JOURNAL OF CELLULAR PHYSIOLOGY 145:16-23 (1990)

Thrombospondin Gene Expression I s Associated With Mitogenesis in 3T3 Cells: Induction by Basic Fibroblast Growth Factor DORlT B. DONOVIEL, SHARON L. AMACHER, KEVIN W. JUDGE,AND PAUL BORNSTEIN*

Departments of Biochemistry (D.B.D., S.I.A., K.W.)., P.B.), and Medicine (K.W.).,P.B.), University of Washington, Seattle, Washington 98 I95 Growth factor-depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF)with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the a1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0-2 h), “superinduction“ of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed, and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4-8 h) the combination of cyclohexirnide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short-lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect o n 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation.We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage-dependent cells through the cell cycle. ThrombosDondin (TS) is the major constituent of platelet a-granules and DarticiDates in the secondary. irreversible phase of piatelet aggregation (Santoro; 1987; Silverstein et al., 1986a). TS is also synthesized and secreted by a wide variety of cells in culture (reviewed in Lawler, 1986 and Majack and Bornstein, 1987), but its extravascular function is obscure. TS clearly supports the attachment and spreading of human keratinocytes (Varani et al., 1988), squamous carcinoma cells (Varani et al., 1986), and melanoma cells (Roberts et al., 1987; Riser et al., 1988). On the other hand, there is less agreement regarding the ability of TS to serve as an attachment factor for fibroblasts, endothelial cells, and smooth muscle cells (SMC) (Tuszynski et al., 1987; Lahav, 1988; Murphy-Ullrich and Mosher, 1987; Murphy-Ullrich and Hook, 1989). Lawler et al. (1988) have identified an integrin complex, related to glycoprotein IIb-IIIa, as an Arg-GlyAsp-dependent receptor for TS in endothelial and SMC; however, while these cells attached to TS-coated substrates, less than 5%of such anchorage-dependent cells spread on TS. There is increasing evidence for a more dynamic role for TS in cell-substratum interactions, one that is dependent both on the nature of the cell surface and on the conformation of the protein (Kaesberg et al., 1989). Evidence from this laboratory has implicated cell surface-associated TS in augmenting the mitogenesis of rat aortic SMC, stimulated by epidermal growth factor (Majack et al., 1986; Majack and Bornstein, 1987). A

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A role for TS in SMC growth is also suggested by the recent observation that monoclonal antibodies to TS inhibit SMC proliferation while, at the same time, markedly reducing the abundance of TS on the cell surface (Majack et al., 1988). In keeping with a proposed role for TS in cell cycle progression, it has been observed that both SMC (Majack et al., 1985) and glial cells (Asch et al., 1986) are stimulated to synthesize TS by platelet-derived growth factor (PDGF).The kinetics of the response of TS mRNA levels to PDGF (Kobayashi et al., 1986; Majack et al., 1987) resemble those seen for c-fos and c-myc (Greenberg and Ziff, 1984; Curran et al., 1985). Furthermore, transcription of the TS gene is induced by PDGF, as demonstrated by nuclear run-on experiments (P. Framson and P. Bornstein, manuscript in preparation), and TS mRNA levels are “superinduced” by the addition of the protein synthesis inhibitor, cycloheximide, to cells in the presence of PDGF (Majack et al., 1987). These findings place the TS gene in the category of “immediate-early’’ PDGFresponse genes (Cochran et al., 1983; Lau and Nathans, 1987). In this paper we report that basic fibroblast growth factor (bFGF) induces the TS gene in Swiss 3T3 cells, which respond mitogenically to the growth factor (Olwin and Hauschka, 1986), but not in BC3H1 cells Received April 2, 1990; accepted June 12, 1990.

*To whom reprint requestskorrespondence should be addressed.

bFGF INDUCES THROMBOSPONDIN GENE EXPRESSION

for which bFGF is not a mitogen (Lathrop et al., 1985; Spizz et al., 1986). We show that the elevated TS mRNA levels seen in 3T3 cells in response to bFGF result both from a n increase in the rate of transcription of the TS gene and from stabilization of TS mRNA. Our findings add support to the hypothesis that TS acts in a n autocrine fashion to facilitate the growth of some anchorage-dependent cells.

MATERIALS AND METHODS Growth factors Bovine brain bFGF and recombinant human bFGF were generously provided by Bradley Olwin and Stephen Hauschka, University of Washington, and were used at a concentration of 300 pM (or about 5 ng/ml&a dose which elicited a maximal mitogenic response in confluent monolayers of Swiss 3T3 cells (Olwin and Hauschka, 1986). No difference in the effects of the two preparations was observed. In some experiments with BC3H1 cells a dose of 3 nM was used. Cell culture Swiss 3T3 cells (clone D1) were grown to a subconfluent state in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum. The cells were made quiescent by culture for 24 h in 2% CMS (calf serum depleted of growth factors by passage over a column of CM-Sephadex). Cycloheximide was used a t 10 kg/ml. RNA synthesis was inhibited by the addition to the culture medium of 60 pM 5,6-dichloro-l-P-Dribofuranosylbenzimidazole (DRB), a n inhibitor of transcriptional initiation (Zandomeni et al., 1983). BC3H1 cells (Schubert et al., 1974; obtained from Stephen Hauschka, University of Washington) were cultured in Dulbecco’s modified Eagle’s medium containing 15%calf serum. Cells were made quiescent by culture in 0.5% calf serum-containing medium for 48 h. The labeling index, measured by incorporation of [3H]thymidine during the last 24 h, was less than 10%. Isolation of RNA and blot hybridization (Northern) analysis Tissue culture plates were washed twice with icecold phosphate-buffered saline and the cells were scraped on ice. RNA was extracted and purified as described by Chomczynscki and Sacchi (1987). RNA was quantitated by measurement of optical density a t A260, and 10 pg of RNA was fractionated on 1% agarose gels in the presence of 18%(vol/vol)formaldehyde. Nucleic acids were transferred to nitrocellulose and probed with a random prime-labeled human TS cDNA probe, TS33 (Kobayashi e t al., 1986). Autoradiograms of RNA blot hybridizations were analyzed by densitometry of appropriate exposures. To ascertain that equal quantities of RNA were loaded and transferred from each lane, ethidium bromide staining of ribosomal RNA bands on both the gel and the blot was examined. In addition, in some experiments the blot was probed with sequences directed to glyceraldehyde 3-phosphate dehydrogenase (G3PD). Each experiment was repeated a t least twice, and in most cases 3 or more times, with essentially the same results.

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Analysis for TS mRNA levels by solution hybridization and RNase protection Mouse TS mRNA levels were analyzed using a n RNase protection assay with total cellular RNA, as previously described (Melton et al., 1984; Bornstein and McKay, 1988). The riboprobe was constructed with a 492 bp TS fragment extending from a PstI site in exon 4 to a PstI site in exon 5 in the mouse TS gene (Bornstein et al., submitted for publication). This riboprobe would be expected to protect fragments of 177 bp and 70 bp in mouse TS mRNA. Nuclear run-on analysis Subconfluent, quiescent 3T3 cells were treated with bFGF for various times, after which the plates were washed once with ice-cold phosphate-buffered saline and immediately placed on ice. The cells were scraped in lysis buffer and the nuclei isolated as described by Groudine et al. (1981), except that NP40 was omitted from the lysis buffer. Approximately 1x lo7 nuclei were used for each run-on reaction according to the protocol of McKnight and Palmiter (1979). A total of 3.4-5.6 x lo6 cpm, precipitable by trichloroacetic acid and ethanol, were added, in a volume of 100 pl of hybridization buffer (50% vol/vol formamide), to probes immobilized on nitrocellulose discs. Each hybridization tube contained four discs on each of which 5 p.g of denatured double-stranded plasmid had been blotted. After 72 h a t 45”C,the filters were washed and counted for 20 min in 10 ml Aquasol (Dupont). Values are expressed as parts per million of input cpm, corrected for background (hybridization to vector probe), and normalized to 1 kbp of specific sequences in the immobilized probe. DNA probes The human TS cDNA probe was a 1.3 kbp fragment cloned into pGEM2 (Kobayashi et al., 1986);the v-fos probe was a 950 bp PstI fragment of the mouse v-fos gene (Curran e t al., 1982) cloned into pUC18. The collagen probe was a 1.6 kb PstI fragment of rat al(1) cDNA (Genovese et al., 1984) cloned into pUC19. The fibronectin probe was a 2.1 kb PstI fragment of human fibronectin cDNA cloned into pUC13 (Sekiguchi et al., 1986). The G3PD probe was a 1.3 kbp PstI fragment of chicken cDNA. RESULTS TS mRNA levels are increased by bFGF in cells that respond mitogenically to this growth factor When Swiss 3T3 cells were made quiescent by culture in growth factor-depleted medium for 24 h, and then treated with bFGF, a substantial increase in TS mRNA levels was observed, with a maximum effect approximately 2 h after initiation of treatment (Fig. 1). Densitometric scans of appropriately exposed autoradiograms indicated a stimulation of 10-15-fold. In the continued presence of bFGF, TS mRNA levels gradually declined. The addition of cycloheximide to bFGF resulted in no significant change in TS mRNA levels 30 min after treatment, in comparison with that observed with bFGF alone; however, treatment of cells with cycloheximide alone increased TS mRNA levels to almost the same degree a s that seen with the combination of

DONOVIEL ET AL.

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Fig. 1. Blot hybridization analysis of total RNA from bFGF-stimulated Swiss 3T3 cells, fractionated on a 1 4 agarose gel. Cells were deprived of growth factors for 24 h. bFGF (300 pM) was added for the indicated times and cycloheximide (10kg/ml) was added at the time of bFGF addition. The 6 kb thrombospondin mRNA is identified. 10 pg of RNA was loaded in each lane.

bFGF and cycloheximide (Fig. 1).We therefore conclude that, within the time span examined in these experiments, basal levels of TS mRNA can be increased by inhibition of protein synthesis but that the increase observed with bFGF requires ongoing protein synthesis. Alternatively, FGF and cycloheximide may stimulate TS gene expression by a similar mechanism, e.g., inactivation of a short-lived transcriptional repressor. These findings are in contrast to the stimulation of TS mRNA levels observed in SMC with PDGF since the addition of cycloheximide to PDGF led t o a “superinduction” of TS mRNA levels (Majack et al., 1987). As shown in Figure 1, TS mRNA levels returned almost to baseline 4 h after addition of bFGF, despite the continued presence of active growth factor. However, if cycloheximide was added to bFGF, and TS mRNA levels measured 4, 6, or 8 h later, a substantial increase in mRNA was detected, in comparison with levels determined in 3T3 cells treated with either bFGF or cycloheximide alone (Fig. 2). The RNase protection analysis for mRNA shown in Figure 2 reveals protected bands of 180 and 70 bp. These values are very close to those predicted from the exonhntron structure of the mouse TS gene. The experiment shown is representative of four experiments that gave comparable results. Comparable results were also obtained when mRNA levels were determined by Northern analysis (data not shown). These findings suggest that, in response to conditions that elevate TS mRNA levels, cells produce a labile transcriptional repressor andlor rapidly degrade TS mRNA and that steady-state levels of such inhibitory factors can be reduced by inhibition of protein synthesis. Collagen a n d fibronectin mRNA levels are not significantly increased by bFGF To determine whether Swiss 3T3 cells respond generally to bFGF with an increase in mRNA levels for secreted cell surface-associated proteins, quiescent 3T3 cells were stimulated with bFGF and mRNA levels for the a1 chain of type I collagen and fibronectin were determined by Northern analysis. As can be seen in

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Fig. 2. Autoradiogram of TS mRNA analyzed by solution hybridization. Total RNA (10 pg) from Swiss 3T3 cells was hybridized with a 492 bp 3’P-1abeled, antisense mouse TS genomic probe. After RNase digestion, ethanol-precipitable material was analyzed by electrophoresis in a 6%acrylamide denaturing gel. Lanes represent analyses of RNA from cells treated for 4, 6, and 8 h with bFGF, bFGF plus cycloheximide, or cycloheximide alone. The arrows indicate protected bands of 180 and 70 bp. RNA from cells treated for 8 h was electrophoresed for a slightly longer time, accounting for the discrepancy in the position of the 70 bp bands in these lanes. The positions of size markers obtained from an MspI digest of pBR322 are shown to the left. The relative values for the 180 bp band, determined by scintillation counting of the excised bands, are as follows. 4 h bFGF, 1.0; bFGF and cycloheximide, 5.9; cycloheximide, 2.8. 6 h: bFGF, 1.0; bFGF and cycloheximide, 7.3; cycloheximide, 2.4. 8 h bFGF, 1.0; bFGF and cycloheximide, 5.4; cycloheximide, 1.6.

Figure 3, there was no significant increase in mRNA levels for either protein over a period of 24 h after bFGF stimulation, and the addition of cycloheximide also had no substantial effect on either the basal or

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bFGF INDUCES THROMBOSPONDIN GENE EXPRESSION

-- +- +- +- ++ -+ 4-- +- Cycloheximide FGF -Fibronectin

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Fig. 3. Blot hybridization analysis of total RNA from bFGF-stimulated Swiss 3T3 cells, fractionated on a 1%agarose gel. Fibronectin and the 5.7 and 4.7 kb mRNAs for the cul(1) chain of type I collagen are identified. See legend to Figure 1 for additional details.

bFGF-treated mRNA level. Quiescent Swiss 3T3 cells are, however, capable of increasing collagen and fibronectin mRNA levels since a 10-20-fold increase was observed 8-16 h after addition of TGF-P (Penttinen e t al., 1988). Thus, the response of the TS gene to bFGF differs from t ha t of the genes for some other matrix proteins over the time course studied. A similar conclusion had been reached in rat aortic SMC in which TS, but not collagen or fibronectin, mRNA levels were increased by PDGF (S. Kobayashi and P. Bornstein, unpublished observations).

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hours 16 8 4 0 2 4 8 The elevation of TS mRNA levels in response to bFGF correlates with mitogenic activity 4. Blot hybridization analysis of total RNA from bFGF or seBC3Hl cells were derived from a nitrosourea- Fig. rum-stimulated BC3H1 cells, fractionated on a 1% agarose gel. Cells induced mouse brain neoplasm (Schubert et al., 1974) were deprived of growth factors for 48 h and treated with either 5 nM and are now thought to be a skeletal muscle cell line bFGF (lanes 1-3) or 15% calf serum (lanes 5-7) for the indicated (Taubman et al., 1989). These cells do not respond mi- times. The mRNAs for TS and G3PD are identified. RNA from control togenically to bFGF, but bFGF inhibits differentiation cells was run in lane 4. 10 pg of RNA was loaded in each lane. as shown by a marked decrease in mRNA levels for muscle creatine kinase (Spizz et al., 1986).If elevated TS mRNA levels are associated with progression of SMC and fibroblasts and cell density (and thus presumcells through the cell cycle, one would predict that ably a correlation with the rate of cell division) had treatment of BC3H1 cells with bFGF would not lead to been made previously by Mumby et al. (1984). a n increase in TS mRNA levels. (One could, alternabFGF increases the transcription rate of the tively, propose that the response of the TS gene to TS gene bFGF in BC?.Hl cells was preserved but that a mitoTranscription rates were determined by nuclear rungenic response was lacking for additional, unrelated reasons.) When quiescent BC3Hl cells were treated on analyses in 3T3 cells that had been quiescent for with high levels of bFGF (10 times those required to 24 h. As shown in Figure 5, a 10-fold increase in the elicit a mitogenic response in 3T3 cells), no increase in rate of nuclear TS RNA synthesis was observed 30 min TS mRNA levels was observed over a period of 16 h. after addition of bFGF to cells, with a gradual reduc(Fig. 4). In other experiments, no response was ob- tion to basal levels over a period of 3 h. The rate of served even at a n earlier 2 h time point (data not transcription of the c-fos gene also increased by a factor shown). BC3H1 cells are, however, capable of increas- of approximately 10 within 15 min after addition of ing TS mRNA levels within 2 h after addition of 15% bFGF, with a gradual reduction thereafter (Fig. 5). Litcalf serum (Fig. 4). Thus, at least in these two cell lines, tle or no increase in the transcription rate of the a1 an association exists between elevated TS levels and gene of type I collagen was observed in response to cell proliferation. An inverse correlation between the bFGF, a finding consistent with the lack of suostantial level of TS in the culture medium of endothelial cells, increase in mRNA levels for this protein (Fig. 3).

DONOVIEL ET AL.

20 1001

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lime, min Fig. 5. Nuclear run-on analysis in Swiss 3T3 cells treated with bFGF. Cells were deprived of growth factors for 24 h. Labeled nuclear RNA was isolated after the indicated times of treatment with 300 pM bFGF and hybridized to double-stranded DNA immobilized on nitre cellulose discs. Values are expressed as parts per million input cpm. TS: human TS cDNA; fos: mouse v-fos genomic DNA. Colul(1): rat al(1) collagen cDNA.

An increase in TS mRNA stability contributes to elevated mRNA levels following bFGF stimulation Although an increase in the transcription rate of the TS gene in 3T3 cells can be shown in response to bFGF (Fig. 5), this increase was transient; we were therefore led to consider additional post-transcriptional effects of bFGF. As shown in Figure 6, when RNA synthesis was largely inhibited in 3T3 cells by the inhibitor of transcriptional initiation, DRB, TS mRNA levels decayed with a half-life of approximately 3-4 h. The addition of bFGF led to a doubling in the half-life of the mRNA. Thus, the increase in TS mRNA levels in response to bFGF results from a combination of transcriptional and post-transcriptional effects. A similar observation was made by Penttinen et al. (1988) in relation to the elevation of collagen mRNA levels induced in Swiss 3T3 cells by TGF-P. In the latter case, stabilization of collagen mRNA could be shown in confluent but not in subconfluent cells. The effect of cell density on the stability of TS mRNA that is induced by bFGF has not been assessed. As noted in Figure 6, the ratio of TS mRNA levels between bFGF-treated and untreated cells is less than that seen in Figure 1. This difference is due, in large part, to the higher basal TS levels in the cells used in Figure 6. Some variability in the rapidity with which Swiss 3T3 cells become quiescent after growth factor depletion, and in the response of these cells to bFGF, has been observed in these experiments, which spanned a period of almost 2 years. We do not know all of the factors that contribute to this variability but we

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Fig. 6. Temporal pattern of TS mRNA levels in DRB-treated cells. Subconfluent Swiss 3T3 cells were treated with bFGF (300 pM) for 2 h prior to addition of DRB (60 pM) at time 0. The ordinate presents the residual fraction of TS mRNA, normalized to the level at time 0. TS mRNA levels in control ).-.( and bFGF-treated (0-0) cells were determined by densitometric analyses of hybridization blots. An example is shown in the inset. Inset:TS mRNA identified by blot hybridization as a function of time in h in the absence (-1 or presence ( + ) of 300 pM bFGF.

suspect that phenotypic drift (despite the use of a single clone of 3T3 cells), different serum lots and differences in cell density can contribute to such variability.

DISCUSSION With the notable exception of TGF-P, which rapidly induces collagen and fibronectin synthesis in vivo and in vitro (Roberts et al., 1986; Ignotz and Massagub, 19861,growth factors have not been shown to stimulate transcription of genes coding for matrix proteins. In wound chambers implanted subcutaneously in rats, PDGF and bFGF increase production of granulation tissue but this effect is likely to be due to an increased cellularity and an increase in inflammatory cells elicited by those growth factors (Sprugel et al., 1987). Neither PDGF nor EGF increased collagen synthesis by NRK cells in culture (Roberts et al., 19861, although there may be an effect of PDGF on the relative proportion of collagen types synthesized by gingival fibroblasts (Narayanan and Page, 1983). In cultured rat bone bFGF stimulates DNA and total protein synthesis but inhibits collagen synthesis (Canalis et al., 1988). We were led to consider the effects of bFGF on TS gene expression since PDGF was shown to stimulate TS protein synthesis by rat SMC (Majack et al., 1985) and human glial cells (Asch et al., 1986) and to increase TS mRNA levels in SMC (Kobayashi et al.,

bFGF INDUCES THROMBOSPONDIN GENE EXPRESSION

1986; Majack et al., 1987) and in 3T3 cells (P. Framson and P. Bornstein, manuscript in preparation). Acidic FGF and bFGF are homologous heparin-binding mitogens that stimulate division of most mesoderm- and neuroectoderm-derived cells. FGFs also induce angiogenesis and neurite outgrowth (see Gospodarowicz et al., 1987; Thomas, 1987, for reviews). We have found that addition of bFGF to quiescent Swiss 3T3 cells leads to a substantial increase in mRNA levels for TS (Fig. 1).This increase results in large part from an increase in the rate of transcription of the TS gene (Fig. 5), but stabilization of TS mRNA (Fig. 6) contributes to the observed increase in steady-state mRNA levels. The molecular basis for the transcriptional activation of the TS gene, or for that matter of any gene, by bFGF is not known. Experiments with Swiss 3T3 cells suggest that tyrosine phosphorylation may be involved in eliciting the mitogenic effect of bFGF (Coughlin et al., 19881, but neither cis-acting elements nor transcription factors that are induced or activated by bFGF have been identified. Since we and others have isolated and partially sequenced clones that contain the human TS promoter (Donoviel et al., 1988; Laherty et al., 1989) we are in a position to examine this region of DNA for sequences that respond transcriptionally to bFGF. Although transcriptional activation appears to be the major mode of action of bFGF on TS gene expression, a significant increase in mRNA stability was also observed (Fig. 6). Hennessy et al. (1989) have recently reported the sequence of the 3’ untranslated region (3‘ UTR) of human TS mRNA. This region is over 2 kb in length and contains 37 UAUU or AUUU(A) motifs that have been proposed as mediators of rapid mRNA turnover in a number of cytokines and oncogenes (Shaw and Kamen, 1986; Meijlink et al., 1985; Caput et al., 1986). The 3‘ UTR of TS also contains a poly(U) stretch of 42 nucleotides, 40 of which are U (Hennessy et al., 1989). This poly(U) tract has the potential of hybridizing with the poly(A)tail in TS mRNA to form a loop of about 160 nucleotides. It has been proposed that this loop protects against 3’ exonucleolytic attack on mRNAs (Brawerman, 1989). Thus, TS mRNA has the structural potential to be subject to varying rates of turnover, and one basis for the increase in TS mRNA observed after treatment of 3T3 cells with bFGF could be a reduction in the rate of mRNA degradation. It is of interest that the mouse al(1) collagen gene also contains a number of A/T-rich sequences in its 3’ UTR, as well as a poly(T) tract (Mooselehner and Harbers, 1988),and that TGF-P increases al(1) collagen mRNA stability in 3T3 cells (Penttinen et al., 1988). The addition of cycloheximide to cells increases TS mRNA levels (Fig. 1; Majack et al., 1987). This characteristic is also true for the competence family of growth factor-responsive genes, also termed “immediate-early” response genes (Cochran et al., 1983; Lau and Nathans, 1987). Thus, basal levels of TS mRNA may be regulated in part by short-lived ribonucleases or by transcriptional repressors. However, in contrast to the effect of cycloheximide plus PDGF, which leads to “superinduction” of mRNA levels for TS (Majack et al., 1987) as well as for c-myc and c-fos (Kelly et al., 1983; Greenberg and Ziff, 1984), the addition of cycloheximide to bFGF for short time periods (30 min) re-

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duces the stimulation of TS mRNA to levels observed with either cycloheximide or bFGF alone (Fig. 1).We therefore surmise that a labile protein factor is required to mediate bFGF stimulation of TS mRNA levels. Since the major component of the effect of bFGF occurs at the level of transcription (Fig. 51, protein synthesis may be required to induce the synthesis of, or to activate, a transcriptional activator protein. Alternatively, in view of the rapid induction of TS by FGF, a short-lived protein involved in transcriptional regulation of TS may be modified by action of the growth factor. In contrast, prolonged treatment with bFGF and cycloheximide (4-8 h) results in markedly increased TS mRNA levels in comparison with those observed with either bFGF or cycloheximide alone (Fig. 2). We believe that, in response to prolonged stimulation with bFGF, cells down-regulate TS mRNA either by reduced transcription of the gene or by increased turnover of TS mRNA. This possibility is supported by recent observations showing that TS mRNA levels in 3T3 cells, stably transformed with bFGF, are very low and that these levels can be raised substantially by treatment of cells with cycloheximide (K. Judge and P. Bornstein, unpublished observations). The kinetics of the response of the TS gene to bFGF resemble those of the responses of c-fos and c-myc, proto-oncogenes whose expression contributes to progression of cells through the cell cycle (Curran et al., 1985). There are, indeed, a number of reasons to suggest that TS may serve, in some anchorage dependent cells, to facilitate progression of cells through the cell cycle. a) As discussed above, the TS gene responds positively and rapidly to bFGF and PDGF, suggesting that the protein may be required for transition of 3T3 cells and SMC through some part of the cell cycle. b) Interleukin 1,which also stimulates l3H1thymidine incorporation in 3T3 cells (Burch et al., 1989), rapidly increases TS mRNA levels in these cells (Donoviel and Bornstein, 1988). c) The stimulation of TS mRNA production by bFGF and PDGF is unusual since this stimulation does not extend to other cell surface-associated and matrix proteins such as collagen and fibronectin. d) In contrast to the observations in 3T3 cells, bFGF does not increase mRNA levels in BC3H1 cells which also do not respond mitogenically to this growth factor. e) TS acts synergistically with EGF to stimulate mitogenesis in SMC (Majack et al., 1986), and monoclonal antibodies to TS inhibit SMC proliferation and reduce cell surface-associated TS (Majack et al., 1988). f ) Heparin, which is a growth inhibitor for SMC, has varied effects, among them a reduction in cell surface-associated TS (Majack et al., 1985). g) Rapidly growing cells in culture synthesize more TS than do quiescent cells (Mumby et al., 1984). h) TS activates ribosomal protein S6 kinase and increases phosphatidyl inositol turnover in SMC (Scott-Burden et al., 1988). These effects are commonly seen in response to growth factors. If TS acts to facilitate cell cycle progression, how might this effect occur? We presume that TS must act in the environment of the cell surface since it is a secreted protein. It has been shown that TS, plasminogen, and tissue plasminogen activator (TPA) can form a ternary complex and that this complex markedly accelerates the ability of plasminogen to be converted to plasmin by TPA (Silverstein et al., 1985). Further-

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in the 3' untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Sci. USA, 83:1670-1674. Chiquet-Ehrismann, R., Kalla, P., Pearson, C.A., Beck, K., and Chiquet, M. (1988) Tenascin interferes with fibronectin action. Cell, 53:383-390. Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal. Biochem., 162:156-159. Cochran, B.H., Reffel, A.C., and Stiles, C.D. (1983) Molecular cloning of gene sequences regulated by platelet-derived growth factor. Cell, 33:939-947. Coughlin, S.R., Barr, P.J., Lousens, L.S., Fretto, L.J., and Williams, L.T. (1988) Acidic and basic fibroblast growth factor stimulate tyrosine kinase activity in vivo. J. Biol. Chem., 263:988-993. Curran, T., Peters, G., van Beveren, C., Teich, N.M., and Verma, I.M. (1982) FBJ murine osteosarcoma virus: identification and molecular cloning of biologically active proviral DNA. J. Virol., 44:674682. Curran, T., Bravo, R., and Miiller, R. (1985) Transient induction of c-fos and c-myc is a n immediate consequence of growth factor stimulation. Cancer Surveys, 4:655-681. Donoviel, D.B., and Bornstein, P. (1988) The thrombospondin gene is induced by basic fibroblast growth factor (bFGF) and interleukin 1 (IL-1). J. Cell. Biol., 107:596a. Donoviel, D., Framson, P., Eldridge, C., Cooke, M., Kobayashi, S., and Bornstein, P. (1988) Structural analysis and expression of the human thrombospondin gene promoter. J. Biol. Chem., 263:1859018593. Genovese, C., Rowe, D., and Kream, B. (1984) Construction of DNA sequences complementary to rat alpha-1 and alpha-2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry, 23352106216. Gospodarowicz, G., Neufeld, G., and Sehweigerer, L. (1987) Fibroblast growth factor: structural and biological properties. J. Cell. Physiol. LSuppl.1, 5:15-26. Greenberg, M.E., and Ziff, E.B. (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature, 311:433438. Groudine, M., Peretz, M., and Weintraub, H. (1981) Transcriptional regulation of hemoglobin switching in chicken embryos. Mol. Cell. Biol., Ir281-288. Frazier, B.A., Kim, D.D., Deckwerth, T.L., BaumgarHennessy, S.W., tel, D.M., Rotwein. P., and Frazier. W.A. (1989) Comalete thrombospondin mRNA sequence includes'potential regulatdry sites in the 3' untranslated region. J. Cell Biol., 1083729-736, ACKNOWLEDGMENTS Ignotz, R.A., and Massagu6, J. (1986) Transforming growth factorbeta stimulates the expression of fibronectin and collagen and their We thank Bradley Olwin and Stephen Hauschka for incorporation into the extracellular matrix. J . Biol. Chem., 261: bFGF and BC3H1 cells, Roger Kaspar and David Mor4337-4345. ris for the v-fos and G3PD probes, and Kathleen Doeh- Kaesberg, P.R., Ershler, W.B., Esko, J.D., and Mosher, D.F. (1989) ring for preparation of the manuscript. We also thank Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan. Helene Sage and Paul Framson for a critical review of J. Clin. Invest., 83:994-1001. the manuscript. K., Cochran, B.H., Stiles, C.D., and Leder, P. (1983) Cell-speThis work was supported in part by grants AM Kelly, cific regulation of the c-myc gene by lymphocyte mitogens and 11248, HL 18645 and DE 08229. D.B.D. was supported platelet-derived growth factor. Cell, 35:603-610. by grant PHS NRSA T32 GM07270 from NIGMS and Kobayashi, S., Eden-McCutchan, F., Framson, P., and Bornstein, P. (1986) Partial amino acid sequence of human thrombospondin as K.W.J. by a fellowship from the Washington Affiliate determined by analysis of cDNA clones: homology to malarial cirof the AHA. cumsporozoite proteins. Biochemistry, 25:8418-8425. Lahav, J. (1988) Thrombospondin inhibits adhesion of endothelial cells. Exp. Cell Res., 177:199-204. 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more, both TPA and urokinase enhance the binding of TS to plasminogen (Silverstein et al., 1986b). Thus, in a microenvironment in which both plasminogen and TPA are present, the introduction of TS could lead to the localized release of proteolytic activity at the cell surface. In regions of focal adhesions such proteolytic activity could contribute to the ability of an anchoragedependent cell to partially detach itself from a substratum in preparation for the cell rounding that is required for mitosis. In support of this conjecture, it has recently been shown by Murphy-Ullrich and Hook (1989) that the addition of soluble TS to adherent bovine aortic endothelial cells leads to a reduction in focal adhesions in these cells, although cell spreading was not affected. This effect was specific for TS and was inhibited by a monoclonal antibody to the heparinbinding domain of the protein and by heparin. A dynamic function for TS at the cell surface could be provided by the ability of cell surface-associated heparan sulfate proteoglycans to bind to TS and to internalize rapidly the protein (Murphy-Ullrich and Mosher, 1987). The ability of TS to serve as an attachment factor, but the failure of most cells to spread on a TS-coated substratum, would be consistent with a role for TS in destabilizing focal adhesions. Although we favor the view that TS participates in facilitating cell division by its effect on cell-substratum adhesion, TS may alternatively, or in addition, influence the interaction of growth factors with their receptors or may directly generate intracellular signals that complement those produced by growth factors. Finally, TS may be only one of a group of proteins that appears to destabilize cell-substratum adhesions, a group that includes tenascin (cytotactin) (Chiquet-Ehrissmann et al., 1988) and SPARC (osteonectin) (Sage et al., 1989).

bFGF INDUCES THROMBOSI'ONDIN GENE EXPRESSION

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