM Ca2� EMEM as described in the legend to Fig. 1 . At day 6, cellswere trypsinized and resuspended in 0.05 m� Ca2� medium with increas-ing dilutions of GoH3 antibody. Suspended cells were then allowed toreattach to Petri dishes coated with fibronectin (20 g�g/m�, laminin 1 (20pg/mI), orlaminin 5 (10 �g/m�, and attachment assays were performed asdescribed in “Materials and Methods.” Attachment of cells to all matricesis presented as percentage of cell number attached relative to attachmentofcells without the addition ofthe blocking antibody determined as 100%.

newly synthesized receptors, cells in 0.05 m�i Ca2� mediumwere labeled for 3 h with [ssS]methionine and [�#{176}2S]cysteineandchased for 14 h with cold methionine and cysteine. At zero time,immunoprecipitation with anti-as mAb (G0H3) copnecipitateda6 and 134but not 13� integrin subunits (not shown) detected byWestern blotting, confirming the absence of a�3� complexes in

mouse keratinocyte cultunes (Fig. 7). After a chase of 14 h in0.05 m�i Ca2� medium, differential processing of a� and �34

subunits was observed. The turnover of the a� subunit was

slow, and labeled protein was reduced by only 20% at 14 h ofchase. In contrast, the copnecipitating mature form of f3� inte-

grin decreased by more than 80% in 14 h (Fig. 7). The rapiddisappearance of the mature form of the p4 subunit coincided

with the loss of a shghfly smaller protein (Fig. 7, Band A) andincreased intensity of a coimmunoprecipitating band at Mr140,000 (FIg. 7, Band B). The Mr 140�000 proteIn band as well

as lower-molecular-weight protein bands were recognized by a

134antibody directed against the extracellulan domain in West-em blots (seen in Figs. 9 and I OA) and by immunoprecipitation(Figs. 8 and 9). It is surprising that the processing of the a� and

134subunits was not altered substantially when Ca�� concen-tration was increased to 0.12 m� during the chase period(Fig. 7).

When cells were induced to differentiate for 16 h in 0.12mM Ca2� followed by metabolic labeling for 3 h, there weremajor differences in the processing of a� and 134 proteinsdepending on the extnacellular Ca2� (Fig. 8). In differentiatingkenatinocytes, a6 synthesis is reduced by about 50%, andthere appears to be a lower amount of associated labeled p4(Fig. 8A). In the same cell lysates, immunoprecipitation with

the anti-f34 antibody did not indicate a significant reduction in

134 synthesis in 0.1 2 m� Ca2� , but revealed both the matureand the Mr 140,000 134bands, suggesting that the Mr 140,000

band must be formed rapidly after synthesis of �34 in differ-

entiating cells and is an integral part of 134 processing, sug-

gested previously (14, 1 7). Experiments depicted in Fig. 8, Band C, studied the turnover of a6 and p4 proteins whenlabeled for 3 h after 16 h in 0.05 or 0.1 2 m� Ca2� and thenchased. Both metabolically labeled a6 and p4 are lost fastenin differentiating cells cultured in 0.12 m� Ca2� for 16 h.Furthermore, the degradation of (34 is accelerated in 0.12 m�i

Ca2�, as indicated by the early disappearance of both the Mr140,000 and the mature p4 during the chase period. Proc-essing of the f34 protein during the differentiation processwas substantiated additionally in Western blots using anantibody directed against the p4 intracellular domain, cyto227 (Ref. 1 7; Fig. 9A). Multiple bands in the range of Mr45,000-1 00,000 representing processed p4 protein were

abundant both in basal cells and in differentiating kenatino-cytes when probed with antibody cyto 227 (Fig. 9). Specificchanges in the p4 processing profile in differentiating kena-tinocytes were detected, including expression of unique pro-tein bands in the range of Mr 120,000150,000 and in-creased expression of bands also detected in basalkenatinocytes (Fig. 9A). TheMr 140,000 protein that is pro-cessed rapidly with mature �34 in differentiating cells is nec-ognized by antibody 346-1 1A (Fig. 9B) in basal cells, mdi-cating this band contains the extracellulan domain of the p4protein. However, cyto 227 did not recognize this band in p4immunoprecipitates (Fig. 9B), indicating that the Mr 140,000band is a processed p4 lacking the intracellular domain,which is distinct from the p4-related proteins of similar size

detected by the cyto 227 antibody in differentiating kena-tinocytes (Fig. 9A). Together, these results indicate that

decreased synthesis, increased loss from the cell mem-brane, and increased processing of the p4 protein allcontribute to the reduction in a6f34 during keratmnocytedifferentiation.

Suspension-induced Terminal Differentiation ofMouse Keratinocytes Down-Regulates Both 1i� and p�Integrin Complexes. Terminal differentiation of culturedhuman keratinocytes can be induced by forced suspensionof cells in a semisolid medium, and this was shown previ-ously to alter integnin expression (26). Similarly, mouse ke-natinocytes that detach from 0.05 mr�i Ca2� cultures havebeen reported to differentiate in suspension (37). To studysuspension-induced changes, medium was changed at zerotime, and 3 on 24 h later, the cells that had detached spon-taneously were collected and analyzed for mRNA and pro-tein. As seen in Fig. 1OA, there is a major loss of both a and

p subunits 24 h after suspension of cells in 0.05 m� Ca2�medium. This is associated with functional loss of attach-ment to fibnonectin, laminin, and collagen type IV (notshown). As early as 3 h after suspension, 40% of the cellsretained surface p1and p4mntegnin expression as determinedby immunofluonescence (not shown), but mRNA for both 131and p� integnin subunits was almost absent (Fig. 1 OB). Sus-pension induced a modest increase in the expression of theearly differentiation marker K1 and a large increase in trans-glutammnase expression independent of extracellulan Ca2�(Fig. 1 OB). Transglutaminase transcripts increased within 3 hin suspension, but no additional change was observed by 24h. When mitochondnial metabolic capacity was analyzed by

Ca2�(mM) 0.05(h)’O 3 6 14

0.12

0 3 6 14

200- �

67�

45 - � �; � , �

:� M4-


Fig. 6. Northern blot analysis of kera-tinocytes induced to differentiate byCa2 � . Primary keratmnocytes were iso-lated and cultured as described in Fig.1 . After 5 days in culture, Ca2 ‘ con-centrations were elevated to 0.12 m�i(A) or 0.5 mM (B) for the indicated timepoints. ANA was isolated, electro-phoretically separated, and transferredto Nytran filters. Blots were probedwith: 32P-labeled cDNAs for �6’ f34.and I3� integrmn subunits; Ki; trans-glutaminase (TG); and glyceralde-hyde-3-phosphate dehydrogenase(GAPDH), as described in “Materialsand Methods.”

A.

ca2#{176}(mM)q,g� 0.12Tlm.(h)�i�’i 3 612152448’

a6 ‘�..

t�4 � �

131 �

Ki

TG

“. �. S I

C.�

B.

ca2#{176}(mM) QQ� 0.5Tim.(h) 0 1 3 612152448

o6 �

(34 .

(31

Ki

TG ...�..

GAPDH ..� �

the MiT assay, cells retained their metabolic activity during

Ca2 -induced differentiation but rapidly lost metabolic ac-

tivity when suspended (Fig. 1 OC), as detected by MiT as-says. This suggests that the majority of the cells have termi-

nally differentiated, resulting in cell death by 24 h in

suspension.

Growth Inhibition Caused by TGF-1J Down-Regulatesa3131 but not a6134. The induction of terminal differentiation by

Ca24 is associated with the loss of proliferative capability of

keratinocytes (33). Previous studies suggest this is related to

the up-regulation of TGF-13 expression (38). TGF-j3 is a powerful

GAPDH . . . �

Fig. 7. Immunoprecipitation of ‘6(�4

from metabolically labeled primarykeratinocytes maintained in 0.05 m�Ca2 � EMEM. Primary keratinocyteswere isolated and cultured as de-scribed in Fig. 1 . After 6 days in cul-ture, cells were labeled with r5S]me-thionine and cysteine for 3 h. After 3h incubation, the Ca2 ‘ concentrationwas adjusted to 0.1 2 m� in somecultures as indicated, and unlabeledmethionine was added to all culturedishes and cells harvested at the in-dicated time points. After extractionwith detergent buffer containingEDTA (10 mM) and protease inhibi-tors, samples were precipitated withGoH3 antibody and analyzed undernonreducing conditions with 6.5%SDS-PAGE. A and B, additional co-precipitating bands appearing duringthe pulse-chase period along withmature a6and p4 bands. Relativedensitometry of the relevant bands ispresented graphically. The 0 h timepoint was arbitrarily assigned as 1.0.

but reversible inhibitor of keratinocyte proliferation and does not

induce terminal differentiation. The addition of TGF-(3 to cul-

tuned kenatinocytes in 0.05 mt�i Ca2 � differentially regulated the

integnin receptors but in directions opposite to 0.12 mr�i Ca2 �.

As seen in Fig. 1 1 , a3 but not the a6 subunit was down-

regulated by 0.5 ng/ml TGF-131 or TGF-132 within 24 h of treat-

ment in 0.05 mM Ca2� . This corresponded to parallel decreases

in f3� but not 13� hetenodimers (not shown). These changes

indicate that TGF-13 is not the mediator of changes in a6f34during Ca24 -induced differentiation but could contribute to the

down-regulation of a3f31 in suprabasal cells.

A

Ca2�(mM)

a6 (GOH3)BCa2�(rnM) I 0.05 _____________

I 1Time (hrs) 0 5 10 20

� �-�-

�-�---�“----------- ---.

- 200

-116

-97

-67

CCa2 ‘1mM)

Time (hrs)

1�4 -�

a6� �! 1L(..4�C�

-200

-116

-97

-67

622 Integrin Regulation in Keratinocyte Differentiation

a6I II 1.05 .12 .05 .12

�:: I�1 �2OO

a6-” � . . � -116

�

�4 (346-1 1A)I 0.05 0.12 I

I I I I0 5 10 20 0 5 10 20

0.12F

0 5 1020

Fig. 8. Immunoprecipitation of cr�f3� integrin in metabolically labeled keratmnocytes induced to differentiate in 0.1 2 m� Ca2 � . Primary keratinocytes were isolatedand cultured as described in Fig. 1 . After 5 days in culture, Ca2 � was adjusted to 0.1 2 m�, and cells were allowed to differentiate for 16 h. Thereafter, differentiatingkeratmnocytes were labeled with [�5S]methionine and cysteine for 3 h. At the end of 3 h incubation, methionine was added to all culture dishes. Cells were extractedat the end of the labeling period [time 0 (A)] or at the indicated time points (B and C) with detergent buffer containing EDTA (1 0 mM) and protease inhibitors. Samplesadjusted by equal counts (A) or equal protein (B and C) were precipitated with GoH3 antibody (A and B) or 346-i 1A �4 and C) and analyzed under nonreducingconditions by 7.5% SDS-PAGE. #{176},additional coprecipitating bends appearing during the pulse- chase period along with mature � and (34 bands.

DiscussionA critical step in the differentiation process of all epithelialcells is the detachment of the cells from the basement mem-

brane, and this process is likely to involve modification in

integnin-matnix interactions. Our study reveals that the induc-

tion of differentiation in cultured mouse keratinocytes results

in the early loss of the a6j34 integnin complex within 24 h in

association with the induction of the spinous marker K1.

Importantly, this change is linked to a selective loss of affinity

for the laminin family of substrates, which are major compo-

nents of the basement membrane adjoining the basal cells.

Others have shown that attachment to matrix proteins wasinfluenced directly by the extracellulan concentration of di-

valent cations such as Mg2�, Mn2, and Ca2� in several celllines (12, 39-43). However, this is unlikely to be related

directly to our studies, because all our attachment assays

were conducted in 0.05 mr�i Ca2� medium. Moreover, inmouse kenatinocytes, reduced attachment to laminins was

evident only after 24 h and was specific to the spinous cells

expressing K1.

Keratinocytes committed to spinous cell differentiation

maintain a3j31 expression and attach to fibronection andcollagen IV substrates similarly to basal cells. Previous stud-

ies showed that 13� integnins are detected in differentiating

human keratinocytes in vivo and in vitro (23, 31). Miller et a!.

(44) and Adams and Watt (45) suggested that loss of both

a5131 and attachment to fibronectin in basal cells are early

related events in keratinocyte differentiation. However, sub-

sequent studies revealed the coexpression of /3� integnins

and involucnin in differentiating keratinocytes (31 , 46). Our

results suggest that binding to laminins 1 and 5 via a6134

integnin may be more relevant to the basal cell phenotypeand its loss more closely associated with the initiation ofdifferentiation. The loss of binding to both laminins 1 and 5

coincides with the down-regulation of aj34 in differentiating

keratinocytes. Nevertheless, keratinocytes and other epithe-

hal cells expressing a6j34 do not attach or migrate well on

laminin 1 (1 1 , 19, 27, 47), and a6/34has low affinity for laminin

1 binding (8). However, the attachment to other members of

the laminin family, such as the laminin 5 (kalinin/nicein/epili-

A ImmunoblottingCyto 227

B Immunoprecipitation(346-11 A)

t�4lmmunoblotting

I 346-lhA Cyto227I 1 1

0.05 C 0.05 C

� :l� I- : -

- I_lasFig. 9. Immunoblotting of (34 fragments in keratmnocytes. A, keratino-cytes cultured in 0.05 m�i Ca2 � , differentiating keratinocytes in 0.5 m�Ca2 � for 24 h, and 3T3 cells were extracted in radioimmunoprecipitationassay buffer and samples separated on SDS-PAGE gels under reducingconditions and transferred to nitrocellulose. Identical filters were probedwith cyto 227 or with the cyto 227 antibody together with 10 �.tg/mlblocking peptide. B, total cell lysates from basal keratinocytes cultured in0.05 m�i Ca2 � for 5 days and differentiating keratmnocytes maintained in0.12 m� Ca2 � for 24 h were extracted and samples immunoprecipitatedwith an antibody to the extracellular domain of (3� (346-1 1A) or rat lgG (C),and then total lysates and immunoprecipitates were separated on SDS-PAGE gels and transferred to nitrocellulose. Identical filters were probedwith cyto 227 ([34 antibody to the intracellular COOH-terminal domain) orwith (34 antibody to the extracellular domain (346 -1 1A). “, additional bandsappearing during keratinocyte differentiation. ‘4, bands expressed differ-entially in differentiating keratmnocytes.


+Peptide

Ker. � Ker.

ca 2#{176}(mM) 3T3 ‘0.05 0.5 ‘ 3T3 � 0.05 0.5I’ II II UI IC II

200 -

, , *

116- *

97- .�4

67- �

t�4

Immunoblotting

346-hA

Ca 2. (mMl 0.05 0.12

200 -

116 -

97- 4,

67 -

45 -

grin complex), which bind to a6134 with high affinity, could

mediate this matrix interaction (12, 13, 48, 49). In mouse

keratinocytes, attachment to both laminins 1 and 5 was

blocked efficiently by a6 blocking antibody, implying the a6134

integnin is a major receptor mediating keratinocyte attach-

ment to both matrices. This view is consistent with data in

human keratinocytes, where attachment to laminin 5 was

reduced by a6 blocking antibody, although complete inhibi-

tion of attachment to laminin 5 required blocking of both a3131

and a6/34 by specific antibodies (47, 50). Although a complex

interaction with both a6134 and a3f31 mediates cell attachment

to laminin 5, only a3131 influenced cell spreading in human

keratinocytes (1 1 , 47, 50). The specific down-regulation of

aj34 could contribute to reversal of hemidesmosome-medi-

ated adhesion and result in loss of polarized adhesion during

early differentiation, while the cells retain cell adhesion via

a3f31 (11, 13, 22, 23).

The reduction of a6134 in keratinocytes is controlled by both

transcriptional and posttranscniptional events, and both

mechanisms were described previously for /3� integnins (30).

However, whereas the processing of � integnins intracellu-

larly is regulated by glycosylation (26, 51 , 52), a selective

processing of the 13� intracellular domain precedes the turn-

over of the a6134 complex and occurs at the cell surface.

Intracellular processing of the � protein was shown by

Giancotti et a!. (1 7) to be a Ca2�-sensitive process that

occurs in extracts from several cell types. Although intracel-

lular Ca2� rises in differentiating keratinocytes, this is not

likely to be directly responsible for f3� processing, because

metabolically labeled 13� was processed similarly in basal

keratinocytes cultured in 0.05 mr�i Ca2�.

The differentiation state resulting from exposure of the

keratinocytes to 0.12 mr�i Ca2� for 16 h did affect the proc-

essing of 134and a6 proteins in a rather complex manner. This

included a reduction in both a6 and f3� transcripts, reduced

synthesis of the a6 protein and loss of a6 protein from the

membrane, and enhanced degradation of the 13� chain cou-

pled to the appearance of processed � fragments of lower

molecular weight. Given that the putative role of the p�subunit is the interaction with the keratin filaments, the early

selective processing of the 13� subunit could be associated

with the shift from the predominant K5 and K14 cytoskeleton

of basal cells to the Ki and Ki 0 cytoskeleton of spinous

cells, particularly since the basal cell cytokeratins and the /3�

integnin are components of the hemidesmosomes that are

lost during differentiation. Moreover, the processing of the 13�complex in basal cells maintained in 0.05 m� Ca2 � could

contribute to the spontaneous release of basal cells into the

culture medium. This change could be associated with the

expression of Ki in a subpopulation of cells in cultures

maintained in 0.05 mrvi Ca2 � (32) preceding their detachment

from the culture dish. The specific degradation of the intra-

cellular fragments of the 13� subunit was demonstrated byimmunostaining skin in vivo from human (17) and mouse

origins (not shown), suggesting this pathway is operative in

vivo.

Although early spinous differentiation is linked to the se-

lective loss of the cr6134 complex, loss of both f3� and f3�

integrins occurs later in differentiation as cells approach the

terminal phase of maturation leading to cell death, such as

that induced by 0.5 mM Ca2� or suspension. Under these

conditions, keratinocytes lose adhesive potential to all matrix

proteins examined. A functional relationship may exist be-

tween the loss of f3� integrins and terminal differentiation

given that incubation of keratinocytes with blocking antibod-

ies to 13� integrin resulted in the appearance of terminal

differentiation markers in human keratinocytes (50). In cul-

tured human keratinocytes, forced detachment of cells and

suspension into a semisolid medium resulted in an abrupt

200 -

118 - S

78 -

47 -

Att. Susp. Att. Susp.

*� . B

‘““C Att. Susp.Ca2�(mM) 0.05 0.12 0.05

II II U

Time (h) 24 3 24 24‘I .� � �I

Ki

a�n� O.l2rvM

0 8 24 0


A ct6 a3 1�4

Att. Susp. Att. Susp.

�0

I

TG

31

C

I

St�i(h)

(�1 �

f�4 �

Fig. 10. Comparison of mntegrmn expression, differentiation marker expression, and cell viability of suspended keratinocytes in 0.05 m� Ca2 ‘ and attachedkeratinocytes in 0.12 m� Ca2� . Keratinocytes were isolated and cultured as described in Fig. 1. After 5 days, culture dishes were rinsed three times withPBS to assure the elimination of all spontaneously suspended cells. Attached cells from 0.05 and 0. 12 m�i Ca2 � cultures and spontaneously detached cellsfrom 0.05 mM Ca2� cultures were extracted after 24 h. A, membranal fractions from attached and spontaneously suspended cells in 0.05 m�.i Ca2� wereprocessed for integrin expression of a6, (��‘ a3, and f3� protein subunits by Western blot. B, Northern blot analysis, attached cells from 0.05 and 0.1 2 m�Ca2� cultures and suspended cells from 0.05 m� Ca2� cultures were collected and extracted for RNA at 3 and 24 h as indicated. RNA samples wereelectrophoresed and transferred to a Nytran membrane and hybridized with 32P-labeled cDNAto Ki , transglutammnase (713), and f3� and f34 integrmn subunits.285 and 185 ribosomal bands recognized by ethidium bromide staining are presented as control for loading in each lane. RNA for Northern blots andmembranal fractions for Western blots were processed as described in “Materials and Methods.” C, metabolic activity was assayed in a MiT assay onsuspended cells maintained in 0.05 rn� Ca2� for 24 h in comparison with attached basal keratinocytes cultured in 0.05 m� Ca2� (0 time) and keratmnocytesdifferentiating in 0.1 2 mM for 24 h. Twenty thousand spontaneously detached cells were removed from the medium of 0.05 m� Ca2 � cultures, and an equalnumber of attached cells from each Ca2� group was isolated by trypsinization. Cell groups were transferred to a 96-well titer plate, and a MIT assay wascarried out as described in “Materials and Methods.” Data are presented as mean; bars, SD.

loss of adhesive potential and loss of integnin expression on

the cell surface (26, 45, 52). Because keratinocyte diffenen-

tiation in vivo is a sequential program that is coordinated

temporally and spatially, the Ca2�-induced model described

here may reflect more accurately the sequential changes in

integnin processing than suspension cultures.

An integral part of keratinocyte differentiation in addition to

the expression of important structural proteins is the differ-

ential expression of regulatory growth factors. TGF-13 ex-

pression is compartmentalized in mouse and human epider-

mis such that TGF-j31 is expressed in basal cells and TGF-132

is expressed suprabasally (53, 54). Cultured keratinocytes

up-regulate TGF-f32 expression in concert with the induction

of differentiation (38, 53). TGF-13, and TGF-132 specifically

down-regulate a3J31 protein expression in a Ca2 �-independ-

ent manner similarly to results obtained in human keratino-

cytes (55). The suprabasal compartmentalization of TGF-132

suggests this factor could be involved in the down-regulation

of 13� integnins. TGF-13, a growth factor sequestered in the

extracellular matrix, is a prime candidate for mediating inte-

:::::: .�3- S

a6 - � � -�2.O

Cl)

C

0 1.5

C.)

0.5 2.0

0.5 2.0


ng/mI

C TGF�i TGF1�2‘0.5 2’ ‘0.5 2’

Fig. 1 1 . TGF-f3 down-regulates surface ex-pression of n3but not a6 integrin subunits inprimary mouse keratinocytes. Primary kerati-nocytes were isolated and cultured in 0.05mM Ca2 � medium as described in the legendto Fig. 1 . At day 5, TGF-j31 and TGF-�3� wereadded in increasing concentrations (0.5-2 ng/ml) for 24 h, followed by extraction of mem-branal fractions as described in “Materialsand Methods.” Western blot of a3 and a6

integrin subunits was analyzed by ECL Re-suIts illustrate original blots and densitometryof a representative experiment repeated threetimes.

TGFI3I (ng/ml)

TGF1�2 (ng/ml)

grin expression and function. In fibroblasts as well as in

epithelial cells, TGF-13 differentially stimulates matrix protein

synthesis and deposition (56, 57), regulates integrin expres-

sion (58-60), and influences cell proliferation and locomotion

(61 ). In concordance with our results, the absence of TGF-131

in genetically null mice up-regulated f3� integrins in lympho-

cytes, contributing to a massive inflammatory reaction and

early cell death (62). In these mice, injection of fibronectin

delayed the inflammatory response and enhanced longevity.

Presumably, treatment with TGF-j31 or TGF-132 would have

the same effects by reducing I3� integnins.

The ability to study keratinocyte differentiation in a culture

model where Ca2�-induced sequential changes in gene ex-

pression, protein processing, and cell signaling reflect the

temporal and spatial changes measured in epidermis is es-

sential for determining regulatory events in the maturation

process. Certain discrepancies exist in data derived from

human and mouse keratinocyte cultures stimulated by Ca2�

(46, 51 , 63) with regard to integnin expression. These could

reflect essential differences among species, but this seems

unlikely, considering the identical compartmentalization

identified for integnins in mouse and human skin. Alterna-

tively, several studies have reported that subculturing human

keratinocytes results in increased attachment to fibronectin

and laminin 1 (47, 63). Culture conditions for mouse and

human keratinocytes are quite different in most experiments.

Many studies of human keratinocytes employ media en-

riched in growth factors, particularly epidermal growth factor,

hydrocortisone, and insulin. Both epidermal growth factor

and insulin influence specific integrin expression (64-66) and

function. Thus, future studies must consider the contribution

of culture conditions when comparing results from different

laboratories and different species and particularly when ex-

trapolating in vitro findings to in vivo functions. The Ca2�-

induced differentiation of mouse keratinocytes in vitro seemsparticularly useful to evaluate pathways contributing to reg-

ulation of thea6f34 and the j3� integnins. Future studies will

emphasize the contribution of these cell surface receptors to

the regulation of differentiation and proliferation of epidermal

cells.

Materials and MethodsAntibodies. The antibody 6844, specific for the a�A integrin cytoplasmicdomain, was a gift from Dr. V. Quaranta (Scripps Research Institute, La

Jolla, CA; Ref. 67). The rat antimouse mAb a6 (GoH3) was purchased fromAmac (Westbrook, ME). Rat mAb directed against the extracellular domain

of mouse 134(346-1 1A) was a gift from Dr. S. J. Kennel (Oak RidgeNational Laboratory, Oak Ridge, TN; Ref. 68). A rabbit polyclonal antibody

directed against the intracellular domain of mouse f3� (cyto 227) and the

blocking peptide was a gift from Dr. F. Giancotti (New York UniversityMedical Center, New York, NY; Ref. 17). KM16, a rat mAb to the f3�integrin subunit, was a gift from Dr. J. Gimble (Oklahoma Medical Re-

search Foundation, Oklahoma City, OK; Ref. 69). A rabbit polyclonal

antibody to the a3 integrin was purchased from Chemicon International,Inc. (Temecula, CA). Monospecific keratin antibodies to Ki and K14 wereprepared in rabbits against synthetic peptides corresponding to unique

sequences at the carboxyl terminus of mouse keratins (70).

Cell Culture. Primary keratinocytes from newborn BALB/c mice wereprepared as described by Hennings et al. (33). Keratinocytes were cul-tured in a humidified atmosphere at 36#{176}Cand 7% CO2 in EMEM, sup-plemented with 8% FCS and treated with Chelex 100 (Bio-Rad) to remove


Ca2� and 0.2% Antibiotic Solution (Ufe Technologies, Inc., Grand Island,NY). Calcium concentration was adjusted to 0.05 mr�i by the addition ofCaCI2. In certain experiments, the Ca2� concentration was increased to

0.12 or 0.5 mM for various time points to induce differentiation.

Attachment Assays. Ninety-six well Petri plates (Costar) were coated(100 s.d/well) with increasing concentrations (5-30 �g/ml) of larninin,

fibronectin, or collagen type IV (Collaborative Biomedical Products; Bec-ton Dickinson) in PBS for 1 h at 37#{176}C.After incubation, plates were

washed with PBS and incubated with 0.1 % BSA for 30 mm at roomtemperature to block nonspecific binding. Cells from exponentially grow-ing primary keratinocyte cultures were washed in PBS and trypsinized

briefly with 0.25% trypsin. After detachment, cells were resuspended in

0.05 m� Ca2� medium containing 8% FCS and centrifuged at 1000 rpm

for 10 minutes. Cells were resuspended in EMEM without FCS supple-rnented with 0.1 % BSA, and the number of cells was determined using a

Coulter counter (model ZBI). Keratinocytes (5 x 10�) were added to thecoated wells and incubated for 1 h at 37#{176}C(6 wells/matrix concentration).

Nonadherent cells were removed, and the wells rinsed twice with PBS.The fluorogenic substrate 4-methylumbelliferyl heptanoate (Sigma Chern-ical Co., St. Louis, MO; Ref. 71) was added to each well (10 jig/100 �I) for1 0-30 mm, and fluorescence was measured after incubation using the

Microfluor Reader (Dynatech). The nonfluorescent fluorescein ester be-

comes fluorogenic in a living cell after hydrolysis by cell esterases (72). Forattachment to larninin 5 (a gift from Dr. A. Burgeson, Harvard University,

Cambridge, MA), 24-well Petri plates were coated with 10 �g/ml laminin5 in PBS at 4#{176}Cfor 16 h. After incubation, the plates were washed with

PBS and incubated with 0.1 % BSA to block nonspecific binding. Keratin-ocytes (2.5-5.0 x 10�) were added to the coated wells for 10 mm at 37#{176}C.

Nonadherent cells were washed, adherent cells were trypsinized, and cell

number was determined by a Coulter counter.For antibody-blocking experiments, dilutions of anti-a6 integrin (G0H3)

antibody were added to primary keratinocytes in suspension followed byattachment to 24-well plates coated with fibronectin (20 �g/ml), larninin 1

(20 pg/mI), or laminin 5 (10 �g/ml). The number of attached cells wasdetermined by a Coulter counter.

Immunofluorescence. Thirty-five-mm Petri dishes (Falcon; BectonDickinson Labware, Lincoln Park, NJ) were coated with laminin 1 or

fibronectin (Collaborative Biomedical Products; Becton Dickinson) at a

protein concentration of 20 �g/ml in PBS for 1 h at 37#{176}C,washed withPBS, and incubated with 0.1 % BSA for 1 h at room temperature to block

nonspecific binding. Primary keratinocytes induced to differentiate in cul-ture by elevating the Ca2� concentration to 0.1 2 m�i for 24 h were washedin PBS and trypsinized briefly with 0.25% trypsin. Cells were resuspendedin 0.05 mM Ca2� medium with 8% FCS, counted using a Coulter counter,

and centrifuged at 1 000 rpm for 1 0 mm. Cells were also isolated fromnewborn epidermis by floating newborn skins (33) on 0.25% trypsin for 15

h at 4#{176}C.After epidermis separation, freshly isolated keratinocytes wereresuspended in 1 .2 mM Ca2� medium with 8% FCS, counted, and cen-trifuged at 1000 rpm. Cultured keratinocytes as well as freshly isolated

cells were then resuspended in 0.05 m� Ca2� EMEM without FCS sup-plemented with 0.1 % BSA. Suspended cells (0.75 x 106) were plated induplicate dishes and incubated for 1 h at 37#{176}C.Nonadherent cells wereremoved, and the dishes were washed twice with PBS. Adherent cells

were then fixed in methanol:acetone (1:1 , v/v) for 5 mm at room temper-ature and kept at -20#{176}Cuntil analyzed. For analysis, plates were rinsed

with PBS and incubated overnight at 4#{176}Cwith mixtures of primary anti-

bodies from different species [guinea pig antimouse K14 (1:1000) andrabbit antimouse K1 (1 :500) or rabbit antimouse loricrin (1 :500)] diluted in1 % BSA and PBS. After incubation, the dishes were washed twice for 10

mm with PBS and incubated with biotinylated secondary anti-guinea pigantibody for 20 mm. Biotinylated secondary antibody was removed withPBS washes, and cells were exposed to FITC-conjugated secondary

antirabbit antibody mixed together with streptavidin Texas red for 20 mmto visualize the two antigens simuitaneously. The dishes were rinsed withPBS and examined with a Zeiss Axiophot fluorescence microscopeequipped with a fliter for differential detection of FITC and Texas red.

SDS-Page and Western Blot Analysis. For membrane fractions, cellsfrom 100-mm dishes were washed twice with cold PBS and harvested by

scraping into 250 �tI PBS containing 10 �g/ml aprotinin, 10 �g/ml leu-peptin, 2 �g/ml pepstatin (Boehringer Mannheim, Mannheim, Germany), 1mM phenylmethylsulfanyl fluoride, 10 m�,i EDTA (Digene, Silver Spring,MD), 200 �J.M NaVO4, and 10 mr�i NaF. After three freeze-thaw cycles, cells

were spun at 4#{176}Cat 14,000 rpm. The supernatant containing the soluble

protein fraction was transferred to another tube. The pellet was resus-

pended in 250 �l PBS containing 1% Triton X-100 with proteinase andphosphatase inhibitors. Cells were incubated for 30 mm at 4#{176}Cand spunat 14,000 rpm at 4#{176}C.The supernatant containing the Triton-soluble

fraction was transferred to a fresh tube. The remaining pellet containedcytoskeletal proteins. In some experiments, total lysates were extracted

by scraping the cells into 250 �d radioirnmunoprecipitation assay buffer

containing protease and phosphatase inhibitors. Protein concentrations

were measured using a modified Lowry assay (Bio-Rad DC Protein Assay

Kit). Twenty � of protein were loaded onto each lane of a 10% SDS-PAGE gel and separated under reducing or nonreducing conditions (67) as

specified in the figure legends. Separated proteins were transferred to a

nylon membrane, and specific protein bands were detected by incubating

the protein blots with specific antibodies to the different integrin subunits.Equal protein loading was confirmed by Ponceau-S staining (Sigma

Chemical Co.). Bands were visualized by ECL using the Renaissance Kit

(Dupont NEN Research Products, Boston, MA). Densitometric analysis of

linear range exposures of representative blots were quantified using Im-

age Quant software (Molecular Dynamics).Metabolic Labeling of Cellular Proteins and Immunoprecipitation.

Confluent primary keratmnocytes cultured in 100-mm dishes were washedwith PBS and labeled for 2-4 h as indicated with methionine-free EMEM

containing 8% FCS and 100 �.tCVml[�5S]methionine and cysteine. Cells

were then washed with cold (4#{176}C)PBS and incubated in EMEM enriched

with methionine (1 5 mg/mI) and cysteine until harvested. For zero and later

time points, cells were washed with cold PBS and harvested on ice by

scraping with cell lifters (Costar) into 500 �l protein lysis buffer (1 % Triton

x-100, protease inhibitors, and phosphatase inhibitors). Lysates werestored at -70#{176}Cuntil analyzed. Labeled protein lysates were spun at

16,000 x g at 4#{176}Cfor 30 mm. For irnmunoprecipitation, 500 �d of super-

natant (adjusted according to counts/mm and protein content) were trans-

ferredto a fresh tube and precleared by incubating with 10 �tl protein A+G

agarose slurry (Santa-Cruz) at 4#{176}Cfor 30 mm. After spinning samples for

2 mm at 16,000 x g, supernatants were transferred to a new tube, 25 �lprotein A+G agarose slurry and antibody (1 �g mAb GoH3, 20 �g poly-

clonal antibody 6845, and 5 �g mAb 346-hA) were added, and samples

were rotated overnight at 4#{176}C.After incubation, samples were spun at

16,000 x g for 5 mm. The supernatant was aspirated, and bound protein

was eluted from protein A+G agarose by incubating pellet at 1 00#{176}Cin 30

�il loading buffer for 5 mm. Protein samples were loaded onto SDS-PAGE

gels and proteins separated under nonreducing conditions on 7.5% poly-

acrylarnide gels and electrophoretically transferred onto reinforced nitro-

cellulose filters. Protein bands were visualized by autoradiography on

X-ray film.mRNA Isolation and Northern Blotting. Total RNA was isolated from

cultured cells using Trizol isolation solution followed by extraction with

chloroform and precipitation in 2-propanol (Life Technologies, Inc., Gaith-ersburg, MD). Twenty �.tg total RNA were separated and analyzed by gel

electrophoresis in a 1% agarose-formaldehyde denaturing gel followed byNorthern blot transfer onto Nytran nylon membranes (Schelicher &Schuell) and UV cross-linking. Transcripts were identified by hybridizing

with the following cDNAs labeled by 32P nick translation: mouse Ki (73);

rat transglutaminase, a gift from Dr. R. Rice (University of California, Davis,

CA; Ref. 74); 13i integrin, a gift from Dr. E. J. Brown (Washington Univer-sity, St. Louis, MO; Ref. 75); mouse a6 integrin, a gift from Dr V. Quaranta

(15); and mouse �34 integrin (41), a gift from Dr. S. J. Kennel. Rehybrid-

ization with rat glyceraldehyde-3-phosphate dehydrogenase cDNA (76)

was used as an internal standard for RNA loading.

MiT Assay. For the MiT assay, suspended cells or trypsmnized at-

tached cells were resuspended in appropriate medium containing 0.05 or0.12 mr�i Ca2�. Culture medium (100 �l) containing 20,000 cells from eachexperimental group was dispensed into wells of a 96-well Petri dish. The

Cell Titer 96 proliferation assay (Prornega) was performed following man-ufacturer directions, and absorbance was read at 570 nm using an ELISA

plate reader. The different groups assayed included attached cells from

6-day-old primary keratinocytes maintained in 0.05 m� Ca2� or in 0.12

mM Ca2� for 24 h and suspended cells in 0.05 m�i Ca2� medium for the

indicated time points.


AcknowledgmentsPurified laminin 5 was provided generously by Dr. A. Burgeson. We thankDr. F. Giancotti and Dr. S. Kennel for the generous gifts of 134 antibodies.

We thank Margaret Taylor for her editorial assistance.

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selective changes inlaminin adhesion anda634integrin...

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