JOURNAL OF CELLULAR PHYSIOLOGY 151:588-595 (1992)
Regulation of Vascular Smooth Muscle Cell lntegrin Expression by Transforming Growth Factor pl and by Platelet-Derived Growth Factor-BB M. F O U A D JANAT, W. SCOTT ARGRAVES, AND GENE L I A U * Lahoratory of Molecular Biology dnd Biochemistry, American Red Crosc, Jerome H Holland Lahoratory for Bromedical Sciences, Rockville, M a y l a n d ,2085 5
We have examined the ability of transforming growth factor-pl (TGF-p1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the a,p3 integrin complex was induced by both growth factors, although TGF-PI appeared to be the more potent inducer. mRNA level of the p3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the a, integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the a& integrin protein complex after growth factor stimulation was due to increased expression of thc P3 integrin subunit mRNA. The TGF-Pl induced increase in p3 integrin mRNA was delaycd, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in p3mRNA level. By contrast, PDGF-BB induced a more rapid increase in p3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable p3 integrin mRNA remained after 24 h. Interestingly, the PDCF-BB induced elevation of p3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the a5 integrin subunit in response to growth factors was very similar to p3. However, in contrast to p3 and as, neither TCF-pl nor PDGF-BB were able to alter the expression of the pi inlegrin subunit in vascular SMC. However, in TGF-PI treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the p, integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-pl treated fibroblasts. The a , integrin subunit has a M W of approximately 190 kDa and i s capable of complexing with pi. Analysis of the a , integrin subunit mRNA level indicated that it was indeed induced by TGF-P1, but not by PDCFBB, suggesting that the 190 kDa polypeptide may be the mi integrin subunit. Thcsc results indicate that TGF-Pl and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC. o 1992 Wilev-Liss. Inc.
Aortic smooth muscle cell (SMC) migration from the intima and subsequent proliferation is a critical event in arterial wound repair and atherosclerosis (Ross, 1986; Schwartz et al., 1986). Growth factors, as well as cell surface associated proteoglycans and extracellular matrix (ECM) proteins, are believed to be important regulators of SMC proliferation (Ross, 1986; Schwartz et al., 1986; Castellot et al., 1981; Fritze et al., 1985; Hedin et al., 1988). We have previously shown that transforming growth factor-pl (TGF-Pl) induced a large increase in thrombospondin (TSP) and (r,(IV) collagen mRNA level in vascular SMC, but the expression of fibronectin, az(I),( ~ ~ ( 1 1and 1 ) ~a,(V) collagen was unchanged (Liau and Chan, 1989; Janat and Liau, 1992). TSP, a large multifunctional ECM protein, secreted by activated platelets and synthesized by a variety of cells is particularly interesting, since its expression in SMC is also induced by platelet derived-growth factor-BB (PDGF-BB) and angiotensin I1 and is associated with Q
1992 WILEY-TISS. INC.
SMC proliferation in vivo and in vitro (Majack et al., 1985, 1988; Scott-Burden et al., 1990; Lawler, 1986; Frazier, 1987; Raugi et al., 1982, 1990). It is believed that the binding of SMC to TSP plays an important role in the proliferative response of these cells (Majack et al., 1985, 1988). However, the mechanism by which TSP can effect SMC growth is presently unknown. The binding of cells to ECM proteins such as TSP is largely mediated by a number of cell surface molecules termed integrins. They are heterodimers consisting of distinct CY and p subunit (Hynes, 1987; Ruoslahti and Giancotti, 1989).The combination of these subunits defines the specificity of these integrin complexes for
Received November 8,1991; accepted January 14,1992.
* To whom reprint requestskorrespondenceshould be addressed.
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
ECM proteins. To date, the integrins identified on SMC are all members of the p1 and p3 family (Lawler et al., 1988; Clyman et al., 1990; Belkin et al., 1990; Bottger et al., 1989). They consists of a fibronectin receptor, CY&; the alpland aapl integrins that mediate adhesion of laminin and collagens and the a,p3 integrins. The a$, complex is capable of binding to a number of ECM proteins and in human vascular SMC this integrin complex is believed to mediate binding to TSP (Ruoslahti and Giancotti, 1989; Lawler et al., 1988). We have previously found that TGF-Pl induced increase in TSP appears to occur via a different mechanism than that for PDGF and was not associated with a n increase in DNA synthesis (Janat and Liau, 1992). However, TGF-p1 was able to strongly enhance the mitogenic response of SMC to PDGF-BB and addition of both growth factors also resulted in a synergistic increase in TSP expression (Janat and Liau, 19921. We have now examined the effect of TGF-p1 and PDGF-BB on the expression of both the p3 and PI integrin families. We found that expression of the TSP binding integrin complex, a,p3 was stimulated by both growth factors. Increased expression of this integrin complex was due to a n increase in p3subunit mRNA level, since the a, integrin mRNA level was unchanged. Interestingly, expression of the p3 as well a s the a5 integrin subunit mRNAs in response to TGF-p1 and PDGF-BB mirrored the previously reported expression of TSP (Janat and Liau, 1992). By contrast, the p1 integrin subunit was not regulated by TGF-p1 or PDGF-BB, and levels of various p, integrin protein complex correlated with changes in CY integrin subunit mRNA level. Our results suggest that both TGF-P1 and PDGF-BB are potent modulators of the binding of SMC to the ECM. The role of altered cell binding to the ECM after growth factor stimulation is presently unclear.
589
growth factor and either metabolically labeled or used to isolate total RNA.
RNA preparation and hybridization analysis Total cellular RNA were isolated using guanidinium isothiocynate and cesium chloride a s previously described (Liau and Chan 1989). The purified RNA was size fractionated on a 1% denaturing formaldehyde agarose gel, transferred to nitrocellulose, and hybridized under stringent conditions, a s described previously (Liau et al., 1985). Slot blot analysis was carried out using a manifold apparatus (Schleicher & Schuell) and the resulting autoradiograms quantitated using a Hoefer GS-300 scanning densitometer as previously described (Liau et al., 1991). cDNAs used to analyze the PI,a5,and a, integrin subunit mRNAs have been described previously and are 2.4 Kb, 1.7 Kb, and 520 bp, respectively (Argraves e t al., 1987, 1986; Suzuki et al., 1987). A 1.1 Kb p3 cDNA was obtained from Dr. E. Ruoslahti. A 1.8 Kb a1 cDNA was kindly provided by Dr. G. Marcantonio (Columbia University, NY).
Cell labeling and immunoprecipitation Confluent cultures were incubated for two days with methionine-free Dulbecco’s Modified Eagles Medium supplemented with 0.5% FBS, 10 pM insulin, and 5 pgiml transferrin. Growth factor was added for the desired period and labeled for the last six h with a mixture of 25 $2i/ml of L-[35S]methionine and L-[35S]cysteine (specific activity 1138 Ciimmole) [ICN, Irvine, CAI. The immunoprecipitation was carried out a s previously described (Argraves et al., 1990). Briefly, cells were washed twice with cold PBS and lysed in a detergent mixture containing 1% triton X-100, 0.5 M NaC1, 50 mM Tris, pH 7.4, and 0.05% Tween-20. The cell lysate was centrifuged a t 100,OOOg for 30 min. The supernatant fluid was preabsorbed with protein-A sepharose MATERIALS AND METHODS beads and 3 x lo6 cpm was incubated with the approMaterials priate antiserum overnight at 4°C. The immune-comDefined fetal bovine serum was purchased from Hy- plex was precipitated with protein-A Sepharose beads clone Labs, Inc. (Logan, UT). Bovine insulin and trans- and analyzed by electrophoresis under reducing and ferrin were obtained from Sigma (St. Louis, MO). Por- non-reducing conditions on a 7.5% sodium dodecyl sulcine platelet transforming growth factor-pl (TGF-pl), fate-polyacrylamide gel (Lammli, 1970). The gel was and porcine PDGF were purchased from R & D Sys- treated with Enlightening (DuPont-New England Nutems, Inc. (Minneapolis, MN). Rabbit skin fibroblasts clear Research Products, Boston, MA) for 20 min, dried, were obtained from American Type Culture Collection and exposed to X-ray film. (ATCC # CRL 1414, Rockville, MD). Antibody to the RESULTS and p3 integrins were kindly provided by Dr. R.O. TGF-p1 is a specific and potent enhancer of p3 Hynes (MIT, MA) and Dr. E. Ruoslahti (La Jolla Cancer integrin mRNA expression Research Foundation, CA), respectively. We first examined the effect of TGF-p1 on the expresCell culture sion of the p3 and the p1 integrin mRNAs, since they Aortic smooth muscle cells (SMC) were isolated from comprise the two known integrin families on vascular rabbit aorta by enzymatic digestion and propagated as SMC. In addition, because the a,p3 complex can bind previously described (Liau and Chan, 1989). Cells used TSP in vitro, we also determined the expression of the in all the experiments were between 4 to 8 passages. a, mRNA in response to TGF-P1. Cells were treated Fibroblasts were cultured in Basal Medium Eagle sup- with 200 pM of TGF-P1 and harvested a t various times plemented with 10% FBS and 2 mM L-glutamine. SMC for RNA extraction and analysis. The results, shown in were seeded at a density of 2 x lo4 cells/cm2 and al- Figure l a , indicated that cultured SMC contained a lowed to reach confluence (2-3 days). The cultures were high constitutive level of PI integrin subunit mRNA then fed fresh Medium 199 containing 0.5% FBS, that was not modulated by TGF-P1. By contrast, these 10- 6M insulin, and 5 pgiml transferrin supplemented cells contained a very low basal level of p3 integrin with 2 mM L-glutamine (low serum medium). After 2-3 subunit mRNA that was augmented by TGF-p1 at least days, cultures were treated with the appropriate by 24 h and mRNA expression of p3 remain elevated 48
590
dANAT ET AL.
A
TGF-Pi (200 pM)
0 0.5 1
2
6 24 48 hours
p3
2
200
c
c
2;100
*"
P I
\
/
c
\
\
,s.--....
a Ia ,
........
\
/ I I .
2
2 -
\
\
300
p3
c-----o
\
/'
I
a,
P1
.
,*---a 400
\
\ \
-
.... \
0
1
Hours After Incubation with 200pM TGF-P
B
TGF-B1
0
P3
Fig. 2. Analysis of p,, p3, ab,and a, integrin mRNA levels in skin fibroblasts treated with TGF-61. Confluent cultures of quiescent fibroblasts were treated with 200 pM of TGF-P1 for 0.5 to 48 h and RNA was isolated and analyzed by slot blot. The results were quantitated by densitometry and plotted as percentage increase over control levels.
5 50 100200 P M
blasts. Expression of a, integrin subunit mRNA was unchanged, while that of a5paralleled that of PI.
TGF-pl specifically i n d u c e d expression of the p3 polypeptide and a p1 associated 190 kDa a polypeptide in vascular SMC To determine the relationship between mRNA levels Fig. 1. Analysis of PI, P3, and ayintegrin mRNA levels in vascular and protein expression, we performed immunoprecipiSMC treated with TGF-P1. A: Confluent cultures of quiescent SMC were treated with 200 pM of TGF-61 for 0.5 to 48 h and RNA was tation analysis on extracts of metabolically labeled isolated and analyzed by Northern blot. B: SMC were treated with 5 to cells using antibodies directed against the cytoplasmic 200 pM of TGF-p1 and RNA was isolated and analyzed using the p3 domain of the P1 and P3 integrin receptors to examine cDNA probe. their expression. For comparison, parallel immunoprecipitation analyses were also performed on skin fibroblasts. Quiescent cells were incubated with the growth h after growth factor addition. The a, cDNA identified factor for 24 h, metabolically labeled for the last 6 h and three major transcripts of 9.5 Kb, 7 Kb, and 5.5 Kb, as the integrin proteins were immunoprecipitated with well as a minor transcript of 4.5 Kb in rabbit SMC. the appropriate antiserum and analyzed by sodium TGF-p1 did not appreciably alter the level of any of dodecyl sulfate-polyacrylamide gel electrophoresis these a, transcripts. These results indicate that (SDS-PAGE). The immunoprecipitation results showed TGF-P1 selectively enhanced the expression of the p3 that there was a n undetectable level of anti-p, precipiintegrin subunit mRNA, but did not alter the expres- table polypeptide in untreated SMC (Fig. 3A). Howsion of Pl o r n, integrin subunits in vascular SMC. We ever, in TGF-p1 treated cells, two polypeptides that examined the sensitivity of the p3 integrin response to migrated with a n apparent molecular mass of 100 kDa TGF-P1 and found that as little as 5 pM of this factor and 130 kDa under reducing SDS-PAGE were identiwas effective in elevating the expression of the p3 fied by this antiserum. Under nonreducing conditions, they migrated as a 95 kDa and a 150 kDa polypeptide, mRNA (Fig. IB). We next compared integrin mRNA expression in vas- respectively. The size and electrophoretic migration cular SMC with their expression in fibroblasts. We pattern of the 100 kDa polypeptide indicate it is the p3 found that increased expression of both p1 and p3 inte- integrin subunit. The 130 kDa polypeptide is a n (Y integrin subunit mRNA levels were observed in rabbit skin grin subunit co-precipitated with p3 and its size and fibroblast treated with 200 pM of TGF-pl (Fig. 2). In- electrophoretic migration pattern indicate it is likely duction of the p1 integrin was observed by 1 h and the a, integrin subunit (Lawler e t al., 1988).A similar remained elevated until 48 h after TGF-p1 addition. p3 pattern of expression was observed in fibroblasts, exintegrin mRNA level also increased rapidly in response cept that the apparent size of the (Y integrin subunit to TGF-P1, but had returned almost to basal level by 24 was slightly smaller when analyzed under reducing h after growth factor addition. By contrast, in vascular conditions (Fig. 3B). These results indicate that there SMC, only the p3 integrin mRNA level increased in was a concomitant increase in p3 subunit mRNA and response to TGF-P1 and this response was substan- polypeptide level after TGF-p1 treatment and the p3 tially more prolonged than that observed in skin fibro- subunit was complexed with the a, subunit. P1
591
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
TGF-81
(A) 81 P3 NRS -
anti-sera -
- + - + -
TGF-P
+
B1 B3 NRS --- + - + - +
0
(200 PM)
2 6 10 14 24 48 h w s
KD
220-
\
I
97-
68
a5
-
42 -
GAPDH Fig. 4. Analysis of a, and a5 integrin subunit mRNA level in cells treated with TGF-Pl. Quiescent SMC were treated with 200 pM of TGF-p1 for 2 h to 48 h a n d subsequently harvest for RNA. mRNA level of a 1 and a, integrin subunits were assessed by Northern blot. GAPDH level was also determined as a control for RNA loading.
(B) B1 B3 NRS ---
anti-sera TGF-B
- + - + - +
Bi 83 NRS ---
+
-
t
-
+
KD 220-
9766-
42-
Reduced
Nonreduced
Fig. 3. Immunoprecipitation analysis of p, and p3 integrin proteins in vascular SMC and in fibroblasts after TGF-pl treatment. Cultured cells were treated for 24 h in the presence (+) or absence (-) of 200 pM TGF-p1 and labeled for the last 6 h with a mixture of 25 uCi/ml of L-[35S]Methionine and L-[35S]Cysteine.After treatment, integrin proteins were immunoprecipitated with antibodies specific to PI or & integrin subunits and analyzed by SDS-PAGE as described in Materials and Methods. Control immunoprecipitation was also carried out with normal rabbit serum (NRS). A: SMC treated with TGF-P1. B: Fibroblasts treated with TGF-p1.
The anti-p, antiserum specifically immunoprecipitated 2 major polypeptides of approximately 100 kDa and 120 kDa from both SMC and fibroblasts (Fig. 3). These polypeptides had a faster electrophoretic mobility when analyzed on SDS-PAGE under nonreducing conditions. The size and electrophoretic mobility properties indicate the 100 kDa and 120 kDa polypeptides are, respectively, the precursor and mature form of the p1 subunit (Ignotz and Massague, 1987). TGF-p1 had no effect on the amount or on the ratio of the precursor and mature forms in vascular SMC. However, there was a n enhanced amount of the 100 kDa precursor form of the p1 polypcptide in TGF-p1 treated fibroblasts
(Fig. 3B). This film was overexposed to allow visualization of the much less abundant p3 integrin complex. Densitometric analysis of a less exposed autoradiogram indicated there was a %fold enhancement of the p1 precursor form after TGF-p1 treatment. In vascular SMC two a chain subunits were also co-precipitated with the p1 integrin subunit by the anti+, antibody. Particularly striking was a large increase in a 190 kDa (Y chain subunit in SMC treated with TGF-p1. In addition, a 150 kDa a chain observed on nonreducing SDS-PAGE also increased slightly after growth factor treatment. Presumably, under reducing conditions, this a chain comigrated with the p1 integrin subunit. In fibroblasts, the 190 kDa 01 integrin subunit was not detected, while the 150 kDa a integrin subunit was expressed a t a much higher level and was not appreciably altered by TGF-61 treatment. The results indicate that although the p1 integrin subunit level was not detectably altered by TGF-61, expression of a 190 kDa (Y chain integrin subunit associated with pl was induced by TGF-p1 treatment of vascular SMC but not of skin fibroblasts.
Both a1 and 0 1 integrin ~ subunit mRNA levels are increased after TGF-p1 treatment Although a number of a integrin subunits have been demonstrated to interact with the p1 integrin subunit, only the (Y, integrin subunit has a molecular mass corresponding to that of the TGF-61 inducible a polypeptide associated with p1 (Humphries, 1990; Clyman et al., 1990). We were, therefore, interested i n knowing whether or not expression of a1 integrin was enhanced by TGF-Pl and performed RNA hybridization analysis using a cDNA probe to the a1 integrin. We also examined expression of the a5integrin subunit mRNA, since the asp1 integrin complex has also been identified on SMC (Bottger et al., 1989; Clyman et al., 1990; Belkin et al., 1990). The results, shown in Figure 4,indicate that mRNA levels of both a, and a5 integrin subunits increased in response to TGF-Pl addition. Enhanced
592
JANAT ET AL.
PDGF (0.1nM) 0 0.5 1 2 6 24
Pi
anti-sera
hours
PDGF KD 220
P3 a5 NRS
-+-+-+-+
(31 -
P3 a5 NRS
-+-+-+-+
-
97 -
I
\
68
-
42 -
Reduced
Fig. 5. Analysis of PI, p3,a5,and 01, integrin mRNA levels in vascular SMC treated with PDGF-BB. Confluent cultures of SMC were treated with 0.1 nM of PDGF-BB for 0.5 to 24 h and RNA was isolated and analyzed by Northern blot.
expression was observed after 6 h and remained elevated 48 h after TFG-p1 treatment. Interestingly for a5,in addition to the 4.5 Kb message, we also observed a larger transcript that ran just above the 28s marker. We do not know if this second transcript represents a distinct variant in rabbit or was merely due to technical differences in Northern blotting. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA expression was also monitored a s a control and showed no appreciable change. These results suggest that the TGF-61 inducible 190 kDa a integrin subunit associated with the P1 subunit is possibly the a1subunit.
Nonreduced
Fig. 6 . Immunoprecipitation analysis of PI, B3, and as integrin proteins in vascular SMC after PDGF-BB treatment. Cultured SMC were incubated for 10 h in the presence ( + I or absence (-) of 0.1 nM of PDGF-BB and labeled for the last 6 h with a mixture of 25 uCi/ml of LP"1Methionine and L-[:3"S1Cysteine.After treatment, integrin proteins were immunoprecipitated with the specific antibodies and analyzed by SDS-PAGE. Control immunoprecipitation was also carried out with normal rabbit serum (NRS).
We also studied integrin polypeptide expression in response to PDGF-BB. SMC were treated with PDGF-BB for 10 h and the metabolically labeled integrins were immunoprecipitated with antibody directed against the PI,p3, and a5 integrin subunits, respectively. We chose to analyze integrin expression a t 10 h instead of a t 24 h, since PDGF-BB induced a more transient expression of integrin mRNA than TGF-P1. The results, shown in Figure 6, indicated that there was increased expression of the 100 kDa p3 polypeptide after PDGF-BB treatment. Associated with the pn subunit was a 130 kDa a subunit that showed retarded PDGF-BB also increases integrin expression in migration under nonreducing SDS-PAGE conditions. vascular SMC The size and migration pattern of this a subunit is We next examined whether or not PDGF-BB, a po- identical to that previously observed for TGF-p1 tent mitogen and chemoattractant for SMC, is also ca- treated SMC and suggest it is also the a, integrin subpable of modifying integrin expression in these cells unit. PDGF-BB had no effect on PI polypeptide expres(Ross, 1986). Expression of the TSP binding integrin sion or on conversion from the precursor to mature form complex aVp3was of particular interest, since TSP level of this receptor. In addition, PDGF-BB did not elicit a n is rapidly enhanced by PDGF (Majack et al., 1985). increase in the 190 kDa a integrin subunit associated Quiescent SMC were treated with 0.1 nM PDGF-BB for with the p1 integrin subunit as previously observed for various times and analyzed for a",p3, as,a,,and p1 TGF-P1 treated SMC. Since we also observed that the mRNA levels. The result, shown in Figure 5, indicated a1integrin was not induced by PDGF-BB, these results that like TGF-P1, PDGF-BB was also able to induce a n are consistent with the possibility that the 190 kDa increase in P3 mRNA level, but expression of the a, integrin induced by TGF-P1 is the a1integrin subunit. mRNA was not altered. Interestingly, PDGF-BB in- A 150 kDa ct integrin subunit was co-immunoprecipiduced increase in mRNA level occurred by 2 h, tated with the p1 integrin subunit. However, it was peaked by 6 h, and was no longer apparent by 24 h. This unaltered by PDGF-BB treatment. This a integrin subis in great contrast with TGF-P1 enhanced expression unit is apparently not ct5, since a5 antibody identified a of the P3 integrin, which persisted for up to 48 h. The slightly larger polypeptide that increased upon level of a5integrin subunit mRNA essentially mirrored PDGF-BB treatment. These results indicate t h a t PDGF-BB is also capable that of p3 (Fig. 5). Expression of p, integrin subunit mRNA was unaltered by PDGF-BB, while PDGF-BB of increasing the expression of certain integrins in vaswas unable to induce a n increase in a very low al basal cular SMC. Interestingly, both PDGF-BB and TGF-P1 induced increase in a,p3 integrin complex were due to level (Fig. 5 and results not shown).
593
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
(A)
- -
(B) +
+
- + - +
TGF-B1 ( 2 0 0 ~ M )
+
C y c lo h e x im ide (2wIrnl)
- +
P3
P3
B1
P1
+
PDGF (O.lnM) C y c lo he x imide ( 2 U g l r n l )
Fig. 7. Analysis of the effect of cycloheximide on TGF-p1 and PDGF-BB induced increase in integrin mRNA expression. SMC were treated with 2 u g h 1of cycloheximide 30 min prior to addition of: A: 200 pM of TGF-P1 for 24 h. B: 0.1 nM of PDGF-BB for 6 h. Identical Northern blots were used to analyze p,, pa, and as integrin mRNA levels.
a n increase in p3 integrin subunit mRNA expression since the a, integrin mRNA level was unchanged. However, there are a number of differences in integrin expression in response to these growth factors. PDGF-BB was apparently not as potent as TGF-p1 in inducing expression of the a,p3 integrin complex (compare Fig. 3A and Fig. 6). In addition, the induction of both p3 and a5 integrin mRNA expression by PDGF-BB was considerably more rapid and more transient than that observed for TGF-pl (Figs. 1, 4, 5). Finally, unlike TGF-61, PDGF-BB was unable to induce a n increase in expression of a 190 kDa a subunit associated with integrin subunit.
of a5 integrin was superinduced in the presence of either TGF-p1 or PDGF-BB and CHX. PI integrin mRNA level increased slightly in the presence of CHX, but there was no additional increase in the presence of growth factors. Our results indicate that TGF-P1 induced increase in p3 expression occurs via a direct mechanism, while PDGF-BB induced increase. of this integrin subunit is mediated by a second protein. These results strongly suggest that TGF-p1 induced expression of the p3 integrin is not due to prior induction of other autocrine factors, such a s PDGF.
TGF-p, and PDGF-BB induced increase in P3 expression occurs through distinct mechanisms
The expression and regulation of integrin receptors on vascular SMC are not well characterized. To date, the integrins identified are all members of the p1 and p3 family (Lawler et al., 1988; Clyman et al., 1990; Belkin et al., 1990; Bottger et al., 1989). Both a n asp,-like receptor t h a t binds fibronectin and a n a,P,-like receptor that binds laminin and types I and IV collagen have been defined (Bottger et al., 1989; Clyman et al., 1990; Belkin e t al., 1990). We found that expression of the PI integrin subunit was high and not modulated significantly by TGF-p1 or PDGF-BB in vascular SMC. However, expression of both a1 and a5 integrin subunits, which can associate with PI, was enhanced by growth factors. By contrast, both TGF-P1 and PDGF-BB were able to directly stimulate P3 mRNA level. The increase in p3 subunit level is responsible for enhanced a,p3 integrin expression, since the a, integrin mRNA level was constitutively expressed in these cells. Therefore, regulation of the PI and P3 family of integrins by growth factors occurs through two distinct mechanisms, with the family of receptors regulated by change in a subunit levels, while p3 itself is directly responsive to growth factors. The proposed mechanism of regulation of the family of integrins is consistent with available in vivo data. Analysis of alp,integrin
One interpretation of our finding that TGF-p1 induced increase in p3 and a5 integrin subunits is considerably more delayed and prolonged than PDGF is that these two growth factors induce P3 and a5 integrin expression through distinct mechanisms. However, it is also possible that cultured SMC exposed t o TGF-pl may subsequently express PDGF or other proteins, which then enhanced integrin expression through a n autocrine mechanism. TGF-P is capable of inducing PDGF expression in a variety of cell types (Leof et al., 1986; Makela et al., 1987; Majack et al., 1990; Battegay et al., 1990).To distinguish between these possibilities, we examined the effect of the protein synthesis inhibitor, cycloheximide (CHX) on growth factor induced increase in p3 and a5 integrin expression. The result, shown in Figure 7, indicated that CHX was unable to abolish the augmented expression of Pn integrin in response to TGF-P1. Indeed, there was a superinduction of P3 integrin mRNA level in the presence of CHX. But surprisingly, although PDGF-BB induced expression of p3 integrin mRNA was more rapid, the increase was completely abolished by CHX. By contrast, expression
DISCUSSION
594
JANAT ET AL.
expression in the human aorta indicate that P1 expression is relatively unchanged during development and a similar level of p1was expressed in SMC from the aortic media and in intimal thickening (Belkin et al., 1990). By contrast, expression of the a, integrin subunit is developmentally regulated and is high in contractile cells of the media, but low in intimal cells and in cultured SMC which have modulated to a proliferative phenotype (Belkin et al., 1990). These authors concluded t h a t expression of the alp, integrin complex was only associated with differentiated SMC (Belkin et al., 1990). We found that in vitro, TGF-(31 but not PDGFBB, was able to induce a n increase in a, integrin subunit mRNA level, as well as a strong increase in expression of a 190 kDa a,-like integrin subunit in association with PI. This effect of TGF-Pl was not observed with fibroblasts. Our results suggest that TGF-p1 but not PDGF-BB may be one of the factors that is important in regulating a1 integrin subunit expression in SMC. Whether or not TGF-P1 or other related polypeptides are important for maintaining SMC in the contractile phenotype is presently unclear. TGF-P1 induced increase in P3 integrin subunit mRNA level was detected by 6 h and remained elevated for at least 48 h after TGF-(31 treatment, while PDGF-BB enhanced P3 expression was observed by 2 h but p3 expression was undetectable by 24 h after treatment. This temporal difference is very similar to the temporal difference in TSP response to TGF-P1 and PDGF-BB and indicates that expression of TSP and the TSP binding avP3receptor complex is very similarly regulated by these growth factors (Janat and Liau, 1992). Although other cell surface proteins that are able to bind TSP have been identified and characterized (Roberts et al., 1987; Kaesberg et al., 1989; Prater et al., 1991), the close coupling of growth factor response of TSP and the avP3receptor complex is intriguing and a t least consistent with the idea that this integrin complex is functionally important for TSP binding (Lawler et al., 1988). However, our results do not rule out the possibility that other TSP receptors are important and possibly more biologically relevant mediators of TSP action in vascular SMC. There are at least two possible explanations for the similar response of TSP and Pn to the two growth factors: 1. Regulation of TSP and the P3 integrin subunit genes occur through a common or similar pathway. 2. Expression and binding of TSP to the cell, presumably through the a&, receptor, induces increased expression of the p3 integrin subunit mRNA. Consistent with the second possibility is our finding that TSP expression is induced approximately 4 h earlier than p3 by TGF-Pl and 1.5 h earlier than P3 by PDGF-BB (Janat and Liau, 1992). However, this scenario predicts that CHX should be able to abolish p3 expression. CHX is, indeed, able to abolish PDGF-BB induced increase in p3 integrin mRNA expression, but it superinduces p3 expression in TGF-P1 treated cells. This result suggests that two distinct pathways are used to induce p3expression by PDGF-BB and TGF-P1. PDGF-BB induced expression of p3 could be due t o prior TSP expression and binding to SMC, while TGF-Pl may induce both TSP and p3 integrin expression through related pathways.
SMC migration and proliferation have a central role in vascular diseases such as atherosclerosis and hypertension, as well a s in vascular graft failures (ROSS, 1986; Schwartz et al., 1986; Ferns e t al., 1991). There is good evidence to suggest that PDGF plays a n important role in SMC migration (Ferns et al., 1991). What role TGF-(31 might have is presently unclear. Our results indicate that both of these factors are likely capable of modifying the interaction of SMC with its extracellular matrix environment (present manuscript and J a n a t and Liau, 1992). These changes may have a role in altering SMC migration and recent experiments demonstrating that fibronectin is able to activate the NaiH antiporter suggest that altered cell-matrix interactions may also directly modify signal transduction pathways important for cell proliferation (Ingber et al., 1990; Schwartz et al., 1991).
ACKNOWLEDGMENTS This work was supported in part by grant HL37510 (G.L.) and by grant GM42912 (W.S.A.) from the National Institutes of Health. G.L. is a recipient of a Research Career Development Award HL02449.
LITERATURE CITED Albelda, S.M., and Buck, C.A. (1990) Integrins and other cell adhesion molecules. FASEB J., 42868-2880, Argraves, W.S., Pytela, R., Suzuki, S., Millan, J.L., Pierschbacher, M.D., and Ruoslahti, E. (1986) cDNA sequences from the CI subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J. Biol. Chem., 26It12922-12924. Argraves, W.S., Shintaro, S., Arai, H., Thompson, K., Pierschbacher, M.D., and Ruoslahti, E. (1987) Amino acid sequence of the human fibronectin receptor. J. Cell Biol., 105r1183-1190. Argraves, W.S., Ti-an, H., Burgess, W.H., and Dickerson, K. (1990) Fibulin is a n extracellular matrix and plasma glycoprotein with repeated domain structure. J. Cell Biol., 111:3155-3164. Battegay, E.J., Raines, E.W., Seifert, R.A., Bowen-Pope, D.F., and Ross, R. (1990) TGF-p induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell, 63t515-524. Belkin, V.M., Belkin, A.M., and Koteliansky, V.E. (1990) Human smooth muscle VLA-1 integrin: Purification, substrate specificity, localization in aorta, and expression during development. J. Cell Biol., 11I t2159-2170. Bottger, B.A., Heldin, U., Johansson, S., and Thyberg, J. (1989) Integrin-type fibronectin receptors of rat arterial smooth muscle cells: Isolation, partial characterization, and role in cytoskeletal organization and control of differentiated properties. Differentiation, 41 t158-167. Castellot, J., Addonizio, M., Rosenberg, R., and Karnovsky, M. (1981) Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J . Cell Biol., 90t372-379. Clyman, R.I., McDonald, K.A., and Kramer, K.H. (1990) Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen. Circ. Res., 67:175-186. Fritze, L., Reilly, C., and Rosenberg, K. (1985) An antiproliferative heparan sulfate species produced by post-confluent smooth muscle cells. J . Cell Biol., 100:1041-1049. Hedin, U. Bottger, B.A., Forsberg, E., Johansson, S., and Thyberg, J. (1988) Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J . Cell Biol., 107:307-319. Humphries, M.J. (1990) The molecular basis and specificity of integrin-ligand interactions. J . Cell Sci., 97:585-592. Hynes, R.O. (1987) Integrins: A family of cell surface receptors. Cell, 48549-554. Ignotz, R.A., and Massague, J. (1987) Cell adhesion protein receptors as targets for transforming growth factor-p action. Cell, 51r189197. Ingber, D.E., Prusty, D., Frangioni, J., Cragoe, Jr., E.J., Lechene, C.,
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS and Schwartz, M.A. (19901 Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J. Cell Biol., 110:18031811. Janat, M.F., and Liau, G. (1992) Transforming growth factor pl is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. J. Cell. Physiol., 150t232-242. Kaesberg, P.R., Erschler, W.B., Esko, J.D., and Mosher, D.F. (1989) Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparin sulfate proteoglycan. J. Clin. Invest., 83t99P1001. Laemmli, U.K. (1970) Cleavage structural proteins during the assembly of the head bacteriophage T4. Nature, 227t680-685. Lawler, J. (1986) The structural and functional properties of thrombospondin. Blood, 67t1197-1209. Lawler, J., Weinstein, R., and Hynes, R.O. (1988) Cell attachment t o thrombospondin: The role of ARG-GLY-ASP, calcium, and integrin receptors. J. Cell Biol., 107:2351-2361. Liau, G., Yamada, Y., and decrombrugghe, B. 11985)Coordinate regulation of the levels of type I11 and type I collagen mRNA in most but not all mouse fibroblast. J. Biol. Chem., 260531-536. Loef, E.B., Proper, J.A., Goustin, A.C., Shipley, G.D., DiCorleto, P.E., and Moses, H.L. (1986)Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor p: A proposed model for indirect mitogenesis involving autocrine activity. Proc. Natl. Acad. Sci. U.S.A., 83:2453-2457. Majack, R.A., Cook, S.C., and Bornstein, P. (1985) Platelet-derived growth factor and heparin-like glycosaminoglycansregulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J. Cell Biol., 101:1059-1070. Majack, R.A., Goodman, L.V., and Dixit, V.M. (1988) Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J. Cell Biol., 106r415-422. Majack, R.A., Majesky, M.W., and Goodman, L.V. (1990) Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-p. J. Cell Biol., 111:239-247. Makela, T.P., Alito, R., Paulsson, Y., Westermark, B., Heldin C-H., and Alitalo, K. (1987) Regulation of platelet-derived growth factor gene expression by transforming growth factor p and phorbol ester in human leukemia cell lines. Mol. Cell. Biol., 7t36563662.
595
Prater, C.A., Plotkin, J.,Jaye, U., and Frazier, W.A. (1991) The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol., 112:1031-1040. Raugi, G.J., Mumby, S.M.,Abbott-Brown, D., and Bornstein, P. (1982) Thrombospondin: Synthesis and secretion by cells in culture. J. Cell. Biol., 95t351354. Rauei. G.. Mullen. J.S.. Bark. D.H.. Okada. T.. and Mavbere. M.R. (19901Thrombosoondin deoosition in rat carotid arterv"iniu&. " * " Am. J. Pathol., 137tlj9-185. Roberts, D.D.,Sherwood, J.A., and Ginsberg, V. (1987)Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J. Cell Biol., 104:131-139. Ross, R. (1986) The pathogenesis of atherosclerosis-An update. N. Engl. J. Med., 314t488-500. Ruoslahti, E., and Giancotti, F.G. (1989) Integrins and tumor cell dissemination. Cancer Cells, 1t119-126. Ryseck, R.P., MacDonald-Bravo, H., Zerial, M., and Bravo, R. (19x9) Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts. Exp. Cell Res., 180:537-545. Scott-Burden, T., Resink, T.J., Hahn, A.W.A., andBuhler, F.R. (1990) Induction of thrombospondin expression in vascular smooth muscle cells by angiotensin 11. J. Cardiovasc. Pharmacol., 16:S17-S20. Schwartz, S.M., Campbell, G.R., and Campbell, J.M. (1986) Replication of smooth muscle cells in vascular disease. Circ. Res., 58r427444. Schwartz, M.A., Lechene, C., and Ingber, D.E. (1991) Insoluble fibronectin activates the Na/H antiuorter bv clustering and immobilizing integrin independent 0.f cell shape. Proc. Natl. Acad. Sci. U.S.A., 88:7849-7855. Suzuki, S., Armaves, W.S., Arai, H.. Lanmino, L.R.. Pierschbacher. M.D., and Rioslahti, E. (1987) Amino acid sequence of the vitronectin receptor 01 subunit and comparative expression of adhesion receptor mRNAs. J . Biol. Chem., 262:14080-14085. Wight, T.N., Raugi, G.J., Mumby, S.M., and Bornstein, P. (19851 Light microscopic immunolocation of thrombospondin in human tissues. J. Histochem. Cytochem., 33:295-302.
Regulation of Vascular Smooth Muscle Cell lntegrin Expression by Transforming Growth Factor pl and by Platelet-Derived Growth Factor-BB M. F O U A D JANAT, W. SCOTT ARGRAVES, AND GENE L I A U * Lahoratory of Molecular Biology dnd Biochemistry, American Red Crosc, Jerome H Holland Lahoratory for Bromedical Sciences, Rockville, M a y l a n d ,2085 5
We have examined the ability of transforming growth factor-pl (TGF-p1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the a,p3 integrin complex was induced by both growth factors, although TGF-PI appeared to be the more potent inducer. mRNA level of the p3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the a, integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the a& integrin protein complex after growth factor stimulation was due to increased expression of thc P3 integrin subunit mRNA. The TGF-Pl induced increase in p3 integrin mRNA was delaycd, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in p3mRNA level. By contrast, PDGF-BB induced a more rapid increase in p3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable p3 integrin mRNA remained after 24 h. Interestingly, the PDCF-BB induced elevation of p3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the a5 integrin subunit in response to growth factors was very similar to p3. However, in contrast to p3 and as, neither TCF-pl nor PDGF-BB were able to alter the expression of the pi inlegrin subunit in vascular SMC. However, in TGF-PI treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the p, integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-pl treated fibroblasts. The a , integrin subunit has a M W of approximately 190 kDa and i s capable of complexing with pi. Analysis of the a , integrin subunit mRNA level indicated that it was indeed induced by TGF-P1, but not by PDCFBB, suggesting that the 190 kDa polypeptide may be the mi integrin subunit. Thcsc results indicate that TGF-Pl and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC. o 1992 Wilev-Liss. Inc.
Aortic smooth muscle cell (SMC) migration from the intima and subsequent proliferation is a critical event in arterial wound repair and atherosclerosis (Ross, 1986; Schwartz et al., 1986). Growth factors, as well as cell surface associated proteoglycans and extracellular matrix (ECM) proteins, are believed to be important regulators of SMC proliferation (Ross, 1986; Schwartz et al., 1986; Castellot et al., 1981; Fritze et al., 1985; Hedin et al., 1988). We have previously shown that transforming growth factor-pl (TGF-Pl) induced a large increase in thrombospondin (TSP) and (r,(IV) collagen mRNA level in vascular SMC, but the expression of fibronectin, az(I),( ~ ~ ( 1 1and 1 ) ~a,(V) collagen was unchanged (Liau and Chan, 1989; Janat and Liau, 1992). TSP, a large multifunctional ECM protein, secreted by activated platelets and synthesized by a variety of cells is particularly interesting, since its expression in SMC is also induced by platelet derived-growth factor-BB (PDGF-BB) and angiotensin I1 and is associated with Q
1992 WILEY-TISS. INC.
SMC proliferation in vivo and in vitro (Majack et al., 1985, 1988; Scott-Burden et al., 1990; Lawler, 1986; Frazier, 1987; Raugi et al., 1982, 1990). It is believed that the binding of SMC to TSP plays an important role in the proliferative response of these cells (Majack et al., 1985, 1988). However, the mechanism by which TSP can effect SMC growth is presently unknown. The binding of cells to ECM proteins such as TSP is largely mediated by a number of cell surface molecules termed integrins. They are heterodimers consisting of distinct CY and p subunit (Hynes, 1987; Ruoslahti and Giancotti, 1989).The combination of these subunits defines the specificity of these integrin complexes for
Received November 8,1991; accepted January 14,1992.
* To whom reprint requestskorrespondenceshould be addressed.
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
ECM proteins. To date, the integrins identified on SMC are all members of the p1 and p3 family (Lawler et al., 1988; Clyman et al., 1990; Belkin et al., 1990; Bottger et al., 1989). They consists of a fibronectin receptor, CY&; the alpland aapl integrins that mediate adhesion of laminin and collagens and the a,p3 integrins. The a$, complex is capable of binding to a number of ECM proteins and in human vascular SMC this integrin complex is believed to mediate binding to TSP (Ruoslahti and Giancotti, 1989; Lawler et al., 1988). We have previously found that TGF-Pl induced increase in TSP appears to occur via a different mechanism than that for PDGF and was not associated with a n increase in DNA synthesis (Janat and Liau, 1992). However, TGF-p1 was able to strongly enhance the mitogenic response of SMC to PDGF-BB and addition of both growth factors also resulted in a synergistic increase in TSP expression (Janat and Liau, 19921. We have now examined the effect of TGF-p1 and PDGF-BB on the expression of both the p3 and PI integrin families. We found that expression of the TSP binding integrin complex, a,p3 was stimulated by both growth factors. Increased expression of this integrin complex was due to a n increase in p3subunit mRNA level, since the a, integrin mRNA level was unchanged. Interestingly, expression of the p3 as well a s the a5 integrin subunit mRNAs in response to TGF-p1 and PDGF-BB mirrored the previously reported expression of TSP (Janat and Liau, 1992). By contrast, the p1 integrin subunit was not regulated by TGF-p1 or PDGF-BB, and levels of various p, integrin protein complex correlated with changes in CY integrin subunit mRNA level. Our results suggest that both TGF-P1 and PDGF-BB are potent modulators of the binding of SMC to the ECM. The role of altered cell binding to the ECM after growth factor stimulation is presently unclear.
589
growth factor and either metabolically labeled or used to isolate total RNA.
RNA preparation and hybridization analysis Total cellular RNA were isolated using guanidinium isothiocynate and cesium chloride a s previously described (Liau and Chan 1989). The purified RNA was size fractionated on a 1% denaturing formaldehyde agarose gel, transferred to nitrocellulose, and hybridized under stringent conditions, a s described previously (Liau et al., 1985). Slot blot analysis was carried out using a manifold apparatus (Schleicher & Schuell) and the resulting autoradiograms quantitated using a Hoefer GS-300 scanning densitometer as previously described (Liau et al., 1991). cDNAs used to analyze the PI,a5,and a, integrin subunit mRNAs have been described previously and are 2.4 Kb, 1.7 Kb, and 520 bp, respectively (Argraves e t al., 1987, 1986; Suzuki et al., 1987). A 1.1 Kb p3 cDNA was obtained from Dr. E. Ruoslahti. A 1.8 Kb a1 cDNA was kindly provided by Dr. G. Marcantonio (Columbia University, NY).
Cell labeling and immunoprecipitation Confluent cultures were incubated for two days with methionine-free Dulbecco’s Modified Eagles Medium supplemented with 0.5% FBS, 10 pM insulin, and 5 pgiml transferrin. Growth factor was added for the desired period and labeled for the last six h with a mixture of 25 $2i/ml of L-[35S]methionine and L-[35S]cysteine (specific activity 1138 Ciimmole) [ICN, Irvine, CAI. The immunoprecipitation was carried out a s previously described (Argraves et al., 1990). Briefly, cells were washed twice with cold PBS and lysed in a detergent mixture containing 1% triton X-100, 0.5 M NaC1, 50 mM Tris, pH 7.4, and 0.05% Tween-20. The cell lysate was centrifuged a t 100,OOOg for 30 min. The supernatant fluid was preabsorbed with protein-A sepharose MATERIALS AND METHODS beads and 3 x lo6 cpm was incubated with the approMaterials priate antiserum overnight at 4°C. The immune-comDefined fetal bovine serum was purchased from Hy- plex was precipitated with protein-A Sepharose beads clone Labs, Inc. (Logan, UT). Bovine insulin and trans- and analyzed by electrophoresis under reducing and ferrin were obtained from Sigma (St. Louis, MO). Por- non-reducing conditions on a 7.5% sodium dodecyl sulcine platelet transforming growth factor-pl (TGF-pl), fate-polyacrylamide gel (Lammli, 1970). The gel was and porcine PDGF were purchased from R & D Sys- treated with Enlightening (DuPont-New England Nutems, Inc. (Minneapolis, MN). Rabbit skin fibroblasts clear Research Products, Boston, MA) for 20 min, dried, were obtained from American Type Culture Collection and exposed to X-ray film. (ATCC # CRL 1414, Rockville, MD). Antibody to the RESULTS and p3 integrins were kindly provided by Dr. R.O. TGF-p1 is a specific and potent enhancer of p3 Hynes (MIT, MA) and Dr. E. Ruoslahti (La Jolla Cancer integrin mRNA expression Research Foundation, CA), respectively. We first examined the effect of TGF-p1 on the expresCell culture sion of the p3 and the p1 integrin mRNAs, since they Aortic smooth muscle cells (SMC) were isolated from comprise the two known integrin families on vascular rabbit aorta by enzymatic digestion and propagated as SMC. In addition, because the a,p3 complex can bind previously described (Liau and Chan, 1989). Cells used TSP in vitro, we also determined the expression of the in all the experiments were between 4 to 8 passages. a, mRNA in response to TGF-P1. Cells were treated Fibroblasts were cultured in Basal Medium Eagle sup- with 200 pM of TGF-P1 and harvested a t various times plemented with 10% FBS and 2 mM L-glutamine. SMC for RNA extraction and analysis. The results, shown in were seeded at a density of 2 x lo4 cells/cm2 and al- Figure l a , indicated that cultured SMC contained a lowed to reach confluence (2-3 days). The cultures were high constitutive level of PI integrin subunit mRNA then fed fresh Medium 199 containing 0.5% FBS, that was not modulated by TGF-P1. By contrast, these 10- 6M insulin, and 5 pgiml transferrin supplemented cells contained a very low basal level of p3 integrin with 2 mM L-glutamine (low serum medium). After 2-3 subunit mRNA that was augmented by TGF-p1 at least days, cultures were treated with the appropriate by 24 h and mRNA expression of p3 remain elevated 48
590
dANAT ET AL.
A
TGF-Pi (200 pM)
0 0.5 1
2
6 24 48 hours
p3
2
200
c
c
2;100
*"
P I
\
/
c
\
\
,s.--....
a Ia ,
........
\
/ I I .
2
2 -
\
\
300
p3
c-----o
\
/'
I
a,
P1
.
,*---a 400
\
\ \
-
.... \
0
1
Hours After Incubation with 200pM TGF-P
B
TGF-B1
0
P3
Fig. 2. Analysis of p,, p3, ab,and a, integrin mRNA levels in skin fibroblasts treated with TGF-61. Confluent cultures of quiescent fibroblasts were treated with 200 pM of TGF-P1 for 0.5 to 48 h and RNA was isolated and analyzed by slot blot. The results were quantitated by densitometry and plotted as percentage increase over control levels.
5 50 100200 P M
blasts. Expression of a, integrin subunit mRNA was unchanged, while that of a5paralleled that of PI.
TGF-pl specifically i n d u c e d expression of the p3 polypeptide and a p1 associated 190 kDa a polypeptide in vascular SMC To determine the relationship between mRNA levels Fig. 1. Analysis of PI, P3, and ayintegrin mRNA levels in vascular and protein expression, we performed immunoprecipiSMC treated with TGF-P1. A: Confluent cultures of quiescent SMC were treated with 200 pM of TGF-61 for 0.5 to 48 h and RNA was tation analysis on extracts of metabolically labeled isolated and analyzed by Northern blot. B: SMC were treated with 5 to cells using antibodies directed against the cytoplasmic 200 pM of TGF-p1 and RNA was isolated and analyzed using the p3 domain of the P1 and P3 integrin receptors to examine cDNA probe. their expression. For comparison, parallel immunoprecipitation analyses were also performed on skin fibroblasts. Quiescent cells were incubated with the growth h after growth factor addition. The a, cDNA identified factor for 24 h, metabolically labeled for the last 6 h and three major transcripts of 9.5 Kb, 7 Kb, and 5.5 Kb, as the integrin proteins were immunoprecipitated with well as a minor transcript of 4.5 Kb in rabbit SMC. the appropriate antiserum and analyzed by sodium TGF-p1 did not appreciably alter the level of any of dodecyl sulfate-polyacrylamide gel electrophoresis these a, transcripts. These results indicate that (SDS-PAGE). The immunoprecipitation results showed TGF-P1 selectively enhanced the expression of the p3 that there was a n undetectable level of anti-p, precipiintegrin subunit mRNA, but did not alter the expres- table polypeptide in untreated SMC (Fig. 3A). Howsion of Pl o r n, integrin subunits in vascular SMC. We ever, in TGF-p1 treated cells, two polypeptides that examined the sensitivity of the p3 integrin response to migrated with a n apparent molecular mass of 100 kDa TGF-P1 and found that as little as 5 pM of this factor and 130 kDa under reducing SDS-PAGE were identiwas effective in elevating the expression of the p3 fied by this antiserum. Under nonreducing conditions, they migrated as a 95 kDa and a 150 kDa polypeptide, mRNA (Fig. IB). We next compared integrin mRNA expression in vas- respectively. The size and electrophoretic migration cular SMC with their expression in fibroblasts. We pattern of the 100 kDa polypeptide indicate it is the p3 found that increased expression of both p1 and p3 inte- integrin subunit. The 130 kDa polypeptide is a n (Y integrin subunit mRNA levels were observed in rabbit skin grin subunit co-precipitated with p3 and its size and fibroblast treated with 200 pM of TGF-pl (Fig. 2). In- electrophoretic migration pattern indicate it is likely duction of the p1 integrin was observed by 1 h and the a, integrin subunit (Lawler e t al., 1988).A similar remained elevated until 48 h after TGF-p1 addition. p3 pattern of expression was observed in fibroblasts, exintegrin mRNA level also increased rapidly in response cept that the apparent size of the (Y integrin subunit to TGF-P1, but had returned almost to basal level by 24 was slightly smaller when analyzed under reducing h after growth factor addition. By contrast, in vascular conditions (Fig. 3B). These results indicate that there SMC, only the p3 integrin mRNA level increased in was a concomitant increase in p3 subunit mRNA and response to TGF-P1 and this response was substan- polypeptide level after TGF-p1 treatment and the p3 tially more prolonged than that observed in skin fibro- subunit was complexed with the a, subunit. P1
591
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
TGF-81
(A) 81 P3 NRS -
anti-sera -
- + - + -
TGF-P
+
B1 B3 NRS --- + - + - +
0
(200 PM)
2 6 10 14 24 48 h w s
KD
220-
\
I
97-
68
a5
-
42 -
GAPDH Fig. 4. Analysis of a, and a5 integrin subunit mRNA level in cells treated with TGF-Pl. Quiescent SMC were treated with 200 pM of TGF-p1 for 2 h to 48 h a n d subsequently harvest for RNA. mRNA level of a 1 and a, integrin subunits were assessed by Northern blot. GAPDH level was also determined as a control for RNA loading.
(B) B1 B3 NRS ---
anti-sera TGF-B
- + - + - +
Bi 83 NRS ---
+
-
t
-
+
KD 220-
9766-
42-
Reduced
Nonreduced
Fig. 3. Immunoprecipitation analysis of p, and p3 integrin proteins in vascular SMC and in fibroblasts after TGF-pl treatment. Cultured cells were treated for 24 h in the presence (+) or absence (-) of 200 pM TGF-p1 and labeled for the last 6 h with a mixture of 25 uCi/ml of L-[35S]Methionine and L-[35S]Cysteine.After treatment, integrin proteins were immunoprecipitated with antibodies specific to PI or & integrin subunits and analyzed by SDS-PAGE as described in Materials and Methods. Control immunoprecipitation was also carried out with normal rabbit serum (NRS). A: SMC treated with TGF-P1. B: Fibroblasts treated with TGF-p1.
The anti-p, antiserum specifically immunoprecipitated 2 major polypeptides of approximately 100 kDa and 120 kDa from both SMC and fibroblasts (Fig. 3). These polypeptides had a faster electrophoretic mobility when analyzed on SDS-PAGE under nonreducing conditions. The size and electrophoretic mobility properties indicate the 100 kDa and 120 kDa polypeptides are, respectively, the precursor and mature form of the p1 subunit (Ignotz and Massague, 1987). TGF-p1 had no effect on the amount or on the ratio of the precursor and mature forms in vascular SMC. However, there was a n enhanced amount of the 100 kDa precursor form of the p1 polypcptide in TGF-p1 treated fibroblasts
(Fig. 3B). This film was overexposed to allow visualization of the much less abundant p3 integrin complex. Densitometric analysis of a less exposed autoradiogram indicated there was a %fold enhancement of the p1 precursor form after TGF-p1 treatment. In vascular SMC two a chain subunits were also co-precipitated with the p1 integrin subunit by the anti+, antibody. Particularly striking was a large increase in a 190 kDa (Y chain subunit in SMC treated with TGF-p1. In addition, a 150 kDa a chain observed on nonreducing SDS-PAGE also increased slightly after growth factor treatment. Presumably, under reducing conditions, this a chain comigrated with the p1 integrin subunit. In fibroblasts, the 190 kDa 01 integrin subunit was not detected, while the 150 kDa a integrin subunit was expressed a t a much higher level and was not appreciably altered by TGF-61 treatment. The results indicate that although the p1 integrin subunit level was not detectably altered by TGF-61, expression of a 190 kDa (Y chain integrin subunit associated with pl was induced by TGF-p1 treatment of vascular SMC but not of skin fibroblasts.
Both a1 and 0 1 integrin ~ subunit mRNA levels are increased after TGF-p1 treatment Although a number of a integrin subunits have been demonstrated to interact with the p1 integrin subunit, only the (Y, integrin subunit has a molecular mass corresponding to that of the TGF-61 inducible a polypeptide associated with p1 (Humphries, 1990; Clyman et al., 1990). We were, therefore, interested i n knowing whether or not expression of a1 integrin was enhanced by TGF-Pl and performed RNA hybridization analysis using a cDNA probe to the a1 integrin. We also examined expression of the a5integrin subunit mRNA, since the asp1 integrin complex has also been identified on SMC (Bottger et al., 1989; Clyman et al., 1990; Belkin et al., 1990). The results, shown in Figure 4,indicate that mRNA levels of both a, and a5 integrin subunits increased in response to TGF-Pl addition. Enhanced
592
JANAT ET AL.
PDGF (0.1nM) 0 0.5 1 2 6 24
Pi
anti-sera
hours
PDGF KD 220
P3 a5 NRS
-+-+-+-+
(31 -
P3 a5 NRS
-+-+-+-+
-
97 -
I
\
68
-
42 -
Reduced
Fig. 5. Analysis of PI, p3,a5,and 01, integrin mRNA levels in vascular SMC treated with PDGF-BB. Confluent cultures of SMC were treated with 0.1 nM of PDGF-BB for 0.5 to 24 h and RNA was isolated and analyzed by Northern blot.
expression was observed after 6 h and remained elevated 48 h after TFG-p1 treatment. Interestingly for a5,in addition to the 4.5 Kb message, we also observed a larger transcript that ran just above the 28s marker. We do not know if this second transcript represents a distinct variant in rabbit or was merely due to technical differences in Northern blotting. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA expression was also monitored a s a control and showed no appreciable change. These results suggest that the TGF-61 inducible 190 kDa a integrin subunit associated with the P1 subunit is possibly the a1subunit.
Nonreduced
Fig. 6 . Immunoprecipitation analysis of PI, B3, and as integrin proteins in vascular SMC after PDGF-BB treatment. Cultured SMC were incubated for 10 h in the presence ( + I or absence (-) of 0.1 nM of PDGF-BB and labeled for the last 6 h with a mixture of 25 uCi/ml of LP"1Methionine and L-[:3"S1Cysteine.After treatment, integrin proteins were immunoprecipitated with the specific antibodies and analyzed by SDS-PAGE. Control immunoprecipitation was also carried out with normal rabbit serum (NRS).
We also studied integrin polypeptide expression in response to PDGF-BB. SMC were treated with PDGF-BB for 10 h and the metabolically labeled integrins were immunoprecipitated with antibody directed against the PI,p3, and a5 integrin subunits, respectively. We chose to analyze integrin expression a t 10 h instead of a t 24 h, since PDGF-BB induced a more transient expression of integrin mRNA than TGF-P1. The results, shown in Figure 6, indicated that there was increased expression of the 100 kDa p3 polypeptide after PDGF-BB treatment. Associated with the pn subunit was a 130 kDa a subunit that showed retarded PDGF-BB also increases integrin expression in migration under nonreducing SDS-PAGE conditions. vascular SMC The size and migration pattern of this a subunit is We next examined whether or not PDGF-BB, a po- identical to that previously observed for TGF-p1 tent mitogen and chemoattractant for SMC, is also ca- treated SMC and suggest it is also the a, integrin subpable of modifying integrin expression in these cells unit. PDGF-BB had no effect on PI polypeptide expres(Ross, 1986). Expression of the TSP binding integrin sion or on conversion from the precursor to mature form complex aVp3was of particular interest, since TSP level of this receptor. In addition, PDGF-BB did not elicit a n is rapidly enhanced by PDGF (Majack et al., 1985). increase in the 190 kDa a integrin subunit associated Quiescent SMC were treated with 0.1 nM PDGF-BB for with the p1 integrin subunit as previously observed for various times and analyzed for a",p3, as,a,,and p1 TGF-P1 treated SMC. Since we also observed that the mRNA levels. The result, shown in Figure 5, indicated a1integrin was not induced by PDGF-BB, these results that like TGF-P1, PDGF-BB was also able to induce a n are consistent with the possibility that the 190 kDa increase in P3 mRNA level, but expression of the a, integrin induced by TGF-P1 is the a1integrin subunit. mRNA was not altered. Interestingly, PDGF-BB in- A 150 kDa ct integrin subunit was co-immunoprecipiduced increase in mRNA level occurred by 2 h, tated with the p1 integrin subunit. However, it was peaked by 6 h, and was no longer apparent by 24 h. This unaltered by PDGF-BB treatment. This a integrin subis in great contrast with TGF-P1 enhanced expression unit is apparently not ct5, since a5 antibody identified a of the P3 integrin, which persisted for up to 48 h. The slightly larger polypeptide that increased upon level of a5integrin subunit mRNA essentially mirrored PDGF-BB treatment. These results indicate t h a t PDGF-BB is also capable that of p3 (Fig. 5). Expression of p, integrin subunit mRNA was unaltered by PDGF-BB, while PDGF-BB of increasing the expression of certain integrins in vaswas unable to induce a n increase in a very low al basal cular SMC. Interestingly, both PDGF-BB and TGF-P1 induced increase in a,p3 integrin complex were due to level (Fig. 5 and results not shown).
593
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS
(A)
- -
(B) +
+
- + - +
TGF-B1 ( 2 0 0 ~ M )
+
C y c lo h e x im ide (2wIrnl)
- +
P3
P3
B1
P1
+
PDGF (O.lnM) C y c lo he x imide ( 2 U g l r n l )
Fig. 7. Analysis of the effect of cycloheximide on TGF-p1 and PDGF-BB induced increase in integrin mRNA expression. SMC were treated with 2 u g h 1of cycloheximide 30 min prior to addition of: A: 200 pM of TGF-P1 for 24 h. B: 0.1 nM of PDGF-BB for 6 h. Identical Northern blots were used to analyze p,, pa, and as integrin mRNA levels.
a n increase in p3 integrin subunit mRNA expression since the a, integrin mRNA level was unchanged. However, there are a number of differences in integrin expression in response to these growth factors. PDGF-BB was apparently not as potent as TGF-p1 in inducing expression of the a,p3 integrin complex (compare Fig. 3A and Fig. 6). In addition, the induction of both p3 and a5 integrin mRNA expression by PDGF-BB was considerably more rapid and more transient than that observed for TGF-pl (Figs. 1, 4, 5). Finally, unlike TGF-61, PDGF-BB was unable to induce a n increase in expression of a 190 kDa a subunit associated with integrin subunit.
of a5 integrin was superinduced in the presence of either TGF-p1 or PDGF-BB and CHX. PI integrin mRNA level increased slightly in the presence of CHX, but there was no additional increase in the presence of growth factors. Our results indicate that TGF-P1 induced increase in p3 expression occurs via a direct mechanism, while PDGF-BB induced increase. of this integrin subunit is mediated by a second protein. These results strongly suggest that TGF-p1 induced expression of the p3 integrin is not due to prior induction of other autocrine factors, such a s PDGF.
TGF-p, and PDGF-BB induced increase in P3 expression occurs through distinct mechanisms
The expression and regulation of integrin receptors on vascular SMC are not well characterized. To date, the integrins identified are all members of the p1 and p3 family (Lawler et al., 1988; Clyman et al., 1990; Belkin et al., 1990; Bottger et al., 1989). Both a n asp,-like receptor t h a t binds fibronectin and a n a,P,-like receptor that binds laminin and types I and IV collagen have been defined (Bottger et al., 1989; Clyman et al., 1990; Belkin e t al., 1990). We found that expression of the PI integrin subunit was high and not modulated significantly by TGF-p1 or PDGF-BB in vascular SMC. However, expression of both a1 and a5 integrin subunits, which can associate with PI, was enhanced by growth factors. By contrast, both TGF-P1 and PDGF-BB were able to directly stimulate P3 mRNA level. The increase in p3 subunit level is responsible for enhanced a,p3 integrin expression, since the a, integrin mRNA level was constitutively expressed in these cells. Therefore, regulation of the PI and P3 family of integrins by growth factors occurs through two distinct mechanisms, with the family of receptors regulated by change in a subunit levels, while p3 itself is directly responsive to growth factors. The proposed mechanism of regulation of the family of integrins is consistent with available in vivo data. Analysis of alp,integrin
One interpretation of our finding that TGF-p1 induced increase in p3 and a5 integrin subunits is considerably more delayed and prolonged than PDGF is that these two growth factors induce P3 and a5 integrin expression through distinct mechanisms. However, it is also possible that cultured SMC exposed t o TGF-pl may subsequently express PDGF or other proteins, which then enhanced integrin expression through a n autocrine mechanism. TGF-P is capable of inducing PDGF expression in a variety of cell types (Leof et al., 1986; Makela et al., 1987; Majack et al., 1990; Battegay et al., 1990).To distinguish between these possibilities, we examined the effect of the protein synthesis inhibitor, cycloheximide (CHX) on growth factor induced increase in p3 and a5 integrin expression. The result, shown in Figure 7, indicated that CHX was unable to abolish the augmented expression of Pn integrin in response to TGF-P1. Indeed, there was a superinduction of P3 integrin mRNA level in the presence of CHX. But surprisingly, although PDGF-BB induced expression of p3 integrin mRNA was more rapid, the increase was completely abolished by CHX. By contrast, expression
DISCUSSION
594
JANAT ET AL.
expression in the human aorta indicate that P1 expression is relatively unchanged during development and a similar level of p1was expressed in SMC from the aortic media and in intimal thickening (Belkin et al., 1990). By contrast, expression of the a, integrin subunit is developmentally regulated and is high in contractile cells of the media, but low in intimal cells and in cultured SMC which have modulated to a proliferative phenotype (Belkin et al., 1990). These authors concluded t h a t expression of the alp, integrin complex was only associated with differentiated SMC (Belkin et al., 1990). We found that in vitro, TGF-(31 but not PDGFBB, was able to induce a n increase in a, integrin subunit mRNA level, as well as a strong increase in expression of a 190 kDa a,-like integrin subunit in association with PI. This effect of TGF-Pl was not observed with fibroblasts. Our results suggest that TGF-p1 but not PDGF-BB may be one of the factors that is important in regulating a1 integrin subunit expression in SMC. Whether or not TGF-P1 or other related polypeptides are important for maintaining SMC in the contractile phenotype is presently unclear. TGF-P1 induced increase in P3 integrin subunit mRNA level was detected by 6 h and remained elevated for at least 48 h after TGF-(31 treatment, while PDGF-BB enhanced P3 expression was observed by 2 h but p3 expression was undetectable by 24 h after treatment. This temporal difference is very similar to the temporal difference in TSP response to TGF-P1 and PDGF-BB and indicates that expression of TSP and the TSP binding avP3receptor complex is very similarly regulated by these growth factors (Janat and Liau, 1992). Although other cell surface proteins that are able to bind TSP have been identified and characterized (Roberts et al., 1987; Kaesberg et al., 1989; Prater et al., 1991), the close coupling of growth factor response of TSP and the avP3receptor complex is intriguing and a t least consistent with the idea that this integrin complex is functionally important for TSP binding (Lawler et al., 1988). However, our results do not rule out the possibility that other TSP receptors are important and possibly more biologically relevant mediators of TSP action in vascular SMC. There are at least two possible explanations for the similar response of TSP and Pn to the two growth factors: 1. Regulation of TSP and the P3 integrin subunit genes occur through a common or similar pathway. 2. Expression and binding of TSP to the cell, presumably through the a&, receptor, induces increased expression of the p3 integrin subunit mRNA. Consistent with the second possibility is our finding that TSP expression is induced approximately 4 h earlier than p3 by TGF-Pl and 1.5 h earlier than P3 by PDGF-BB (Janat and Liau, 1992). However, this scenario predicts that CHX should be able to abolish p3 expression. CHX is, indeed, able to abolish PDGF-BB induced increase in p3 integrin mRNA expression, but it superinduces p3 expression in TGF-P1 treated cells. This result suggests that two distinct pathways are used to induce p3expression by PDGF-BB and TGF-P1. PDGF-BB induced expression of p3 could be due t o prior TSP expression and binding to SMC, while TGF-Pl may induce both TSP and p3 integrin expression through related pathways.
SMC migration and proliferation have a central role in vascular diseases such as atherosclerosis and hypertension, as well a s in vascular graft failures (ROSS, 1986; Schwartz et al., 1986; Ferns e t al., 1991). There is good evidence to suggest that PDGF plays a n important role in SMC migration (Ferns et al., 1991). What role TGF-(31 might have is presently unclear. Our results indicate that both of these factors are likely capable of modifying the interaction of SMC with its extracellular matrix environment (present manuscript and J a n a t and Liau, 1992). These changes may have a role in altering SMC migration and recent experiments demonstrating that fibronectin is able to activate the NaiH antiporter suggest that altered cell-matrix interactions may also directly modify signal transduction pathways important for cell proliferation (Ingber et al., 1990; Schwartz et al., 1991).
ACKNOWLEDGMENTS This work was supported in part by grant HL37510 (G.L.) and by grant GM42912 (W.S.A.) from the National Institutes of Health. G.L. is a recipient of a Research Career Development Award HL02449.
LITERATURE CITED Albelda, S.M., and Buck, C.A. (1990) Integrins and other cell adhesion molecules. FASEB J., 42868-2880, Argraves, W.S., Pytela, R., Suzuki, S., Millan, J.L., Pierschbacher, M.D., and Ruoslahti, E. (1986) cDNA sequences from the CI subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J. Biol. Chem., 26It12922-12924. Argraves, W.S., Shintaro, S., Arai, H., Thompson, K., Pierschbacher, M.D., and Ruoslahti, E. (1987) Amino acid sequence of the human fibronectin receptor. J. Cell Biol., 105r1183-1190. Argraves, W.S., Ti-an, H., Burgess, W.H., and Dickerson, K. (1990) Fibulin is a n extracellular matrix and plasma glycoprotein with repeated domain structure. J. Cell Biol., 111:3155-3164. Battegay, E.J., Raines, E.W., Seifert, R.A., Bowen-Pope, D.F., and Ross, R. (1990) TGF-p induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell, 63t515-524. Belkin, V.M., Belkin, A.M., and Koteliansky, V.E. (1990) Human smooth muscle VLA-1 integrin: Purification, substrate specificity, localization in aorta, and expression during development. J. Cell Biol., 11I t2159-2170. Bottger, B.A., Heldin, U., Johansson, S., and Thyberg, J. (1989) Integrin-type fibronectin receptors of rat arterial smooth muscle cells: Isolation, partial characterization, and role in cytoskeletal organization and control of differentiated properties. Differentiation, 41 t158-167. Castellot, J., Addonizio, M., Rosenberg, R., and Karnovsky, M. (1981) Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J . Cell Biol., 90t372-379. Clyman, R.I., McDonald, K.A., and Kramer, K.H. (1990) Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen. Circ. Res., 67:175-186. Fritze, L., Reilly, C., and Rosenberg, K. (1985) An antiproliferative heparan sulfate species produced by post-confluent smooth muscle cells. J . Cell Biol., 100:1041-1049. Hedin, U. Bottger, B.A., Forsberg, E., Johansson, S., and Thyberg, J. (1988) Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J . Cell Biol., 107:307-319. Humphries, M.J. (1990) The molecular basis and specificity of integrin-ligand interactions. J . Cell Sci., 97:585-592. Hynes, R.O. (1987) Integrins: A family of cell surface receptors. Cell, 48549-554. Ignotz, R.A., and Massague, J. (1987) Cell adhesion protein receptors as targets for transforming growth factor-p action. Cell, 51r189197. Ingber, D.E., Prusty, D., Frangioni, J., Cragoe, Jr., E.J., Lechene, C.,
INTEGRIN EXPRESSION IN SMOOTH MUSCLE CELLS and Schwartz, M.A. (19901 Control of intracellular pH and growth by fibronectin in capillary endothelial cells. J. Cell Biol., 110:18031811. Janat, M.F., and Liau, G. (1992) Transforming growth factor pl is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. J. Cell. Physiol., 150t232-242. Kaesberg, P.R., Erschler, W.B., Esko, J.D., and Mosher, D.F. (1989) Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparin sulfate proteoglycan. J. Clin. Invest., 83t99P1001. Laemmli, U.K. (1970) Cleavage structural proteins during the assembly of the head bacteriophage T4. Nature, 227t680-685. Lawler, J. (1986) The structural and functional properties of thrombospondin. Blood, 67t1197-1209. Lawler, J., Weinstein, R., and Hynes, R.O. (1988) Cell attachment t o thrombospondin: The role of ARG-GLY-ASP, calcium, and integrin receptors. J. Cell Biol., 107:2351-2361. Liau, G., Yamada, Y., and decrombrugghe, B. 11985)Coordinate regulation of the levels of type I11 and type I collagen mRNA in most but not all mouse fibroblast. J. Biol. Chem., 260531-536. Loef, E.B., Proper, J.A., Goustin, A.C., Shipley, G.D., DiCorleto, P.E., and Moses, H.L. (1986)Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor p: A proposed model for indirect mitogenesis involving autocrine activity. Proc. Natl. Acad. Sci. U.S.A., 83:2453-2457. Majack, R.A., Cook, S.C., and Bornstein, P. (1985) Platelet-derived growth factor and heparin-like glycosaminoglycansregulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J. Cell Biol., 101:1059-1070. Majack, R.A., Goodman, L.V., and Dixit, V.M. (1988) Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J. Cell Biol., 106r415-422. Majack, R.A., Majesky, M.W., and Goodman, L.V. (1990) Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-p. J. Cell Biol., 111:239-247. Makela, T.P., Alito, R., Paulsson, Y., Westermark, B., Heldin C-H., and Alitalo, K. (1987) Regulation of platelet-derived growth factor gene expression by transforming growth factor p and phorbol ester in human leukemia cell lines. Mol. Cell. Biol., 7t36563662.
595
Prater, C.A., Plotkin, J.,Jaye, U., and Frazier, W.A. (1991) The properdin-like type I repeats of human thrombospondin contain a cell attachment site. J. Cell Biol., 112:1031-1040. Raugi, G.J., Mumby, S.M.,Abbott-Brown, D., and Bornstein, P. (1982) Thrombospondin: Synthesis and secretion by cells in culture. J. Cell. Biol., 95t351354. Rauei. G.. Mullen. J.S.. Bark. D.H.. Okada. T.. and Mavbere. M.R. (19901Thrombosoondin deoosition in rat carotid arterv"iniu&. " * " Am. J. Pathol., 137tlj9-185. Roberts, D.D.,Sherwood, J.A., and Ginsberg, V. (1987)Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J. Cell Biol., 104:131-139. Ross, R. (1986) The pathogenesis of atherosclerosis-An update. N. Engl. J. Med., 314t488-500. Ruoslahti, E., and Giancotti, F.G. (1989) Integrins and tumor cell dissemination. Cancer Cells, 1t119-126. Ryseck, R.P., MacDonald-Bravo, H., Zerial, M., and Bravo, R. (19x9) Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts. Exp. Cell Res., 180:537-545. Scott-Burden, T., Resink, T.J., Hahn, A.W.A., andBuhler, F.R. (1990) Induction of thrombospondin expression in vascular smooth muscle cells by angiotensin 11. J. Cardiovasc. Pharmacol., 16:S17-S20. Schwartz, S.M., Campbell, G.R., and Campbell, J.M. (1986) Replication of smooth muscle cells in vascular disease. Circ. Res., 58r427444. Schwartz, M.A., Lechene, C., and Ingber, D.E. (1991) Insoluble fibronectin activates the Na/H antiuorter bv clustering and immobilizing integrin independent 0.f cell shape. Proc. Natl. Acad. Sci. U.S.A., 88:7849-7855. Suzuki, S., Armaves, W.S., Arai, H.. Lanmino, L.R.. Pierschbacher. M.D., and Rioslahti, E. (1987) Amino acid sequence of the vitronectin receptor 01 subunit and comparative expression of adhesion receptor mRNAs. J . Biol. Chem., 262:14080-14085. Wight, T.N., Raugi, G.J., Mumby, S.M., and Bornstein, P. (19851 Light microscopic immunolocation of thrombospondin in human tissues. J. Histochem. Cytochem., 33:295-302.
