Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.     

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.     

Effect of actin filament distribution on tensile properties of smooth muscle cells obtained from rat thoracic aortas

Tensile properties and actin filament distribution of rat aortic smooth muscle cells (SMCs) were measured in the same cells to correlate the mechanical properties of cells with their cytoskeleton. The cells freshly isolated from rat thoracic aorta with enzymatic dispersion (FSMCs), cultured cells (CSMCs), and CSMCs treated with cytochalasin D to disrupt their actin filaments (CSMCs-CYD) were stretched in a Ca(2+)- Mg(2+) -free Hank's balanced salt solution at 37 degrees C with an originally designed micro tensile tester. Some of CSMCs and CSMCs-CYD were fixed and stained with rhodamine phalloidin for actin filament after the tensile test while they remained attached to the tester. The force-elongation curves were almost linear for all of the three groups. Normalized stiffness E(all) obtained from the slope of the curves was significantly different among groups and was 11.0 +/- 1.9 kPa (mean+/-SEM, n = 8), 2.6 +/- 0.5 kPa (n = 21), 1.5 +/- 0.2 kPa (n = 13), for FSMCs, CSMCs, and CSMCs-CYD, respectively. Relative concentration of the actin filament in the central region of the cell F has significant positive correlation with E(all) both for CSMCs and CSMCs-CYD. The slope of the regression line DeltaE(all)/DeltaF was much higher in the CSMCs than in the CSMCs-CYD. These results indicate that elastic properties of smooth muscle cells may be affected not only by the amount of their actin filaments, but also by their organization and distribution in cells.     

The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle

Contractile stimulation induces actin polymerization in smooth muscle tissues and cells, and the inhibition of actin polymerization depresses smooth muscle force development. In the present study, the role of Cdc42 in the regulation of actin polymerization and tension development in smooth muscle was evaluated. Acetylcholine stimulation of tracheal smooth muscle tissues increased the activation of Cdc42. Plasmids encoding wild type Cdc42 or a dominant negative Cdc42 mutant, Asn-17 Cdc42, were introduced into tracheal smooth muscle strips by reversible permeabilization, and tissues were incubated for 2 days to allow for protein expression. Expression of recombinant proteins was confirmed by immunoblot analysis. The expression of the dominant negative Cdc42 mutant inhibited contractile force and the increase in actin polymerization in response to acetylcholine stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type Cdc42 had no significant effect on force, actin polymerization, or myosin light chain phosphorylation. Contractile stimulation increased the association of neuronal Wiskott-Aldrich syndrome protein with Cdc42 and the Arp2/3 (actin-related protein) complex in smooth muscle tissues expressing wild type Cdc42. The agonist-induced increase in these protein interactions was inhibited in tissues expressing the inactive Cdc42 mutant. We conclude that Cdc42 activation regulates active tension development and actin polymerization during contractile stimulation. Cdc42 may regulate the activation of neuronal Wiskott-Aldrich syndrome protein and the actin related protein complex, which in turn regulate actin filament polymerization initiated by the contractile stimulation of smooth muscle.     

Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder

The morphological characteristics of smooth muscle cells (SMCs) and their innervation of the suburothelial microvasculature of the mouse bladder were investigated by immunohistochemistry. Whole mount bladder mucosal preparations were immune-stained for α-smooth muscle actin (α-SMA) and/or neuronal markers and examined using confocal laser scanning microscopy. Suburothelial arterioles consisted of α-SMA-immunopositive circular smooth muscle cells, while the venular wall composed of α-SMA-positive SMCs that displayed several processes which extended from their cell bodies to form an extensive meshwork. In larger venules, a complex meshwork of stellate-shaped SMCs were observed. NG2 chondroitin sulphate proteoglycan-immunoreactive cell bodies of capillary pericytes were not immunoreactive for α-SMA. In the rat bladder suburothelial venules, circular SMCs were the dominant cell type expressing α-SMA-immunoreactivity. Since α-SMA-positive SMCs in suburothelial arterioles and venules in the mouse bladder had quite distinct morphologies, the innervation of both vessels could be examined by double labelling for α-SMA and various neuronal markers. Varicose nerve bundles immunoreactive for tyrosine hydroxylase (sympathetic nerves), choline acetyltransferase (cholinergic nerves) or substance P (primary afferent nerves) were all detected along side suburothelial arterioles. Single varicose nerve fibres positive for these three neuronal markers were also detected around the venules. Thus, whole mount preparations are useful when examining the morphology of α-SMA-positive SMCs of the microvasculature in the suburothelium of mouse bladder as well as their relationship with their innervations. In conclusion, arterioles and venules of the bladder suburothelium are the target of sympathetic, cholinergic and primary afferent nerve fibres.     

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains.

We have previously demonstrated that alpha-smooth muscle (alpha-SM) actin is predominantly distributed in the central region and beta-non-muscle (beta-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and alpha-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha-SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with alpha-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.     

CCN5 Expression in mammals. II. Adult rodent tissues

CCN5 is a secreted heparin- and estrogen-regulated matricellular protein that inhibits vertebrate smooth muscle cell proliferation and motility. CCN5 is expressed throughout murine embryonic development in most organs and tissues. However, after embryonic development is complete, we hypothesized that CCN5 distribution would be largely restricted to small set of tissues, including smooth muscle cells of the arteries, uterus, airway, and digestive tract. Because CCN5 inhibits proliferation of smooth muscle cells in vitro, it might function to prevent excessive growth in vivo. In contrast, another member of the CCN family, CCN2, promotes smooth muscle cell proliferation in vitro, and thus it was expected that its expression levels would be low in uninjured normal adult tissues. Frozen sections from adult tissues and organs were analyzed immunohistochemically using anti-CCN5 and anti-CCN2 antibodies. Both proteins were detected in arteries, the uterus, bronchioles, and the digestive tract as expected, and also in many other tissues including the pancreas, spleen, liver, skeletal muscle, ovary, testis, thymus, brain, olfactory epithelium, and kidney. CCN5 and CCN2 protein was found in smooth muscle, endothelial cells, epithelial cells, skeletal muscle, cells of the nervous system, and numerous other cell types. In many cells, both CCN5 and CCN2 was present in the nucleus. Rather than having opposite patterns of localization, CCN5 and CCN2 often had similar sites of expression. The wide distribution of both CCN5 and CCN2 suggests that both proteins have additional biological functions beyond those previously identified in specific cellular and pathological models.     

The adapter protein CrkII regulates neuronal Wiskott-Aldrich syndrome protein, actin polymerization, and tension development during contractile stimulation of smooth muscle

Actin polymerization has been shown to occur in tracheal smooth muscle tissues and cells in response to contractile stimulation, and there is evidence that the polymerization of actin is required for contraction. In tracheal smooth muscle, agonist-induced actin polymerization is mediated by activation of neuronal Wiskott-Aldrich syndrome protein (N-WASp) and the Arp (actin-related protein) 2/3 complex, and activation of the small GTPase Cdc42 regulates the activation of N-WASp. In the present study, the role of the adapter protein CrkII in the regulation of N-WASp and Cdc42 activation, actin polymerization, and tension development in smooth muscle tissues was evaluated. Stimulation of tracheal smooth muscle tissues with acetylcholine increased the association of CrkII with N-WASp. Plasmids encoding wild type CrkII or a CrkII mutant lacking the SH3 effector-binding ability, CrkII SH3N, were introduced into tracheal smooth muscle tissues, and the tissues were incubated for 2 days to allow for protein expression. Expression of the CrkII SH3N mutant in smooth muscle tissues inhibited the association of CrkII with N-WASp and the activation of Cdc42. The CrkII SH3N mutant also inhibited the increase in the association of N-WASp with Arp2, a major component of the Arp2/3 complex, in response to contractile stimulation, indicating inhibition of N-WASp activation. Expression of the CrkII SH3N mutant also inhibited tension generation and actin polymerization in response to contractile stimulation; however, it did not inhibit myosin light chain phosphorylation. These results suggest that CrkII plays a critical role in the regulation of N-WASp activation, perhaps by regulating the activation of Cdc42, and that it thereby regulates actin polymerization and active tension generation in tracheal smooth muscle. These studies suggest a novel signaling pathway for the regulation of N-WASp activation and active contraction in smooth muscle tissues.     

The Small GTPase RhoA Regulates the Contraction of Smooth Muscle Tissues by Catalyzing the Assembly of Cytoskeletal Signaling Complexes at Membrane Adhesion Sites

The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/βPIX. Paxillin tyrosine phosphorylation and its association with βPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.     

Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.     

Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors

E-box/basichelix-loop-helix (bHLH)-dependent regulation of promoters for skeletalmuscle-specific genes is well established, but similar regulation ofsmooth muscle-selective promoters has not been reported. Usingtransient transfection assays of smooth muscle -actin (SMA)promoter-chloramphenicol acetyltransferase (CAT) reporter constructs inrat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, weidentified two activator elements, smE1 and smE2, with sequencescorresponding to E-box (5'-CAnnTG-3') motifs. In L6myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completelyabolished promoter activity. In contrast, mutation of smE1 and smE2 wasrequired to reduce promoter activity in SMCs. Supershift analysesidentified a myogenin-containing complex as the predominant smE1 andsmE2 binding activity in skeletal muscle, and myogenin overexpressiontransactivated the promoter. Supershift analyses with SMC extractsdemonstrated that the bHLH protein upstream stimulatory factor (USF)bound smE1, and USF overexpression transactivated the promoter in ansmE1-dependent manner. In summary, our results provide novel evidenceimplicating E-box elements in directing expression of the SMApromoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.