Thromboxane/Prostaglandin Endoperoxide–Induced Hypertrophy of Rat Vascular Smooth Muscle Cells Is Signaled by Protein Kinase C–Dependent Increases in Transforming Growth Factor-β
In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-β (TGF-β) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-β in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-β was supported by the following observations: (1) exogenous human TGF-β1 increased protein synthesis; (2) antibody to TGF-β blocked both TGF-β– and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-β bioactivities; and (4) the actions of U46619 on protein content and TGF-β bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-β and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-β (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-β that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-β bioactivity and stimulated protein synthesis. The hypertrophic response to bFGF was blocked by anti–TGF-β. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-β–induced suppression of proliferation, as evidenced by the ability of antibody to TGF-β to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-β bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-β. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-β bioactivity via protein kinase C. Increases in both bFGF and TGF-β are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-β occurs through a protein kinase C–independent pathway.
- protein kinase C
- transforming growth factor beta
- fibroblast growth factor, basic
- muscle, smooth, vascular
The vasoconstrictor agonists thromboxane and Ang II have been reported to stimulate either hypertrophy or proliferation in cultured VSMCs.1 2 3 4 5 6 7 8 In the case of Ang II, the hypertrophic response depends on autocrine production of TGF-β.3 4 By contrast, bFGF has been implicated as a mediator of the hypertrophic response to the thromboxane/prostaglandin endoperoxide analogue U46619 and the proliferative response to Ang II.3 4 The role of autocrine TGF-β in the expression of the hypertrophic response to U46619 and the potential interelationships between TGF-β and bFGF in the expression of vasoconstrictor hormone action on VSMC growth have not been adequately addressed. In the present study, we examined the effects of U46619 on hypertrophy and proliferation of VSMCs cultured from the aorta of normal rat and the role of TGF-β as mediator of these responses to U46619. Since bFGF has been reported to be elevated in VSMCs exposed to U46619 and to mediate the hypertrophic response to U46619,2 we also assessed the relationship between bFGF and TGF-β with respect to expression of U46619 actions. Since some of the actions of U46619 are likely mediated by inositol phospholipid hydrolysis and activation of PKC9 and PKC has been implicated in both the expression of bFGF actions on VSMC hypertrophy10 and the induction of TGF-β mRNA and bioactivity in response to vasoconstrictor hormones,4 11 we examined the potential role of PKC in the expression of the actions of U46619 and bFGF to stimulate hypertrophy of VSMCs. The results demonstrate that U46619-induced increases in TGF-β bioactivity mediate the hypertrophic response to U46619 and further indicate that activation of PKC by U46619 signals both increases in TGF-β bioactivity and VSMC protein synthesis. bFGF appears to participate in expression of the hypertrophic actions of U46619 by its combined actions to stimulate cell cycle progression and increase endogenous TGF-β through a PKC-dependent mechanism.
Three male Wistar-Kyoto rats were obtained from Taconic Farms, Germantown, NY, at 14 weeks of age. VSMCs were obtained for culture by a slight modification of the method of Ross.12 Briefly, rats were anesthetized with ether, the abdomen coated with betadine, and the thoracic aorta removed through a midline incision with sterile instruments. Aortas were cut longitudinally, placed in sterile Dulbecco's modified Eagle's medium (DMEM) plus 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine. The adventitia and intima were then removed under a dissecting microscope and discarded. The aortic media was cut into 2.5×2.5-mm pieces, washed in three changes of the above medium, and placed in a 38-mm-diameter Petri dish, luminal side down. Ten milliliters of DMEM that contained 20% FBS plus 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine was then added slowly over several hours, and the aortic explants were allowed to incubate for 10 days undisturbed. The VSMCs that grew out from the explants were passed at confluence into the same medium except that FBS was lowered to 15%, penicillin to 5 U/mL, and streptomycin to 5 μg/mL. FBS was again lowered to 10% on the next passage. The standard growth medium thus was DMEM plus 5 U/mL penicillin, 5 μg/mL streptomycin, 2 mmol/L glutamine, and 10% FBS. VSMCs grew with a typical hill-and-valley morphology and stained positive for smooth muscle actin and negative for factor VIII. Mouse monoclonal antibody to human smooth muscle actin was obtained from Sigma Chemical Co. Rabbit polyclonal antibody to human factor VIII was obtained from Dako Corp. Cells were used between passages 4 and 15.
Determination of Proliferation and Protein Synthesis
Cells were seeded in standard culture medium as described above. After 24 hours, the medium was changed to the same except that FBS was reduced to 0.5%. After an additional 48 hours, the medium was changed to serum-free plus 10 μg/mL each of insulin and transferrin. As previously reported,13 cells remained viable in this serum-free medium and responded to addition of platelet-derived growth factor with an increase in cell number. Test agents were added to the serum-free medium for the times indicated in the figure legends. Medium was changed daily except in the studies illustrated in Fig 2⇓. At the end of the incubation, cell number was determined by counting of cells in a hemocytometer. Protein content was determined with bicinchoninic acid (Sigma).
In some experiments, [35S]methionine (4 μCi/mL) or [3H]dThd was present for the final 4 hours of incubation. At the end of the incubation, cells were washed twice with cold phosphate-buffered saline and incubated with cold 6% trichloroacetic acid for 5 minutes. The cells were then washed with cold 6% trichloroacetic acid and solubilized in 0.3 mL of 5% sodium dodecyl sulfate at 90°C for 5 minutes. An aliquot was then counted in a scintillation counter.
VSMC Incubation for Assay of Medium TGF-β Bioactivity
VSMCs were seeded and made quiescent as described above. Except where indicated, medium was changed daily and collected for assay of TGF-β at various times after addition of test agents as indicated in the figure legends. During the last 24 hours of incubation, medium was changed to minimum essential medium (MEM) Eagle with nonessential amino acids and Earle's balanced salt solution plus glutamine (2 mmol/L), penicillin (5 U/mL), streptomycin (5 μg/mL), insulin (10 μg/mL), transferrin (10 μg/mL), and test agents. MEM with nonessential amino acids was substituted for DMEM during the final 24 hours of incubation because the former was optimal for culture of MLECs (MV1 Lu). In preliminary studies, we assessed optimal conditions for harvest and storage of experimental media for assay of TGF-β. Storage of conditioned media for 24 hours at 0°C to 4°C in the absence of protease inhibitors resulted in a threefold increase in active TGF-β bioactivity compared with that observed in the presence of protease inhibitors. Dialysis of conditioned media (Spectra/Por2, 12 000 D to 14 000 D cutoff) had no effect on TGF-β bioactivity. Freezing and thawing led to variable activation of latent TGF-β. Accordingly, at the completion of the experiment, media were centrifuged to remove any particulate material, and albumin and protease inhibitors were added to give a final concentration of 1 mg/mL bovine serum albumin, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin. An aliquot of the medium was then transferred directly to MLECs for bioassay of TGF-β.
Assay of TGF-β
Active TGF-β content of conditioned media was determined by comparison of the ability of conditioned media to suppress [3H]dThd incorporation into DNA of MV1 Lu cells with that of a range of concentrations of exogenous TGF-β. Addition of human platelet TGF-β1 (R&D Systems) (0.1 to 5 pmol/L) to conditioned media that contained protease inhibitors resulted in dose-dependent suppression of MV1 Lu cell proliferation that was indistinguishable from that obtained when TGF-β was added to control unconditioned media. The IC50 averaged 0.75 pmol/L. Total TGF-β (active plus latent) content was determined in conditioned media that had been heated at 80°C for 20 minutes.14 Appropriate control media containing test agents were included in each assay. Neither U46619 nor bFGF influenced MLEC proliferation. Specificity of the TGF-β assay was established with affinity-purified pan-specific neutralizing antibody to TGF-β (R&D Systems). The assay was conducted as previously reported.15 16 The assay does not distinguish between TGF-β1 and TGF-β2.15 MV1 Lu cells (American Type Culture Collection) were seeded in 48-well plates at 104/cm2 in MEM Eagle with nonessential amino acids and Earle's balanced salt solution and 10% FBS plus glutamine (2 mmol/L), penicillin (5 U/mL), and streptomycin (5 μg/mL). After 6 hours, the medium was changed to MEM and 0.2% FBS plus glutamine (2 mmol/L), penicillin (5 U/mL), streptomycin (5 μg/mL), insulin (10 μg/mL), and transferrin (10 μg/mL). After 16 hours, the medium was again changed to conditioned medium or control medium with or without standard TGF-β (0.1 to 5 pmol/L), and the final concentration of FBS was adjusted to 0.2%. The incubation was continued for 24 hours. [3H]dThd (0.06 μCi per well) was then added for the final 4 hours of incubation. At the end of the incubation, the cells were washed twice with cold phosphate-buffered saline and incubated with cold 6% trichloroacetic acid for 5 minutes. The cells were then washed with cold 6% trichloroacetic acid followed by cold 90% ethanol and were solubilized in 0.3 mL of 5% sodium dodecyl sulfate at 90°C for 5 minutes. The extract was then counted in a scintillation counter. Preincubation of conditioned media with TGF-β antibody (10 μg/mL) for 60 minutes at 37°C abolished the suppressive effects of conditioned media on MV1 Lu cell proliferation. Preincubation with affinity-purified control IgG (R&D Systems) under the same conditions had no effect.
Determination of PKC Activity
Cells from one 10-cm Petri dish were used for each condition. Cells were grown to confluence in standard culture medium containing 10% serum. Cells were then made quiescent by culture in serum-free medium plus insulin and transferrin (10 μg/mL each) for 24 hours. They were then incubated for 15 minutes at 37°C with vehicle or 0.1 μmol/L U46619. After 15 minutes, dishes were placed on ice, rinsed with cold phosphate-buffered saline, and extracted as previously described17 with sonication for 5 seconds on ice into lysis buffer that contained 1 mmol/L NaHCO3, 5 mmol/L MgCl2, 25 μg/mL leupeptin, and 25 μg/mL aprotinin, pH 7.5.
After centrifugation at 500g for 5 minutes at 4°C, the supernatant was centrifuged at 100 000g for 10 minutes. The pellet was resuspended in 50 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2, 2 μmol/L CaCl2, 200 μmol/L vanadate, 200 μmol/L sodium pyrophosphate, 25 mmol/L NaF, 25 μg/mL aprotinin, and 25 μg/mL leupeptin (pH 7.5) as previously described17 ; PKC was assayed with a kit obtained from GIBCO. All assays contained 50 μmol/L Ac-myelin basic protein,4 5 6 7 8 9 10 11 12 13 14 20 μmol/L ATP, 1 mmol/L CaCl2, 20 mmol/L MgCl2, 4 mmol/L Tris (pH 7.5), 10 μmol/L phorbol myristate acetate, and 0.28 mg/mL phosphatidylserine in Triton X-100–mixed micelles. PKC activity was defined as the difference between activity measured in the presence and absence of a selective peptide inhibitor of PKC.17
Results shown are mean±SE of at least three separate experiments. Each experimental condition was represented by triplicate wells in a single experiment, and values were averaged and entered as one for the purposes of statistical analysis. Significance of differences between any two mean values was determined by Student's t test. Comparisons of multiple values with a single control were analyzed by ANOVA followed by the Fisher multiple comparison method.
Fig 1⇓ illustrates the time course of effects of 0.1 μmol/L U46619 on protein synthesis and proliferation in VSMCs between 24 and 72 hours after addition of U46619. U46619 increased protein content and [35S]methionine incorporation into protein by 40% to 60% at 24, 48, and 72 hours after addition. U46619 transiently increased [3H]dThd incorporation into DNA at 24 hours, but the increase was not sustained at 48 or 72 hours and was not associated with an increase in cell number at any of the time points studied.
Fig 2⇓ shows the time course of actions of U46619 on active and total TGF-β bioactivities in the culture media of VSMCs. In these studies, media were not changed during the 24- to 72-hour experimental period after addition of U46619. As shown, basal accumulation of total TGF-β bioactivity in the media was higher at 48 hours compared with values at 24 hours. In the absence of U46619, total TGF-β accumulation did not increase further between 48 and 72 hours. Compared with corresponding control values, U46619 increased total TGF-β bioactivity at 72 hours after addition but not at earlier time points. In contrast to results obtained with total TGF-β, active TGF-β bioactivity in the media under basal incubation conditions did not change between 24 and 72 hours. However, U46619 increased active TGF-β bioactivity by 24 hours after exposure, and this increase was sustained at 48 and 72 hours.
Fig 3⇓ illustrates the concentration-response relationship between U46619 and both active TGF-β and protein synthesis in VSMCs. VSMCs were exposed to the indicated concentration of U46619 for 48 hours before harvest of media for determination of TGF-β and of [35S]methionine incorporation into protein. Significant increases in both active TGF-β and protein synthesis were observed at 10−8 mol/L U46619, with optimal increases observed at 10−7 mol/L U46619.
Fig 4⇓ illustrates the effects of SQ 29,548 on U46619-induced increases in the protein content of VSMCs and on active and total TGF-β bioactivities in culture media from these same cells. U46619 increased protein content of VSMCs approximately 40% and increased active and total TGF-β by 60% to 75%. Addition of anti–TGF-β abolished the increases in active TGF-β induced by U46619, whereas isotypic control rabbit IgG had no effect (not shown). The thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548 blocked U46619-induced increases in VSMC protein content and in active and total TGF-β bioactivities in the media.
Fig 5⇓ shows the concentration-response relationship between TGF-β and [35S]methionine incorporation into protein and cell number in VSMCs. In these studies, VSMCs were exposed to the indicated TGF-β1 concentration for 72 hours. The minimum effective concentration of exogenous TGF-β was 0.5 pmol/L, which increased protein synthesis of VSMCs by approximately 20%. Optimal (60%) increases were observed with 10 pmol/L exogenous TGF-β. The response to 10 pmol/L TGF-β was blocked by 30 μg/mL anti–TGF-β but not control IgG (not shown). Under basal conditions, VSMCs accumulated 0.4 pmol/L active TGF-β in culture media; this value rose to 0.7 pmol/L after incubation of VSMCs with 0.1 μmol/L U46619 (Fig 4⇑). Thus, the concentration of endogenous active TGF-β that accumulated in the media under conditions that led to a maximal increase in protein synthesis in response to U46619 (0.7 pmol/L) was less than the concentration of exogenous TGF-β (10 pmol/L) required to produce a comparable protein synthetic response. As is also shown in Fig 5⇓, TGF-β1 did not influence VSMC cell number at any of the concentrations shown.
Fig 6⇓ illustrates the time course of effects of 25 pmol/L TGF-β on cell number in VSMCs incubated with or without serum. As shown, TGF-β1 had no effect on cell number when added to VSMCs grown in serum-free medium but suppressed the stimulatory effect of serum on cell number at 48 and 72 hours.
The influence of anti–TGF-β on protein content and [3H]dThd incorporation into DNA of VSMCs is shown in Fig 7⇓. In the presence of affinity-purified control IgG, U46619 increased the protein content of VSMCs 55% over control. This response and basal protein content shown in Fig 4⇑ in the presence of control IgG did not differ from corresponding values observed in the absence of added antibodies. Exposure of VSMCs to anti–TGF-β for 24 hours did not significantly suppress basal protein content. However, anti–TGF-β blocked U46619-induced increases in this parameter (Fig 7⇓). As also illustrated in Fig 7⇓, U46619 increased [3H]dThd incorporation into DNA by 24 hours in VSMCs incubated in the presence of control IgG. The increase in [3H]dThd incorporation into DNA in response to U46619 was enhanced in the presence of antibody to TGF-β.
Fig 8⇓ illustrates the concentration-response relationship between bFGF and both [3H]dThd incorporation into DNA and protein synthesis. As illustrated, bFGF stimulated both [3H]dThd incorporation into DNA and protein synthesis in VSMCs; however, the concentration-response relationships differed. bFGF progressively increased [3H]dThd incorporation into DNA between 0.25 and 5 ng/mL. A higher concentration of bFGF (20 ng/mL) was less effective at stimulating [3H]dThd incorporation than was 5 ng/mL. By contrast, 20 ng/mL bFGF was required to increase protein synthesis.
Fig 9⇓ illustrates the effects of antibody to TGF-β on increases in [3H]dThd incorporation into DNA and protein content in response to 20 ng/mL bFGF. As illustrated and analogous to results obtained with U46619 (Fig 7⇑), antibody to TGF-β enhanced [3H]dThd incorporation into DNA observed with bFGF and blocked the hypertrophic response to bFGF. Thus, the reduced DNA synthetic response to a high concentration (20 ng/mL) of bFGF illustrated in Fig 8⇑ appears to be due to endogenous production by TGF-β.
Fig 10⇓ compares the effects of bFGF and U46619 on protein synthesis and total TGF-β accumulation in media of VSMC cultures. bFGF at the concentration added to VSMC culture media had a modest stimulatory effect on MLEC proliferation. Accordingly, active TGF-β in response to bFGF was not assessed. The bFGF present in dilutions of VSMC-conditioned media used (×10) for assessment of total TGF-β was without effect on MLEC proliferation. As shown, bFGF increased total TGF-β accumulation and protein synthesis to a similar extent as did U46619. The role of PKC in the expression of the actions of bFGF and U46619 to increase TGF-β and protein synthesis was assessed with two relatively selective inhibitors of PKC activity, GFX and staurosporine. The influence of the PKC inhibitors on increases in protein synthesis induced by exogenous TGF-β1 was also determined.
GFX does not inhibit receptor tyrosine kinases and has an IC50 for phosphorylase kinase and cAMP-dependent protein kinase that are 70 and 200 times greater than the IC50 for PKC.18 Staurosporine is also a potent and relatively selective inhibitor of PKC activity (IC50, 0.7 nmol/L).19 As illustrated in Fig 10⇑, U46619 (0.1 μmol/L), bFGF (20 ng/mL), and TGF-β (10 pmol/L) increased VSMC protein synthesis approximately 50%. GFX (5 μmol/L) and staurosporine (50 nmol/L) abolished the stimulatory effect of U46619 and bFGF on protein synthesis but had no effect on basal values or TGF-β–induced increases in this parameter. As also illustrated in Fig 10⇑, GFX and staurosporine reduced basal TGF-β bioactivity to undetectable levels and abolished U46619-induced increases in this parameter. Moreover, both PKC inhibitors reduced total TGF-β by 40% and abolished U46619- and bFGF-induced increases in total TGF-β. GFX did not suppress increases in [3H]dThd incorporation into DNA induced by addition of serum to quiescent cells (without serum, 152±12 disintegrations per minute per well; 10% FBS, 6232±715; 5 μmol/L GFX without serum, 148±18; 5 μmol/L GFX with serum, 7859±812). Similar results were obtained with staurosporine. The lack of effect of GFX and staurosporine on increases in protein synthesis induced by exogenous TGF-β (Fig 10⇑) or serum-induced increases in proliferation and the ability of cells to exclude trypan blue in the presence of GFX or staurosporine (not shown) imply that the suppressive effects of GFX and staurosporine on U46619- and bFGF-induced increases in protein synthesis and TGF-β are not due to nonspecific toxic effects.
The effects of exposure of intact VSMCs to U46619 on PKC activity were determined in 100 000g particulate fractions by the incorporation of [32P]ATP into the myelin basic protein in the presence of optimal concentrations of Ca2+, phosphatidylserine, and phorbol ester as described in “Methods.” U46619 increased particulate PKC activity in VSMCs from Wistar-Kyoto rats approximately 60% (vehicle, 840±65 pmol/min per milligram protein; 0.1 μmol/L U46619, 1330±145) within 15 minutes. The increase in PKC activity declined to basal values by 24 hours after addition of U46619 (not shown).
The results of the present study demonstrated that U46619 stimulated [35S]methionine incorporation into protein and increased the protein content of VSMCs. The thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548 suppressed U46619-induced increases in protein content per cell, consistent with mediation of these actions of U46619 via the thromboxane/prostaglandin endoperoxide receptor. SQ 29,548 had no effect on the protein content of VSMCs in the absence of added U46619, suggesting that endogenous thromboxane production was not modulating protein content under basal incubation conditions. By contrast, under the conditions used in the present study, although U46619 modestly increased [3H]dThd incorporation into DNA, this agent did not stimulate proliferation of VSMCs as assessed by an increase in cell number. These results differ from some earlier findings5 6 7 8 that have demonstrated proliferative effects of U46619 on VSMCs from control rats. However, they are in general agreement with researchers who have reported that U46619 stimulates hypertrophy but not proliferation in VSMCs from normal rats.1 2 The reasons for these discrepancies are not known. However, results of the present and previous2 10 studies suggest that the concentration of bFGF produced in response to U46619 by VSMCs under different incubation conditions may be an important determinant. Thus, the present results demonstrate that at low concentrations (0.25 to 5 ng/mL), bFGF is a potent inducer of proliferation, whereas at higher concentrations (20 ng/mL), bFGF stimulates TGF-β production, resulting in an increase in protein synthesis and a reduced proliferative response to bFGF. In the present study, U46619 had only a weak stimulatory effect on [3H]dThd incorporation into DNA and did not increase cell number for up to 72 hours. Analogous to previous studies with Ang II,3 4 the suppressed proliferative response to U46619 was abrogated by antibody to TGF-β, implying mediation by autocrine TGF-β. Previous studies have demonstrated both stimulation and suppression of proliferation in response to mitogens depending on the concentration of TGF-β1 used or the density of cells.20 21 22 In the present study we found evidence of suppression by TGF-β of proliferation in response to serum, U46619, or bFGF. Moreover, exogenous TGF-β1 failed to stimulate proliferation when present in serum-free VSMC cultures for up to 3 days at a wide range of concentrations (0.5 to 100 pmol/L).
Several observations strongly support a role for TGF-β in the expression of the VSMC hypertrophic response to U46619. U46619 increased TGF-β bioactivity in the media of cultured VSMCs. The thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548 blocked the actions of U46619 to increase the protein content of VSMCs and enhance TGF-β bioactivity, implying mediation of both responses by the thromboxane/prostaglandin endoperoxide receptor. As previously reported,20 exogenous TGF-β increased protein synthesis of VSMCs in a concentration-dependent fashion. Addition of anti–TGF-β to VSMCs incubated with either U46619 or exogenous TGF-β blocked the increases in protein content induced by these agents. Anti–TGF-β had no significant effect on VSMC protein content in the absence of U46619 or exogenous TGF-β, implying that the endogenous TGF-β production was not a major determinant of protein synthesis under basal incubation conditions. Addition of affinity-purified control IgG was without effect on basal protein content or the increases induced by U46619 on exogenous TGF-β, supporting the selectivity of action of the anti–TGF-β. The results obtained with the TGF-β antibody strongly support a role for increased TGF-β bioactivity in the expression of the action of U46619 to increase VSMC protein content. However, a comparison of the concentration of exogenous TGF-β which optimally increased protein synthesis of VSMCs with that which accumulated in media of VSMC incubates stimulated with U46619 indicated that the concentration of TGF-β bioactivity produced in response to U46619 (0.7 pmol/L) was well below that which produced an optimal increase in protein synthesis when added exogenously (10 pmol/L). U46619 has been shown to stimulate bFGF production by VSMCs, and both antibody to bFGF and bFGF antisense were found to suppress the hypertrophic response to U46619.2 Accordingly, a possible explanation for our findings is that low concentrations of TGF-β that accumulate in the medium in response to U46619 synergize with bFGF to mediate the hypertrophic response to U46619. When added alone, higher concentrations of TGF-β are required to stimulate protein synthesis in serum-free medium.
Results of the present study demonstrate that bFGF increases total TGF-β bioactivity in the media of VSMC cultures and that the hypertrophic response to bFGF is blocked by antibody to TGF-β. In this regard, previous studies have demonstrated that bFGF increases TGF-β mRNA23 in VSMCs. When combined with previous data which demonstrated that U46619 increases bFGF and that bFGF is required for the hypertrophic response to U46619,10 the present results suggest that bFGF participates in the expression of the hypertrophic response to U46619 at least in part by increasing TGF-β production.
Studies of the time course of U46619 action on TGF-β indicated that U46619 may have distinct stimulatory effects on active versus total TGF-β bioactivity. Thus, U46619 increased total TGF-β bioactivity in the culture media of VSMCs after 72 hours of incubation but not after 24 or 48 hours. By contrast, U46619 increased active TGF-β production at all time points studied after addition of U46619 (24 to 72 hours). Thus, U46619 may influence more than one step in the pathway leading to increased TGF-β bioactivity and may initially enhance conversion of preexisting latent TGF-β to the active form as well as subsequently increasing accumulation of latent TGF-β. The early increase in active TGF-β may also contribute to the later increase in total TGF-β. In this regard, TGF-β has been shown to autoinduce its own gene expression through the same target sequences used by phorbol ester.24 Accordingly, the delayed increase in total versus active TGF-β accumulation could be partly due to autoinduction of TGF-β gene expression.
U46619- and bFGF-induced increases in TGF-β bioactivity and VSMC protein synthesis were linked to activation of PKC. Thus, addition of U46619 to intact VSMCs activated PKC in isolated particulate fractions. Moreover, GFX and staurosporine prevented U46619- and bFGF-induced increases in active and total TGF-β bioactivities and in VSMC protein synthesis. By contrast, GFX and staurosporine had no effect on increases in VSMC protein synthesis induced by exogenous TGF-β, indicating that suppression of U46619 responses by these agents did not reflect a nonselective toxic action. These results strongly imply that PKC signals the hypertrophic response to U46619 and bFGF through an action to increase TGF-β bioactivity. By contrast, the hypertrophic response to TGF-β does not appear to require PKC activation.
Recent studies by Ali et al10 have demonstrated that PKC inhibitors suppress the hypertrophic response to U46619 in VSMCs at a step distal to bFGF production. The present results further implicate PKC-dependent increases in TGF-β in the expression of the hypertrophic response to U46619 and bFGF. PKC has been implicated in the induction of TGF-β gene expression or bioactivity in response to both Ang II and U46619,4 11 and phorbol ester activators of PKC have been shown to increase TGF-β mRNA expression in several cell systems, including VSMCs.4 24 25 26 Accordingly, results of the current study that demonstrate U46619- and bFGF-induced increases in active and total TGF-β bioactivities and suppression of these actions by GFX and staurosporine may be explained at least in part by a PKC-dependent action of U46619 to induce TGF-β gene expression. However, U46619 may also have direct effects on the conversion of latent to active TGF-β, as suggested by the different time course of action of U46619 on total versus active TGF-β. The mechanism by which U46619 increases the conversion of latent to active TGF-β and the role of PKC, if any, in these events are not known. In this regard, phorbol ester activators of PKC have been shown to increase plasminogen activator gene expression in VSMCs.27 Moreover, plasmin activates TGF-β in vitro,27 and the induction of plasminogen activator by bFGF in endothelial cells28 and during cocultures of endothelial cells with pericytes or smooth muscle cells has been shown to mediate an increase in active TGF-β.29 30 Accordingly, it is possible that U46619 may induce plasminogen activator through a bFGF- and/or PKC-mediated pathway. Increased plasmin may then enhance conversion of latent to active TGF-β. Whatever the precise mechanism involved, the present and previous results are consistent with the following sequence in expression of the action of U46619 to stimulate VSMC hypertrophy: U46619 increases bFGF expression2 and rapidly activates PKC by a receptor-mediated mechanism.9 Activation of PKC is not required for the U46619-induced increase in bFGF.2 U46619, bFGF, or both increase TGF-β bioactivity through a PKC-dependent mechanism. TGF-β in turn participates in a subsequent hypertrophic response by a process that does not involve PKC activation. bFGF and TGF-β act together to induce hypertrophy. By contrast, the proliferative effect of bFGF, which has previously been shown to depend on PKC activation,6 is suppressed by an autocrine feedback loop involving TGF-β.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|bFGF||=||basic fibroblast growth factor|
|FBS||=||fetal bovine serum|
|MLEC||=||mink lung epithelial cell|
|PKC||=||protein kinase C|
|TGF-β||=||transforming growth factor-β|
|VSMC||=||vascular smooth muscle cell|
This work was supported by a grant from the American Heart Association, Pennsylvania Affiliate, and the General Medical Research Service of the Department of Veterans Affairs Medical Center. SQ 29,548 was a kind gift of The Squibb Institute, Princeton, NJ. The authors are indebted to Jacquelynn Johnston and Rhonda Spiess for excellent technical assistance and to JoAnn Orbin for typing the manuscript.
- Received December 1, 1995.
- Revision received January 30, 1996.
- Accepted January 30, 1996.
Dorn GW, Becker MW, Davis MG. Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle. J Biol Chem.. 1992;267:24897-24905.
Ali S, Davis MG, Becker MW, Dorn GW. Thromboxane A2 stimulates vascular smooth muscle hypertrophy by up-regulating the synthesis and release of endogenous basic fibroblast growth factor. J Biol Chem.. 1993;286:17397-17403.
Itoh H, Mukoyama M, Pratt RE, Gibbons GH, Dzau VJ. Multiple autocrine growth factors modulate vascular smooth muscle cell growth response to angiotensin II. J Clin Invest.. 1993;91:2268-2274.
Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs hyperplasia: autocrine transforming growth factor β expression determines growth response to angiotensin II. J Clin Invest.. 1992;90:456-461.
Ishimitsu T, Uehara Y, Ishii M, Ikeda T, Matsuoka H, Sugimoto T. Thromboxane and vascular smooth muscle cell growth in genetically hypertensive rats. Hypertension. 1988;12:46-51.
Nagata T, Uehara Y, Numabe A, Ishimitsu T, Hirawa N, Ikeda T, Matsuoka H, Sugiomoto T. Regulatory effect of TXA2 on proliferation of vascular smooth muscle cells from rats. Am J Physiol.. 1992;263:H1331-H1338.
Morinelli TA, Zhang LM, Newman WH, Meier KE. Thromboxane A2/prostaglandin H2-stimulated mitogenesis of coronary artery smooth muscle cells involves activation of mitogen-activated protein kinase and S6 kinase. J Biol Chem.. 1994;269:5693-5698.
Dorn GW, Becker MW. Thromboxane A2 stimulated signal transduction in vascular smooth muscle. J Pharmacol Exp Ther.. 1993;265:447-456.
Ali S, Becker MW, Davis MG, Dorn GW. Dissociation of vasoconstrictor stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells: relevant roles of protein kinase C. Circ Res.. 1994;75:836-843.
Ross R. The smooth muscle cell, II: growth of smooth muscle in culture. J Cell Biol.. 1971;50:172-186.
Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Bandet V, Boissin P, Boursier E, Loriolle F, Duhame L, Charon D, Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem.. 1991;266:15771-15781.
Owens GK, Geisterfer AAT, Yang YW-H, Komoriya A. Transforming growth factor β induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol.. 1988;107:771-780.
Majack RA. Beta type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures. J Cell Biol.. 1987;105:465-471.
Agrotis A, Saltis J, Bobik A. Transforming growth factor-β1 gene activation and growth of smooth muscle from hypertensive rats. Hypertension. 1994;23:593-599.
Kim SJ, Denhez F, Kim KY, Holt JT, Sporn MB, Roberts AB. Activation of the second promoter of the transforming growth factor beta-1 gene by transforming growth factor beta-1 and phorbol ester occurs through the same target sequences. J Biol Chem.. 1989;264:19373-19378.
Au YPT, Kenagy RD, Clowes AW. Heparin selectivity inhibits the transcription of tissue type plasminogen activator in primate arterial smooth muscle cells during mitogenesis. J Biol Chem.. 1992;267:3438-3444.
Flaumenhaft R, Abe M, Mugnatti P, Rifkin DB. Basic FGF-induced activation of latent TGFβ1 in endothelial cells: regulation of plasminogen activator activity. J Cell Biol.. 1992;118:901-909.
Antonelli-Orlidge A, Sauders KB, Smith SR, D'Amore PA. An activated form of TGFβ is produced by co-culture of endothelial cells and pericytes. Proc Natl Acad Sci U S A. 1989;86:4544-4548.
Sato Y, Rifkin DB. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-β1 like molecule by plasmin during co-culture. J Cell Biol. 1989;109:309-315.