Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.     

RhoA modulates Smad signaling during transforming growth factor-beta-induced smooth muscle differentiation

We recently reported that transforming growth factor (TGF)-beta induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-beta actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-beta-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-beta. We demonstrate here that RhoA signaling is critical to TGF-beta-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle alpha-actin, SM22alpha, and calponin in TGF-beta-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-beta-treated cells. RhoA signaling was activated as early as 5 min following TGF-beta addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-beta-induced SMC differentiation.     

Smad3-mediated myocardin silencing: a novel mechanism governing the initiation of smooth muscle differentiation

Both TGF-β and myocardin (MYOCD) are important for smooth muscle cell (SMC) differentiation, but their precise role in regulating the initiation of SMC development is less clear. In TGF-β-induced SMC differentiation of pluripotent C3H10T1/2 progenitors, we found that TGF-β did not significantly induce Myocd mRNA expression until 18 h of stimulation. On the other hand, early SMC markers such as SM α-actin, SM22α, and SM calponin were detectable beginning 2 or 4 h after TGF-β treatment. These results suggest that Myocd expression is blocked during the initiation of TGF-β-induced SMC differentiation. Consistent with its endogenous expression, Myocd promoter activity was not elevated until 18 h following TGF-β stimulation. Surprisingly, Smad signaling was inhibitory to Myocd expression because blockade of Smad signaling enhanced Myocd promoter activity. Overexpression of Smad3, but not Smad2, inhibited Myocd promoter activity. Conversely, shRNA knockdown of Smad3 allowed TGF-β to activate the Myocd promoter in the initial phase of induction. Myocd was activated by PI3 kinase signaling and its downstream target Nkx2.5. Interestingly, Smad3 did not affect PI3 kinase activity. However, Smad3 physically interacted with Nkx2.5. This interaction blocked Nkx2.5 binding to the Myocd promoter in the early stage of TGF-β induction, leading to inhibition of Myocd mRNA expression. Moreover, Smad3 inhibited Nkx2.5-activated Myocd promoter activity in a dose-dependent manner. Taken together, our results reveal a novel mechanism for Smad3-mediated inhibition of Myocd in the initiation phase of SMC differentiation.     

Vascular smooth muscle cells spontaneously adopt a skeletal muscle phenotype: a unique Myf5(-)/MyoD(+) myogenic program.

Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.(J Histochem Cytochem 48:1173-1193, 2000)     

HOXB7 overexpression promotes differentiation of C3H10T1/2 cells to smooth muscle cells

The presence of immature smooth muscle cells and ectopic tissues such as fully-formed bone in atherosclerotic lesions, may result from recapitulation of embryonic mechanisms in the artery wall. We hypothesized that expression of homeobox genes is triggered in atherogenesis and that these regulate proliferation and differentiation of multipotential progenitor cells along one or more specific lineages. We identified expression of the homeobox gene HOXB7 in clones of bovine aortic medial cells previously shown to be multipotent. HOXB7 was subsequently detected in human atherosclerotic plaques by RT-PCR and in situ hybridization. Expression was localized to areas adjacent to calcification and scattered in media and neointima, which may be reflective of a role in either osteoblastic or smooth muscle cell differentiation. To differentiate between these possibilities, we overexpressed HOXB7 in C3H10T1/2 cells, a multipotent cell line able to differentiate into vascular smooth muscle cells (SMC), as well as osteogenic and chondrogenic lineages. Results showed that overexpression of HOXB7 increased proliferation 3.5-fold, and induced an SMC-like cell morphology. In addition, expression of the early SMC markers calponin and SM22alpha increased 4-fold and 3-fold respectively by semi-quantitative RT-PCR. Expression of the intermediate SMC marker smooth muscle myosin heavy chain (SM-MHC) did not change. No increase in osteogenic or chondrogenic differentiation was detected, neither in the C3H10T1/2 cells nor in M2 cells, a bone marrow stromal cell line used to confirm this result. These findings suggest that HOXB7 plays a role in expansion of immature cell populations or dedifferentiation of mature cells.     

Orai1 and Ca2+-independent phospholipase A2 are required for store-operated Icat-SOC current, Ca2+ entry, and proliferation of primary vascular smooth muscle cells

Store-operated Ca(2+) entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca(2+)-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca(2+)-independent phospholipase A(2) (iPLA(2)β, or PLA2G6) in activation of cat-SOC current (I(cat-SOC)), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA(2)β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA(2)β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.     

Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) alpha-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299-306). Results of this study show that, although there is a 6-fold increase in SM alpha-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM alpha-actin mRNA levels are not different between these cells. This suggests that the SM alpha-actin gene is constitutively active under both of these conditions and that accumulation of SM alpha-actin in postconfluent cells is due to translational and/or post-translational controls. The relationship between growth and cytodifferentiation was further explored by examining the effects of platelet-derived growth factor (PDGF)- or serum-induced growth on actin expression in postconfluent, quiescent cultures maintained in a defined serum-free media. Although both factors have been shown to stimulate proliferation and decrease fractional SM alpha-actin synthesis (Blank et al., 1988), their effects on actin mRNA levels were quite different. PDGF was found to induce a dramatic drop in SM alpha-actin steady state mRNA level but had no effect on nonmuscle beta-actin mRNA level. In contrast, serum stimulation was shown to increase nonmuscle beta-actin mRNA level, whereas SM alpha-actin mRNA level remained constant. Taken together these results indicate that PDGF is a specific and potent repressor of SM alpha-actin expression in vascular SMC and implicate a possible developmental role for PDGF in control of SMC differentiation. In addition, the observation that the level of SM alpha-actin mRNA is unaltered in serum-stimulated cells indicates that an absolute decrease in SM alpha-actin mRNA is not obligatory for cell cycle entrance.