©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Requirements for Transforming Growth Factor- Regulation of the Pro-2(I) Collagen and Plasminogen Activator Inhibitor-1 Promoters (*)

(Received for publication, November 1, 1994; and in revised form, January 3, 1995)

Enoch Chang (1) Howard Goldberg (1)(§)

From the Division of Nephrology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Toronto M5G 1X8, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Experiments were designed to clarify the role of several proteins, junB, retinoblastoma protein (RB), and the transforming growth factor-beta (TGF-beta) receptors that are potential intermediates in TGF-beta activation of the alpha2(I) collagen promoter. Treatment of NIH-3T3 cells with TGF-beta increased the activity of a transiently transfected murine alpha2(I) collagen promoter (nucleotides -350 to +54) fused to a luciferase reporter gene 9-fold. Co-transfection of a junB stimulated the basal activity of the alpha2(I) collagen promoter 93-fold, respectively. Expression of antisense junB RNA attenuated the effect of TGF-beta. Simian virus 40 large T antigen, an inhibitor RB function, did not prevent TGF-beta effects on the alpha2(I) collagen promoter. A chimeric receptor containing the extracellular domain of the colony-stimulating factor-1 receptor and the intracellular domain of the type I TGF-beta receptor enhanced alpha2(I) collagen promoter activity 4.8-fold, whereas a similar chimera containing the type II receptor intracellular domain had much weaker effects. Similar results were obtained with a plasminogen activator inhibitor-1 promoter, previously shown to be activated by TGF-beta through AP-1 elements. We conclude that TGF-beta activates the alpha2(I) collagen and plasminogen activator inhibitor-1 promoters in NIH-3T3 cells through junB and the type I TGF-beta receptor kinase domain.


INTRODUCTION

Excessive deposition of extracellular matrix (ECM) (^1)in progressive renal disease, liver cirrhosis, and lung fibrosis results in permanent organ dysfunction (1, 2, 3, 4) . One of the major regulators of ECM deposition in fibrotic disease is transforming growth factor-beta (TGF-beta)(1, 2, 3, 4) , a 25-kDa homodimeric protein that belongs to a superfamily of multifunctional cytokines(5) . Among the effects of TGF-beta that enhance ECM deposition are increased transcription of the type I collagen genes (6, 7, 8) and a protease inhibitor that reduces ECM degradation, plasminogen activator inhibitor-1 (PAI-1)(9) . However, the mechanism whereby TGF-beta affects the above genes is incompletely understood. The goal of the present study was to determine the role of potential intermediates, junB, retinoblastoma protein (RB), and the type I and type II TGF-beta-receptor kinase domains, in TGF-beta action on the alpha2(I) collagen and PAI-1 promoters.

Binding sites for the transcription factor AP-1 were classically described as being protein kinase C response elements(10) . More recently, TGF-beta has also been shown to activate AP-1 elements in the PAI-1(11) , TGF-beta1(12) , TIMP-1(13) , and artificial promoters(14, 15) . TGF-beta induces the expression of c-fos, junB, and c-jun which bind to AP-1 sites(14, 16, 17, 18, 19, 20, 21) . Interestingly, junB expression was increased to a greater extent than c-jun expression by TGF-beta in several reports(14, 16, 17, 18, 19, 20) , including two out of three studies in NIH-3T3 cells(14, 20) . junB, which is about 50% homologous to c-jun(22) , could mediate both the positive and negative effects of TGF-beta on gene expression(23, 24) . However, junB has not been previously reported to transactivate the alpha2(I) collagen promoter. Rather, the effects of TGF-beta on type I collagen promoters have been attributed to nuclear factor-1(25) , SP1 (26, 27) , and unidentified transcription factors(26, 28) . Thus, it is of interest to determine whether junB might be involved in mediating the effects of TGF-beta on the alpha2(I) promoter.

TGF-beta-mediated growth arrest of epithelial cells in the G(1) phase of the cell cycle is manifested by inhibition of retinoblastoma protein (RB) phosphorylation(29) . Altered phosphorylation of RB causes it to release the proteins bound to it, including the transcription factor E2F-1 (30) and an inhibitor of SP1 (31) . Thus, changes in RB phosphorylation, in response to TGF-beta, could influence the expression of ECM genes. Experiments utilizing simian virus 40 (SV40) transformed cell lines, in which RB function is blocked(32) , or in cells lacking RB (33) show that TGF-beta is still able to stimulate PAI-1 gene expression. Nevertheless, it is important to study the role of RB in TGF-beta activation of type I collagen promoters, since TGF-beta modulates the activity of these promoters partly through SP1(26, 27) .

Three TGF-beta receptors designated types I-III were originally detected by I-TGF-beta cross-linking and subsequently cloned. The type III TGF-beta receptor is a proteoglycan, betaglycan, that is involved in ligand presentation(34) . The type I and II TGF-beta receptors, found on most cell types, possess intrinsic serine-threonine kinase activity(35, 36, 37) . TGF-beta-resistant, mutant mink lung cells developed by Massague and colleagues(15) , demonstrate that both the type I and type II TGF-beta receptors are required for TGF-beta-mediated changes in PAI-1 gene expression and growth inhibition. Furthermore, the type II TGF-beta receptor has recently been shown to phosphorylate the type I TGF-beta receptor in response to TGF-beta(38) . However, these experiments do not indicate whether the type I or type II receptor or both are able to phosphorylate the downstream substrates that participate in the activation of type I collagen genes by TGF-beta.

We report here that junB is necessary for TGF-beta to increase transcription from the a2(I) collagen and PAI-1 promoters and that inhibition of RB function by SV40 large T antigen did not prevent TGF-beta from acting on either promoter. Construction of chimeric receptors showed that the type I TGF-beta-receptor kinase domain was able to activate both promoters.


MATERIALS AND METHODS

Cell Culture

NIH-3T3 cells, obtained from American Type Culture Collection (ATCC, Rockville, MD), were grown in Dulbecco's modified Eagle's medium with 10% calf serum (Life Technologies, Inc.).

Transient Transfection and Luciferase Measurement

NIH-3T3 cells were transfected as described(39) . A beta-galactosidase expression vector, TKbeta (Clontech, Palo Alto, CA) for the experiments in Fig. 1Fig. 2Fig. 3Fig. 4and Table 1or CMVbeta (Clontech) for some early experiments in Fig. 3and Table 1, was included to control for transfection efficiency. After 16 h the cells were washed three times with phosphate-buffered saline and then 10 ml of serum free media, which was composed of Dulbecco's modified Eagle's medium/F12 (Life Technologies, Inc.), 1 g/liter albumax (Life Technologies, Inc.), 60 mg/liter mannose 6-phosphate, 10 mg/liter transferrin, and trace metals (Life Technologies, Inc.), was added. Mannose 6-phosphate was included to inhibit the activation of any latent TGF-beta that might be secreted by the fibroblasts(40) . Three h latter TGF-beta, 4 ng/ml (R and D Systems, Minneapolis), or vehicle (1 mg/ml bovine serum albumin, 4 mM HCl) was added. After a further 21 h, luciferase and beta-galactosidase activity was measured as described(39) . The results are expressed relative to the luciferase activity in vehicle-treated cells transfected with the indicated construct after taking into account differences in transfection efficiency. Each transfection was repeated at least three times with three dishes/group.


Figure 1: Effect of TGF-beta on deletion mutants of the alpha2(I) collagen promoter. NIH-3T3 cells were transfected with 14 µg of plasmid DNA containing the indicated segments of the alpha2(I) collagen promoter fused to a luciferase reporter gene along with a beta-galactosidase expression vector (TKbeta) and then stimulated with TGF-beta (4 ng/ml) or vehicle. Luciferase was assayed after 21 h in serum-free media and the results expressed relative to that in cells treated with vehicle for each construct. Each value (average ± S.E.) represents the results from three independent experiments. The asterisk indicates luciferase activity from TGF-beta-stimulated cells is statistically greater than the luciferase activity measured in vehicle-treated cells transfected with the same construct (Student's t test, p < 0.01).




Figure 2: Effect of junB on deletion mutants of the alpha2(I) collagen promoter. NIH-3T3 cells were co-transfected with 14 µg of plasmid DNA containing the indicated segments of the alpha2(I) collagen promoter fused to a luciferase reporter gene, either pRcCMV or the junB expression vector pJUNB, and a beta-galactosidase expression vector, TKbeta. Luciferase was assayed after 21 h in serum-free media and the results expressed relative to each construct transfected with the pRcCMV vector. Each value (average ± S.E.) represents the results from three independent experiments. The asterisk indicates luciferase activity from pJUNB transfected cells is statistically greater than the luciferase activity measured in pRcCMV-transfected cells that were also co-transfected with the same alpha2(I) collagen reporter gene construct (Student's t test, p < 0.05).




Figure 3: Lack of inhibition of TGF-beta effects on the alpha2(I) collagen and PAI-1 promoters by SV40 large T antigen. A, NIH-3T3 cells were co-transfected with 14 µg of pH 6 (nt -350 to +54 of the alpha2(I) collagen promoter fused to luciferase), either pSG5, expression vectors for SV40 T antigen, pTAG, or a non-RB binding SV40 T antigen mutant (pMUTTAG), and a beta-galactosidase expression vector (TKbeta or CMVbeta). The cells were stimulated with TGF-beta (4 ng/ml) for 21 h in serum-free media and luciferase measured. The results are expressed relative to the luciferase activity in vehicle-treated cells transfected with pSG5. Each value (average ± S.E.) represents the results from three independent experiments. The asterisk indicates luciferase activity from TGF-beta-stimulated cells is statistically greater than the luciferase activity measured in vehicle-treated cells transfected with the same constructs (Student's t test, p < 0.01). B, similar experiments were performed with pPAI-1 (nt -699 to +54 of the PAI-1 promoter fused to luciferase).




Figure 4: Comparison of the function of the type I and type II TGF-beta receptor intracellular domains. NIH-3T3 cells were co-transfected with 6 µg of pH 6 (nt -350 to +54 of the alpha2(I) collagen promoter fused to a luciferase reporter gene) or pPAI-1 (nt -699 to +54 of the PAI-1 promoter fused to a luciferase reporter gene), 7 or 14 µg of either pCHIMONE, a chimera containing the extracellular and transmembrane domains of the CSF-1 receptor, and the type I TGF-beta-receptor kinase domain, pCHIMTWO a chimera containing the type II TGF-beta-receptor kinase domain or pKN, a chimera containing a kinase-negative mutant of the type I TGF-beta receptor intracellular domain, and 5 µg of the beta-galactosidase expression vector TKbeta. Luciferase was measured after 21 h in serum-free media, and the results are expressed relative to pH 6 or the PAI-1 promoter co-transfected with a similar amount of pKN. Each value (average ± S.E.) represents the results from three independent experiments. The asterisk indicates luciferase activity from pCHIMONE- or pCHIMTWO-transfected cells is statistically greater than the luciferase activity measured in pKN-transfected cells that were also co-transfected with the same reporter gene construct (Student's t test, p < 0.05)





Plasmids

The plasmids, pH 6, 8, 10, and 39, containing the following segments -350 to +54, -224 to +54, -108 to +54, and -41 to +54, respectively, of the murine alpha2(I) collagen promoter fused to a luciferase reporter gene in the vector, pA(3)LUC, have been previously described(39) . pA(3)LUC (41) was the generous gift of Dr. E. Chester Ridgeway and Dr. William Wood. The -699 to +64 segment of the human PAI-1 promoter (42) was amplified by polymerase chain reaction (PCR) from human genomic DNA (Novagen, Madison, WI) and cloned into pA(3)LUC between Asp and HindIII to form pPAI-1. All PCRs used a low concentration of dNTPs (50 µM) (43) and Ultma DNA polymerase (Perkin Elmer) to reduce PCR errors.

Expression vectors were prepared by ligating the relevant cDNA into a blunt-ended HindIII site of pRcCMV (Invitrogen, San Diego, CA). The cDNAs for pJUN, pJUNB, pFOS, and pTAG were obtained as follows: a 2.6-kilobase EcoRI c-jun cDNA from pJAC.1 (44) (ATCC), a 1570-base pair HindIII-EcoRI junB cDNA fragment from ATCC clone 465.20(22) , a 2.1-kilobase EcoRI rat c-fos cDNA from pc-fos (45) , a kind gift of Dr. Peter Maxwell and Dr. Tom Curran, and a 2.9-kilobase BamHI SV40 T antigen from pBRSV (ATCC). pREVJUN, pREVJUNB, and pREVFOS express c-jun, jun B, and c-fos cDNA in the reverse orientation, respectively. For pREVFOS c-fos cDNA was amplified by PCR from a murine fibroblast cDNA library (Stratagene, La Jolla, CA).

Site-directed mutagenesis was performed as described(39) . Mutant sequences were confirmed by dideoxy sequencing with a Sequenase kit (United States Biochemical, Cleveland, OH).The following mutations were produced: pNFN, in which 5`-TGGCA-3`, bases -305 to [minus[301 from the murine alpha2(I) collagen promoter, was changed to 5`-TTTAA-3`; pMUTTAG, in which lysine 107 from SV40 large T antigen was changed to glutamine(46) ; pKN, in which lysine 232 from the ALK5 (35) type I TGF-beta-receptor kinase domain incorporated in the chimeric receptor described below was changed to arginine; and pMUTPLA2, in which serine-505 of murine cPLA(2)(47) (a generous gift of J. Knopf, The Genetics Institute, Cambridge, MA) was changed to alanine.

pCSF was created by ligating cDNA encoding the extracellular and transmembrane domains of the murine colony stimulating factor-1 (CSF-1) receptor (48) amplified by PCR from a murine fibroblast cDNA library (Stratagene) into the EcoRI and BglII sites of the expression vector, pSG5 (Stratagene). A BamHI site was included in the 3` end of the CSF-1 cDNA. To create pCHIMONE and pCHIMTWO, cDNA encoding the intracellular domain of the type I TGF-beta receptor, ALK-5 (also called R4)(35, 37) , and the type II receptor (36) , respectively, were amplified by PCR from a human placenta cDNA library (Stratagene) and cloned into the BamHI site of pCSF.


RESULTS

TGF-beta Regulates the alpha2(I) Collagen and PAI-1 Promoters Through JunB

The -350 to +54 segment of the murine alpha2(I) collagen promoter was selected for analysis, since this fragment was sufficient for tissue-specific expression in transgenic mice (with the exception of aberrant brain expression)(39, 49) , and earlier investigations mapped a TGF-beta-response element to this fragment(25) . This alpha2(I) collagen promoter segment was fused to a luciferase reporter gene that has three SV40 poly(A) sites upstream of the multiple cloning site to prevent read-through transcription(41) . Transient transfection of the resulting reporter gene construct into serum starved NIH-3T3 cells was performed, in the presence or absence of TGF-beta (4 ng/ml).

Exposure of NIH-3T3 cells to TGF-beta enhanced luciferase activity driven by the -350 to +54 segment of the alpha2(I) collagen promoter and two smaller segments (-224 to +54 and -108 to +54) 9-, 5-, and 5-fold, respectively (Fig. 1), consistent with previous reports on type I promoters (25, 26, 27, 28) and nuclear run-off studies of the type I collagen genes(6, 7, 8) . The specificity of the response is shown by the minimal effect of TGF-beta on the -41 to +54 segment of the alpha2(I) collagen promoter (Fig. 1). Furthermore, TGF-beta did not increase the activity of the thymidine kinase promoter linked to beta-galactosidase that was used to control for transfection efficiency. Thus, TGF-beta affected two regions of the alpha2(I) collagen promoter, -350 to -224 and -108 to -41.

To determine the role of junB, c-jun, and c-fos in TGF-beta signaling, we co-transfected the alpha2(I) collagen promoterreporter gene noted above with vectors directing the expression of these transcription factors in the sense or the antisense orientation. As depicted in Table 1, co-expression of c-jun or junB with the alpha2(I) collagen promoter resulted in 4- and 93-fold increases in luciferase activity, respectively. TGF-beta did not increase the activity of the alpha2(I) collagen promoter co-transfected with the junB expression vector further. Expression of c-fos did not affect the basal activity of the alpha2(I) collagen promoter, but augmented the effect of TGF-beta from 3.8- to 6.2-fold.

Antisense RNA effectively reduces the expression of endogenous genes (50) . The results of experiments aimed at inhibiting junB expression are summarized in Table 1. Co-transfection of an expression vector for antisense junB RNA diminished the action of TGF-beta on the alpha2(I) collagen promoter from 3.8- to 1.2-fold (Table 1). Expression of antisense c-jun or c-fos RNA had no effect. Luciferase activity driven by the alpha2(I) collagen promoter was elevated 11-fold by expression of antisense junB, but only 1.8-fold by expression of antisense c-jun RNA. This effect is discussed below. To evaluate for nonspecific effects, an inactive mutant of cPLA(2), in which serine 505 was mutated to alanine(48) , was co-transfected. It had no effect on basal or TGF-beta-stimulated expression of the reporter gene (Table 1). Similar results were obtained with the human PAI-1 promoter (nucleotides (nt): -699 to +64), except that junB had a slightly smaller effect (53-fold) and c-jun had a greater effect (22-fold) (Table 1).

The effect of junB on several deletion mutants of the alpha2(I) collagen promoter was tested. As shown in Fig. 2, co-transfection of junB increased activity of the -350 to +54, -224 to +54, -108 to +54, and -41 to +54 segments of the alpha2(I) collagen promoter 50-, 62-, 22-, and 2.7-fold, respectively. Thus, the effects of junB are specific and involve one of the segments, (-108 to -41), of the alpha2(I) collagen promoter that is affected by TGF-beta.

It was previously reported that a 3-base pair mutation in an nuclear factor-1-binding site in the murine alpha2(I) collagen promoter abolished TGF-beta effects on this promoter(25) . We tested the same mutation under the present experimental conditions. In accordance with previous results, the 3-base pair mutation reduced basal promoter activity to 26%, but the mutation did not alter the effect of TGF-beta on the alpha2(I) collagen promoter (not shown).

TGF-beta Activates the alpha2(I) Collagen and PAI-1 Promoter Independently of the Retinoblastoma Protein

To examine the the role of RB in TGF-beta action, we co-transfected the alpha2(I) collagen promoter-reporter gene with an expression vector for SV40 large T antigen, which inhibits RB function by binding to it(32) . As illustrated in Fig. 3A, luciferase activity from the alpha2(I) collagen promoter was elevated 6-fold by SV40 large T antigen, but TGF-beta was still able to further increase promoter activity to a similar degree as in vector-transfected cells. Similar results were obtained with the PAI-1 promoter (Fig. 3B). A mutant SV40 large T antigen in which lysine 107 is changed to glutamine (50) is unable to bind RB. Expression of this mutant with the plasmid pMUTTAG had no effect on either promoter (Fig. 3, A and B).

The Type I TGF-beta Receptor Kinase Domain Activates the alpha2(I) Collagen and PAI-1 Promoters

To determine the relative roles of the TGF-beta type I and type II receptor kinase domains in activating the alpha2(I) collagen and PAI-1 promoters, we constructed chimeric receptors. The extracellular and transmembrane domains of the CSF-1 receptor were fused to either the human type I or type II TGF-beta-receptor cytoplasmic kinase domains. The type I TGF-beta receptor used in these studies, ALK-5 (also referred to as R4), is the only type I TGF-beta receptor that mediates increased gene expression in response to TGF-beta(35, 37) . Expression of the extracellular domain of the CSF-1 receptor alone resulted in increased luciferase activity from either the alpha2(I) collagen or PAI-1 promoters in NIH-3T3 fibroblasts (not shown). Therefore, the results were expressed relative to a chimeric receptor containing a kinase negative mutant of the type I receptor, pKN. As shown in Fig. 4, transfection of 7 or 14 µg of the type I receptor chimera (pCHIMONE) stimulated the alpha2(I) collagen promoter 3.4- or 4.8-fold compared to pKN. The chimera containing the type II kinase (pCHIMTWO) had much smaller effects, 1.08- or 2.52-fold stimulation when 7 or 14 µg were transfected. Co-transfection of pCHIMONE and pCHIMTWO increased the activity of the alpha2(I) collagen promoter 12-fold compared to pKN. Addition of macrophage colony-stimulating factor, the ligand for the CSF-1 receptor, had no further effects in the cells transfected with the chimeric receptors (not shown). The lack of effect of macrophage colony-stimulating factor may be due to secretion of macrophage colony-stimulating factor by fibroblasts or receptor autoactivation when high levels of receptor are expressed. Similar results were found with the PAI-1 promoter (Fig. 4).


DISCUSSION

We have shown that junB is a mediator of TGF-beta action on the murine alpha2(I) collagen promoter. First, junB or c-jun transactivated the alpha2(I) collagen reporter gene. Consistent with a role for junB in TGF-beta action, expression of c-fos augmented the effect of TGF-beta from 3.8- to 6.2-fold, presumably by dimerizing with junB (Table 1). Importantly, expression of antisense junB RNA, but not antisense c-jun or c-fos RNA, diminished the effect of TGF-beta (Table 1). The relevance of the effects of junB on the alpha2(I) collagen promoter are further reinforced by the similar results obtained when a promoter previously shown to be regulated by TGF-beta through AP-1 sites that can bind junB, the PAI-1 promoter, was tested (Table 1).

The results of the present study do not distinguish between a requirement for new junB transcription versus dephosphorylation and activation of pre-existing junB. Cycloheximide, a protein synthesis inhibitor, abolishes the effect of TGF-beta on type I collagen mRNA levels in some studies(6, 51) , but not others(52, 53) . These observations suggest that depending on the experimental conditions, TGF-beta is able to exert its effects by increasing the synthesis of new transcription factors, such as junB, or by modifying pre-existing transcription factors. By analogy with other agonists, it is possible that TGF-beta may activate junB by dephosphorylating it near its DNA-binding domain (54) .

Other factors, in addition to junB, may be involved in TGF-beta activation of the alpha2(I) collagen promoter. The effects of c-jun and junB on the alpha2(I) collagen promoter may be indirect, since the murine alpha2(I) collagen promoter fragment tested has no consensus AP-1 sites, unlike the PAI-1 promoter. Furthermore, a role for other transcription factors, in addition to junB, in mediating the effects of TGF-beta is not excluded by the present experiments.

Phorbol esters (activators of protein kinase C) and platelet-derived growth factor (PDGF) are known to induce the expression of both c-jun and junB(55) but not the type I collagen genes. Addition of phorbol esters or epidermal growth factor, which also activates a receptor tyrosine kinase like PDGF, to fibroblasts inhibits type I collagen gene expression(56, 57) . Therefore, the ability of PDGF or phorbol esters to elevate c-jun and junB may be opposed by other effects that inhibit alpha2(I) collagen gene expression. Alternatively, TGF-beta may provide a second signal to the alpha2(I) collagen gene, in addition to altered junB activity, which is not available to cells exposed to PDGF or phorbol esters.

Expression of antisense junB RNA increased alpha2(I) collagen or PAI-1 promoter activity. This paradoxical effect may be explained by the release of c-jun from inactive c-jun-junB heterodimers, when the level of junB is reduced. The cells used in the present experiments probably contained significant levels of c-jun and junB, since they were serum starved for only 3 h prior to the addition of TGF-beta. However, the effect of antisense junB was larger than that of expressing c-jun indicating there is an additional unexplained effect of expressing junB cDNA.

The TGF-beta1 gene contains a functional AP-1 site in its promoter(12) . However, the effects of c-jun and junB are not likely to be due to increased release of active TGF-beta from NIH-3T3 fibroblasts. Most cells release TGF-beta in a latent form that requires activation by proteases(40) . In particular, NIH-3T3 fibroblasts transformed by p21 were found to secrete only latent TGF-beta (58) . Furthermore, mannose 6-phosphate, which has previously been shown to inhibit TGF-beta processing, was included in the cell culture media during the transfections(40) .

The present results differ from some previous reports on the alpha2(I) collagen promoter. A mutation in the alpha2(I) collagen promoter nuclear factor-1 site around -300, previously described to eliminate the effect of TGF-beta(25) , did not prevent TGF-beta activation of the alpha2(I) collagen promoter under the present experiments. We observed a 2-fold decrease in the effect of TGF-beta when the alpha2(I) collagen promoter was deleted from -350 to -224 to +54 consistent with a recent report on the human alpha2(I) collagen promoter(26) . However, we also detected an additional effect of TGF-beta in the -108 to -39 region, which appeared to be mediated through junB. The discrepancies between the present data and previous results with the alpha2(I) collagen promoter are probably due to differences in the experimental conditions and the cell types studied.

The results in Fig. 3demonstrate that inhibition of RB function does not block TGF-beta action on the alpha2(I) collagen or the PAI-1 promoters. Indeed, the enhancement of alpha2(I) collagen and PAI-1 promoter activity by SV40 T antigen, but not a non-RB-binding SV40 large T antigen mutant, raises the possibility that RB may tonically inhibit these promoters, perhaps by modifying SP1 activity(31) . Since SV40 large T antigen has many potential effects, further experiments will be required to settle this point.

This is the first report concerning an active chimera between the extracellular domain of a tyrosine kinase receptor and the intracellular domain of a serine-threonine kinase receptor. The results indicated that the type I TGF-beta-receptor kinase domain is sufficient to convey the TGF-beta signal to the alpha2(I) and PAI-1 promoters (Fig. 4). A similar chimera containing the kinase domain from the type II TGF-beta receptor had a lesser effect and was active only when large amounts were transfected. The recently reported phosphorylation of the type I TGF-beta receptor in response to TGF-beta (38) and the apparent determination of the specificity of responses to TGF-beta family members by the type I rather than the type II TGF-beta receptor (38) are consistent with the present experiments. However, the data presented here do not contradict the observed requirement for intact type I and type II TGF-beta receptors in vivo as demonstrated by TGF-beta signaling in mutant cell lines(15) . Under normal circumstances, the type II TGF-beta receptor is needed for ligand binding to the type I receptor and to phosphoylate the type I receptor(38) . In fact, the increased activation of the a2(I) collagen or PAI-1 promoter noted when both chimeric receptors were transfected together could be due to phosphorylation of the type I receptor by the type II receptor(38) ,

In summary, the results we obtained suggest that TGF-beta action on the alpha2(I) collagen and PAI-1 promoters involves the type I TGF-beta-receptor kinase domain and junB, but not changes in RB function.


FOOTNOTES

*
This work was supported by grants from the Baxter Healthcare Extramural Grant Program and The Kidney Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Nephrology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada.

(^1)
The abbreviations used are: ECM, extracellular matrix; TGF-beta, transforming growth factor-beta; PAI-1, plasminogen activator inhibitor-1; nt, nucleotides; PCR, polymerase chain reaction; RB, retinoblastoma protein; cPLA(2), cytosolic phospholipase A(2); CSF-1, colony-stimulating factor-1; PDGF, platelet-derived growth factor; SV40, simian virus 40.


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