The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle

Transforming growth factor (TGF) β1 increases pro‐inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3′ kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro‐inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110β inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL‐6, calponin or smooth muscle α‐actin (α‐SMA) expression. However, TGFβ1 increased calponin and α‐SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFβ1 induced IL‐6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α‐SMA and the proinflammatory cytokine IL‐6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. J. Cell. Physiol. 227: 3044–3052, 2012. © 2011 Wiley Periodicals, Inc.     

Expression of calponin in rabbit and human aortic smooth muscle cells

Summary Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture,ccalponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.     

Immunoelectron microscopic evidence of contractile proteins in the cellular and acellular components of mouse kidney glomeruli

Actin and/or actin-like protein have been localized in the cellular and acellular components of the glomerular walls of mouse kidney by means of immunoelectron microscopy, employing human antibodies to smooth muscle (SMA). Contractile antigens have been confirmed to be present in the cytoplasm of podocytes and mesangial cells in association with fine filaments which are considered of importance in the control of blood flow, intravascular pressure, and filtration rate within the glomerulus. The extracellular presence of contractile proteins in the mesangial matrix and glomerular basement membrane can be related to cell movement in a frictional environment. This latter phenomenon, which is strictly interdependent with cell adhesion and aggregation, is most evident in the mesangial cells in a form of luminar pseudopodia, cytoplasmic projections, and phagocytosis.     

Bindarit Inhibits Human Coronary Artery Smooth Muscle Cell Proliferation,Migration and Phenotypic Switching

Bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs) synthesis, reduces neointimal formation in animal models of vascular injury and recently has been shown to inhibit in-stent late loss in a placebo-controlled phase II clinical trial. However, the mechanisms underlying the efficacy of bindarit in controlling neointimal formation/restenosis have not been fully elucidated. Therefore, we investigated the effect of bindarit on human coronary smooth muscle cells activation, drawing attention to the phenotypic modulation process, focusing on contractile proteins expression as well as proliferation and migration. The expression of contractile proteins was evaluated by western blot analysis on cultured human coronary smooth muscle cells stimulated with TNF-α (30 ng/mL) or fetal bovine serum (5%). Bindarit (100–300 µM) reduced the embryonic form of smooth muscle myosin heavy chain while increased smooth muscle α-actin and calponin in both TNF-α- and fetal bovine serum-stimulated cells. These effects were associated with the inhibition of human coronary smooth muscle cell proliferation/migration and both MCP-1 and MCP-3 production. The effect of bindarit on smooth muscle cells phenotypic switching was confirmed in vivo in the rat balloon angioplasty model. Bindarit (200 mg/Kg/day) significantly reduced the expression of the embryonic form of smooth muscle myosin heavy chain, and increased smooth muscle α-actin and calponin in the rat carodid arteries subjected to endothelial denudation. Our results demonstrate that bindarit induces the differentiated state of human coronary smooth muscle cells, suggesting a novel underlying mechanisms by which this drug inhibits neointimal formation.     

Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells

Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β(1). As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β(1) induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β(1). The failure by TGF-β(1) to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β(1) signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.     

Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development

Abstract. Calponin and SM 22 are two proteins related in sequence that are particularly abundant in smooth muscle cells. Here, the distribution patterns of calponin and SM 22 were compared with that of other smooth muscle contractile and cytoskeletal components in the avian embryo using immunofluorescence microscopy and immunoblotting. Like myosin-light-chain kinase and heavy caldesmon, both calponin and SM 22 were more or less exclusively found in smooth muscle cells, during embryonic development and in the adult. Labelling of other cell types including striated muscle was not observed. In contrast, tropomyosin, smooth muscle α-actin, filamin and desmin could also be detected in many other cell types in addition to smooth muscles, at least during part of embryonic life. Calponin and SM 22 appeared almost synchronously during the differentiation of all smooth muscle cell populations, though with a slight time difference in the case of the aorta. The appearance of calponin, SM22 and heavy caldesmon was generally delayed in relation to desmin, tropomyosin, smooth muscle α-actin, myosin-light-chain kinase and filamin and a marked increase in abundance of these proteins was observed in the late embryo and in the adult. From these observations we can conclude that both calponin and SM 22 belong to a group of late differentiation determinants in smooth muscle and may constitute convenient and reliable markers to follow the differentiation of most, if not all, smooth muscle cell populations.     

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.     

Effect of Oxidative Stress on the Expression of Thin Filament-Associated Proteins in Gastric Smooth Muscle Cells

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague–Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H₂O₂) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H₂O₂ or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.     

DJ‐1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle‐like Cells in Response to Sphingosylphosphorylcholine

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox‐sensitive molecules including a multifunctional protein DJ‐1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)‐stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α‐smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC‐stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ‐1 in hMSCs in response to SPC. The increased abundance of oxidized DJ‐1 in SPC‐stimulated hMSCs was validated by immunoblot analysis. The SPC‐induced increase in the expression of α‐SMA was stronger in DJ‐1‐knockdown hMSCs than in control cells. Moreover, the expression of α‐SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ‐1‐overexpressing hMSCs. Exogenous H₂O₂ mimicked the responses induced by SPC treatment. These results indicate that the ROS‐related DJ‐1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.     

microRNA let‐7g suppresses PDGF‐induced conversion of vascular smooth muscle cell into the synthetic phenotype

Platelet‐derived growth factor (PDGF) can promote vascular smooth muscle cells (VSMCs) to switch from the quiescent contractile phenotype to synthetic phenotype, which contributes to atherosclerosis. We aimed to investigate the role of microRNA let‐7g in phenotypic switching. Bioinformatics prediction was used to find let‐7g target genes in the PDGF/mitogen‐activated protein kinase kinase kinase 1 (MEKK1)/extracellular signal‐regulated kinase (ERK)/Krüppel‐like factor‐4 (KLF4) signalling pathway that affects VSMC phenotypic switching. The luciferase reporter assay and let‐7g transfection were used to confirm let‐7g target genes. Two contractile proteins alpha‐smooth muscle actin (α‐SMA) and calponin were VSMC‐specific genes and were measured as the indicators for VSMC phenotype. Lentivirus carrying the let‐7g gene was injected to apolipoprotein E knockout (apoE^?/?) mice to confirm let‐7g's effect on preventing atherosclerosis. Through the PDGF/MEKK1/ERK/KLF4 signalling pathway, PDGF‐BB can inhibit α‐SMA and calponin. The PDGFB and MEKK1 genes were predicted to harbour let‐7g binding sites, which were confirmed by our reporter assays. Transfection of let‐7g to VSMC also reduced PDGFB and MEKK1 levels. Moreover, we showed that let‐7g decreased phosphorylated‐ERK1/2 while had no effect on total ERK1/2. KLF4 can reduce VSMC‐specific gene expression by preventing myocardin–serum response factor (SRF) complex from associating with these gene promoters. The immunoprecipitation assay showed that let‐7g decreased the interaction between KLF4 and SRF. Further experiments demonstrated that let‐7g can increase α‐SMA and calponin levels to maintain VSMC in the contractile status. Injection of lentivirus carrying let‐7g gene increased let‐7g's levels in aorta and significantly decreased atherosclerotic plaques in the apoE^?/? mice. We demonstrated that let‐7g reduces the PDGF/MEKK1/ERK/KLF4 signalling to maintain VSMC in the contractile status, which further reduce VSMC atherosclerotic change.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

Heterogeneity of smooth muscle-associated proteins in mammalian brain microvasculature

In the brain, the microvascular system is composed of endothelial cells surrounded by a layer of pericytes. The lack of smooth muscle cells in this tissue suggests that any contractile function must be performed by one or both of these cell types. The present study was undertaken in order to identify cells in terminal blood vessels that contain smooth muscle-like contractile machinery. Endothelial cells were reactive with antibodies against smooth muscle myosin but showed no other smooth muscle-related features. In contrast, pericytes of intact microvessels showed a pattern of protein expression similar to that of smooth muscle cells. Pericytes also behaved in tissue culture like cultured smooth muscle cells, with regard to the changes in expression of smooth muscle-related proteins. These data confirm the close relationship between smooth muscle cells and pericytes, and point to their contractile function in the brain microvessels.     

Distribution of Caldesmon and of the Acidic Isoform of Calponin in Cultured Cerebellar Neurons and in Different Regions of the Rat Brain: An Immunofluorescence and Confocal Microscopy Study

Caldesmon and calponin are two F-actin-binding and calcium-calmodulin-dependent proteins. In smooth muscle and nonmuscle cells both proteins are localized on actin filaments. Using one- or two-dimensional gel electrophoresis followed by the Western blot technique, and by immunofluorescence studies, we have given evidence that calponin is also present in rat and pig brain. In the present study, for the first time, we demonstrate caldesmon- and calponin-specific immunoreactivities in cerebellar cultured neurons. In the rat central nervous system these antibodies mainly stain neuronal cell bodies and dendrites. By confocal analysis we observed that calponin and caldesmon are located in the actomyosin domain although the total actin and myosin were not saturated. In many cases it is clear that these two proteins are adjacent rather than superimposed in the same domain of the cell. These results are compatible with the functional role of caldesmon and calponin in the regulation of the actomyosin activity as described by others and suggest that they are part of the contractile apparatus of neural cells.     

Smooth muscle calponin. Inhibition of actomyosin MgATPase and regulation by phosphorylation

Calponin isolated from chicken gizzard smooth muscle inhibits the actin-activated MgATPase activity of smooth muscle myosin in a reconstituted system composed of contractile and regulatory proteins. ATPase inhibition is not due to inhibition of myosin phosphorylation since, at calponin concentrations sufficient to cause maximal ATPase inhibition, myosin phosphorylation was unaffected. Furthermore, calponin inhibited the actin-activated MgATPase of fully phosphorylated or thiophosphorylated myosin. Although calponin is a Ca2(+)-binding protein, inhibition did not require Ca2+. Furthermore, although calponin also binds to tropomyosin, ATPase inhibition was not dependent on the presence of tropomyosin. Calponin was phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II, but not by cAMP- or cGMP-dependent protein kinases, or myosin light chain kinase. Phosphorylation of calponin by either kinase resulted in loss of its ability to inhibit the actomyosin ATPase. The phosphorylated protein retained calmodulin and tropomyosin binding capabilities, but actin binding was greatly reduced. The calponin-actin interaction, therefore, appears to be responsible for inhibition of the actomyosin ATPase. These observations suggest that calponin may be involved in regulating actin-myosin interaction and, therefore, the contractile state of smooth muscle. Calponin function in turn is regulated by Ca2(+)-dependent phosphorylation.