Alpha-smooth Muscle Actin mRNA Research Articles

Triiodothyronine induces over-expression of alpha-smooth muscle actin, restricts myofibrillar expansion and is permissive for the action of basic fibroblast growth factor and insulin-like growth factor I in adult rat cardiomyocytes.

Effects of triiodothyronine (T3) on the expression of cytoskeletal and myofibrillar proteins in adult rat cardiomyocytes (ARC) were followed during two weeks of culture in the presence of 20% T3-depleted (stripped) FCS. Control cultures expressed mainly beta-myosin heavy chain (MHC) mRNA. T3 caused a switch to alpha-MHC expression and a dose-dependent increase of alpha-smooth muscle (alpha-sm) actin mRNA and protein. In parallel, the number of alpha-sm actin immunoreactive cells increased from 1% in controls to 29 and 62% in ARC treated with 5 and 100 nM T3. In the presence of T3, cells exhibited a higher beating rate than controls. The distribution of myofibrils in T3-treated cells was restricted to the perinuclear area with a sharp boundary. Only 5% of the control cells but 30 and 62% of the T3-treated (5 and 100 nM) ARC showed this restricted myofibrillar phenotype. Basic fibroblast growth factor (bFGF) which restricts myofibrillar growth and upregulates alpha-sm actin in ARC cultured with normal FCS had no effect on alpha-sm actin in ARC cultured in stripped FCS, but potentiated the effect of T3. In contrast, insulin-like growth factor I (IGF I), which suppresses alpha-sm actin and stimulates myofibrillogenesis in the presence of normal FCS suppressed T3-induced alpha-sm actin expression in stripped FCS. Thus, T3 appears to be permissive for the action of bFGF and IGF I on alpha-sm actin expression.

Advanced glycation end products modulate transcriptional regulation in mesangial cells

Advanced glycation end products (AGEs) stimulate synthesis of extracellular matrix (ECM) in a receptor-mediated manner on mesangial cells. In the present study, we examined the transcriptional regulation of the gene for type IV collagen [(IV)collagen], which is one of the major components of mesangial sclerosis, after stimulation of AGEs on mesangial cells. The methylation pattern of the promoter/enhancer region of (IV)collagen gene was similar in AGE-treated and control cells. AGEs significantly increased the transcriptional activity of the (IV)collagen gene, as measured by transient transfection assays using the reporter gene construct containing (IV)collagen promoter/enhancer and the chloramphenicol acetyltransferase gene. AGEs also increased smooth muscle alpha-actin mRNA levels as well as its transcriptional activity. Nuclear factor binding of the promoter of (IV)collagen gene was stimulated by AGEs. Furthermore, AGEs dramatically decreased the mRNA levels of (IV)collagen promoter binding protein (MSW), a larger subunit of DNA replication complex, AP1. These results suggest that AGEs increase expression of (IV)collagen gene by modulating the levels of promoter binding proteins. These transcriptional events may play a critical role in ECM accumulation in response to AGEs.

Differentiation of smooth muscle phenotypes in mouse mesangial cells

Smooth muscle alpha-actin (SMA) mRNA, a marker of vascular smooth muscle cells, was identified in the normal glomerular mesangium both in vivo and in vitro. Several populations of mesangial cells were studied to determine if SMA and basement membrane collagen were regulated together. The levels of SMA expression, which could be linked to the stage of differentiation, were different for the differing cell populations. One cell population had high SMA and type IV collagen levels at its early passages. The others expressed both interstitial and basement membrane collagens. The first population developed these phenotypic features at later passages. The levels of SMA and alpha 1(IV) collagen expression were regulated together in concert, whereas the alpha 2(I) collagen levels were expressed inversely to SMA and alpha 1(IV) collagen. Both SMA and type IV collagen were controlled by the methylation states of the cis-regulators; however, type I collagen was mainly regulated by the trans-acting regulators. Treatment with 5-azacytidine converted the cells of a fibroblast-phenotype to a smooth muscle cell-like phenotype. These cell lines may be useful for studying the differentiation process in vitro.

Proteoglycan biosynthesis is required in BC3H1 myogenic cells for modulation of vascular smooth muscle α‐actin gene expression in response to microenvironmental signals

Induction of vascular smooth muscle (VSM) alpha-actin mRNA expression during cytodifferentiation of mouse BC3H1 myogenic cells coincides with the accumulation of cell surface- and extracellular matrix-associated sulfated proteoglycans. Inhibition of proteoglycan biosynthesis in myogenic cells using an artificial beta-D-xyloside glycosaminoglycan acceptor was accompanied by a reduction in cell surface/extracellular matrix proteoglycans and VSM alpha-actin mRNA expression while enhancing the secretion of free chondroitin sulfate/dermatan sulfate and heparan sulfate glycosaminoglycans into the culture medium. Maximum inhibition of VSM alpha-actin mRNA expression required that proteoglycan biosynthesis be blocked during the early phase of cytodifferentiation when myoblasts were fully confluent and quiescent. The inhibitory effect of beta-D-xyloside on alpha-actin mRNA expression resulted from attenuation at both the transcriptional and post-transcriptional control points. Sustained proteoglycan biosynthesis was required for induction of VSM alpha-actin mRNA in quiescent myoblasts in response to cytodifferentiation-permissive, substrate-associated macromolecules (SAM) or upon exposure to soluble serum factors capable of transiently stimulating VSM alpha-actin gene transcription. The results suggested that efficient myoblast cytodifferentiation and modulation of VSM alpha-actin mRNA levels depended on intact cell surface proteoglycans to convey signals generated as a consequence of cellular interaction with substrate components and serum factors.

Overexpression of transforming growth factor-beta 1 mRNA is associated with up-regulation of glomerular tenascin and laminin gene expression in nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop immune-mediated insulin-dependent diabetes mellitus and nephropathy, providing an opportunity to study the early molecular events in a model of diabetic glomerulosclerosis. The expression of several genes coding for growth factors and extracellular matrix was examined in microdissected glomeruli, by the use of reverse transcription-competitive polymerase chain reaction, in diabetic NOD mice (mean duration of diabetes, 28.5 +/- 7 days) and age-matched nondiabetic NOD mice with normal glucose tolerance. The levels of mRNA coding for transforming growth factor-beta 1, tenascin, and laminin B1 increased 1.9-, 2.0-, and 1.7-fold, respectively, whereas platelet-derived growth factor (PDGF)-B, alpha 1(IV) collagen, 72-kd collagenase, alpha-smooth muscle actin, and beta-actin mRNA remained stable in the diabetic mice. The kidney advanced glycosylation end-products levels increased 2.1-fold in the diabetic mice, and the diabetic glomeruli showed an accumulation of tenascin and laminin but not of type IV collagen by immunofluorescence microscopy. There was no increase in cell number per glomerulus after the onset of diabetes, a finding consistent with stable PDGF-B and alpha-smooth muscle actin mRNA levels. These findings provide evidence that increased glomerular transforming growth factor-beta 1, but not PDGF-B, mRNA is associated with the up-regulation of tenascin and laminin expression after advanced glycosylation endproduct accumulation, early after the onset of diabetes.

Distribution of gelsolin in mouse ovary.

The distribution of gelsolin, a calcium-dependent actin-modulating protein, and the expression of the corresponding gene, have been characterized with respect to morphogenetic processes in mouse ovary. Substantial amounts of gelsolin have been detected in the ovary and uterus of the mouse by immunoblot analysis. The similar relative ratio of mRNA of alpha-smooth muscle actin (alpha-SM actin) and gelsolin in the two organs suggests that expression of these two genes is coordinated at the transcriptional level. Immunofluorescence has demonstrated gelsolin predominantly in three types of cells in the ovary: (1) cells of the theca externa and stroma, (2) endothelial cells lining blood vessels, and (3) cells of the superficial epithelium of ovary. In the smooth-muscle-like cells of the theca externa, gelsolin appears tightly associated with the microfilamentous cytoskeleton, which is also rich in alpha-SM actin. The presence of gelsolin in myoid cells suggests that this protein, possibly by modulation of the activity of the actomyosin ATPase, plays a critical role in contractile and morphogenetic processes, e.g., during growth and maturation of the follicle or during ovulation. In cells of the endothelium, intracellular gelsolin is associated with the F-actin cytoskeleton around the nucleus. The circumferential belt lining the lateral cell membranes in cells of the superficial epithelium at the ovarian surface is also rich in gelsolin. Our observations indicate that the function of gelsolin as a calcium- and phospholipid-dependent modulator of actin assemblies is pivotal for the regulation of the dynamic alterations of the actin cytoskeleton in the superficial epithelium when cells become attenuated and retract their microvilli during growth of the follicle.

Deregulated expression of the c-myc oncogene abolishes inhibition of proliferation of rat vascular smooth muscle cells by serum reduction, interferon-gamma, heparin, and cyclic nucleotide analogues and induces apoptosis.

We have investigated the requirement for c-myc downregulation in the growth arrest of vascular smooth muscle cells (VSMCs). Rat VSMCs were infected with a retrovirus vector directing constitutive expression of either the complete human c-Myc protein (VSM-myc cells) or the c-Myc deletion mutant D106-143, which is inactive in cotransformation and autosuppression assays (VSM-D106-143 myc cells). Clones of transfected VSM-myc cells were isolated that constitutively expressed a range of levels of c-Myc protein from that observed in normal proliferating VSMCs to approximately seven times normal. The growth rates of these clones and their responses to growth inhibitors were then assessed. VSM-myc clones possessed a shorter mean intermitotic time than normal cells, which was inversely correlated (P < .05) with the level of c-Myc protein expressed. VSM-myc cells also expressed lower levels of alpha-smooth muscle actin mRNA and protein and exhibited an altered morphology. The proliferation of normal VSMCs and VSM-D106-143 myc cells was inhibited by serum reduction (0.5% fetal calf serum) and also by treatment with interferon-gamma (100 IU/mL), heparin (50 micrograms/mL), 8-bromo-cAMP (0.1 mmol/L), or 8-bromo-cGMP (0.1 mmol/L). In contrast, proliferation of VSM-myc cells was not inhibited by any of these agents, even if present at 10-fold higher concentrations. However, approximately 75% of VSM-myc cells expressing levels of c-Myc protein seen in normal proliferating VSMCs underwent apoptosis after 4 days of serum reduction or treatment with interferon-gamma. The results show that constitutive c-myc expression induces continuous cell proliferation, reduction in alpha-smooth muscle actin expression and apoptosis in VSMCs. We conclude that downregulation of c-myc is a prerequisite for growth arrest and subsequent survival of VSMCs. Conversely, deregulated c-myc expression may be important in the proliferation and death of VSMCs--characteristics of the pathogenesis of atherosclerosis.

Alterations in the expression of the beta-cytoplasmic and the gamma-smooth muscle actins in hypertrophied urinary bladder smooth muscle.

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle gamma-actin, shows over 200% increase in the gamma-actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in gamma-actin, since the 2D gel electrophoresis does not distinguish gamma-smooth muscle actin from gamma-cytoplasmic actin. The bladder smooth muscle alpha-actin and the smooth muscle alpha-actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the beta-cytoplasmic actin (37%) and a concomitant diminution in the beta-cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.

ECGF and heparin determine differentiation of cloned cerebral endothelial cells in vitro.

Protein expression patterns of morphologically different cloned capillary endothelial cells from porcine and murine brain cortices were examined. Type I cells, grown in medium containing heparin and endothelial cell growth factor (ECGF), exhibited a polygonal, cobblestone appearance and appeared to replicate the cells of the blood-brain barrier endothelium. Type II cells, grown in medium without heparin and ECGF, were elongated and appeared to replicate capillaries in central nervous system tissue. Cells of both phenotypes stained positive by the specific endothelial cell marker Bandeiraea simplicifolia lectin. The expression of alpha smooth-muscle actin (mRNA and protein) was taken as a marker for type II cells. By use of 2-D gel images and the GELLAB II system, a data base was created revealing that two proteins (90 kDa, pI 5.1, and 35 kDa, pI 5.7) were exclusively expressed in type I cells. Furthermore, the synergistic action of ECGF and heparin in respect to the phenotypic determination of cerebral endothelial cells was demonstrated.

Age-related changes in sarcoplasmic reticulum Ca(2+)-ATPase and alpha-smooth muscle actin gene expression in aortas of normotensive and spontaneously hypertensive rats.

The expression of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase gene and the SR Ca2+ pump function were investigated in thoracic aortas of 5- and 17-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The relative level of the two isoforms of SR Ca(2+)-ATPase mRNA expressed in the aorta (i.e., SERCA 2a and SERCA 2b) was determined by quantitative S1 nuclease protection analysis and normalized to the level of alpha-smooth muscle (alpha-Sm) actin mRNA. The level of alpha-Sm actin mRNA itself was normalized to the level of 18S ribosomal RNA using slot-blot hybridization assays. Total SR Ca2+ pump activity was estimated by measuring the rate of oxalate-supported Ca2+ uptake in homogenates. At 5 weeks, the amount of SERCA 2a and SERCA 2b mRNA, normalized to 18S ribosomal RNA, and the ratio of alpha-Sm actin mRNA to 18S RNA were identical in SHR and WKY rats. The Ca2+ pump activity was similar in the two strains of rats at 5 weeks. From 5 to 17 weeks, the amount of SERCA 2a mRNA increased in both strains while the level of SERCA 2b mRNA remained constant. The Ca2+ pump activity was unchanged in SHRs and tended to decrease in WKY rats. Accordingly, the change in the ratio of the SR Ca(2+)-ATPase mRNA isoforms does not appear to influence SR function. The level of alpha-Sm actin mRNA and SERCA 2a mRNA increased in parallel from 5 to 17 weeks in both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Dietary-induced atherosclerotic lesions have increased levels of acidic FGF mRNA and altered cytoskeletal and extracellular matrix mRNA expression.

Growth factor and extracellular matrix gene expression by vessel wall cells influence the development of arterial lesions. In this study, we compared the level of acidic and basic fibroblast growth factor mRNA expression in aortic vessels from normal swine and from swine with dietary-induced vascular lesions. There was a striking increase in the level of acidic fibroblast growth factor mRNA within the lesions while the level of basic fibroblast growth factor mRNA decreased. Swine fed an atherosclerotic diet supplemented with L-arginine developed atherosclerotic plaques that also contained increased levels of acidic fibroblast growth factor mRNA. We also examined the expression level of a number of extracellular matrix and cytoskeletal mRNAs to compare the biosynthetic state of normal arteries and atherosclerotic plaques. Compared with the normal artery, the level of alpha-smooth muscle actin mRNA decreased, and there was a concomitant increase in vimentin, fibronectin and thrombospondin mRNA levels. Surprisingly, alpha 1(I), alpha 2(I) and alpha 1(III) collagen mRNA levels were decreased in the atherosclerotic lesions when compared with the normal artery. These results indicate that vascular lesion formation in hypercholesterolemic swine is accompanied by alterations in growth factor, cytoskeletal and extracellular matrix gene expression.

Density-dependent modulation of vascular smooth muscle alpha-actin biosynthetic processing in differentiated BC3H1 myogenic cells.

The expression of vascular smooth muscle (VSM) alpha-actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non-proteolytic dissociation of cell-cell and cell-matrix contacts in post-confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA-dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal alpha-actin mRNA. Muscle alpha-actin mRNA levels in EDTA-dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse-labeling techniques using L-[35S]cysteine to identify muscle actin biosynthetic intermediates revealed that EDTA-dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle alpha-actin polypeptide chains. However EDTA-dispersed myocytes were less efficient in the post-translational processing of immature VSM alpha-actin compared to non-dispersed myocytes. Simple cell-to-cell contact may mediate VSM alpha-actin processing efficiency since high-density preparations of EDTA-dispersed myocytes processed more VSM alpha-actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mechanisms capable of discriminating between different isoform classes of nascent actin polypeptide chains.

Disorganization of microfilaments is accompanied by downregulation of alpha-smooth muscle actin isoform mRNA level in cultured vascular smooth muscle cells.

In the previous report, we demonstrated that cAMP negatively regulated alpha-smooth muscle actin mRNA levels through destabilization of the mRNA [O. Ohara et al. (1991) J. Biochem. 109, 834-839]. We here report that the decline in the alpha-smooth muscle actin mRNA levels was well correlated with disorganization of microfilaments but not necessarily with the rise in intracellular cAMP levels in rat cultured vascular smooth muscle cells. The decrease in the alpha-smooth muscle actin mRNA induced by microfilament-disorganizing agents also resulted from enhancement of the turnover rate of the mRNA. These results raise the possibility that the post-transcriptional control of the alpha-smooth muscle actin expression is linked to the organization of actin filaments in smooth muscle cells.

The vascular smooth muscle alpha-actin gene is reactivated during cardiac hypertrophy provoked by load.

Cardiac hypertrophy triggered by mechanical load possesses features in common with growth factor signal transduction. A hemodynamic load provokes rapid expression of the growth factor-inducible nuclear oncogene, c-fos, and certain peptide growth factors specifically stimulate the "fetal" cardiac genes associated with hypertrophy, even in the absence of load. These include the gene encoding vascular smooth muscle alpha-actin, the earliest alpha-actin expressed during cardiac myogenesis; however, it is not known whether reactivation of the smooth muscle alpha-actin gene occurs in ventricular hypertrophy. We therefore investigated myocardial expression of the smooth muscle alpha-actin gene after hemodynamic overload. Smooth muscle alpha-actin mRNA was discernible 24 h after coarctation and was persistently expressed for up to 30 d. In hypertrophied hearts, the prevalence of smooth muscle alpha-actin gene induction was 0.909, versus 0.545 for skeletal muscle alpha-actin (P less than 0.05). Ventricular mass after 2 d or more of aortic constriction was more highly correlated with smooth muscle alpha-actin gene activation (r = 0.852; P = 0.0001) than with skeletal muscle alpha-actin (r = 0.532; P = 0.009); P less than 0.0005 for the difference in the correlation coefficients. Thus, smooth muscle alpha-actin is a molecular marker of the presence and extent of pressure-overload hypertrophy, whose correlation with cardiac growth at least equals that of skeletal alpha-actin. Induction of smooth muscle alpha-actin was delayed and sustained after aortic constriction, whereas the nuclear oncogenes c-jun and junB were expressed rapidly and transiently, providing potential dimerization partners for transcriptional control by c-fos.

Mechanisms of angiotensin II- and arginine vasopressin-induced increases in protein synthesis and content in cultured rat aortic smooth muscle cells. Evidence for selective increases in smooth muscle isoactin expression.

Previous studies from this laboratory have demonstrated that angiotensin II (Ang II) and arginine vasopressin (AVP) are potent hypertrophic agents in cultured rat aortic smooth muscle cells. The present study identified major proteins that accumulate in Ang II-induced and AVP-induced hypertrophic cells and initiated studies of the mechanisms that contribute to their accumulation. Smooth muscle cell hypertrophy induced by Ang II and/or AVP (1 microM each) was associated with widespread increases in the content of many cellular proteins that were resolved by one- and two-dimensional gel electrophoresis. However, increases were also selective in nature, with increases in certain individual proteins, including actin (twofold to threefold), vimentin (2.5-fold to sevenfold), tropomyosin (threefold to sixfold), and myosin heavy chain, far exceeding overall increases in cellular protein content (20-40%). Increases in actin content were due largely to increased expression of smooth muscle alpha-actin (3.6- to 7.5-fold), as opposed to nonmuscle beta-actin (1.7- to 2.5-fold). Increases in smooth muscle alpha-actin were accompanied by a fivefold to eightfold increases in smooth muscle alpha-actin mRNA, indicating that these changes were not due exclusively to translational controls. Results demonstrate that contractile agonist-induced hypertrophy in cultured smooth muscle cells is due, in part, to increased expression of smooth muscle contractile proteins. Furthermore, the fact that Ang II and AVP induced selective increases in smooth muscle alpha-actin suggests that these agonists may not only regulate growth of vascular smooth muscle but may also promote expression of smooth muscle-specific contractile proteins during differentiation of vascular smooth muscle.

Platelet‐derived growth factor‐induced destabilization of smooth muscle alpha‐actin mRNA

We have previously shown that treatment of postconfluent, quiescent rat vascular smooth muscle cells (SMC) with platelet-derived growth factor (PDGF) dramatically reduced smooth muscle (SM) alpha-actin synthesis and SM alpha-actin mRNA abundance, suggesting a role for this mitogen in the control of SMC differentiation. In the present studies, we explored the molecular mechanisms whereby PDGF decreases SM alpha-actin mRNA levels. Treatment of postconfluent SMC with both platelet PDGF and recombinant PDGF-BB resulted in a dramatic and concentration-dependent decrease in SM alpha-actin mRNA levels. We observed no differences in efficacy between platelet PDGF and PDGF-BB, indicating that the PDGF-A chain is not required for the effect. The rate of decrease in SM alpha-actin mRNA abundance in PDGF-treated SMC was greater than that observed in cells treated with the transcriptional inhibitor, actinomycin D, with or without PDGF, indicating that PDGF induced a transcriptionally dependent destabilization of the cytosolic SM alpha-actin mRNA pool. This effect appeared selective for SM alpha-actin, in that there was no evidence of a similar change in non-muscle (NM) beta-actin mRNA stability following PDGF treatment. Results of nuclear run-on analyses showed no differences in SM alpha-actin transcription between PDGF- and vehicle-treated SMC at either 4 or 24 hours following treatment, demonstrating that decreases in transcription of the SM alpha-actin gene did not contribute to PDGF-induced changes in SM alpha-actin mRNA abundance. Results of these studies support a possible role for PDGF in regulation of SMC differentiation via a post-transcriptional control mechanism.

Regulation of α‐smooth muscle actin and other polypeptides in proliferating and density‐arrested vascular smooth muscle cells

We have examined alpha-smooth muscle actin (alpha-SM actin) protein and mRNA levels in proliferating and density-arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two-dimensional (2-D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2-D gel analysis, we consistently detected increased expression of 12 polypeptides in growth-arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth-arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high-density growth-arrested cells. Under these conditions we observed no change in relative alpha-SM actin protein content as determined by 2-D gel analysis and Western blots. This was corroborated by high levels of alpha-SM actin mRNA levels in both proliferating and high-density growth-arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (alpha-SM actin) in a proliferation- and density-independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically- and explant-derived SMC contained high levels of alpha-SM actin as determined by immunofluorescence and by Northern analysis.

Effect of intestinal bypass on the expression of actin mRNA in ileal smooth muscle

In this study, messenger RNAs (mRNAs) for actin isoforms were assessed in longitudinal smooth muscle from the ileum of unoperated rats and from rats that had undergone bypass of the middle 70% of the small intestine. The plasmid clone pGEM 10C, which contains a DNA insert complementary to the 3' untranslated region and the region of mRNA that codes for the synthesis of alpha-smooth muscle actin protein, was used to synthesize two riboprobes. One probe, complementary to the coding region of the insert, hybridizes to most, if not all, actin isoform mRNAs. The second probe, complementary to the 3' untranslated region of the insert, hybridizes only to alpha-smooth muscle actin mRNA. RNA was isolated from animals 4 to 5 days after operation, size fractionated by denaturing gel electrophoresis, transferred to nylon membranes, and exposed to the two 32P-labeled riboprobes. Both probes hybridized to RNA of about 1.3 kilobases long. Longitudinal muscle from both groups of animals contained alpha-smooth muscle actin mRNA as well as mRNA for other actin isoforms. Dot blots of varying amounts of RNA were hybridized to the riboprobes to determine the proportions of actin mRNAs. The content and concentration of mRNAs for all actins, and of mRNA for alpha-smooth muscle actin, were significantly greater in muscle from the functioning ileum of bypassed animals 4-5 days after the operation. Thus the operation induces a rapid, specific activation of these contractile protein genes.

Interferon gamma inhibits both proliferation and expression of differentiation-specific alpha-smooth muscle actin in arterial smooth muscle cells.

Differentiation of muscle cells is characterized morphologically by the acquisition of contractile filaments and characteristic shape changes, and on the molecular level by induction of the expression of several genes, including those for the muscle-specific alpha-actin isoforms. IFN-gamma is an inhibitor of proliferation for several cells, including vascular smooth muscle, and is also an inducer of differentiated properties for several hematopoietic cells. We have therefore investigated whether IFN-gamma affects the expression of alpha-smooth muscle actin in cultured arterial smooth muscle cells. Cells exposed to IFN-gamma show a reduction of alpha-smooth muscle actin-containing stress fibers, as detected by immunofluorescence. The effect was observed in all phases of the cell cycle, and was caused by a reduction of the synthesis of alpha-smooth muscle actin protein as revealed by two-dimensional electrophoretic analysis of actin isoforms. RNA hybridization using a cRNA probe that hybridizes to all actin mRNAs showed that IFN-gamma-treated cells have a reduced content of the 1.7-kb mRNA that codes for alpha-smooth muscle actin, and to a lesser extent, also of the 2.1-kb mRNA encoding the beta and gamma-cytoplasmic actins. The reduction of alpha-smooth muscle actin mRNA was confirmed using an alpha-smooth muscle actin-specific cRNA probe. The reduction of alpha-smooth muscle actin mRNA occurs within 12 h, and is dependent on protein synthesis, since cycloheximide treatment reversed the effect. The inhibition of this mRNA species was dose dependent, and detectable by RNA hybridization at a dose of 50 U/ml IFN-gamma. These results suggest that the differentiation of arterial smooth muscle cells is not necessarily coupled to an inhibition of cellular proliferation. Instead, IFN-gamma may regulate the expression of several genes that control both proliferation and expression of differentiation markers.

Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Correlation with cell density and proliferative state.

To investigate a possible correlation between cytodifferentiation, proliferation, and actin expression, smooth muscle cells from the 9-week-old rabbit aortic media were enzyme-dispersed into single cells and were plated in primary culture at different initial seeding densities. The volume fraction of myofilaments (Vv myo) in cells seeded moderately densely fell from 39.5% +/- 1.2% in the intact aortic media to 11.5% +/- 1.6% on Day 5, one day before the onset of logarithmic growth. The Vv myo remained low over the next 3 days, then began to rise as the density of cells increased, returning almost to the original levels after confluency and 1.84 cumulative population doublings (CPD). The expression of alpha-smooth muscle actin mRNA followed a similar time course of change, falling from 84.7% +/- 1.2% of total actin mRNA in freshly isolated cells to 54.0% +/- 6.5% on Day 5, returning to 87.5% +/- 0.5% after confluency. In these cultures, the alpha-smooth muscle actin protein content was 93.7% +/- 2.9% of total actin in freshly isolated cells, 68.7% +/- 3.1% on Day 5, and 73.3% +/- 2.5% 3 days after confluency. In densely seeded cultures, the Vv myo and expression of alpha-smooth muscle actin mRNA fell only slightly on Day 5 and rose to original levels upon confluency after 0.33 CPD. However, at the protein level, alpha-smooth muscle actin decreased on Day 5 and remained low on Day 12. The Vv myo, alpha-smooth muscle actin mRNA, and actin protein of sparsely seeded cells fell on Day 5 and then remained low throughout the culture period, including 5 days after confluency (Day 24), when the cells had undergone 5.37 CPD. Cells that were maintained subconfluent but quiescent on Day 7 in culture had the same low Vv myo, low alpha-actin mRNA expression, and low alpha-actin protein content as actively proliferating cells. The results show that Vv myo and alpha-smooth muscle actin mRNA undergo parallel changes during primary culture according to seeding density, but not to replication, and that alpha-smooth muscle actin protein decreases in culture then remains low irrespective of culture conditions.