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Hypertension. 1998;31:986-994

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(Hypertension. 1998;31:986-994.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Transforming Growth Factor-ß and Receptor Tyrosine Kinase–Activating Growth Factors Negatively Regulate Collagen Genes in Smooth Muscle of Hypertensive Rats

Paula Bray; Alex Agrotis; ; Alex Bobik

From Baker Medical Research Institute, Alfred Hospital, Prahran, Victoria, Australia.

Correspondence to Dr A. Bobik, Baker Medical Research Institute, Commercial Road, PO Box 348, Prahran, Victoria 3181 Australia. E-mail Alex.Bobik{at}alice.baker.edu.au


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract—Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-ß (TGF-ß) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-ß on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-ß isoforms -ß1, -ß2, or -ß3 induced rapid, transient elevations in mRNAs encoding collagens {alpha}1(I), {alpha}2(I), and {alpha}1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen {alpha}1(III)] to ninefold [collagen {alpha}1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-ß isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-ß1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-ß isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-ß did not affect the time course or magnitude of the changes in collagens {alpha}1(I), {alpha}2(I), or {alpha}1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-ß1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-ß, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-ß responsiveness.


Key Words: : • transforming growth factor-ß • receptor protein–tyrosine kinase • collagen • genes, c-myc • muscle, smooth, vascular • rats, inbred SHR


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Primary hypertension in humans and experimental animals is frequently accompanied by hypertrophy of the arterial vasculature.1 2 3 Multiple mechanisms contribute to this hypertrophy, and in vessels of SHR the contributing processes include smooth muscle cell proliferation,3 4 DNA endoreduplication leading to polyploidy,5 6 and cellular hypertrophy.4 6 Extracellular matrix accumulation due to increases in collagens and other extracellular proteins can also contribute to the vascular hypertrophy,7 but in vessels of SHR, collagen content is lower than in similarly aged normotensive WKY rats.8 9 10 However, other extracellular matrix proteins such as elastin and the electrolyte content in vessels of SHR are normal.

The reasons as to why vessel collagen content, such as that of collagen type I, is lower in SHR than in WKY rats are not known but could involve defects in TGF-ß–VSMC signaling. The closely related isoforms of TGF-ß, -ß1, -ß2, and -ß3, known to be produced by mammalian cells, are multipotential peptide growth and differentiation factors, which can stimulate many cell types to produce a variety of extracellular matrix proteins, including fibronectins, proteoglycans,11 and various collagens.12 13 14 TGF-ß1 is known to be produced by vessels of normotensive and genetically hypertensive animals, both during the development of hypertension and with increasing age.15 There is also substantial evidence that indicates that TGF-ß–induced growth responses in VSMCs from SHR are abnormal compared with those in WKY. For example, TGF-ß1 does not inhibit the mitogenic effects of growth factors that activate receptor tyrosine kinases such as platelet-derived growth factors (PDGFs), bFGF, or EGF on VSMCs of SHR; rather it enhances their mitogenic effects.16 TGF-ß1 also fails to autoactivate its own gene in these cells.16 Whether collagen genes are also differentially affected by TGF-ß1 or the other two isoforms in VSMCs of SHR and WKY is not known.

In this study, we examined the possibility that TGF-ß–induced collagen gene activation was impaired in VSMCs of SHR. Furthermore, because the temporal effects of TGF-ß on collagen gene activation in VSMCs have been incompletely defined, our aims were twofold: (1) to define the effects of the three TGF-ß isoforms on vascular smooth muscle collagen gene expression and (2) to determine whether the previously observed differential effects of TGF-ß1 on proliferation and TGF-ß1 gene activation in VSMCs of SHR and WKY16 extend to collagen gene responses. Specifically, we evaluated the effects of TGF-ß1, -ß2, and -ß3 on procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels in VSMCs of SHR and WKY. Their effects were compared with those induced by receptor tyrosine kinase (RTK)-activating growth factors, which can stimulate TGF-ß1 gene activation.16 Our results indicate that the TGF-ß isoforms transiently reduce mRNAs encoding these respective procollagens in VSMCs of SHR to below basal levels while causing transient upregulation in VSMCs of WKY. Similar effects on collagen mRNA levels were observed with EGF and bFGF. The differences in TGF-ß–regulated collagen gene expression between VSMCs of WKY and SHR appeared to be associated with overexpression of the c-myc oncoprotein in the latter cells.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
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Animals
Male SHR and WKY rats (weight, 250 to 300 g) were obtained from the Baker Medical Research Institute Biology Research Unit. They were bred from stock originally supplied by Y. Yamori. The animals were deeply anesthetized with halothane before opening of the abdominal and thoracic cavities and removing the aorta. This procedure was approved by the Baker Institute-Alfred Hospital Animal Experimentation Committee and conformed to the guidelines of the Australian National Health and Medical Research Council.

Materials
Fetal calf serum (FCS), penicillin G, and Dulbecco's phosphate-buffered saline (PBS) were purchased from the Commonwealth Serum Laboratories. Dulbecco's modified Eagle's medium (DMEM) and tissue culture dishes were obtained from FLOW Laboratories and Sterilin Ltd, respectively. Collagenase type 1, elastase, and EGF were purchased from Sigma Chemical Co. Porcine TGF-ß1 was obtained from British Bio-Technology Ltd and TGF-ß2 and TGF-ß3 were from Celtrix. bFGF, bovine serum albumin (BSA), and the random primed DNA labeling kit were purchased from Boehringer-Mannheim. Rat procollagen {alpha}1(I) and {alpha}2(I) cDNAs were provided by Dr D. Rowe, University of Connecticut Health Center. A rat procollagen {alpha}1(III) cDNA was obtained from Dr E. Vuori, University of Turku, Finland. The c-myc probe, a 1.45 kb Sac I/Hind III fragment from mouse cDNA, was obtained from Dr P. Fuller, Prince Henry's Institute for Medical Research, Melbourne, Australia.

Culture of Aortic Smooth Muscle Cells
Primary cultures of VSMCs were prepared by enzyme dispersion of aortic media from 12- to 14-week-old SHR and WKY rats as previously described.17 Examination of the confluent cell cultures by phase-contrast microscopy indicated formation of a "hill-and-valley" pattern, a well-known growth characteristic of VSMCs in culture. Their identity as VSMCs was confirmed by immunocytochemical staining, using antibodies to smooth muscle {alpha}-actin. VSMCs from the primary cultures were passaged every week in 10% FCS/DMEM and used in experiments up to the 4th passage. DMEM without serum was used during incubation of VSMCs with growth factors.

RNA Isolation and Northern Blot Analyses
Total RNA was extracted from confluent VSMC cultures (60-mm dishes) by the guanidinium thiocyanate method,18 and 5 to 15 µg was electrophoresed in 1% agarose/(2.2 mol/L) formaldehyde gels. After electrophoresis, the gels were treated for 20 minutes with 50 mmol/L NaOH, neutralized by soaking for 20 minutes in a 1.5 mol/L NaCl, pH 7.4, solution containing 0.5 mol/L Tris, and then equilibrated with 20x SSC (20x SSC=3 mol/L NaCl and 0.3 mol/L Na citrate, pH 7.0) for 20 to 40 minutes. The RNA was then transferred to 0.45-µm Biotrans membranes (ICN), UV fixed for 5 minutes and then baked at 80°C under vacuum for 1 to 2 hours. Prehybridization was carried out at 65°C for 1 to 4 hours in a solution of 7% SDS, 1% bovine serum albumin, 0.5 mol/L NaHPO4, and 1 mmol/L EDTA. Hybridization was carried out at 65°C for 15 hours in the same buffer containing 32P-labeled cDNA probes (1 to 1.5x106cpm/mL), prepared using the random priming method as previously described.19 Filters were subsequently washed three times at 65°C for 30 minutes (each wash) in 1% SDS, 40 mmol/L NaHPO4, and 1 mmol/L EDTA before being sealed in plastic and exposed to Kodak X-Omat AR film with intensifying screens for 4 to 48 hours at -70°C. The resulting autoradiographs were analyzed by laser densitometry at 600 nm or a visual phosphorimage analysis system. The intensity of ribosomal 28s and 18s bands visualized under UV light were used to monitor equal loading of RNA onto the agarose gels.

Immunohistochemical Visualization of Nuclear c-myc Protein Accumulation
WKY ({approx}2x103 cells/cm2) and SHR VSMCs ({approx}.07x103 cells/cm2) were plated onto 30-mm tissue culture plates containing 22-mm sterile glass coverslips in 2 mL of 10% FCS/DMEM. The cells were grown for an identical time, reaching in both instances {approx}70% to 80% confluence before deprivation in DMEM for 48 hours. Two to 6 hours after addition of growth factors (see "Results"), the cells were fixed in acetone at -20°C for 30 minutes and then washed twice with PBS, pH 7.4, for 5 minutes. c-myc–immunoreactive proteins were detected with a c-myc mouse monoclonal IgG (Santa Cruz), the Vectastain ABC kit (Vector Laboratories), and preabsorbed anti-mouse IgG (Sigma Chemicals). The slides were mounted in Depex before microscopic examination for accumulation of c-myc in nuclei of the VSMCs. Only c-myc–immunoreactive cells were counted and the percentage containing immunoreactive peptides in the nucleus determined.

Statistical Analyses
The significance of differences within or between SHR and WKY were assessed either by one-way ANOVA followed by unpaired t tests20 or Kruskal-Wallis ANOVA on ranks. Results are the mean±SEM of three to five independent experiments.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
TGF-ß Isoforms and Collagen mRNA Regulation in SHR and WKY
Because collagen {alpha}1(I) and collagen {alpha}2(I) genes contain AP-1 binding sequences that can be activated by TGF-ß,21 22 we initially investigated the effects of the three TGF-ß isoforms (ß1, ß2, ß3) on collagen mRNA levels in confluent, quiescent VSMCs of SHR. All three TGF-ß isoforms, used at a concentration (1 ng/mL) that we have previously shown for TGF-ß1 to enhance the mitogenic effects of growth factors on these cells,16 failed to elevate collagen mRNA levels over a 24-hour period. Rather, only reductions were apparent, detected during prolonged exposure of the Northern blot filters to x-ray film (Fig 1Down, top). Such reductions were induced by all three TGF-ß isoforms and were most apparent 6 to 12 hours after exposure of the cells to the TGF-ß isoforms; however, the falls were greatest and most sustained with procollagen {alpha}2(I) mRNA, averaging 45% to 60% and persisting for at least 24 hours (Fig 1Down). With procollagen {alpha}1(I) mRNA, the reductions induced by the TGF-ß isoforms ranged from 15% to 35%, were more transient in nature, and by 24 hours were no longer significantly different from controls (P>.05; Fig 1Down). Transient reductions were also observed with procollagen {alpha}1(III) mRNA levels during exposure of the VSMCs to the TGF-ß isoforms; maximal reductions occurred at 6 hours, and by 24 to 48 hours mRNA amounts had returned to control levels.



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  		Figure 1. TGF-ß–induced changes in procollagen mRNAs in VSMCs of SHR (top). Northern blots of mRNA encoding procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) after exposure of confluent, quiescent cell cultures to TGF-ß1, -ß2, or -ß3 (1 ng/mL) for 2 and 6 hours; 28s and 18s represent ribosomal RNA monitoring equivalent gel loading and C represents control, unstimulated VSMCs. Lower panels represent the early time-dependent reductions in {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels induced by the three TGF-ß isoforms, with the subsequent recovery in the ensuing 42-hour time period.

When confluent, quiescent cell cultures of WKY rats were exposed to the TGF-ß isoforms, marked early increases in procollagen mRNA levels were observed (Fig 2Down). The increases in {alpha}1(I) and {alpha}2(I) mRNAs were greatest at {approx}2 hours; the magnitude of such increases in mRNAs and their temporal pattern of responses were essentially similar between the TGF-ß isoforms. The effects of the TGF-ß isoforms were, however, greatest on procollagen {alpha}1(I) mRNA, in which the 2-hour increases in mRNAs were up to ninefold. All three TGF-ß isoforms induced smaller increases in procollagen {alpha}2(I) and {alpha}1(III) mRNA levels, which averaged approximately threefold and fourfold, respectively (Fig 2Down). After 24 to 48 hours of exposure to the TGF-ß isoforms, the levels of mRNAs encoding all three collagens had largely returned to levels present in control, unexposed cells.



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  		Figure 2. TGF-ß–induced increases in procollagen mRNAs in VSMCs of WKY (top). Northern blots of mRNA encoding procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) after exposure of confluent, quiescent cell cultures to TGF-ß1, -ß2, or -ß3 (1 ng/mL) for 2 and 6 hours; 28s and 18s represent ribosomal RNA monitoring equivalent gel loading and C represents control, unstimulated VSMCs. Lower panels represent the early time-dependent increases in {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels induced by the three TGF-ß isoforms, with the subsequent return to basal levels in the ensuing 42-hour period.

Interactions Between TGF-ß and Tyrosine Kinase–Activating Growth Factors on Collagen Gene Expression
Because growth factors capable of activating RTKs and stimulating TGF-ß1 biosynthesis16 23 in VSMCs are known to activate collagen genes in some cell lines,24 25 we also investigated in VSMCs from SHR and WKY whether such growth factors, particularly bFGF and EGF, could also influence collagen mRNA levels and whether their effects were different in the two cell lines. Both bFGF (25 ng/mL) and EGF (25 ng/mL) induced rapid elevations in collagen {alpha}1(I) mRNA levels in VSMCs derived from WKY, 7- and 8.5-fold, respectively, 2 hours after their addition to quiescent cells (Figs 3Down and 4Down); subsequently, levels declined and were close to basal amounts by 24 hours. Simultaneous exposure of the cells to these growth factors and the TGF-ß isoforms had no greater effect on the elevation in collagen {alpha}1(I) mRNA induced by either bFGF or EGF; also the time course of changes in collagen {alpha}1(I) mRNA expression was unaltered (Figs 3Down and 4Down). In contrast, in VSMCs of SHR, both bFGF and EGF only caused reductions in the levels of collagen {alpha}1(I) mRNA, which were most apparent at 6 hours (Figs 5Down and 6Down). When these VSMCs were simultaneously exposed to one of the TGF-ß isoforms together with either bFGF or EGF the reductions in mRNA levels tended to be somewhat smaller, but there were still no increases in mRNA amounts above basal levels (Figs 5Down and 6Down).



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  		Figure 3. Northern blot analysis and time course of effects of bFGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels over a 48-hour period in WKY VSMCs. Left panels, Increases in mRNA levels encoding procollagen {alpha}1(I), {alpha}2(I), or {alpha}1(III) in VSMCs of WKY exposed for 2 and 6 hours to bFGF (25 ng/mL) alone or in combination with either TGF-ß1, -ß2, or -ß3 (1 ng/mL) (F/ß); 28s and 18s represent ribosomal RNA monitoring equivalent gel loading and C represents control, unstimulated VSMCs. Right panels, Time course of effects of bFGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I) (top), {alpha}2(I) (middle), and {alpha}1(III) (bottom) mRNA levels over a 48-hour period in VSMCs of WKY. Results are typical of three to five experiments measured from Northern blots by an optical densitometric system.



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  		Figure 4. Time course of effects of EGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I) (top), {alpha}2(I) (middle), and {alpha}1(III) (bottom) mRNA levels over a 48-hour period in VSMCs of WKY. Results are typical of three to five experiments measured from Northern blots by an optical densitometric system.



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  		Figure 5. Northern blot analysis and time course of effects of bFGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels over a 48-hour period in SHR VSMCs. Left panels, Reductions in mRNA levels encoding procollagen {alpha}1(I), {alpha}2(I), or {alpha}1(III) in VSMCs of SHR exposed for 2 and 6 hours to bFGF (25 ng/mL) alone or in combination with either TGF-ß1, -ß2, or -ß3 (1 ng/mL) (F/ß); 28s and 18s represent ribosomal RNA monitoring equivalent gel loading and C represents control, unstimulated VSMCs. Right panels, Time course of effects of bFGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I) (top), {alpha}2(I) (middle), and {alpha}1(III) (bottom) mRNA levels over a 48-hour period in VSMCs of SHR. Results are typical of three to five experiments measured from Northern blots by an optical densitometric system.



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  		Figure 6. Time course of effects of EGF alone or with the TGF-ß isoforms on procollagen {alpha}1(I) (top), {alpha}2(I) (middle), and {alpha}1(III) (bottom) mRNA levels over a 48-hour period in VSMCs of SHR. Results are typical of three to five experiments measured from Northern blots by an optical densitometric system.

Differential temporal changes in collagen {alpha}2(I) mRNA levels were also induced by bFGF and EGF in VSMCs of WKY and SHR. In VSMCs of WKY, transient elevations in collagen {alpha}2(I) mRNA levels were induced by these growth factors, averaging approximately twofold and threefold, respectively, for bFGF and EGF (Figs 3Up and 4Up), substantially less than their effects on collagen {alpha}1(I) mRNA levels. Peak effects on these two mRNAs were observed at 2 hours, then the mRNA levels returned to basal amounts by 12 hours (Fig 4Up). Again, the simultaneous presence of the TGF-ß isoforms with either bFGF or EGF had no greater effect (P>.05) on either the magnitude or the time course of changes induced by either bFGF or EGF alone. In VSMCs of SHR, neither bFGF nor EGF caused any elevations in mRNA levels over a 48-hour period (Figs 5Up and 6Up). Rather, they consistently reduced (P<.05) collagen {alpha}2(I) mRNA levels in a manner similar to that observed in the presence of the TGF-ß isoforms (Fig 1Up). These changes in mRNA levels were not altered by the simultaneous presence of the TGF-ß isoforms (Figs 5Up and 6Up). In WKY and SHR VSMCs the effects of the growth factors on collagen {alpha}1(III) mRNA levels were qualitatively similar to those observed with collagen {alpha}2(I) mRNA. Peak stimulatory effects of bFGF and EGF on collagen {alpha}1(III) mRNA in VSMCs derived from WKY were similar in magnitude (twofold elevation) but in the simultaneous presence of TGF-ß and EGF the stimulation was approximately fourfold (Fig 4Up). In VSMCs of SHR the reductions in collagen {alpha}1(III) mRNA were more pronounced with bFGF than with EGF, the former reducing mRNA levels 70% on average in the 6- to 24-hour period. The magnitude of this reduction was unaffected by the simultaneous presence of TGF-ß (Fig 5Up) and was greater than in the presence of both EGF and TGF-ß (Fig 6Up).

TGF-ß1 and c-myc Oncoprotein mRNA
Because the oncoprotein c-myc has the potential to inhibit collagen gene transcription,26 27 affect TGF-ß1 growth responses,28 and is elevated in VSMCs of SHR exposed to TGF-ß1,29 we investigated whether the induction of c-myc mRNA could be related to the differential effects of TGF-ß1 on collagen mRNA levels in VSMCs of SHR and WKY. Incubation of VSMCs of SHR with TGF-ß1 elevated c-myc mRNA levels (Fig 7Down); at 2 hours the rise in mRNA was approximately fivefold and at 4 hours approximately sixfold. In these cells EGF also elevated c-myc mRNA to the same extent as TGF-ß1 at 2 hours, but the elevation at 4 hours was higher, {approx}10-fold. Simultaneous addition of TGF-ß1 and EGF elevated c-myc mRNA eightfold and sixfold at 2 hours and 4 hours. In contrast, in VSMCs of WKY, TGF-ß1 did not elevate c-myc mRNA at 2-hour exposure and at 4 hours the elevation in mRNA levels was just detectable. In contrast, EGF alone or together with TGF-ß1 elevated c-myc mRNA approximately threefold and fivefold in these VSMCs at 2 and 4 hours (Fig 7Down).



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  		Figure 7. Northern blots representing 2.5 kb c-myc mRNA transcript levels in VSMCs of SHR and WKY rats 2 hours and 4 hours after exposure to EGF (E) (25 ng/mL), TGF-ß1 (ß1) (1 ng/mL), or a combination of the two growth factors (E/ß1); 28s and 18s represent ribosomal RNA monitoring equivalent gel loading and C represents control, unstimulated VSMCs. Results are typical of three experiments.

Nuclear Accumulation of c-myc Oncoprotein
Because the ability of c-myc oncoprotein to affect gene transcription is dependent on its binding to DNA,30 31 we examined whether c-myc peptide accumulation in cell nuclei was related to the differential effects of TGF-ß1 and EGF on collagen gene expression in VSMCs of SHR and WKY. In quiescent VSMC cultures from SHR the proportion of c-myc-immunopositive cells exhibiting c-myc peptide associated with their nuclei averaged 7% (TableDown). After 2 hours of exposure to TGF-ß1 the number of cells with c-myc-immunopositive nuclei increased to 57% (P<.0.5). The increases in nuclear-associated c-myc peptides 2 hours after cell exposure to EGF alone or together with TGF-ß1 were similar, as were those at 6 hours (TableDown). When VSMC cultures from WKY were exposed to TGF-ß1, an essentially similar pattern of relative increases in nuclear c-myc-immunoreactive peptides were observed; however, the relative magnitude of the increases tended to be smaller. In these studies we did not investigate whether proportionally the same number of VSMCs from SHR and WKY responded to the growth factors to increase the nuclear c-myc-immunoreactive peptide levels.


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  		Table 1. Nuclear Accumulation of c-myc


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study demonstrates that TGF-ß differentially regulates collagen gene expression in VSMCs from SHR and WKY rats. All three TGF-ß isoforms, -ß1, -ß2, and -ß3, elevate the levels of mRNA encoding procollagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) in VSMCs from WKY rats, but in VSMCs from SHR only reductions were observed. A differential pattern of c-myc gene expression was also apparent when the VSMCs from the two strains of rats were exposed to TGF-ß1, with increases in c-myc mRNA levels in the SHR-derived VSMCs. Despite the differential regulation of its mRNA by TGF-ß1, it is likely that c-myc oncoprotein contributes to downregulation of all three collagen genes in both SHR and WKY. Similar patterns of effects on collagens and c-myc were observed in VSMCs of SHR and WKY during exposure to growth factors that activate RTKs and that can elevate TGF-ß1 production.16 23 Together, these results indicate that differences in TGF-ß responsiveness between VSMCs from SHR and WKY are not restricted to effects on cell proliferation16 17 but rather are likely to be of a more general nature, affecting the ability of TGF-ß to induce VSMCs from SHR to produce extracellular matrix proteins such as collagen.

Although TGF-ß is a potent activator of many collagen genes in fibroblasts32 and mesangial cells,24 its effects on collagen gene activation in VSMCs are far less clear.33 34 35 36 TGF-ß1 has been reported to increase collagen types I and III biosynthesis34 36 whereas other reports indicate little or no change in basal collagen {alpha}1(I), {alpha}1(III), or {alpha}2(I) mRNA levels.33 35 Our findings in VSMCs of WKY indicate that all three TGF-ß isoforms are capable of inducing large coordinate but transient increases in mRNAs encoding these three collagens. Peak increases in the mRNAs stimulated by the TGF-ß isoforms occur at {approx}2 hours after their addition to quiescent cell cultures, consistent with activation by early growth factor signaling mechanisms involving AP-1 sequences on these collagen genes.21 We have previously shown that TGF-ß1 is capable of rapidly activating specific genes in WKY VSMCs, most likely through AP-1 sequences.16 These effects of TGF-ß are also consistent with the possible involvement of other recently defined early TGF-ß–initiated intracellular signals such as extracellular signal–regulated kinase (ERK), which in some cell types phosphorylates Elk-1 and enhances its binding to the serum response element in the c-fos promoter.37 Other TGF-ß responsive elements likely to be involved in the growth factor responses include {alpha}2-TAE38 39 and {alpha}1-TAE40 sequences within collagen {alpha}2(I) and {alpha}1(I) and the SP1 transcription factors.39 41 Our findings that mRNA levels encoding the three collagens in VSMCs of WKY are barely elevated 24 hours after exposure to TGF-ß are consistent with earlier reports indicating little if any effect of TGF-ß1 on collagen mRNA levels during prolonged exposure to the growth factor,33 in marked contrast to persistent mRNA elevations observed in cultured fibroblasts. Presumably, in VSMCs, not only positive but also negative regulators of collagen gene activity are induced by TGF-ß during long-term exposure, modulating either transcription or mRNA stability.

Negative regulators of collagen gene activity predominate when VSMCs of SHR are exposed to the TGF-ß isoforms. Because in these VSMCs TGF-ß1 rapidly elevates c-myc mRNA levels and increases the proportion of cells containing nuclear-associated c-myc proteins, it is likely that it contributes not only to the late downregulation of collagen mRNA levels in WKY VSMCs but also to the early downregulation of the collagen genes in SHR VSMCs. Overexpression of c-myc has been reported to alter CCAAT transcription factor/nuclear factor 1 (CTF/NF-1), suppressing the activity of CTF/NF-1 promoters and downregulating basal collagen gene activity in 3T3-L1 cells.26 27 Because in VSMCs of SHR c-myc mRNA levels remain elevated for at least 24 hours during exposure to TGF-ß1,29 it is possible that its protein product persistently influences the activities of collagen genes. Because nuclear accumulation is also seen in VSMCs from WKY during exposure to TGF-ß1 and EGF, it is also possible that c-myc contributes to the transient nature of the elevations in mRNAs encoding the three collagens. Whether in the nucleus other proteins complexing with c-myc also influence its activity is unknown. For example, the proteins mad and max, when complexed, are capable of antagonizing myc transcriptional activity.42 Similarly, b-myb has been implicated in the downregulation of promoter activity of type I collagens,43 but its contribution to TGF-ß downregulation of these genes would be expected to be minor because its levels are low in quiescent cells, increasing only when the cells approach and enter S-phase of the mitotic cell cycle.43 Similarly, an involvement of c-jun would also be expected to be minimal in reducing basal collagen {alpha}2(I) promoter activity because its levels are low in quiescent VSMCs and are not induced by TGF-ß1.21 Precisely how these factors may contribute to the differential regulation of collagen gene activities in SHR and WKY VSMCs clearly requires further study.

Recently a number of RTK-activating growth factors have been shown to be fibrogenic.25 With PDGF and monocyte chemoattractant protein-1, the ability to elevate collagen mRNA has been attributed to stimulation of TGF-ß1 production and secretion.24 36 44 We have also recently reported that PDGF, bFGF,16 and EGF (unpublished) are capable of elevating TGF-ß1 production in VSMCs of SHR and WKY and have previously shown that the characteristics of the membrane receptors for these growth factors are similar in VSMCs from the two rat strains.45 In the present study both bFGF and EGF transiently elevated collagen {alpha}1(I), {alpha}2(I), and {alpha}1(III) mRNA levels in VSMCs of WKY but reduced levels in SHR, in a manner similar to the TGF-ß isoforms. Because these effects were neither antagonized or potentiated by the TGF-ß isoforms, it is tempting to speculate that with RTK-activating growth factors, transcriptional factors that are dependent on TGF-ß production are likely to be important in the differential regulation of the collagen genes in VSMCs of SHR and WKY. Clearly, further experimentation is required to identify these transcription factors. In addition, our studies with TGF-ß, bFGF, and EGF indicate that rather than cell quiescence or proliferation being important in regulating collagen production through mRNA synthesis in VSMCs as previously proposed,33 it is the specific nature of the transcription factors that interact with the collagen genes that determines their level of activity.

Our findings on the differential effects of TGF-ß on various collagen mRNAs and c-myc mRNA in VSMCs of SHR and WKY are consistent with its other differential effects in these cells, particularly on cell proliferation. TGF-ß1 enhances bFGF-, EGF-, and PDGF-stimulated cell proliferation in SHR-derived VSMCs but inhibits proliferation of VSMCs derived from WKY rats.16 Also, TGF-ß1 fails to elevate mRNA encoding TGF-ß1 through gene autoinduction in VSMCs of SHR but not WKY; rather, in the latter cells there is a time-dependent elevation in TGF-ß1 mRNA levels after its addition to quiescent cultures. Together these observations are indicative of differences in very early TGF-ß–induced intracellular signals in the two cell lines occurring at, or just distal to, the signaling TGF-ß receptors. Because in SHR vascular hypertrophy precedes the development of hypertension,3 it is tempting to speculate that such a difference in early signaling has the potential to contribute to differences in vessel structure between the two strains of rats, possibly affecting the structure of vessels in SHR independent of their hypertension through effects on VSMC number and extracellular matrix proteins. In the SHR, vascular hypertrophy has been attributed to a greater number of VSMCs in the media of vessels2 ; also VSMC proliferation accounts for much of the aortic hypertrophy that occurs in renal hypertensive SHR but not in similarly hypertensive WKY rats4 or in one-kidney, one-clip (renal) hypertension.46 In addition, extracellular matrix content and type I collagen have been reported to be lower in vessels of SHR compared with WKY.8 9 10 Theoretically, all three TGF-ß isoforms could contribute to such changes. TGF-ß2 and ß3 have been reported to colocalize at sites of type I procollagen synthesis in some vessels,47 and TGF-ß1 is known to stimulate collagen deposition and even vessel fibrosis.48

In summary, our findings indicate that TGF-ß and RTK-activating growth factors have the capacity to be fibrogenic when acting on VSMCs from WKY but not SHR. These differential effects are not related to the proliferative state of the VSMCs but rather would appear more dependent on the types of transcription factors induced by the growth factors. Because TGF-ß1 production can be induced by RTK-activating growth factors in a number of cell lines including VSMCs,16 23 it is also likely that the prime mechanism responsible for the differential collagen responses is an altered TGF-ß signaling in VSMCs of SHR. The extent to which the differences we observe on collagen gene induction by TGF-ß in VSMCs of SHR and WKY might contribute to differences in the blood vessel wall structure and/or the greater susceptibility of SHR, particularly stroke-prone SHR, to hemorrhagic stroke when their hypertension becomes severely elevated remains to be evaluated.


*    Selected Abbreviations and Acronyms
 
bFGF = basic fibroblast growth factor
EGF = epidermal growth factor
SHR = spontaneously hypertensive rat(s)
TGF-ß = transforming growth factor-ß
VSMC = vascular smooth muscle cell(s)


*    Acknowledgments
 
This study was supported by a grant-in-aid from the National Heart Foundation of Australia (Dr Bobik).

Received July 21, 1997; first decision August 14, 1997; accepted November 10, 1997.


*    References
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