Proc. Nati. Acad. Sci. USAVol. 85, pp. 2161-2165, April 1988Cell Biology

Cell-cell and cell-matrix interactions differentially regulate theexpression of hepatic and cytoskeletal genes in primarycultures of rat hepatocytesAVRI BEN-ZE'EV*t, GREGORY S. ROBINSON*, NANCY L. R. BUCHERt, AND STEPHEN R. FARMER*Departments of *Biochemistry and tPathology, Boston University School of Medicine, Boston, MA 02118

Communicated by Sheldon Penman, November 12, 1987

ABSTRACT Freshly isolated adult rat hepatocytes exhibita flat, extended morphology when cultured on dried rat tailcollagen in the presence of growth factors; they activelysynthesize DNA and express high levels of cytoskeletal mRNAsand proteins (actin, tubulin, cytokeratins, vinculin, a-actinin,and desmoplakin), while exhibiting low levels of liver-specificmRNAs (albumin, a,-inhibitor III, and a1-antitrypsin) andlimited synthesis and secretion of albumin. Hepatocytes cul-tured on hydrated gel matrix from the Engelbreth-Holm-Swarm (EHS) mouse tumor form small spherical aggregatesand exhibit low DNA, cytoskeletal mRNA, and protein syn-thesis, while at the same time exhibiting elevated liver-specificmRNAs and albumin production; these cells, therefore, morenearly conform to the program of gene expression seen withinthe normal animal. Hepatocytes on hydrated rat tail collagenresemble those on dry collagen when cultured at low density,but at high density they form compact trabecular aggregates,synthesize negligible amounts of DNA, and maintain a patternof gene expression resembling that of hepatocytes seeded onthe EHS matrix. If cell morphology is compact, as on EHS oron hydrated rat tail collagen when densely populated, DNAsynthesis and expression of cytoskeletal genes are low, whileliver-specific mRNAs are abundant. When cells are extendedthe opposite is the case. Without the growth supplement DNAsynthesis is low throughout but gene expression is little af-fected. These studies point to the importance of cell-cell andcell-matrix interactions in determining the differentiated phe-notype of hepatocytes, and they reveal an inverse relationshipbetween cytoskeletal and liver-specific protein expression.

Studies in a variety of cell culture systems suggest thatchanges in cell morphology are involved in the regulation ofgrowth and gene expression and in the maintenance ofdifferentiated functions (for reviews, see refs. 1 and 2). Cellmorphology reflects the organization of the intracellularcytoskeletal network, which is partially determined by theinteraction between the cells and their surrounding extracel-lular matrix (ECM) (for reviews, see refs. 3-5). This suggeststhat the ECM influences the expression of differentiatedfunctions through organization of the cytoskeleton (6, 7).Several studies in vitro have demonstrated cytoskeletalinvolvement in transmitting extracellular signals (mitogenicand differentiation-specific) that regulate various programsof gene expression (for reviews see refs. 1 and 2). It is notknown as yet whether mechanisms that regulate the cytos-keleton, both at the level of gene expression and synthesisand at the level of organization of its proteins, are importantcomponents of the differentiation programs of highly spe-cialized cells.

It has been demonstrated that the expression of certaindifferentiated functions in cultured hepatocytes is dependent

on the interaction of these cells with constituents of theECM (8-13). Evidence suggests that particular cytoskeletalgenes may undergo extensive changes in expression alongwith alterations in hepatocyte differentiation (14-16). Wehave therefore exploited the hepatocyte culture system tostudy the influence of the ECM on the expression of adefined set of genes. In this study we show that the cultureof hepatocytes on a reconstituted gel ECM from the Engel-breth-Holm-Swarm (EHS) mouse tumor (17) differentiallyregulates the expression of the major cytoskeletal genes(those for actin, tubulin, and cytokeratins) and of severalliver-specific genes. DNA synthesis, which is also stronglyinfluenced by the ECM and by cell density, is not closelycoordinated with this differential regulation of gene expres-sion.

METHODSCell Culture. Hepatocytes were isolated by perfusion of

rat livers with collagenase (18), in buffers (19) that weremodified to contain 1 mM calcium when collagenase wasadded. They were cultured in Williams E medium, modifiedby substitution of ornithine for arginine, and addition ofdexamethasone (5 nM), gentamycin (30 Rg/ml), ascorbicacid (50 pug/ml, fresh daily), and a growth supplementconsisting of insulin (20 milliunits/ml), epidermal growthfactor (10 ng/ml), and sodium pyruvate (20 mM). Hepa-tocytes were plated at 105 (sparse) or 106 (dense) cells in 1.5ml of culture medium into 35-mm Lux plastic dishes coatedwith (i) rat tail tendon dissolved in acetic acid and allowed todry (crude type I collagen, 0.6 mg per dish) (18), (ii) the samematerial in the form of a gel (prepared as described in ref.20), or (iii) a gel prepared from the EHS tumor (17), which iscomposed primarily of laminin, type IV collagen, and hepa-ran sulfate proteoglycan (1.2 mg of protein per dish). Themedium was changed after 1 hr and at 24-hr intervalsthereafter.DNA synthesis was determined after 24-hr incorporation

of [3H]thymidine [1 ,uCi/ml, 360 ,uCi/mmol; 2.75 ,uM thymi-dine (1 Ci = 37 GBq)] (18).

Cell Labeling, Fractionation, and Polyacrylamide Gel Elec-trophoresis. At selected times after plating, cells were la-beled for 2 hr with [35S]methionine (100 ,Ci/ml, 1121,uCi/mmol) in otherwise methionine-free medium, to whichthe growth supplement was added. The medium was col-lected and the secreted proteins were recovered by precipi-tation with ethanol (2 vol). The cells were then removedfrom the various matrices by incubation at 37°C in Dul-becco's phosphate-buffered saline (PBS) containing trypsin(0.02%), EDTA (5 mM), and collagenase (2 mg/ml), washedin PBS, and lysed in a buffer containing Triton X-100 (0.5%),

Abbreviations: ECM, extracellular matrix; EHS, Engelbreth-Holm-Swarm; a113, a,-inhibitor III.tPermanent address: Department of Genetics, Weizmann Instituteof Science, Rehovot 76100, Israel.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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KCl (0.6 M), 2-mercaptoethanol (14 mM), EGTA (2.5 mM),phenylmethylsulfonyl fluoride, and Hepes, pH 7.4 (10 mM),and mixed vigorously. Centrifugation at 15,000 x g for 2 minyielded a Triton X-100/high-salt-soluble fraction and aninsoluble fraction, enriched in intermediate filaments.Samples were analyzed by sodium dodecyl sulfate (Na-

DodSO4)/gel electrophoresis (21) or by two-dimensional gelelectrophoresis as described by O'Farrell (22).The identification of the actin-associated proteins, vincu-

lin and a-actinin, and the desmosomal plaque protein, des-moplakin, was obtained by means of immunoblotting, wherespecific proteins were separated on two-dimensional gelsand detected with monoclonal antibodies as demonstratedpreviously (7, 23).RNA Extraction, Blot Transfer, and Filter Hybridization.

Isolation and blot hybridization analysis of total RNA wereperformed as described elsewhere (24). The various RNAblots were probed with the following: actin, 1.5-kilobase (kb),8-actin cDNA (25); RBT3, 1.7-kb /8-tubulin cDNA (26);histone 3.2, Sal I-Bgl I fragment of a 5.54-kb fragmentgenomic clone (27); a1-inhibitor III (a113), 2.5-kb cDNAclone (24); albumin, 2.0-kb cDNA (a gift from DouglasCooper, Whitehead Institute, Cambridge, MA); cytokeratin18, 1.5-kb cDNA (28); a1-antitrypsin, 1.35-kb cDNA (a giftfrom Harvey Lodish, Whitehead Institute).

RESULTSCulture of Hepatocytes on Dried Rat Tail Collagen En-

hances the Synthesis of Cytoskeletal Proteins but Reduces theSynthesis of Albumin. Freshly isolated hepatocytes wereinitially cultured in dishes coated with dried rat tail collagenand analyzed after 24-48 hr. Synthesis of the major cyto-skeletal proteins, including actin, tubulin, cytokeratins 8 and18, vinculin, a-actinin, and desmoplakin, was markedlyincreased, as was replication of DNA (Fig. 1). In contrast,the level of newly synthesized albumin, a liver-specificprotein, declined significantly between the early (Fig. 1 JA)and late (Fig. 1 ID) time periods in culture.

Analysis by immunofluorescence microscopy of the cyto-skeletal elements in hepatocytes cultured for 48 hr revealed

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that actin was organized into a cortical ring at areas ofcell-cell contact as well as into elaborate stress fibers at theventral aspects of the cells. These cells also contained anextensive microtubular network. Desmoplakin and cytoke-ratins were evident at points of cell-cell contact (not shown),whereas vimentin was not detected.We conclude that the culture of hepatocytes on dried rat

tail collagen in growth factor-supplemented medium resultsin a coordinate increase in the synthesis of the majorcytoskeletal elements, accompanied by their organizationinto elaborate cytoplasmic networks.

Hepatocytes Cultured on EHS Matrix Maintain a Low Levelof Cytoskeletal Protein Synthesis but High Levels of HepaticProtein Synthesis and Secretion. An extracellular matrixderived from the EHS tumor (17) has been successfully usedto preserve high levels of albumin secretion in culturedhepatocytes (12). We found that whereas hepatocytes cul-tured on dried rat tail collagen acquired a flat and extendedmorphology (Fig. 2A), on the EHS matrix they remainedround and formed small aggregates that adhered strongly tothe matrix (Fg. 2C). Kinetic studies showed that, withincreasing times in culture on rat tail collagen, there was agradual increase in the synthesis of actin and cytokeratins(Fig. 3 1, lanes A, C, and E), while between 24 and 48 hr inculture hepatocytes on EHS matrix synthesized these cyto-skeletal proteins at a low rate (Fig. 3 1, lanes B, D, and F).Two-dimensional gel analysis of [35S]methionine pulse-labeled proteins revealed that, in addition to the synthesis ofcytokeratins (Fig. 3 1, gels E' and F') in cells cultured onEHS matrix, the synthesis of actin and tubulin was alsosignificantly lower (Fig. 3 2). Furthermore, compared withcells cultured on rat tail collagen, hepatocytes cultured onEHS matrix synthesized (Fig. 3 2) and secreted (Fig. 3 3)substantially increased amounts of albumin.

Hepatocytes Cultured on EHS Matrix Maintain Low Levelsof Cytoskeletal mRNAs but High Levels of Liver-SpecificmRNAs, Similar to the Levels of These mRNAs in the Liver.The results of blot hybridization analysis (Fig. 4) clearlydemonstrate that, in sparse cultures of hepatocytes culturedon rat tail collagen, there was a dramatic increase in thecontent ofmRNAs coding for actin, tubulin, and cytokeratin

IEF

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FIG. 1. Increase in DNA and cytoskeletal protein synthesis, but decrease in albumin synthesis, in hepatocytes plated on rat tail collagen.Sparsely seeded hepatocytes (105 cells per 35-mm dish) were labeled for 2 hr with [35S]methionine after 2 hr (JA, 2A'), 8 hr (IB), 24 hr (IC, 2C'),or 48 hr (ID, 2D') in culture. The Triton X-100/high-salt-soluble fraction (1) and -insoluble fraction (2), which is enriched in intermediatefilaments, were analyzed by the methods of O'Farrell (22) and Laemmli (21), respectively. The amount of DNA synthesis after various timesin culture was measured by [3H]thymidine incorporation (3). IEF, isoelectric focusing; alb, albumin; aa, a-actinin; v, vinculin; t, tubulin; d,desmoplakin, CK 8 and CK 18, cytokeratins as described by Franke et al. (29); M, molecular mass markers (kDa).

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A JX-

* jtAart~

C FoiP . .

FIG. 2. Morphology of hepatocytes cultured at two densities on

the different substrata. Hepatocytes were seeded at iO5 (sparse,

A-C) or 106 (dense, D-F) cells per 35-mm dish on dry'rat tail

collagen (A, D), on hydrated rat tail collagen gel (B, E), and on EHS

matrix (C, F). (Phase contrast; x 190.)

18 (Fig. 4, lanes 2). There was also a high rate of DNAsynthesis (Fig. 5), along with histone mRNA accumulation,while expression of the liver-specific mRNAs for a113,a1-antitrypsin, and albumin decreased significantly (Fig. 4,lanes 2). In contrast, the RNA from normal adult liver (Fig.4, lanes 1) and from cells cultured on EHS matrix (Fig. 4,lanes 3) contained low levels of cytoskeletal protein mRNAsand histone mRNA but high levels of the liver-specificmRNAs.

Population Density of Hepatocytes Plated on HydratedCollagen Gels Influences the Synthesis of Cytoskeletal andLiver-Specific Proteins. To define the nature of the signalresponsible for the maintenance of a more differentiatedphenotype on particular ECMs, hepatocytes were culturedon uncoated dishes and on dishes coated with various ECMcomponents (laminin, fibronectin, collagen types I or IV),and also with poly(L-lysine). On all of these substratahepatocytes had an extended flat morphology and synthe-sized actin, tubulin, cytokeratins, and liver-specific proteinsat levels similar to those found in cells plated on dried rat tailcollagen (data not shown).

Since the EHS matrix was presented to the cells in theform of a hydrated gel, hepatocytes were also cultured for 48hr, at high and low densities, on hydrated rat tail collagengels. Unlike hepatocytes cultured on dry rat tail collagen(Fig. 2D), hepatocytes seeded at high density on hydratedcollagen gels displayed a compact trabecular organization(Fig. 2E); these cells expressed a pattern of cytoskeletalprotein synthesis (Fig. 6 d and e-dense, lane 2) and albuminsynthesis (Fig. 6d) and secretion (Fig. 6 f-dense, lane 2)similar to those of cells cultured on the EHS matrix (Fig. 6 band e- and f-dense, lanes 3). In contrast, hepatocytes cul-tured at low density on hydrated collagen gels had anextended morphology (Fig. 2B) and exhibited high levels ofcytoskeletal protein synthesis (Fig. 6 c and e-sparse, lane 2)

A BC D E F

:3

.alb

FIG. 3. Hepatocytes plated on EHS matrix synthesize lowlevels of cytoskeletal proteins but synthesize and secrete high levelsof albumin. Abbreviations as in Fig. 1. Hepatocytes were seeded ondried rat tail collagen (1, lanes A, C, and E, gel E'; 2A; 3, lane A) andon EHS matrix (1, lanes B, D, and F, gel F'; 2B; 3, lane B) at adensity of 3 x 105 cells per 1.5-ml culture, labeled for 2 hr with[5S]methionine, and fractionated as described for Fig. 1. (1) Inter-mediate filament-enriched fraction; (2) soluble-cytoplasmic fraction;(3) secreted proteins. Cells were labeled after 6 hr (1, lanes A and B),24 hr (1, lanes C and D), or 48 hr (1, lanes E and F, gels E' and F';2 A and B; 3, lanes A and B) in culture.

but low levels of albumin synthesis and secretion (Fig. 6 c

and f-sparse, lane 2) similar to cells cultured on dry rat tailcollagen (Fig. 6 a and e-sparse, lane 1).

In sparse cultures DNA synthesis was high on either dryor hydrated collagen gels but low on EHS matrix (Fig. 5A);in dense cultures it was low on all three substrata. Omissionof the growth factor supplement dramatically reduced thelevel of DNA synthesis under all conditions (Fig. SB) with-out significantly influencing either cytoskeletal or hepaticprotein synthesis; therefore DNA synthesis and expressionof these genes are not closely coordinated (data not shown).Taken together, these results suggest that plating of hepa-

tocytes at high density, under conditions that allow exten-sive cell-cell contact as well as cell contact with hydratedcollagen gels, promotes maintenance of the differentiatedphenotype in vitro, at least over the short term.

DISCUSSIONStudies by others have indicated that maintenance of thedifferentiated phenotype of hepatocytes in cell culture isdependent upon a combination of particular ECM compo-nents and hormonally defined media (12, 13). The resultspresented here are consonant with these findings, but they

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l__ow111 31|S~~~~~~~Nactin"1--------

2163Cell Biology: Ben-Ze'ev et al.

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1 2 3 1 2 3

B-Tubulin

Actin U.I'-A~~~~~~~....

Albumin

oc,- 1(3)

CK18 ocJo(1-AT

Histone

FIG. 4. Cytoskeletal and histone mRNAs and liver-specificmRNAs are inversely regulated in hepatocytes cultured on rat tailcollagen and in hepatocytes maintained on EHS matrix or in normalliver. RNA extracted from normal liver (lanes 1), from hepatocytescultured on dried rat tail collagen (lanes 2), and on EHS matrix(lanes 3) was analyzed by blot hybridization with cDNA probes forcytoskeletal proteins. CK, cytokeratin. These probes were washedoff and the blots were reprobed with cDNAs for liver-specificproteins. a,-AT, a1-antitrypsin.

also demonstrate the importance of overall cell morphologyin regulating tissue-specific gene expression. In particular,we show that cell-cell as well as cell-matrix interactionsplay a central role in these processes, a finding supported byother studies (10, 16, 30, 31). The data further reveal aninverse relationship between the overall expression of thecytoskeleton (i.e., regulation of production of mRNA andsynthesis as well as organization of the corresponding pro-teins) and the expression of the hepatocyte phenotype.Taken together, these results strongly support the idea thatthe organization of the cytoskeleton, which is dictated by theextent of cell-cell and cell-matrix interaction, is intimatelyassociated with mechanisms that regulate tissue-specificgene expression.The use of the different cell culture systems has revealed

a significant degree of plasticity within the program ofmechanisms that regulate hepatocyte gene expression. Itappears that we can control the same pattern of geneexpression either (i) by manipulating the extent of cell-cellinteraction by plating hepatocytes at different densities onhydrated collagen gels or (ii) by culturing cells at any densityon a reconstituted basement membrane extracted from the

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FIG. 5. Cell DNA synthesis is inhibited in the absence of growthfactors, on EHS matrix at all densities, and at high density on allmatrices. Cells were seeded at 10- cells per 35-mm dish (sparse) and106 cells per 35-mm dish (dense) on dried rat tail collagen (bar 1), onhydrated rat tail collagen (bar 2), or on EHS matrix (bar 3) andcultured for 48 hr. The cells were labeled with [PH]thymidine for thefinal 24 hr. (A) Growth factors present throughout the 48-hr periodas usual. (B) Cells were cultured in basic medium without the growthsupplement.

FIG. 6. Hepatocytes cultured on hydrated collagen gels at highdensity maintain low cytoskeletal protein synthesis and high albu-min synthesis and secretion. Abbreviations as for Fig. 1. Hepa-tocytes were seeded at 106 (a, b, d, e-dense, andf-dense) and 10' (c,e-sparse, andf-sparse) cells per 1.5-ml culture on rat tail collagen (a,e lanes 1, f lanes 1), on hydrated collagen gels (c, d, e lanes 2, andf lanes 2), or on EHS matrix (b, e lanes 3, andf lanes 3). The cellswere labeled with ["S]methionine 48 hr after plating for 2 hr andfractionated as described for Fig. 1. (a-d) Triton X-100/high-salt-soluble fraction; (e) intermediate filament-enriched fraction; (I)secreted proteins.

EHS tumor. One common parameter that both systemsshare is a dramatic change in cell shape, implying that cellshape may be a primary regulator of phenotypic expression.Similar findings have been reported in a variety of otherexperimental systems (1, 2, 5). Since the EHS matrix iscapable of preserving the differentiated phenotype in hepa-tocytes at all culture densities, it is possible that differenti-ation is the result of the interaction of cells with specificcomponents of the ECM [e.g., glycosaminoglycans andheparan sulfate proteoglycans, as suggested by Spray et al.(32)], thereby inducing the required morphological change. Itis also possible that cell shape is simply a manifestation ofthe culture conditions and plays no direct role in regulatinggene 'expression. Instead, the predominant mode of regula-tion may depend upon mechanisms that are intimately asso-ciated with the extent of cell-cell and/or cell-matrix inter-action. There is now accumulating evidence that theseinteractions are dependent upon specific transmembranereceptors that link extracellular and intracellular elements(e.g., integrin connects fibronectin to the microfilamentsystem for review, see ref. 33). Signals may therefore betransmitted from these various receptors through a commonset of cytoskeletal elements and converge on similar regula-tory systems.The results presented here provide a dramatic example of

the coordinate regulation of the expression of all majorcytoskeletal proteins during differentiation. Whether suchregulation is directly involved in mechanisms that determinethe differentiated phenotype of hepatocytes is presentlydifficult to assess. It is possible that the dramatic changes in

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cytoskeletal gene expression may initiate the reorganizationof the cytoskeleton and in doing so affect the expression ofliver-specific genes, as suggested in other differentiationsystems (34). Alternatively, this regulation may be totallyunrelated to the differentiation program and may result fromthe alteration of cell morphology. These alterations could inturn induce a change in the organization of the cytoskeleton,triggering feedback or autoregulation mechanisms that con-trol cytoskeletal gene expression (35-41).

During the short culture periods analyzed in this study,hepatocytes cultured either on hydrated collagen gels at highdensity or on the EHS matrix appeared to express the samegeneral pattern of gene regulation. It is possible, however,that these two cell culture systems do regulate gene expres-sion by totally different means (i.e., transcriptional vs.posttranscriptional) and that the cell culture period was notlong enough to detect these differences. The EHS matrix isknown to preserve the differentiated phenotype over longerperiods of culture (12), and therefore, it is necessary toextend our culture period to study the later stages of thededifferentiation program.Recent studies have revealed that there are multiple

mechanisms that regulate the various liver-specific proteins,and these mechanisms differ from protein to protein. Forinstance, while the decrease in albumin expression on dry rattail collagen is transcriptional, significant levels of albuminmRNA are maintained by RNA stabilization (14, 24). This isnot the case, however, for the expression of a113. Thedecrease in transcription of this gene, resulting from thededifferentiation process, is accompanied by a correspond-ing rapid decrease in mRNA in the cytoplasm (24). Expres-sion of a1I3 appears to be hormonally controlled. It is likely,therefore, that complete maintenance of the liver-specificphenotype of hepatocytes will require many interactingsystems, including both extracellular matrices and a plethoraof hormones and factors. Nevertheless, a continued study ofthe effect of extracellular components on the expression of adefined set of genes will contribute significantly to our basicunderstanding of the problem. For instance, it will beimportant to determine whether, in hepatocytes cultured onthe EHS matrix for longer periods of time, the transcriptionof albumin and other liver-specific proteins is restored to thelevels found in the liver and whether proteoglycans andglycosaminoglycans are the ECM components responsiblefor the restoration of such regulation, as suggested by Fujitaet al. (42).What role can the cytoskeleton play in such regulation? It

may be involved in the posttranscriptional regulation ofmRNA half-lives by modulating the interaction of the poly-ribosomes with various elements of the cytoskeleton, assuggested by previous studies (for review, see refs. 3 and43).The molecular mechanisms by which changes in cell-cell

and cell-matrix contacts affect the organization and expres-sion of tlfe cytoskeleton, in such a way as to maintain thedifferentiated phenotype, will have to be determined. Theidentification and characterization of transmembrane recep-tors that link extracellular and cytoskeletal elements willprovide us with additional tools to address questions regard-ing the initial steps in this process at the molecular level.

We thank Ms. Wendy Zimmerman for excellent technical assist-ance, Dr. Hynda K. Kleinman for the EHS mouse tumors, and Dr.D. Montgomery Bissell for valuable advice regarding preparationand use of EHS gels for hepatocyte culture. The support of Dr. J.Folkman for A.B.-Z. is gratefully acknowledged. This work wassupported by National Institutes of Health Grant GM2%30 awardedto S.R.F. and National Cancer Institute Grant CA39099 awarded toN.L.R.B.

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