- Email: [email protected]
Regulation of adherens junction protein expression in growth-activated 3T3 cells and in regenerating liver
EXPERIMENTAL
CELL R.ESEARCH
202,477-486
(19%)
dherens Junction Protein Expression in iver T3 Cells and in Regeneratin
ulatio
URSULAGL~JCK, Jo& Department
tiv
LUISRODR~GUEZFERN~DEZ,ROUMENPANKOV,~AND
of Molecular
Genetics and Virology,
The Weizmann
The expression of the adherens junction proteins vinculin, cu-actinin, and talin was compared in serum-stimuated 3T3 cells and in regenerating rat liver following partial hepatectomy. The levels of vinculin RNA and protein synthesis were rapidly and transiently elevated in growth-activated fibroblasts (peaking at 2-3 h) and in regenerating liver (at 4-8 h), preceeding the replicative stage. cr-Actinin expression was also induced, but more slowly (peaking at 6-8 h in 3T3 cells and at 28 h in regenerating liver), and remained elevated when DNA synthesis was proceeding in both systems. The expression of talin RNA was only slightly elevated in 3T3 cells following serum stimulation, and it remained largely unchanged in regenerating liver. The levels of RNA coding for fibronectin and for the fi,-integrin subunit were transiently and extensively induced during liver regeneration (fibronectin with a peak at 8 h and @,-integrin at 12 h). The uvomorulin RNA level, and the expression of the liver-specific genes albumin and transthyretin, decreased in regenerating liver. The results suggest a physiologically significant regulation in the expression of structural components which link the extracellular matrix to the microfilament system in growth-activated fibroblasts and in regenerating G! 1992 Academic Press, be. liver.
INTRQDUCTION The proliferation of fibroblasts in culture depends on the presence of growth factors and nutrients in the culture medium and on the adhesion and spreading of the cells on a solid surface [l--6]. The loss of dependence on adhesion for cell proliferation is often associated with cell transformation [7-IO], and it is the best correlate in vitro to the tumorigenic ability of transformed fibroblasts [ll]. Cell adhesion to the substrate, or to the extracellular matrix (ECM), is mediated by a transmembrane Linkage via cell surface integrin receptors and an ' Permanent address: University of Sofia, Faculty of Biology, Department of Cytology, 8 Dragan Tzankov Street, 1421 Sofia, Bulgaria. *To whom correspondence and reprint requests should be addressed at the above address. Fax: (972).g-344-108.
Institute
of Science, Rehouot 76106, Israel
association with the microfilament system through components of the adhesion plaque 142, IS]. The stimulation of quiescent cell cycle is characterized by t group of immediate early genes regulators [14--161 and for proteins comprising the structural link between the ECM and the a~t~~l-c~~ta~ni~~ filaments (fibronectin, PI-integrin, actin, and a-tropomyosin) [14, 17-191. More recently, a transient induction in the transcription of vincukin, a major component of adherens type junctions (AJ) found in both adhesion plaques and cell-cell adhesions [13], was demonstrated in 3T3 cells stimulated with various grotvf,h factors [ZO, 211. These results suggested that the expression of proteins forming the EC complex may reflect a necessary ste the transition through the cell cycle. The structural proteins of AJ, like other cytoskeletai and ECM proteins, are considered ab~~~a~t and stable constituents of the protein repertoire in the cell. The inductisn in t physiol.ogical relevance of the transient expression of these proteins during growth activation 3T3 fibroblasts is not known Regenerating liver, following a ‘10% hepatectomy, is often used as a model for studying processes associated with growth activation in vivo, as ing hepatocytes in viva The rem synchronously triggered to enter resting G, state, and the cells start 48 h, restoring the mass of the li P week [22-241. The sequence of indufcti growth-related genes c-,fos, cregenerating liver, was shown netics as in 3T3 cells stimul [25-291. In order to address the ~hys~o~o~~a~ significance of the induction of AJ proteins in growth-activated ceils, we compared the expression of talin, v~nc~~in and a-actinin in serum-stimulate nerating s zip3 Biver. In addition, we de in r ing liver the expression of a cellreceptor subunit &-rntegrin and that of the cell-cell adhesion molecule uvomoruhn (E-cadherin) which colocahzes with AJ in vim and in cultured cells [30, 31f. 477
‘0014-4827192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproductim in any form reserved.
GLfiCK
MATERIALS
AND
METHODS
Cell culture. Balb/C3T3 clone A31 cells were cultured in Dulbecco’s-modified Eagle’s medium in the presence of 10% calf serum (GIBCO, Grand Island, NY). For serum stimulation experiments
ET AL. cells were seeded at a density of 5 X lo5 cells per 15-cm-diameter dish and allowed to grow to confluence over 7-9 days. During this period, the confluent cultures depleted the medium of growth factors and became arrested at the G, state. The cells were stimulated to progress synchronously into the cell cycle by the addition of 20% calf serum
FIG. 1. Pattern of newly synthesized proteins in quiescent 3T3 cells stimulated with serum. Quiescent 3T3 cells (A) were treated with 20% calf serum for different times (0.5 to 24 h) (B-H), and the cells were pulse labeled for 30 min with [?S]methionine. Total cell radioactive proteins (5 X lo5 cpm) were separated by 2-D gel electrophoresis followed by autoradiography. Q, quiescent; a, actin; the large arrowhead marks the position of vinculin; the small arrowheads point to a-actinin isoforms.
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47
(GIBCQ). Cells were also stimulated by the addition of PDGF, 100 U/ml (R&D Systems, Minneapolis, MN); FGF, 100 rig/ml (Biochemical Technologies, Stoughton, MA); or TPA, 0.25 pg/ml (Sigma Chemical Co., St. Louis, MO). Cell labeling and two-dimensional (2-D) gel electrophoresis. Cells grown in 35-mm-diameter dishes were labeled with 200 &i/ml of [%]methionine (800 Ci/mmol) (Amersham, Arlington Heights, IL) and extracted directly into O’Farrell’s lysis buffer A [32]. Between 2 X lo5 and 5 X lo6 cpm of trichloroacetic acid (TCA) precipitable proteins were analyzed by 2-D gel electrophoresis as described [33]. The
FIG. 3. Kinetics of vinculin and a-actinin synthesis in serumstimulated 3T3 cells. The autoradiograms shown in Fig. 1 were scanned by a laser densitometer and automatically quantitated using the QUEST computer program [34]. (A) Greyscale image of the gel shown in Fig. 1A generated by the computer. (3) Tbe values given for the vinculin and cc-actinin spots were normalized ag,sinst 50 random spots on each gel. The symbols are as in the Fig. 1. Similar results were obtained in four independent experiments.
FIG. 2. Localization of vinculin and a-actinin on the 2-D gel pattern. Exponentially growing 3T3 cells were labeled for 16 h with [%Imethionine and 5 X IO7 cpm of total cell radioactive proteins were analyzed by 2-D gel electrophoresis and electroblotted onto nitrocellulose. The protein pattern was visualized by autoradiography of the nitroceilulose blot (A). The positions of vinculin (B) and a-actinin (C) were identified by incubation of identical blots with monoclonal antibodies to these proteins followed by alkaline phosphatase-conjugated anti-mouse IgG. The blot used in (B) is the one shown in (A). The bracket marks the position of oc-actinin isoforms; v, vinculin; a, actin.
autoradiograms of the 2-D gels were scanned with a laser densitometer and the intensity of the spots was automatically quantitated using the QUEST computer program as described ]34]. lmmunoblot analysis. [36S]Methionine-labeled proteins (5 X lo7 cpm) were separated by 2-D ge! electrophoresis and transferred to nitrocellulose by electroblotting. The pattern of total radioactive proteins was visualized by autoradiography of the blot. The positions of a-actinin and vinculin on the 2-D gels were identified with monoclonal anti-a-actinin and anti-vinculin antibodies (BioMakor, Rehovot, Israel), followed by alkaline phosphatase-conjugated anti-mouse IgG (Promega, Madison, WI). Partial hepatectomy. Male SPD rats (150-160 g) were subjectedto partial hepatectomy. Seventy percent of the liver mass was removed
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by a surgical procedure performed under ether anesthesia through a small midabdominal incision [35]. The externalized portion of the liver was resected, and the animals were returned to normal activity in minutes. After various time periods, the animals were sacrificed, and the whole regenerating liver tissue was removed and immediately frozen and pulverized in liquid nitrogen. Sham-operated control animals were treated in an identical manner except that no liver tissue was removed. Total RNA was extracted by RNA extraction and hybridization. the guanidinium isothiocyanate method from the liver tissue [36]. The RNA was dissolved in a buffer containing 50% formamide, 6% formaldehyde, 20 mM Mops, pH 7.2,5 mM NaAc, 1 mM EDTA. Samples were treated at 60°C for 10 min. Twenty-microgram samples of total RNA were fractionated on 1% agarose-formaldehyde (2.2 M) gels and blotted onto nitrocellulose filters (Schleicher and Schuell, Dassel, Germany). Prehybridization and hybridization were performed at 42°C in 50% formamide, 5X SSC, 0.5% SDS, 5X Denhardt’s solution, 20 mA4 sodium phosphate (pH 6.5), 250 fig/ml of denaturated salmon sperm DNA. The following cDNA probes were used: vinculin [20], o-actinin [37], fibronectin [38], uvomorulin [39], PI-integrin (D. W. DeSimone, V. Patel, H. Lodish, and R. Hynes, unpublished), albumin, and transthyretin [40]. The talin cDNA was isolated in our laboratory from a 3T3 cDNA library using a chicken talin cDNA obtained from M. Beckerle (University of Utah, Salt Lake City), and the histone H4 probe was a genomic clone [41]. Probes were labeled with o-[32P]dCTP by the random priming technique [42].
ET AL.
tated after separation on 2-D gels revealed no differences in the half-lives of vinculin, Lu-actinin, and other cytoskeletal proteins between quiescent and serumstimulated cells (data not shown). The levels of RNA coding for talin, vinculin, and aactinin in 3T3 cells treated with serum for various times was determined by Northern blot analysis followed by hybridization with the respective cDNAs (Fig. 4). The results shown in Fig. 4 demonstrate a transient increase in vinculin RNA level, with a sharp rise at 2 h after serum-stimulation and a decrease by 6 to 12 h (Fig. 4A). The kinetics of vinculin RNA regulation were reminiscent of the transient induction of c-fos which occurred 1.5 h earlier (Fig. 4A). The level of ol-actinin RNA also increased starting at 2 h after serum stimulation; it peaked around 6 hours and remained elevated at 12 h (Fig. 4A), in agreement with the levels of cr-actinin protein synthesis (Figs. 1 and 3B). After serum stimulation, talin RNA abundance increased, but unlike vinculin its level remained elevated 12 h after the addition of serum
RESULTS
Regulation of Adhesion Activated 3T3 Cells
Plaque Proteins
in Growth-
The pattern of proteins synthesized at various times after stimulation of quiescent Balb/C 3T3 cells with serum was determined by pulse labeling the cells for 30 min with [35S]methionine and 2-D gel electrophoresis of equal amounts of total radioactive proteins followed by autoradiography (Fig. 1). The positions of vinculin and a-actinin on the 2-D gel pattern were identified by immunoblotting of the separated proteins with monoclonal antibodies against vinculin (Fig. 2B) and a-actinin (Fig. 2C). The total protein pattern on the same blot was obtained by exposure of the nitrocellulose filter to X-ray film (Fig. 2A). Quantitative, computerized densitometry (Fig. 3) of the autoradiograms shown in Fig. 1 revealed that the synthesis of both vinculin and ol-actinin was induced following stimulation of quiescent 3T3 cells with serum. The kinetics of vinculin synthesis induction was transient, peaking around 3 h and reduced to baseline by 8 h after serum stimulation (Fig. 3B), in agreement with previous studies [20, 211. The kinetics of induction of or-actinin was different; the peak of synthesis was around 8 h following serum stimulation, and at 24 h ol-actinin synthesis was still higher than that in quiescent cells. To determine possible differences in the halflives of vinculin and a-actinin between quiescent and growing cells, cells treated with serum for 3 and 8 h and quiescent cells were labeled, for 30 min with [35S]methionine, followed by incubation in nonradioactive medium (“chase”) for up to 72 h. Protein levels quanti-
B
FIG. 4. Regulation of adhesion plaque component RNA levels in serum-stimulated 3T3 cells. Quiescent 3T3 cells (Q) were treated with serum and total RNA was extracted from cells at different times after serum stimulation. The RNA was analyzed by Northern blotting followed by hybridization with 32P-labeled cDNAs for vinculin, ol-actinin, talin, and c-fos (A). (B) Methylene blue staining of the blot used for hybridization.
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ized in a belt-like submembranal organization characteristic of AJ in epithelial cells f ata not shown). In primary cultures of hepatocytes, vi din was also localized in adhesion plaques at the termini of actin filaments (results not shown) STo study possible changes in AJ protein expression following gro viuo, we have determined the levels o AJ proteins in regenerating rat liver. tracted from adult rat liver and from regenerating liver at different times following 70% ~e~at~~~~~~y. RNA extracted from sham-operated animals was included as hich verere follow control in Northern blot by hybridization with va and 7). The results summarized in vinculin RNA levels were ra ing liver, peaking between 4 h following hepateetomy. e onset sf the replicavinculin RNA levels preced an increase in bistone Live stage which was marke H4 RNA levels (Fig. SC). Unlike vinculin, the expression of the liver-specific gene al umin gradually de-
(Fig. 4A). The results thus demonstrated an increased expression of talin, vinculin, and a-actinin following serum stimulation of 3T3 cells, but with different kinetics for each protein. A similar regulation in the levels of these RNAs was obtained when quiescent 3T3 cells were treated with PDGF, FGF, or with the tumor promoter TPA, although the magnitude of the response was less dramatic than with whole serum (data not shown), in agreement with previous studies on vinculin expression [20]. egulation of AJ Component Regenerating Liver
GROWTH
RNA Levels in
The regenerating rat liver is widely used as a model system for studying the regulation of genes expressed during early stages of cell proliferation in vivo [43]. In particular, studies on the induction of protooncogene expression demonstrated a correlation with similar kinetics between results obtained with growth-activated 3T3 cells and regenerating liver 125-29, 44, 451. Immunostaining of frozen sections of adult rat liver has shown that vinculin, a-actinin, and actin are local-
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FIG. 5. Induction of vinculin expression in regenerating liver. RNA was prepared from rat livers at various times (Q-48 h) after partial hepatectomy, after sham operation (4s), and from intact rats (0). Twenty micrograms of total RNA per lane was separated and analyzed by Northern blotting, followed by staining of the blot with methylene blue to reveal the position of 18s and 28s rRNAs (D). The same blot was sequentially hybridized with 32P-labeled cDNAs to vinculin (A), albumin (B), and histone H4 (C). The times after partial hepatectomy are labeled under each lane.
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ET AL.
[12, 13, 30, 311. We have therefore determined, during liver regeneration, the expression of fibronectin, that of a fibronectin receptor subunit Pi-integrin, and that of the cell-cell adhesion molecule uvomorulin (E-cadherin). The results summarized in Fig. ‘7 demonstrate that the expression of fibronectin was transiently induced, peaking around 8 h after hepatectomy (Fig. 7A) and that of /3,-integrin with a peak at 12 h (Fig. 7B). In contrast, the level of uvomorulin RNA (Fig. 7C) was reduced during liver regeneration. The RNA content of another protein expressed mainly in the liver, transthyretin, was also reduced during regeneration (Fig. 7D), as was the expression of albumin (Fig. 5B). The changes observed in the abundance of RNAs of the ECM-AJ complex in regenerating liver (Fig. 8) were not seen in sham-operated animals (Figs. 5-7).
creased following hepatectomy (Fig. 5B). The level of a-actinin RNA was transiently induced at a later time after hepatectomy (at 24 to 28 h) and was elevated during the replicative state (Fig. 6A). The expression of talin did not appear to be induced in regenerating liver (Fig. 6B). In addition, talin RNA was significantly more abundant in normal adult liver than vinculin RNA (Fig. 6C, first lane, compare to Fig. 6B). This result correlated with the higher level of talin RNA found in quiescent 3T3 cells as compared to that of vinculin RNA (Fig. 4A). AJ components are suggested to be involved in the structural link between the microfilament system, via transmembrane integrin-receptors to the ECM, and in the adhesion between neighboring cells through the homophylic cell contact receptors of the cadherin family
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FIG. 6. Expression of talin, a-actinin, and vinculin RNA in regenerating liver. RNA was prepared from regenerating liver and analyzed by Northern blotting as described in the legend to Fig. 5, followed by hybridization with cDNAs to cu-actinin (A), talin (B), and vinculin (C). (D) Methylene blue staining of the blot used in (B).
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FIG. 7. Regulation of fibronectin, Pi-integrin, and uvomorulin expression in regenerating liver. RNA was extracted from regenerating liver as described in Fig. 5 and hybridized with cDNAs to fibronectin (A), &-integrin (B), uvomorulin (C), and transthyretin (D). (E) Methyiene blue staining of the blot used in (A-C). S, samples from sham-operated animals. Similar results were obtained with RNA prepared from three different experiments.
DISCUSSION
The objective of this study was to determine the physiological relevance of regulating the expression of the ECM-microfilament complex in growth-stimulated cells Regenerating liver, following partial hepatectomy, was chosen as an in vim model for growth activation, as it is widely used for studying the induction of growthassociated genes [25-29, 42, 431. We chose to compare the control of AJ component expression of regenerating rat liver in uiuo to that of quiescent 3T3 cells stimulated with serum.
We have demonstrated a rapi transient inducin ‘both systems, tion in the abundance of vinculin s occurred (Figs. before the major -wave of DNA sy 3B, 8). The expression of au-actinin was also elevated in serum-stimulated 3T3 cells and rege differed from the kinetics of vinculin cells, as ol-actinin expression increased later and remained elevated during the DNA synthetic phase of these cells [46]. In regenerating liver, the cw-actinin RNA level was induced after that of vinculin RNA (Fig. 6), and it coincided with the DNA lreplicstive stage of liver cells, measured by the increase in histone II4 RNA level (Fig. 8).
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--Oa-actinin --+--vinculin . ;&.. . .. ta,in
0
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20 Hours
--++-
0
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fibronectin integrin
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albumin
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FIG. 8. Kinetics of gene expression in regenerating liver. The Northern blots shown in Figs. 5-7 and an additional Northern blot hybridized with talin cDNA (not shown) were scanned with a laser densitometer using the “Image Quant” program, and the optical density (in arbitrary units) is shown, to allow comparison of the kinetics of regulation for the various RNAs.
The expression of fibronectin and a fibronectin receptor subunit, ,B,-integrin, was also elevated at aprereplicative stage in regenerating liver (Fig. 8). The regulation of RNA levels for these ECM-AJ assembly proteins in regenerating liver was specific, as it was not observed in sham-operated animals, and was not shared by tissue-specific genes and the cell adhesion molecule uvomorulin. It remains to be determined whether this increase in ECM-AJ RNA levels resulted from in-
ET AL.
creased transcription or by increases in RNA half-lives, mechanisms shown to be extensively active in regenerating liver 140,431. In primary cultures of hepatocytes isolated from adult rat liver, the synthesis of vinculin, a-actinin [47], and fibronectin [45,48] was also elevated following stimulation with EGF and insulin. In a recent study [45], the levels of other ECM molecules (vitronectin, laminin, and collagen) were reported to increase in regenerating liver. Taken together, these results imply that induction of the proliferative state in regenerating liver in uiuo, and in primary cultures of hepatocytes in vitro, includes the elevated expression of ECM and AJ components. The level of talin RNA appeared to be the least regulated among the adhesion plaque components in growth-activated 3T3 cells and in regenerating liver. In this respect, it is interesting to note that in quiescent 3T3 cells treated with growth factors, vinculin is rapidly and transiently dissociated from adhesion plaques, while talin remains associated with focal contacts under these conditions [49]. Talin and vinculin therefore differ in their response to stimulation by growth factors in the assembly of these proteins, and in the regulation of the respective RNA levels. Vinculin and talin also vary in their subcellular distribution; talin is localized at cell-ECM contact sites [12], while vinculin is found, in addition, in cell-cell AJ [13]. The increase of fibronectin and &-integrin RNA in regenerating liver correlates with the reported induction in the transcription and mRNA levels of these genes in quiescent fibroblasts stimulated with serum factors [18, 19, 211. These results thus point to a possible physiological significance for the elevated expression of genes coding for the structural complex which links the ECM to the microfilament system in processes associated with growth activation of cultured fibroblast cell lines and regenerating liver. What could be the role of regulating the expression of cytoskeletal and ECM proteins in growth-stimulated cells? This question is especially intriguing as these proteins are ubiquitous, abundant, and stable cellular constituents. Recent studies, using the cDNA transfection approach to stably elevate the expression of such genes, have demonstrated that their overexpression results in significant physiological changes in the cell; for example, in 3T3 cells where the expression of the actinbinding protein gelsolin was elevated by only 25%, the chemotactic motility of the cells was markedly increased [50]. Moreover, raising the expression of vinculin, following transfection, by 20% over the endogenous vinculin level in 3T3 cells resulted in a drastic suppression of the motile ability of the cells [51]. Furthermore, the elevation of vinculin expression in tumorigenic SV40-transformed 3T3 cells and in rat adenocarcinoma cells, up to that found in 3T3 cells, brought about a dramatic suppression in the tumorigenic ability of these
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cells ]~a]. Similarly, overexpression of the 01~/&integrins in CHC cells suppressed the motility and the tumorigenie phenotype of these cells [53]. These results demonstrate that cellular processes associated with growth stimulation, such as adhesion, motility, and tumorigenicity can be affected, in a major way, by modulating the expression of individual proteins of the ECM-microfilament complex. Changes in the expression of microfilament components were also noted in various differentiating cell systems, suggesting that this regulation may have an important role in the control of cellular differentiation 1541; for example, in freshly isolated hepatocytes the differentiated phenotype is maintained by culturing the cells on ECM components [47,48, F&57], and is apparently mediated through necessary changes in cell shape [47, 481 and in cytoskeletal gene expression [47]. uture studies directed at the molecular analysis of gene expression for proteins of the ECM-microfilament complex will provide an understanding of the mechanisms whereby changes in the abundance of these proteins affect growth, differentiation, and cell transformation We thank R. Hynes, R. Kemfer, J. Stein, D. Kwiatkowski, M. Beckerle, and J. Darnell, JF. for generously providing cDNA probes. This study was supported by grants from the NCRD, Israel-DKFC, Germany, collaboration program and from the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science. R.P. was supported by a short term FEBS fellowship and J.L.R.F. by a scholarship from the Conserjeria de Education (Comunidad Autonoma de Las Islas Canarias). A.B.-Z. holds the LunenfeldKunin Professorial Chair in Genetics and Cell Biology.
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2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Benecke, B.-J., Ben-Ze’ev, A., and Penman, S. (1978) Cell 14, 931-939. Farmer, S. R., Ben-Ze’ev, A., Benecke, B.-J., and Penman, S. (1978) Cell l&627-637. DeLarco, J. E., and Todaro, G. (1978) Proc. Natl. Acad. Sci. USA 75,4001-4005. Folkman, J., and Moscona, A. (1978) Nature 346, 760-763. Stiles, C. D., Isberg, R. R., Pledger, W. J., Antoniades, H. N., and Scher, 6. D. (1979) J. Cdl. Physiol. 99, 395-406. Ben-Ze’ev, A., Farmer. S. R., and Penman, S. (1980) Cell 21, 365-372. Stoker, M., O’Neill, C., Berryman, S., and Waxman, S. (1968) Int. J. Cancer 3, 683-695. Tucker, R. W., Butterfield, 6. E., and Folkman, J. (1981) J. Supramol. Struct. Cell. Biochem. 15, 29-40. Wittelsberger, S. C., Kleene, K., and Penman, S. (1981) Cell 29, 859-866. Raz, A., and Ben-Ze’ev, A. (1982) int. J. Cancer 29, 711-715. Shin, S., Freedman, V. H., Risser, R., and Pollack, R. (1975) Proc. Nutl. Acad. Sci. USA 72,4435-4439. Burridge, K., Fath, C. K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Bid. 4, 487-525. Geiger, B., and Ginsberg, D. (1991) CeEl Motil. Cytosheleton 20, 1-6.
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14. Greenberg, M. E., and Zii, E. B. (1984) Nature @11,433-437. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25.
Lau, L. F., and Nathans; D. (1987) Proc. ~“barl. .Acor$. sci. uX.4 84,1182-1186. Almendral, J. M., Sommer, D., MacDonald-Bravo, II., hard& J., Perera, J., and Bravo, R. (1988) Mol. Cell. 2140-2148. Elder, P. K., Schmidt, L. O., One, T., and Getz, M. J. (1984) Proc. Natl. Acad. Sci. USA 81, 7476-7480. Blatti, S. P., Foster, D. N., Ranganathan, G Gets, M. J. (1988) Proc. N&l. Acad. Sci. u.§ Rysek, R.-P., MacDonald-Bravo, I-I., Zesial, (1989) Exp. Cell Res. 180, 537-545. Ben-Ze’ev, A., Reiss, R., Bendori, R., and Goradecki, B. (1996) Cd Regul. lb, 621-636. Bellas, R. E., Bendori, R., and Farmer, S. R. (1991) J. Biol. Chem. 266, L2008-12014. p 1464-1469. Grisham, J. W. (1962) Cancer Res. Bucher, N. L. R., and Malt, R. A. (1971). Regeneration of Liver and Kidney, Little, Brown, Boston, MA. Rabes, II. M., Wirshing, R., Tuczek, H. V., and Iseler, G. (1976) Cell Tissue Kinet. 6, 537-532. Goyette, M., Petropoulos, C. J., Shank, P. R., and Fausto, N. (1983) Science 219,510~512.
26.
Makino, R., Hayashi, 697-698.
27.
Kruijer, W., Skelly, II., Botteri, F., van der Putten, II., B J. R., Verma, I. M., and Leffert, II. L. (1986) cj. Bioi. Chem. 7929-7933.
28.
Sobczak, J., Tomnier, (1989) Eur. J. Biochem.
K., and Sugimura,
M.-F., Lotti, P80,49-53.
T. (1984) Nature
$1
A.-
abinet: C., and Fausto, N. (1990)
29. 30.
Belier, K., Vestweber, 1 ,327-332.
3i.
Hirano, S., Nose, A., Hatta, K., Kawakami, (1987). J. Cell Biol. 105, 2501-2510.
REFERENCES 1.
GROWTH
32.
@Farrell,
33.
Ben-Ze’ev,
D., and Kemler,
P. H. (1975) 9. Viol, &em.
R. (1985). J. Cell Biol. A., and Takeichi,
M.
1BB9,4007-4021.
A. (1990) Electrophores
34.
Garells, J. I. (1989) 9. Biol. Chem.
35.
Higgins, 6. M., and Anderson, 186-202.
36.
Chirgwin, J. M., Przybyla, w. J. (1979) Biochemistry
37.
Youssoufian, II., M&fee, Am. J. Hum. Genet. 47,627;.
38.
Schwartzbauer, J. E., Tamkun, J. W., Hynes, R. 0. (1983) Cell 35, 421-431.
39.
Ringwald, M., Scbuh, R., Vestweber, D., Eis,tetter, II., Lottspeich, F., Engel, J., Diilz, R., Jahnig, F., Epplen, J., Mayer, S., Miiller, C., and Kemler, R. (1987) g?&BO vi B:, 3647-3653.
40.
Dermann, E., Kramer, K., Wallig, L., Weinberger, and Darnell, 3. E., Jr. (1981) CeEl23, 731-739.
41.
Plumb, M., Stein, J., and Stein, 6. (1983) Pu’wleic Acids Res. 4 E, 2391-2410.
42.
Feinberg, 266-267.
43.
Friedman, J. M., Chung, E. Y., and Darnell, Mol. Biol. B79, 37-53.
44.
Thompson, N. L., Mead, J. E., Braun, L., Goyette, M., Shank, P. R., and Fausto, N. (1986) Cancer IZes. 46, Sill-3117.
R. M. (1931) Arch. puafhol. 12,
A. E., MacDonaid, 24, 5294-5299.
A. P., and Vogelstein,
R. J., and Rutter,
and Kwistkowski, Lemisch~a,
D. J. (1990) 1. R.,
and
C., Ray, M.,
B. (1983) Anal. &o&em.
137,
J. E., Jr. (1984) J.
486 45. 46. 47. 48. 49. 50.
GLfJCK Jakowlew, S. B., Mead, J. E., Danielpour, D., Wu, J., Roberts, A. B., and Fausto, N. (1991) Cell Regul. 2,535X48. Shaulsky, G., Ben-Ze’ev, A., and Rotter, V. (1991) Oncogene 6, 1707-1711. Ben-Ze’ev, A., Robinson, G. S., Bucher, N. L. R., and Farmer, S. R. (1988) Proc. Natl. Acad. Sci. USA 85, 2161-2165. Mooney, D., Hansen, L., Vacanti, J., Langer, R., Farmer, S., and Ingber, D. (1992) J. Cell Physiol. 151, 497-505. Herman, B., and Pledger, W. J. (1985) J. Cell Biol. 100,10311040. Cunningham, C. C., Stossel, T. P., and Kwiatkowski, D. J. (1991)
Scieme251,1233-1236. Received April 1, 1992. Revised version received June 2, 1992
ET AL. 51.
Rodriguez Fernlndez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1992) Cell Motil. Cytoskeleton 22, 127-134.
52.
Rodriguez H., Ziiller,
Fernlndez, J. L., Geiger, B., Salomon, D., Sabanay, M., and Ben-Ze’ev, A. J. Cell Biol., in press.
53.
Giancotti,
F. G., and Ruoslahti,
54.
Ben-Ze’ev,
A. (1991) BioEssays
55.
Michalopoulos, 70-78.
56.
Bissell, M. D., Arenson, D. M., Maher, J. J., and Roll, F. J. (1987) J. Clin. Invest. 79, 801-812.
57.
Reid, L. M. (1990) Cut-r. Opin. Cell Biol. 2, 121-130.
G., and Pitot,
E. (1990) Cell 60,849-859.
13, 207-212. H. C. (1975) Exp. Cell Res. 94,
CELL R.ESEARCH
202,477-486
(19%)
dherens Junction Protein Expression in iver T3 Cells and in Regeneratin
ulatio
URSULAGL~JCK, Jo& Department
tiv
LUISRODR~GUEZFERN~DEZ,ROUMENPANKOV,~AND
of Molecular
Genetics and Virology,
The Weizmann
The expression of the adherens junction proteins vinculin, cu-actinin, and talin was compared in serum-stimuated 3T3 cells and in regenerating rat liver following partial hepatectomy. The levels of vinculin RNA and protein synthesis were rapidly and transiently elevated in growth-activated fibroblasts (peaking at 2-3 h) and in regenerating liver (at 4-8 h), preceeding the replicative stage. cr-Actinin expression was also induced, but more slowly (peaking at 6-8 h in 3T3 cells and at 28 h in regenerating liver), and remained elevated when DNA synthesis was proceeding in both systems. The expression of talin RNA was only slightly elevated in 3T3 cells following serum stimulation, and it remained largely unchanged in regenerating liver. The levels of RNA coding for fibronectin and for the fi,-integrin subunit were transiently and extensively induced during liver regeneration (fibronectin with a peak at 8 h and @,-integrin at 12 h). The uvomorulin RNA level, and the expression of the liver-specific genes albumin and transthyretin, decreased in regenerating liver. The results suggest a physiologically significant regulation in the expression of structural components which link the extracellular matrix to the microfilament system in growth-activated fibroblasts and in regenerating G! 1992 Academic Press, be. liver.
INTRQDUCTION The proliferation of fibroblasts in culture depends on the presence of growth factors and nutrients in the culture medium and on the adhesion and spreading of the cells on a solid surface [l--6]. The loss of dependence on adhesion for cell proliferation is often associated with cell transformation [7-IO], and it is the best correlate in vitro to the tumorigenic ability of transformed fibroblasts [ll]. Cell adhesion to the substrate, or to the extracellular matrix (ECM), is mediated by a transmembrane Linkage via cell surface integrin receptors and an ' Permanent address: University of Sofia, Faculty of Biology, Department of Cytology, 8 Dragan Tzankov Street, 1421 Sofia, Bulgaria. *To whom correspondence and reprint requests should be addressed at the above address. Fax: (972).g-344-108.
Institute
of Science, Rehouot 76106, Israel
association with the microfilament system through components of the adhesion plaque 142, IS]. The stimulation of quiescent cell cycle is characterized by t group of immediate early genes regulators [14--161 and for proteins comprising the structural link between the ECM and the a~t~~l-c~~ta~ni~~ filaments (fibronectin, PI-integrin, actin, and a-tropomyosin) [14, 17-191. More recently, a transient induction in the transcription of vincukin, a major component of adherens type junctions (AJ) found in both adhesion plaques and cell-cell adhesions [13], was demonstrated in 3T3 cells stimulated with various grotvf,h factors [ZO, 211. These results suggested that the expression of proteins forming the EC complex may reflect a necessary ste the transition through the cell cycle. The structural proteins of AJ, like other cytoskeletai and ECM proteins, are considered ab~~~a~t and stable constituents of the protein repertoire in the cell. The inductisn in t physiol.ogical relevance of the transient expression of these proteins during growth activation 3T3 fibroblasts is not known Regenerating liver, following a ‘10% hepatectomy, is often used as a model for studying processes associated with growth activation in vivo, as ing hepatocytes in viva The rem synchronously triggered to enter resting G, state, and the cells start 48 h, restoring the mass of the li P week [22-241. The sequence of indufcti growth-related genes c-,fos, cregenerating liver, was shown netics as in 3T3 cells stimul [25-291. In order to address the ~hys~o~o~~a~ significance of the induction of AJ proteins in growth-activated ceils, we compared the expression of talin, v~nc~~in and a-actinin in serum-stimulate nerating s zip3 Biver. In addition, we de in r ing liver the expression of a cellreceptor subunit &-rntegrin and that of the cell-cell adhesion molecule uvomoruhn (E-cadherin) which colocahzes with AJ in vim and in cultured cells [30, 31f. 477
‘0014-4827192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproductim in any form reserved.
GLfiCK
MATERIALS
AND
METHODS
Cell culture. Balb/C3T3 clone A31 cells were cultured in Dulbecco’s-modified Eagle’s medium in the presence of 10% calf serum (GIBCO, Grand Island, NY). For serum stimulation experiments
ET AL. cells were seeded at a density of 5 X lo5 cells per 15-cm-diameter dish and allowed to grow to confluence over 7-9 days. During this period, the confluent cultures depleted the medium of growth factors and became arrested at the G, state. The cells were stimulated to progress synchronously into the cell cycle by the addition of 20% calf serum
FIG. 1. Pattern of newly synthesized proteins in quiescent 3T3 cells stimulated with serum. Quiescent 3T3 cells (A) were treated with 20% calf serum for different times (0.5 to 24 h) (B-H), and the cells were pulse labeled for 30 min with [?S]methionine. Total cell radioactive proteins (5 X lo5 cpm) were separated by 2-D gel electrophoresis followed by autoradiography. Q, quiescent; a, actin; the large arrowhead marks the position of vinculin; the small arrowheads point to a-actinin isoforms.
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47
(GIBCQ). Cells were also stimulated by the addition of PDGF, 100 U/ml (R&D Systems, Minneapolis, MN); FGF, 100 rig/ml (Biochemical Technologies, Stoughton, MA); or TPA, 0.25 pg/ml (Sigma Chemical Co., St. Louis, MO). Cell labeling and two-dimensional (2-D) gel electrophoresis. Cells grown in 35-mm-diameter dishes were labeled with 200 &i/ml of [%]methionine (800 Ci/mmol) (Amersham, Arlington Heights, IL) and extracted directly into O’Farrell’s lysis buffer A [32]. Between 2 X lo5 and 5 X lo6 cpm of trichloroacetic acid (TCA) precipitable proteins were analyzed by 2-D gel electrophoresis as described [33]. The
FIG. 3. Kinetics of vinculin and a-actinin synthesis in serumstimulated 3T3 cells. The autoradiograms shown in Fig. 1 were scanned by a laser densitometer and automatically quantitated using the QUEST computer program [34]. (A) Greyscale image of the gel shown in Fig. 1A generated by the computer. (3) Tbe values given for the vinculin and cc-actinin spots were normalized ag,sinst 50 random spots on each gel. The symbols are as in the Fig. 1. Similar results were obtained in four independent experiments.
FIG. 2. Localization of vinculin and a-actinin on the 2-D gel pattern. Exponentially growing 3T3 cells were labeled for 16 h with [%Imethionine and 5 X IO7 cpm of total cell radioactive proteins were analyzed by 2-D gel electrophoresis and electroblotted onto nitrocellulose. The protein pattern was visualized by autoradiography of the nitroceilulose blot (A). The positions of vinculin (B) and a-actinin (C) were identified by incubation of identical blots with monoclonal antibodies to these proteins followed by alkaline phosphatase-conjugated anti-mouse IgG. The blot used in (B) is the one shown in (A). The bracket marks the position of oc-actinin isoforms; v, vinculin; a, actin.
autoradiograms of the 2-D gels were scanned with a laser densitometer and the intensity of the spots was automatically quantitated using the QUEST computer program as described ]34]. lmmunoblot analysis. [36S]Methionine-labeled proteins (5 X lo7 cpm) were separated by 2-D ge! electrophoresis and transferred to nitrocellulose by electroblotting. The pattern of total radioactive proteins was visualized by autoradiography of the blot. The positions of a-actinin and vinculin on the 2-D gels were identified with monoclonal anti-a-actinin and anti-vinculin antibodies (BioMakor, Rehovot, Israel), followed by alkaline phosphatase-conjugated anti-mouse IgG (Promega, Madison, WI). Partial hepatectomy. Male SPD rats (150-160 g) were subjectedto partial hepatectomy. Seventy percent of the liver mass was removed
480
GLUCK
by a surgical procedure performed under ether anesthesia through a small midabdominal incision [35]. The externalized portion of the liver was resected, and the animals were returned to normal activity in minutes. After various time periods, the animals were sacrificed, and the whole regenerating liver tissue was removed and immediately frozen and pulverized in liquid nitrogen. Sham-operated control animals were treated in an identical manner except that no liver tissue was removed. Total RNA was extracted by RNA extraction and hybridization. the guanidinium isothiocyanate method from the liver tissue [36]. The RNA was dissolved in a buffer containing 50% formamide, 6% formaldehyde, 20 mM Mops, pH 7.2,5 mM NaAc, 1 mM EDTA. Samples were treated at 60°C for 10 min. Twenty-microgram samples of total RNA were fractionated on 1% agarose-formaldehyde (2.2 M) gels and blotted onto nitrocellulose filters (Schleicher and Schuell, Dassel, Germany). Prehybridization and hybridization were performed at 42°C in 50% formamide, 5X SSC, 0.5% SDS, 5X Denhardt’s solution, 20 mA4 sodium phosphate (pH 6.5), 250 fig/ml of denaturated salmon sperm DNA. The following cDNA probes were used: vinculin [20], o-actinin [37], fibronectin [38], uvomorulin [39], PI-integrin (D. W. DeSimone, V. Patel, H. Lodish, and R. Hynes, unpublished), albumin, and transthyretin [40]. The talin cDNA was isolated in our laboratory from a 3T3 cDNA library using a chicken talin cDNA obtained from M. Beckerle (University of Utah, Salt Lake City), and the histone H4 probe was a genomic clone [41]. Probes were labeled with o-[32P]dCTP by the random priming technique [42].
ET AL.
tated after separation on 2-D gels revealed no differences in the half-lives of vinculin, Lu-actinin, and other cytoskeletal proteins between quiescent and serumstimulated cells (data not shown). The levels of RNA coding for talin, vinculin, and aactinin in 3T3 cells treated with serum for various times was determined by Northern blot analysis followed by hybridization with the respective cDNAs (Fig. 4). The results shown in Fig. 4 demonstrate a transient increase in vinculin RNA level, with a sharp rise at 2 h after serum-stimulation and a decrease by 6 to 12 h (Fig. 4A). The kinetics of vinculin RNA regulation were reminiscent of the transient induction of c-fos which occurred 1.5 h earlier (Fig. 4A). The level of ol-actinin RNA also increased starting at 2 h after serum stimulation; it peaked around 6 hours and remained elevated at 12 h (Fig. 4A), in agreement with the levels of cr-actinin protein synthesis (Figs. 1 and 3B). After serum stimulation, talin RNA abundance increased, but unlike vinculin its level remained elevated 12 h after the addition of serum
RESULTS
Regulation of Adhesion Activated 3T3 Cells
Plaque Proteins
in Growth-
The pattern of proteins synthesized at various times after stimulation of quiescent Balb/C 3T3 cells with serum was determined by pulse labeling the cells for 30 min with [35S]methionine and 2-D gel electrophoresis of equal amounts of total radioactive proteins followed by autoradiography (Fig. 1). The positions of vinculin and a-actinin on the 2-D gel pattern were identified by immunoblotting of the separated proteins with monoclonal antibodies against vinculin (Fig. 2B) and a-actinin (Fig. 2C). The total protein pattern on the same blot was obtained by exposure of the nitrocellulose filter to X-ray film (Fig. 2A). Quantitative, computerized densitometry (Fig. 3) of the autoradiograms shown in Fig. 1 revealed that the synthesis of both vinculin and ol-actinin was induced following stimulation of quiescent 3T3 cells with serum. The kinetics of vinculin synthesis induction was transient, peaking around 3 h and reduced to baseline by 8 h after serum stimulation (Fig. 3B), in agreement with previous studies [20, 211. The kinetics of induction of or-actinin was different; the peak of synthesis was around 8 h following serum stimulation, and at 24 h ol-actinin synthesis was still higher than that in quiescent cells. To determine possible differences in the halflives of vinculin and a-actinin between quiescent and growing cells, cells treated with serum for 3 and 8 h and quiescent cells were labeled, for 30 min with [35S]methionine, followed by incubation in nonradioactive medium (“chase”) for up to 72 h. Protein levels quanti-
B
FIG. 4. Regulation of adhesion plaque component RNA levels in serum-stimulated 3T3 cells. Quiescent 3T3 cells (Q) were treated with serum and total RNA was extracted from cells at different times after serum stimulation. The RNA was analyzed by Northern blotting followed by hybridization with 32P-labeled cDNAs for vinculin, ol-actinin, talin, and c-fos (A). (B) Methylene blue staining of the blot used for hybridization.
ADHERENS
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ized in a belt-like submembranal organization characteristic of AJ in epithelial cells f ata not shown). In primary cultures of hepatocytes, vi din was also localized in adhesion plaques at the termini of actin filaments (results not shown) STo study possible changes in AJ protein expression following gro viuo, we have determined the levels o AJ proteins in regenerating rat liver. tracted from adult rat liver and from regenerating liver at different times following 70% ~e~at~~~~~~y. RNA extracted from sham-operated animals was included as hich verere follow control in Northern blot by hybridization with va and 7). The results summarized in vinculin RNA levels were ra ing liver, peaking between 4 h following hepateetomy. e onset sf the replicavinculin RNA levels preced an increase in bistone Live stage which was marke H4 RNA levels (Fig. SC). Unlike vinculin, the expression of the liver-specific gene al umin gradually de-
(Fig. 4A). The results thus demonstrated an increased expression of talin, vinculin, and a-actinin following serum stimulation of 3T3 cells, but with different kinetics for each protein. A similar regulation in the levels of these RNAs was obtained when quiescent 3T3 cells were treated with PDGF, FGF, or with the tumor promoter TPA, although the magnitude of the response was less dramatic than with whole serum (data not shown), in agreement with previous studies on vinculin expression [20]. egulation of AJ Component Regenerating Liver
GROWTH
RNA Levels in
The regenerating rat liver is widely used as a model system for studying the regulation of genes expressed during early stages of cell proliferation in vivo [43]. In particular, studies on the induction of protooncogene expression demonstrated a correlation with similar kinetics between results obtained with growth-activated 3T3 cells and regenerating liver 125-29, 44, 451. Immunostaining of frozen sections of adult rat liver has shown that vinculin, a-actinin, and actin are local-
-2.3
8
12
16
20
24
28
32
36
40
48
4s
FIG. 5. Induction of vinculin expression in regenerating liver. RNA was prepared from rat livers at various times (Q-48 h) after partial hepatectomy, after sham operation (4s), and from intact rats (0). Twenty micrograms of total RNA per lane was separated and analyzed by Northern blotting, followed by staining of the blot with methylene blue to reveal the position of 18s and 28s rRNAs (D). The same blot was sequentially hybridized with 32P-labeled cDNAs to vinculin (A), albumin (B), and histone H4 (C). The times after partial hepatectomy are labeled under each lane.
482
GLiiCK
ET AL.
[12, 13, 30, 311. We have therefore determined, during liver regeneration, the expression of fibronectin, that of a fibronectin receptor subunit Pi-integrin, and that of the cell-cell adhesion molecule uvomorulin (E-cadherin). The results summarized in Fig. ‘7 demonstrate that the expression of fibronectin was transiently induced, peaking around 8 h after hepatectomy (Fig. 7A) and that of /3,-integrin with a peak at 12 h (Fig. 7B). In contrast, the level of uvomorulin RNA (Fig. 7C) was reduced during liver regeneration. The RNA content of another protein expressed mainly in the liver, transthyretin, was also reduced during regeneration (Fig. 7D), as was the expression of albumin (Fig. 5B). The changes observed in the abundance of RNAs of the ECM-AJ complex in regenerating liver (Fig. 8) were not seen in sham-operated animals (Figs. 5-7).
creased following hepatectomy (Fig. 5B). The level of a-actinin RNA was transiently induced at a later time after hepatectomy (at 24 to 28 h) and was elevated during the replicative state (Fig. 6A). The expression of talin did not appear to be induced in regenerating liver (Fig. 6B). In addition, talin RNA was significantly more abundant in normal adult liver than vinculin RNA (Fig. 6C, first lane, compare to Fig. 6B). This result correlated with the higher level of talin RNA found in quiescent 3T3 cells as compared to that of vinculin RNA (Fig. 4A). AJ components are suggested to be involved in the structural link between the microfilament system, via transmembrane integrin-receptors to the ECM, and in the adhesion between neighboring cells through the homophylic cell contact receptors of the cadherin family
-wtb
18s
01
4
8
12
16
20
24
48
FIG. 6. Expression of talin, a-actinin, and vinculin RNA in regenerating liver. RNA was prepared from regenerating liver and analyzed by Northern blotting as described in the legend to Fig. 5, followed by hybridization with cDNAs to cu-actinin (A), talin (B), and vinculin (C). (D) Methylene blue staining of the blot used in (B).
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0.5
1
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2
4
8
12
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20
24
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32
48
72
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45
483
16S
FIG. 7. Regulation of fibronectin, Pi-integrin, and uvomorulin expression in regenerating liver. RNA was extracted from regenerating liver as described in Fig. 5 and hybridized with cDNAs to fibronectin (A), &-integrin (B), uvomorulin (C), and transthyretin (D). (E) Methyiene blue staining of the blot used in (A-C). S, samples from sham-operated animals. Similar results were obtained with RNA prepared from three different experiments.
DISCUSSION
The objective of this study was to determine the physiological relevance of regulating the expression of the ECM-microfilament complex in growth-stimulated cells Regenerating liver, following partial hepatectomy, was chosen as an in vim model for growth activation, as it is widely used for studying the induction of growthassociated genes [25-29, 42, 431. We chose to compare the control of AJ component expression of regenerating rat liver in uiuo to that of quiescent 3T3 cells stimulated with serum.
We have demonstrated a rapi transient inducin ‘both systems, tion in the abundance of vinculin s occurred (Figs. before the major -wave of DNA sy 3B, 8). The expression of au-actinin was also elevated in serum-stimulated 3T3 cells and rege differed from the kinetics of vinculin cells, as ol-actinin expression increased later and remained elevated during the DNA synthetic phase of these cells [46]. In regenerating liver, the cw-actinin RNA level was induced after that of vinculin RNA (Fig. 6), and it coincided with the DNA lreplicstive stage of liver cells, measured by the increase in histone II4 RNA level (Fig. 8).
484
GLijCK
--Oa-actinin --+--vinculin . ;&.. . .. ta,in
0
10
20 Hours
--++-
0
0
10
10
30
fibronectin integrin
20 Hours
30
-
albumin
-
H4
20 Hours
40
30
40
40
FIG. 8. Kinetics of gene expression in regenerating liver. The Northern blots shown in Figs. 5-7 and an additional Northern blot hybridized with talin cDNA (not shown) were scanned with a laser densitometer using the “Image Quant” program, and the optical density (in arbitrary units) is shown, to allow comparison of the kinetics of regulation for the various RNAs.
The expression of fibronectin and a fibronectin receptor subunit, ,B,-integrin, was also elevated at aprereplicative stage in regenerating liver (Fig. 8). The regulation of RNA levels for these ECM-AJ assembly proteins in regenerating liver was specific, as it was not observed in sham-operated animals, and was not shared by tissue-specific genes and the cell adhesion molecule uvomorulin. It remains to be determined whether this increase in ECM-AJ RNA levels resulted from in-
ET AL.
creased transcription or by increases in RNA half-lives, mechanisms shown to be extensively active in regenerating liver 140,431. In primary cultures of hepatocytes isolated from adult rat liver, the synthesis of vinculin, a-actinin [47], and fibronectin [45,48] was also elevated following stimulation with EGF and insulin. In a recent study [45], the levels of other ECM molecules (vitronectin, laminin, and collagen) were reported to increase in regenerating liver. Taken together, these results imply that induction of the proliferative state in regenerating liver in uiuo, and in primary cultures of hepatocytes in vitro, includes the elevated expression of ECM and AJ components. The level of talin RNA appeared to be the least regulated among the adhesion plaque components in growth-activated 3T3 cells and in regenerating liver. In this respect, it is interesting to note that in quiescent 3T3 cells treated with growth factors, vinculin is rapidly and transiently dissociated from adhesion plaques, while talin remains associated with focal contacts under these conditions [49]. Talin and vinculin therefore differ in their response to stimulation by growth factors in the assembly of these proteins, and in the regulation of the respective RNA levels. Vinculin and talin also vary in their subcellular distribution; talin is localized at cell-ECM contact sites [12], while vinculin is found, in addition, in cell-cell AJ [13]. The increase of fibronectin and &-integrin RNA in regenerating liver correlates with the reported induction in the transcription and mRNA levels of these genes in quiescent fibroblasts stimulated with serum factors [18, 19, 211. These results thus point to a possible physiological significance for the elevated expression of genes coding for the structural complex which links the ECM to the microfilament system in processes associated with growth activation of cultured fibroblast cell lines and regenerating liver. What could be the role of regulating the expression of cytoskeletal and ECM proteins in growth-stimulated cells? This question is especially intriguing as these proteins are ubiquitous, abundant, and stable cellular constituents. Recent studies, using the cDNA transfection approach to stably elevate the expression of such genes, have demonstrated that their overexpression results in significant physiological changes in the cell; for example, in 3T3 cells where the expression of the actinbinding protein gelsolin was elevated by only 25%, the chemotactic motility of the cells was markedly increased [50]. Moreover, raising the expression of vinculin, following transfection, by 20% over the endogenous vinculin level in 3T3 cells resulted in a drastic suppression of the motile ability of the cells [51]. Furthermore, the elevation of vinculin expression in tumorigenic SV40-transformed 3T3 cells and in rat adenocarcinoma cells, up to that found in 3T3 cells, brought about a dramatic suppression in the tumorigenic ability of these
ADHERENS
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cells ]~a]. Similarly, overexpression of the 01~/&integrins in CHC cells suppressed the motility and the tumorigenie phenotype of these cells [53]. These results demonstrate that cellular processes associated with growth stimulation, such as adhesion, motility, and tumorigenicity can be affected, in a major way, by modulating the expression of individual proteins of the ECM-microfilament complex. Changes in the expression of microfilament components were also noted in various differentiating cell systems, suggesting that this regulation may have an important role in the control of cellular differentiation 1541; for example, in freshly isolated hepatocytes the differentiated phenotype is maintained by culturing the cells on ECM components [47,48, F&57], and is apparently mediated through necessary changes in cell shape [47, 481 and in cytoskeletal gene expression [47]. uture studies directed at the molecular analysis of gene expression for proteins of the ECM-microfilament complex will provide an understanding of the mechanisms whereby changes in the abundance of these proteins affect growth, differentiation, and cell transformation We thank R. Hynes, R. Kemfer, J. Stein, D. Kwiatkowski, M. Beckerle, and J. Darnell, JF. for generously providing cDNA probes. This study was supported by grants from the NCRD, Israel-DKFC, Germany, collaboration program and from the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science. R.P. was supported by a short term FEBS fellowship and J.L.R.F. by a scholarship from the Conserjeria de Education (Comunidad Autonoma de Las Islas Canarias). A.B.-Z. holds the LunenfeldKunin Professorial Chair in Genetics and Cell Biology.
DURING
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Benecke, B.-J., Ben-Ze’ev, A., and Penman, S. (1978) Cell 14, 931-939. Farmer, S. R., Ben-Ze’ev, A., Benecke, B.-J., and Penman, S. (1978) Cell l&627-637. DeLarco, J. E., and Todaro, G. (1978) Proc. Natl. Acad. Sci. USA 75,4001-4005. Folkman, J., and Moscona, A. (1978) Nature 346, 760-763. Stiles, C. D., Isberg, R. R., Pledger, W. J., Antoniades, H. N., and Scher, 6. D. (1979) J. Cdl. Physiol. 99, 395-406. Ben-Ze’ev, A., Farmer. S. R., and Penman, S. (1980) Cell 21, 365-372. Stoker, M., O’Neill, C., Berryman, S., and Waxman, S. (1968) Int. J. Cancer 3, 683-695. Tucker, R. W., Butterfield, 6. E., and Folkman, J. (1981) J. Supramol. Struct. Cell. Biochem. 15, 29-40. Wittelsberger, S. C., Kleene, K., and Penman, S. (1981) Cell 29, 859-866. Raz, A., and Ben-Ze’ev, A. (1982) int. J. Cancer 29, 711-715. Shin, S., Freedman, V. H., Risser, R., and Pollack, R. (1975) Proc. Nutl. Acad. Sci. USA 72,4435-4439. Burridge, K., Fath, C. K., Kelly, T., Nuckolls, G., and Turner, C. (1988) Annu. Rev. Cell Bid. 4, 487-525. Geiger, B., and Ginsberg, D. (1991) CeEl Motil. Cytosheleton 20, 1-6.
ACTIVATION
14. Greenberg, M. E., and Zii, E. B. (1984) Nature @11,433-437. 15. 16.
17. 18. 19. 20. 21. 22. 23. 24. 25.
Lau, L. F., and Nathans; D. (1987) Proc. ~“barl. .Acor$. sci. uX.4 84,1182-1186. Almendral, J. M., Sommer, D., MacDonald-Bravo, II., hard& J., Perera, J., and Bravo, R. (1988) Mol. Cell. 2140-2148. Elder, P. K., Schmidt, L. O., One, T., and Getz, M. J. (1984) Proc. Natl. Acad. Sci. USA 81, 7476-7480. Blatti, S. P., Foster, D. N., Ranganathan, G Gets, M. J. (1988) Proc. N&l. Acad. Sci. u.§ Rysek, R.-P., MacDonald-Bravo, I-I., Zesial, (1989) Exp. Cell Res. 180, 537-545. Ben-Ze’ev, A., Reiss, R., Bendori, R., and Goradecki, B. (1996) Cd Regul. lb, 621-636. Bellas, R. E., Bendori, R., and Farmer, S. R. (1991) J. Biol. Chem. 266, L2008-12014. p 1464-1469. Grisham, J. W. (1962) Cancer Res. Bucher, N. L. R., and Malt, R. A. (1971). Regeneration of Liver and Kidney, Little, Brown, Boston, MA. Rabes, II. M., Wirshing, R., Tuczek, H. V., and Iseler, G. (1976) Cell Tissue Kinet. 6, 537-532. Goyette, M., Petropoulos, C. J., Shank, P. R., and Fausto, N. (1983) Science 219,510~512.
26.
Makino, R., Hayashi, 697-698.
27.
Kruijer, W., Skelly, II., Botteri, F., van der Putten, II., B J. R., Verma, I. M., and Leffert, II. L. (1986) cj. Bioi. Chem. 7929-7933.
28.
Sobczak, J., Tomnier, (1989) Eur. J. Biochem.
K., and Sugimura,
M.-F., Lotti, P80,49-53.
T. (1984) Nature
$1
A.-
abinet: C., and Fausto, N. (1990)
29. 30.
Belier, K., Vestweber, 1 ,327-332.
3i.
Hirano, S., Nose, A., Hatta, K., Kawakami, (1987). J. Cell Biol. 105, 2501-2510.
REFERENCES 1.
GROWTH
32.
@Farrell,
33.
Ben-Ze’ev,
D., and Kemler,
P. H. (1975) 9. Viol, &em.
R. (1985). J. Cell Biol. A., and Takeichi,
M.
1BB9,4007-4021.
A. (1990) Electrophores
34.
Garells, J. I. (1989) 9. Biol. Chem.
35.
Higgins, 6. M., and Anderson, 186-202.
36.
Chirgwin, J. M., Przybyla, w. J. (1979) Biochemistry
37.
Youssoufian, II., M&fee, Am. J. Hum. Genet. 47,627;.
38.
Schwartzbauer, J. E., Tamkun, J. W., Hynes, R. 0. (1983) Cell 35, 421-431.
39.
Ringwald, M., Scbuh, R., Vestweber, D., Eis,tetter, II., Lottspeich, F., Engel, J., Diilz, R., Jahnig, F., Epplen, J., Mayer, S., Miiller, C., and Kemler, R. (1987) g?&BO vi B:, 3647-3653.
40.
Dermann, E., Kramer, K., Wallig, L., Weinberger, and Darnell, 3. E., Jr. (1981) CeEl23, 731-739.
41.
Plumb, M., Stein, J., and Stein, 6. (1983) Pu’wleic Acids Res. 4 E, 2391-2410.
42.
Feinberg, 266-267.
43.
Friedman, J. M., Chung, E. Y., and Darnell, Mol. Biol. B79, 37-53.
44.
Thompson, N. L., Mead, J. E., Braun, L., Goyette, M., Shank, P. R., and Fausto, N. (1986) Cancer IZes. 46, Sill-3117.
R. M. (1931) Arch. puafhol. 12,
A. E., MacDonaid, 24, 5294-5299.
A. P., and Vogelstein,
R. J., and Rutter,
and Kwistkowski, Lemisch~a,
D. J. (1990) 1. R.,
and
C., Ray, M.,
B. (1983) Anal. &o&em.
137,
J. E., Jr. (1984) J.
486 45. 46. 47. 48. 49. 50.
GLfJCK Jakowlew, S. B., Mead, J. E., Danielpour, D., Wu, J., Roberts, A. B., and Fausto, N. (1991) Cell Regul. 2,535X48. Shaulsky, G., Ben-Ze’ev, A., and Rotter, V. (1991) Oncogene 6, 1707-1711. Ben-Ze’ev, A., Robinson, G. S., Bucher, N. L. R., and Farmer, S. R. (1988) Proc. Natl. Acad. Sci. USA 85, 2161-2165. Mooney, D., Hansen, L., Vacanti, J., Langer, R., Farmer, S., and Ingber, D. (1992) J. Cell Physiol. 151, 497-505. Herman, B., and Pledger, W. J. (1985) J. Cell Biol. 100,10311040. Cunningham, C. C., Stossel, T. P., and Kwiatkowski, D. J. (1991)
Scieme251,1233-1236. Received April 1, 1992. Revised version received June 2, 1992
ET AL. 51.
Rodriguez Fernlndez, J. L., Geiger, B., Salomon, D., and BenZe’ev, A. (1992) Cell Motil. Cytoskeleton 22, 127-134.
52.
Rodriguez H., Ziiller,
Fernlndez, J. L., Geiger, B., Salomon, D., Sabanay, M., and Ben-Ze’ev, A. J. Cell Biol., in press.
53.
Giancotti,
F. G., and Ruoslahti,
54.
Ben-Ze’ev,
A. (1991) BioEssays
55.
Michalopoulos, 70-78.
56.
Bissell, M. D., Arenson, D. M., Maher, J. J., and Roll, F. J. (1987) J. Clin. Invest. 79, 801-812.
57.
Reid, L. M. (1990) Cut-r. Opin. Cell Biol. 2, 121-130.
G., and Pitot,
E. (1990) Cell 60,849-859.
13, 207-212. H. C. (1975) Exp. Cell Res. 94,