Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental intimal thickening, and culture

The expression of actin-isoform mRNAs in the smooth muscle cells (SMC) of the aortic media in rats has been studied by Northern-blot hybridization, using a general actin-cRNA probe, and two cRNA probes specific for beta- and gamma-cytoplasmic actins, during: (1) development, (2) intimal thickening after endothelial injury induced by balloon catheterization, and (3) growth in culture. In 5-day-old rats, the ratio between alpha-smooth-muscle-actin mRNA and beta- and gamma-cytoplasmic-actin mRNAs was close to 1. It increased to about 4 in 6-week-old rats. Replicating SMC from regions of intimal thickening 15 days after endothelial injury, and SMC growing in culture contained a predominance of cytoplasmic actin mRNAs. Intimal SMC 60 days after endothelial injury (at which time the endothelium had fully regenerated) demonstrated a pattern of actin mRNAs similar to that of normal media. Functional mRNA measured by translation in a reticulocyte lysate showed increases in the level of alpha-actin and decreases in beta-actin in rats from 5 days to 6 weeks of age. These results suggest that during development, under pathological conditions, and in cell culture, the expression of actin isoforms in arterial SMC depends on many factors, including the amount and translation efficiency of mRNAs, and the relative stabilities of the proteins involved.     

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.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

Pericyte growth and contractile phenotype: Modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2–1.7-fold. Heparin (100 μg/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth ～50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 μg/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (αVSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas αVSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17–30 times the steady-state levels of αVSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in αVSMA and β actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (α:β). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8–10:1; α:β). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset. © 1993 Wiley-Liss, Inc.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

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.     

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.     

Differential regulation of actin and myosin isoenzyme synthesis in functionally overloaded skeletal muscle.

Overload hypertrophy of the chicken anterior latissimus dorsi muscle is accompanied by a replacement of one myosin isoenzyme (slow myosin-1, SM1) by another (slow myosin-2, SM2). To investigate the molecular mechanisms by which these changes occur, we measured the fractional synthesis rates (ks) in vivo of individual myosin-heavy-chain isoenzymes, total actin and total protein during the first 72 h of muscle growth. Although the ks of total protein and actin were doubled at 24 h, the ks for SM1 and SM2 were depressed. However, the ks of both isomyosins were nearly tripled by 72 h. Despite the increase in muscle size observed at 72 h, the amount of SM1 was reduced by half, indicating increased degradation of SM1. Results of translation of polyribosomes in vitro paralleled the results obtained in vivo. The proportion of total polyadenylylated mRNA in total RNA was increased at 48 and 72 h, but unchanged at 24 h despite the increase in protein synthesis at 24 h. Nuclease-protection analyses indicate that the level of specific SM1 and SM2 mRNAs change in a reciprocal fashion during overload. We conclude that gene-specific and temporal differences exist in the regulatory mechanisms that control overload-induced muscle growth.     

Gene expression in regenerating liver in relation to cell proliferation and stress

When hepatocyte proliferation is stimulated in the liver by partial hepatectomy, messenger RNAs coding for fibrinogen, actin, c-myc and topoisomerase I are rapidly accumulated. We distinguish an early phase of accumulation (0-3 h after partial hepatectomy) which is also observed after a sham operation for the four genes, and during inflammation produced by Freund's adjuvant in the case of fibrinogen and c-myc genes. The hepatic response to inflammation appears therefore to mimic events characteristic of the G0/G1 transition, such as the accumulation of the c-myc mRNA. The late phase of mRNA accumulation (beyond 3 h after partial hepatectomy) is typical of liver regeneration. The level of c-myc mRNA is transiently increased (20-fold over normal) 20 h after partial hepatectomy, that is, at the time of DNA synthesis. Topoisomerase-I mRNA level increases between 3 and 24 h after partial hepatectomy (5-10-fold over normal). These results suggest that accumulation of c-myc and topoisomerase-I mRNAs is associated with DNA replication in regenerating liver.     

Expression of myosin heavy chains during thyroid hormone-induced cardiac growth

The expression of mRNAs for two cardiac myosins has been studied in the ventricles of hypo- and hyperthyroid rabbits by using cloned cDNA sequences corresponding to the mRNAs of the alpha- and beta-myosin heavy chains (HCs). The temporal change in relative levels of the alpha and beta HC mRNAs after triiodothyronine (T3) treatment of hypothyroid rabbits was determined by nuclease S1 mapping. In the hypothyroid state, only NC beta-mRNA was expressed in the ventricles. The HC alpha-mRNA was first detectable 4 h after administration of T3 (200 micrograms/kg) to hypothyroid animals. By 12, 24, and 72 h, HC alpha-mRNA represented 20, 50, and 90% of total myosin mRNA. The relationship between the relative mRNA levels and relative synthesis rates of myosin HCs was evaluated in 5- to 6-wk-old normal and thyrotoxic rabbits. Myosin synthesis rates were determined by labeling of protein in vivo with [2H]leucine. The V1 (HC alpha) and V3 (HC beta) isomyosins were separated by immune affinity chromatography and the HCs were isolated electrophoretically. In a normal euthyroid group of animals and in animals 12 and 24 h after administration of 200 micrograms of thyroxine, the relative mRNA levels and relative synthesis rates of the alpha and beta HCs were not significantly different. Our results show that, first, thyroid hormone causes a rapid accumulation of HC alpha-mRNA and loss of HC alpha-mRNA, and second, in normal and thyrotoxic rabbits, the relative synthesis rates of HC alpha and HC beta reflect the relative abundance of their respective mRNAs. These data are consistent with the thyroid hormones regulating synthesis of ventricular myosin at steps that precede translation of its message.     

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.     

Developmental changes in actin and myosin heavy chain isoform expression in smooth muscle

Smooth muscle cells express isoforms of actin and myosin heavy chains (MHC). In early postnatal animals the nonmuscle (NM) actin and MHC isoforms in vascular (aorta) smooth muscle were present in relatively high percentages. More than 30% of the MHC and 40% of the actin isoforms were NM. The relative percentage of the NM isoforms decreased significantly as the animals reached maturity, with NM MHC less than 10% and NM actin less than 30% of the totals. Concurrent with this decrease in NM isoforms was an increase in the smooth muscle (SM) isoforms. The relative changes and time frame in which these changes occurred were very similar for the actin and MHC isoforms. In arterial tissue there were species differences for changes with development in the two SM MHC isoforms (SM1 and SM2). The ratio of SM1:SM2 in young rat aorta was approximately 0.5, while this same ratio was approximately 3 in young swine carotid. Both adult rats and swine had a SM1:SM2 MHC ratio of approximately 1.2. Rat bladder smooth muscle showed no significant change in NM vs SM ratio between young and old rats, while the SM1:SM2 ratio decreased from 2.7 to 1.7 between these age groups. The shifts in alpha and beta actin were similar to those in the vascular tissue, but of much smaller magnitude.     

Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization

The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and beta isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for alpha-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, beta-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.     

Beta and gamma actin mRNAs are differentially located within myoblasts

Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.