ECM, these same cell types proliferated actively and no longer. that the ECM, which is the natural substrate upon which cells

Transcription

1 Proc. Natl. Acad. Sd. USA Vol. 77, No. 7, pp , July 1980 Cell Bology Permssve effect of the extracellular matrx on cell prolferaton n vtro (fbroblast growth factor/granulosa cells/adrenal cortex cells/vascular endothelal cells/collagens) D. GOSPODAROWCZ, D. DELGADO, AND. VLODAVSKY Cancer Research nsttute and the Departments of Medcne and Ophthalmology, Unversty of Calforna Medcal Center, San Francsco, Calforna Communcated by Clfford Grobsten, March 28,1980 ABSTRACT Corneal endothelal cells mantaned n tssue culture retan the ablty to synthesze and secrete an extracellular matrx (ECM) along ther basal cell surface. Treatment of confluent cultures wth 0.5% Trton X-100 results n the removal of the cell monolayer, thereby exposng the ECM, whch adheres strongly to the tssue culture dsh. Dshes coated wth ECM were used to study the permssve effect of such a substrate on cell prolferaton. The prolferaton of bovne granulosa and adrenal cortex cells mantaned on plastc tssue culture dshes was compared to that on dshes coated wth ECM. Nether cell type, even when exposed to optmal serum concentraton, replcated when seeded at low cell densty on plastc. n contrast, when seeded on ECM they prolferated actvely. None of the cultures mantaned on ECM requred fbroblast growth factor n order to reach confluence, although when mantaned on plastc they were totally dependent on fbroblast growth factor for prolferaton. Because cells mantaned on plastc do not respond to factors present n serum or plasma, although they do so respond when mantaned on ECM, t s lkely that the close contact of the cells wth the ECM restores ther senstvty to agents present n serum and plasma. The extracellular matrx (ECM) or basal lamna produced by cells s the natural substrate upon whch cells mgrate, prolferate, and dfferentate n vvo. Although the exact nature and composton of ECMs are stll to be elucdated, they are composed n large part of dfferent types of collagen (1), glycosamnoglycans, proteoglycan (2), and glycoprotens, among whch s fbronectn-a ubqutous component of varous types of ECMs (3). The noton, frst proposed by Grobsten n 1953 (4), that ECMs promote and stablze the morphogeness of the epthela assocated wth them has snce been confrmed (5, 6). However, although numerous studes have dealt wth the dfferentaton of tssue nduced by the ECMs and basal lamna, few studes have dealt wth ther effect on cell prolferaton. Because cell prolferaton precedes cell dfferentaton n most organs, t s lkely that both prolferaton and dfferentaton could be controlled drectly or ndrectly by the substrate upon whch the cells rest. Ths s partcularly true of tssues that have only one developmental opton. Earler studes n our laboratory on the control of cellular prolferaton by fbroblast growth factor (FGF) have shown that cells that prolferate best n tssue culture retan ther ablty to produce an ECM, whereas cells that prolferate poorly, or not at all, have lost that ablty (7). n ths study, we examned the prolferatve behavor of two dfferent cell types of bovne orgn when mantaned on plastc or on an ECM produced by corneal endothelal cells. When mantaned on plastc and exposed to optmal serum concentraton, bovne adrenal cortex cells and granulosa cells dd not prolferate unless they were The publcaton costs of ths artcle were defrayed n part by page charge payment. Ths artcle must therefore be hereby marked "advertsement" n accordance wth 18 U. S. C solely to ndcate ths fact exposed to FGF. n contrast, when mantaned on a corneal ECM, these same cell types prolferated actvely and no longer requred FGF to reach confluence. These results demonstrated that the ECM, whch s the natural substrate upon whch cells rest n vvo, can greatly affect ther prolferaton n vtro. MATERALS AND METHODS Materals. FGF was purfed as descrbed from bovne brans (8), and fbronectn was purfed as descrbed from bovne plasma (9). Purfed collagen (types,,, and V) was donated by S. Tseng (Unversty of Calforna, San Francsco). Dulbecco's modfed Eagle's medum H-16 and F-12 medum were obtaned from GBCO. Calf serum and fetal calf serum were obtaned from rvne Serum (rvne, CA). Tssue culture dshes were from Falcon Plastcs. Dextran (Mr 40,000) was from Sgma, gentamycn was from Scherng, and Fungzone was from Squbb. Cell Culture Condtons. Cultures of bovne corneal endothelal cells were establshed from steer eyes as descrbed (10, 11). Stock cultures were mantaned on tssue culture dshes n Dulbecco's modfed Eagle's medum supplemented wth 10% (vol/vol) fetal calf serum, 5% (vol/vol) calf serum, gentamycn (50,tg/ml), and Fungzone (2.5 jg/ml). FGF (100 ng/ml) was added every other day untl the cells were nearly confluent. Cultures of bovne granulosa cells were establshed from ovaran follcles as descrbed (12). Stock cultures were mantaned n F-12 medum supplemented wth 10% (vol/vol) calf serum, gentamycn (50,gg/ml), Fungzone (2.5,ug/ml), and FGF (100 ng/ml added every other day). Cultures of bovne adrenal cortex cells were establshed from adrenal cortex as descrbed (13). Stock cultures were mantaned n F-12 medum supplemented wth 10% (vol/vol) horse serum, gentamycn (50,gg/ml), Fungzone (2.5,gg/ml), and FGF (100 ng/ml added every other day). Preparaton of Plates Coated wth an ECM, Dfferent Types of Collagen, or Fbronectn. Corneal endothelal cells were plated at an ntal densty of 104 cells per 35-mm dsh and were mantaned n Dulbecco's modfed Eagle's medum supplemented wth 10% (vol/vol) fetal calf serum, 5% (vol/vol) calf serum, 5% (wt/vol) dextran T-40, gentamycn (50,g/ml), and Fungzone (2.5 Atg/ml). FGF (100 ng/ml) was added every other day. Once the cultures became confluent (ordnarly wthn 6 days), the meda were renewed and the cultures were further ncubated for 6 days. The cultures then were washed wth phosphate-buffered salne and exposed for 30 mn to 0.5% Trton X-100 n the same buffer. Once the nucle and the ECM became vsble, the cultures were washed three tmes wth phosphate-buffered salne. After these washngs, only a few cytoskeletons and nucle were assocated wth the ntact ECM that coated the entre dsh (see Fgs. 1 and 2). To coat dshes Abbrevatons: ECM, extracellular matrx; FGF, fbroblast growth factor.

2 Cell Bology: Gospodarowcz et al. wth dfferent types of collagen, 50,ul of a soluton of collagen n 0.5 M acetc acd (5 mg/ml) was appled to a 35-mm dsh and spread unformly wth a rubber polceman. The plates were exposed to ammona vapor for 30 mn and then washed extensvely wth phosphate-buffered salne. To coat the dsh wth fbronectn, 2 ml of a soluton of fbronectn n 0.05 M urea (30 ug/ml) was added to the dshes. After an ncubaton perod of 60 mn at 220C, the dshes were washed fve tmes wth phosphate-buffered salne (14). Cell Growth Measurement. Cells were plated at an ntal densty of 1 or 2 X 104 cells per 35-mm dsh. FGF (100 ng/ml) was added every other day. Trplcate cultures were trypsnzed, and cells were counted wth a Coulter Counter every other day. The morphologcal appearance of the cultures was analyzed by phase-contrast mcroscopy, and pctures were taken when they became confluent. RESULTS Morphologcal Appearance of the ECM Produced by Corneal Endothelal Cells When confluent cultures of corneal endothelal cells were examned by scannng electron mcroscopy, the cells resembled those n vlvo-closely apposed, hghly flattened, and hexagonal n pattern (Fg. 1A). Exposure of the cell monolayer to phosphate-buffered salne contanng 0.5% Trton X-100 resulted n the permeablzaton of the cells, and the ECM underneath the cells became partally vsble (Fg. 1 B and D). n some areas cytoskeletons and assocated nucle spontaneously detached from t and thereby exposed t (Fg. 1C). When the ECM was examned, after removal of the cytoskeletons and assocated nucle, by washng wth phosphate-buffered salne, two areas were dstngushed. One conssted of a unform layer of amorphous materal that was closely assocated wth the plastc (Fg. 2 A and B). Scratchng the plate wth a needle allowed partal detachment of that amorphous materal and enabled one to apprecate ts thckness and homogenety (Fg. 2A). n other areas, rdges formng a ~ ~ ~ ~ ~ ~ j FG. 1. Scannng electron mcrographs of a monolayer of bovne corneal endothelal cells before and after exposure to Trton X-100. (A) A monolayer composed of polygonal, hghly flattened, and closely apposed cells before Trton treatment. (X250.) (B) Posttreatment monolayer composed of nucle and cytoskeletons that no longer attach frmly to the ECM. (X90.) (C) ECM exposure n some areas. (X250.) (D) Nucle and ther assocated cytoskeletons wth the ECM underneath. Processng of the samples for scannng electron mcroscopy was as descrbed (15). Scannng electron mcrographs were obtaned wth a JOEL scannng electron mcroscope (model 35). Proc. Nat. Acad. Sc. USA 77 (1980) 4095 FG. 2. Scannng electron mcrographs of the ECM produced by corneal endothelal cell cultures. (A) Area of lght depost of the ECM; the matrx has been scratched wth a needle to release t from the tssue culture dsh, whch t coats unformly. (X500.) (B) Same as A, but the area has not been scratched. (X1000.) (C) Area of heavy depost of the ECM, whch unformly coats the tssue culture dsh; polygonal rdges of amorphous materal can be seen supermposed on t. (X150.) (D) Same as C, but at a hgher magnfcaton. (X750.) hexagonal pattern were seen (Fg. 2 C and D). The composton of the ECM coatng the dsh has been analyzed by column chromatography (16) and by mmunofluorescence (17). The collagen dstrbuton n the basement membrane was manly collagen types, V, and V. Fbronectn was also present n large quanttes (18), as well as lamnn (19, unpublshed data). Growth and Appearance of Cultured Cells Mantaned on Plastc or ECM; Effect of Absence or Addton of FGF. Granulosa cells. Low-densty granulosa cell cultures mantaned on plastc and exposed to ether hgh (10%) or low (1%) serum concentratons hardly prolferated (Fg. 3 A and B). After a few days, the cells had enlarged consderably (Fg. 4A), and a hgh percentage (20%) of bnucleated cells was observed, whch suggested that the cells had gone once through the process of endomtoss (unpublshed results). Followng the addton of FGF to the cultures, cells prolferated actvely (Fg. 3 A and B). Wthn a week, ths resulted n the formaton of a monolayer of closely apposed mononucleated cells (Fg. 4B). n contrast to cells mantaned on plastc, cells mantaned on the ECM no longer requred the presence of FGF to prolferate actvely. Wthn 5 days the cultures became confluent (Fg. 3 A and B). Addton of FGF to such cultures dd not decrease the mean doublng tme of the cultures but resulted n a fnal cell densty that was twce as hgh as that of cultures mantaned n the absence of FGF. The dfferences n the growth rate of granulosa cells mantaned wth or wthout FGF on plastc or ECM were seen best when the cultures were mantaned n the medum supplemented wth a low (1%) serum concentraton. Under these condtons, cells mantaned on plastc alone dd not

3 4096 Cell Bology: Gospodarowcz et al. Proc. Natl. Acad. Sc. USA 77 (1980) A. Days FG. 3. Prolferaton of granulosa cells (A and B) and adrenal cortex cells (C and D) when mantaned wth (0, A) or wthout (0, *) FGF on plastc or ECM. Granulosa cells were plated at 1 X 104 cells (A) or 2 X 104 cells (B) per 35-mm dsh and mantaned n Dulbecco's modfed Eagle's medum supplemented wth ether 10% (A) or 1% (B) calf serum. The cells were mantaned on plastc (0, 0) or ECM (A, A). FGF (100 ng/ml) was added every other day to half of the dshes (0, A). Adrenal cortex cells were plated at 1 X 104 cells per 35-mm dsh and mantaned n Dulbecco's modfed Eagle's medum supplemented wth ether 10% (C) or 1% calf serum (D). The cells were mantaned on plastc (0, 0) or ECM (-, A). FGF (100 ng/ml) was added every other day to half of the dshes (0, A). prolferate, whereas addton of FGF caused a 7-fold ncrease n cell number over an 8-day perod. Cells mantaned on the ECM reached a cell densty 22-fold hgher than those mantaned on plastc, and addton of FGF caused a 44-fold ncrease n cell number over that n the control (Fg. 3 A and B). Granulosa cells mantaned on the ECM alone and not exposed to FGF had the same appearance as that of cells mantaned on plastc and exposed to FGF; the cultures were composed of a monolayer of cells that were more tghtly packed when mantaned on the ECM than on plastc (Fg. 4C). Cultures mantaned on ECM and grown n the presence of FGF formed a monolayer composed of extremely small and tghtly packed cells whose cytoplasm contaned numerous lpd nclusons (Fg. 4D). Adrenal cortex cells. The growth characterstcs and appearance of adrenal cortex cells mantaned on plastc or ECM and exposed to hgh (10%) or low (1%) serum concentratons wth or wthout FGF added to the medum were essentally smlar to those already reported for granulosa cells. Adrenal cortex cells mantaned on plastc hardly prolferated, even when exposed to a hgh (10%) serum concentraton, unless FGF FG. 4. Granulosa cells mantaned on plastc substrate (A and B) or on an ECM (C and D). Cultures were plated and mantaned as descrbed n Fg. 3A wth (B and D) or wthout (A and C) FGF. Pctures were taken on day 8. (Phase-contrast; X45.) was added to the cultures (Fg. 3 C and D). As descrbed for granulosa cells, wthn 2-3 days these cells enlarged consderably (Fg. 5A), and by 7 days a large proporton of bnucleated cells could be observed n the cultures. When FGF was added to the meda, actve prolferaton was observed, but the mean doublng tme of the cultures was lower than that of cultures mantaned on the ECM and not exposed to FGF. Addton of FGF to cultures mantaned on the ECM and exposed to hgh serum concentratons dd not result n a faster growth rate but dd result n a hgher (2-fold) fnal cell densty. As wth granulosa cells, the dfferences n growth rate between cultures mantaned wth or wthout FGF on plastc versus ECM were best seen when the cultures were mantaned n low (1%) serum concentraton (Fg. 3D). Cultures mantaned on plastc plus FGF, on the ECM alone, and on the ECM plus FGF showed no obvous changes n cell morphology except that cells mantaned on the ECM (Fg. 5C) or the ECM plus FGF (Fg. 5D) had a smaller average sze and a hgher densty of lpd nclusons than dd cells mantaned on plastc plus FGF (Fg. 5B). Prolferaton of Granulosa Cells and Adrenal Cortex Cells on Dshes Coated wth Dfferent Types of Collagen or wth Fbronectn. To test the possblty that collagen or fbronectn alone could be the component of the ECM responsble for the ncreased rate of prolferaton of cells plated on t, the growths of granulosa and adrenal cortex cells plated on dshes coated wth purfed collagen type,,, or V or wth fbronectn were compared. n no case dd the cultures sgnfcantly n- A. SB p k., FG. 5. Appearance of adrenal cortex cells mantaned on plastc substrate (A and B) or ECM (C and D). Cultures were plated and mantaned as descrbed n Fg. 3C wth (B and D) or wthout (A and C) FGF. Pctures were taken on day 7. (Phase-contrast; X45.) t '

4 Ca r- x Gog Uc) Cell Bology: A Gospodarowcz et al [ 2 1 rb FG. 6. Prolferaton of adrenal cortex (A) and granulosa cells (B) mantaned on dfferent substrates. The cells (2 X 104) were seeded on dshes coated wth collagen type 1,11, m, or V, wth collagen types and, wth fbronectn (Fb), or wth an ECM (EM). After 5 days, the cultures were trypsnzed and the cells were counted. The fnal cell densty was compared to that of cultures mantaned for the same perod of tme on plastc wthout FGF (P) or wth FGF (P + FGF). crease ther rate of growth when mantaned on these dfferent substrates (Fg. 6), and an aberrant appearance was observed n all cases-the cultures were composed of large cells. Only cells mantaned on the ECM prolferated actvely (Fg. 6), reachng confluence wthn 5 days. Ths observaton therefore excludes the possblty that the component n the ECM produced by corneal endothelal cells that could have a permssve effect on the prolferaton of other cell types mantaned on t s ether collagen or fbronectn alone. DSCUSSON Early studes on the control of prolferaton and dfferentaton of embryonc epthela demonstrated a requrement for embryonc mesenchyme f epthela are to prolferate and dfferentate (5), although the mechansm through whch the mesenchyme exerts ts permssve effect s stll largely unknown. t s lkely, however, that one of the mechansms nvolved s to provde a proper substratum for epthelal prolferaton. The possblty has also been rased that drect contact between mesenchymal and epthelal cells could be nvolved (20-22). nvestgaton of the role of extracellular materals at the epthelal-mesodermal nterface has shown that collagens and mucopolysacchardes-major molecular speces at the juncton of nteractng tssues-could be mplcated n epthelal morphogeness (5, 23). Because the mesoderm provdes the bulk of the collagens to the epthelomesoderm surface, t could ndrectly modulate prolferaton and morphogeness of the epthelum (5). Equally possble s that epthela nteractng wth the ECM produced by another tssue could produce ther own collagens and mucopolysacchardes (24). Ths newly produced ECM could n turn be responsble for the control of prolferaton of the basal epthelal cell layer, possbly by affectng the cell shape (25). The need for a proper substratum n order for epthelum to prolferate was frst demonstrated by Dodson (26) and Wessels (27), who used as a model the basal layer of chcken embryonc epderms, whch s normally columnar and mtotcally actve. A loss of mtotc actvty results when the derms and epderms are separated n such a way that ther prevous orentaton to each other s destroyed. n such a case the cells also become 6r EM Proc. Natl. Acad. Sc. USA 77(1980) 4097 flattened and lose ther columnar appearance. f, on the other hand, the cells are mantaned on the proper substrate (n ths case refrozen thawed derms, tropocollagen, or Mllpore flters) and exposed to chcken embryo extract, the cells reman columnar and mtotcally actve. Ths ndcates that the orentaton of the cells, ther shape, and ther prolferatve actvty are closely lnked propertes of the basal germnal populaton and wll depend on the nert substrate upon whch the cells are mantaned. Evdence that the substrate upon whch the cells are mantaned s mportant for ther prolferaton s now plentful (for a revew, see ref. 28). The earler work by Ehrman and Gey showed that varous tssues demonstrate enhanced growth and dfferentaton when cultured on collagen gels (29). n the case of prmary cultures of mouse mammary epthelal cells, the cells have a lmted growth capacty; although they undergo a few rounds of dvson, prolferaton cannot be sustaned nor can these cells be passaged (30). When the same cells are embedded wthn collagen gel, sustaned growth of prmary cells can be observed (30). n avo mplantaton of a collagenous bone matrx results n the prolferaton of adjacent mesenchymal cells whch dfferentate nto chondrocytes, thereby mplyng a role for ths ECM n anchorage-dependent events n cell growth and dfferentaton (31). However, even the use of collagen-coated dshes or collagen gel may not replace the need for a normally produced ECM. n addton to the dffcultes nvolved n gettng proper polymerzaton of purfed collagen, one has to keep n mnd that the ECM s composed not only of collagens but also of proteoglycans, glycosamnoglycans, and glycoprotens, all of whch could affect the prolferaton and subsequent morphogeness of cells and tssue placed n contact wth t (5, 23). Therefore, t may well be that the proper sterc recombnaton n vtro of all these components wll be a dffcult, f not mpossble, task. Corneal endothelal cells n tssue culture produce an extensve ECM whose appearance has been shown to correlate wth the acquston by the cells of ther normal "n vdvo" shape, cell surface polarty, and functon (32). Ths ECM could substtute for the ECM produced by other cell types and could help us to understand ts role n the modulaton of the mtogenc response of other cell types when mantaned on t and exposed to the plethora of growth factors that have been dscovered durng the past 10 years and are now routnely tested on cells mantaned on plastc. When the prolferaton of two dfferent cell types such as bovne granulosa and adrenal cortex cells on plastc s compared to that on ECM, none of the cultures mantaned on plastc and exposed to serum prolferated but when mantaned on an ECM they prolferated qute actvely and no longer requred FGF n order to become confluent. n contrast, when mantaned on plastc, both cell types exhbted an absolute requrement for FGF n order to reach confluence. Therefore, one may conclude that the close contact of the cells wth the ECM can make them responsve to factors present n serum and that adherence to plastc prevents such a response. That ths s a lkely explanaton can be nferred from the observaton that cells mantaned on an ECM had a faster growth rate when mantaned n hgh than n low serum concentratons, thereby exhbtng a dependence on serum n order to prolferate. The smple change of substrate from plastc to ECM restores the normal senstvty of these cells to agents present n serum as well as n plasma (33). Although FGF s clearly mtogenc for a number of mesoderm-derved cells, ts acton on some of the cell types could be ndrect. t could ether replace the cellular requrement for a substrate such as the ECM and thereby make the cells fully

5 4098 Cell Bology: Gospodarowcz et al. responsve to growth factors present n serum or plasma, even when the cells are mantaned on plastc, or t could control the synthess and secreton of the ECM produced by the cells. Such control could n turn make the cells senstve to factors present n serum and plasma. That the latter alternatve could occur s supported by earler observatons whch demonstrate that FGF can control the producton, by vascular endothelal cells, of ther extracellular and cell surface components such as fbronectn and varous types of collagen (15, 34). Evdence that FGF can also drectly affect cell prolferaton s provded by human umblcal endothelal cells whch, even when mantaned on the ECM, stll requred FGF n order to become confluent (35). The ways n whch the ECM exerts ts permssve effect on cell prolferaton can only be the object of speculaton. One possble effect could be to modfy the cell shape n order to make the cells responsve to factor(s) to whch they cannot respond unless they adopt an approprate shape. Recently, Folkman and Moscona (36), usng vascular endothelal cells mantaned on tssue culture dshes coated wth an agent that modfes the adhesveness of the cells to the dsh, were able to control precsely the cellular shape, rangng from hghly flattened to almost spherodal. The extent of cell spreadng was found to be correlated wth DNA synthess or cell growth. Whereas hghly flattened cells responded to serum factors, spherodal cells no longer responded, and ntermedate degrees of response could be observed dependng upon how flattened the cells were. Lkewse, wth corneal epthelal cells, changes n cell shape that depend on the substrate upon whch the cells are mantaned correspond to drastcally altered senstvtes of the cells to growth factors (7, 37, 38). The results presented above emphasze how drastcally one can modfy the prolferatve response of a gven cell type to serum factors, dependng upon the substrate upon whch the cells are mantaned. t s possble that the lack of response of many cell types mantaned under tssue culture condtons to naturally occurrng physologcal agents responsble n vvo for ther prolferaton and dfferentaton may be drectly attrbuted to the artfcal substrate, whether plastc or glass, upon whch the cells rest. t s lkely that the study of cell cultures, whch for the past 50 years, n Wlt's words, s "almost a hstory of the adequacy of meda," may become a hstory of the nadequacy of substrates. We thank Mr. Harvey Scodel for hs nvaluable assstance n the preparaton of ths manuscrpt. Ths work was supported by grants from the Natonal nsttutes of Health (EY and HL 20197). 1. Mller, E. J. (1977) n Cell and Tssue nteractons, eds. Lash, J. W. & Burger, M. M. (Raven, New York), Vol. 32, pp Mur, H. 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