Down-regulation of calponin destabilizes actin cytoskeletal structure in A7r5 cells

The effects of changes in the expression levels of h1 calponin (CaP) on actin cytoskeletal organization were studied in control and phorbol-ester-treated A7r5 smooth muscle cells. Protein association and expression in control and stimulated A7r5 smooth muscle cells were evaluated by Western blotting, laser scanning confocal microscopy (LSCM), and fluorescence resonance energy transfer (FRET) microscopy in cells treated with either 2 x 10(-6 ) mol/L TGF-beta 1 or 2 x 10(-)5 mol/L PDGF-BB to alter h1 calponin expression. Single immunostained samples showed that CaP and alpha-actin, localized in fibers in unstimulated control A7r5 smooth muscle cells, were translocated to podosomes following treatment with phorbol-12,13-dibutyrate (PDBu). Confocal colocalization imaging and FRET analysis both indicated substantial association of CaP with alpha-actin in stress fibers of control cells and in podosomes of PDBu-treated cells. PKC alpha, which showed evidence of only slight association with CaP in control cells, exhibited markedly increased (293%) association in PDBu-contracted cells. Platelet-derived growth factor (PDGF)-BB down-regulated CaP to non-detectable levels, whereas transforming growth factor (TGF)-beta 1 up-regulated (424%) the expression of CaP without affecting the levels of alpha-actin or PKC alpha. PDGF-BB resulted in a significant loss in alpha-actin stress fibers (-47%) and reduced podosome formation (-69%). By comparison, TGF-beta 1 had no effect on stress fibers in control cells but also reduced (-70%) podosome formation. The results suggest that CaP could play a major role in the stabilization of actin stress fibers in resting cells and may contribute to podosome formation in PDBu-treated cells.     

Plectin deposition at podosome rings requires myosin contractility

Metalloproteinase-dependent tissue invasion requires the formation of podosomes and invadopodia for localized matrix degradation. Actin cytoskeleton remodeling via Arp2/3-mediated actin polymerization is essential for podosome formation, and dynamic microtubules have an important role in maintaining podosome turnover in macrophages and osteoclasts. Little is known, however, about the involvement of the intermediate filament cytoskeleton in formation, stabilization, and turnover of podosomes. Here we show that vimentin intermediate filaments colocalize with the early sites of podosome formation at the stress fiber - focal adhesion interface in cultured vascular smooth muscle cells, but do not directly contribute to podosome formation, or stabilization. In unstimulated A7r5 cells the cytolinker protein plectin poorly colocalized with vimentin and the microdomains, but following induction by phorbol ester accumulated in the rings that surround the podosomes. In plectin-deficient A7r5 cells actin stress fiber remodelling is reduced in response to PDBu, and small podosomes remain localized at stable actin stress fibres. Pharmacological inhibition of actomyosin contractility by blebbistatin leads to an aberrant localization of podosomes away from the cell periphery and induces failure of plectin to surround the outer perimeter of these invasive adhesions. Taken together, we conclude that plectin is involved in growth and maturation of podosomes by reducing focal adhesion and stress fiber turnover, and that actomyosin-dependent contractility is required for the peripheral localization and specific deposition of plectin at the podosome rings.     

SM22α在细胞骨架组构及血管重塑中的作用

血管平滑肌细胞（VSMC）表型转化是动脉粥样硬化、高血压和血管成形术后再狭窄等血管重塑性疾病的共同病理生理过程。VSMC表型转化过程中平滑肌特异基因的表达变化和细胞骨架的组构是当前研究的热点问题之一。平滑肌22α（SM22α）是近年发现的一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性,该蛋白作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节。本文就SM22α的结构特征及其在VSMC骨架组构和血管重塑中的作用机制进行综述。     

Characterization of human palladin, a microfilament-associated protein

Actin-containing microfilaments control cell shape, adhesion, and contraction. In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments. In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs. We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies. Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin. The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family. Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation. We identified palladin in a yeast two-hybrid search as an ezrin-associated protein. An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays. The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin. Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments. These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin. Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations. In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments. The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.     

Effects of tyrosine phosphorylation of cortactin on podosome formation in A7r5 vascular smooth muscle cells

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKC inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKC acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation. actin cytoskeleton; Src; protein kinase C     

Bundling of actin filaments by alpha-actinin depends on its molecular length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.     

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.     

血管损伤的新靶点：平滑肌22 alpha

血管平滑肌细胞(vascular smooth muscle cell,VSMC)表型转化是血管损伤性疾病动脉粥样硬化、高血压和血管成形术后再狭窄等的共同病理生理过程.平滑肌22 alpha (smooth muscle 22 alpha, SM22α) 是一种VSMC分化标志物,其表达具有平滑肌组织特异性和细胞表型特异性. 该蛋白不仅作为一种肌动蛋白细胞骨架相关蛋白参与VSMC骨架组构和收缩调节,它还参与VSMC的增殖、炎症和氧化应激等进程. 本文就SM22α 的结构特征及其在VSMC血管损伤中的作用机制进行综述.     

PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a PAK-interacting exchange factor-dependent manner

Remodeling of the vascular smooth muscle cytoskeleton is essential for cell motility involved in the development of diseases such as arteriosclerosis and restenosis. The p21-activated kinase (PAK), which is an effector of the Rho GTPases Rac and Cdc42, has been shown to be involved in cytoskeletal remodeling and cell motility. We show herein that expression of cytoskeletally active constructs of PAK1 is able to induce the formation of dynamic, podosome-like F-actin columns in the A7r5 vascular smooth muscle cell line. Most of these actin columns appear at the junctions between stress fibers and focal adhesions and contain several known podosomal protein markers, such as cortactin, Arp2/3, -actinin, and vinculin. The kinase activity of PAK plays a role in the regulation of the turnover rates of these actin columns but is not essential for their formation. The ability of PAK to interact with the PAK-interacting exchange factor (PIX) but not with Rac or Cdc42, however, is required for the formation of the actin columns as well as for the translocation of PIX and G protein-coupled receptor kinase-interacting protein (GIT) to focal adhesions adjacent to the actin columns. These findings suggest that interaction between PAK and PIX, as well as the recruitment of PIX and GIT to focal adhesions, plays an important role in the formation of actin columns that resemble podosomes induced by phorbol ester in vascular smooth muscle cells. actin cytoskeleton; p21-activated kinase     

Caldesmon phosphorylation in actin cytoskeletal remodeling

Caldesmon is an actin-binding protein that is capable of stabilizing actin filaments against actin-severing proteins, inhibiting actomyosin ATPase activity, and inhibiting Arp2/3-mediated actin polymerization in vitro. Caldesmon is a substrate of cdc2 kinase and Erk1/2 MAPK, and phosphorylation by either of these kinases reverses the inhibitory effects of caldesmon. Cdc2-mediated caldesmon phosphorylation and the resulting dissociation of caldesmon from actin filaments are essential for M-phase progression during mitosis. Cells overexpressing the actin-binding carboxyterminal fragment of caldesmon fail to release the fragment completely from actin filaments during mitosis, resulting in a higher frequency of multinucleated cells. PKC-mediated MEK/Erk/caldesmon phosphorylation is an important signaling cascade in the regulation of smooth muscle contraction. Furthermore, PKC activation has been shown to remodel actin stress fibers into F-actin-enriched podosome columns in cultured vascular smooth muscle cells. Podosomes are cytoskeletal adhesion structures associated with the release of metalloproteases and degradation of extracellular matrix during cell invasion. Interestingly, caldesmon is one of the few actin-binding proteins that is associated with podosomes but excluded from focal adhesions. Caldesmon also inhibits the function of gelsolin and Arp2/3 complex that are essential for the formation of podosomes. Thus, caldesmon appears to be well positioned for playing a modulatory role in the formation of podosomes. Defining the roles of actin filament-stabilizing proteins such as caldesmon and tropomyosin in the formation of podosomes should provide a more complete understanding of molecular systems that regulate the remodeling of the actin cytoskeleton in cell transformation and invasion.     

Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels.

Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.     

Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors

Signaling mechanisms forstretch-dependent growth and differentiation of vascular smooth musclewere investigated in mechanically loaded rat portal veins in organculture. Stretch-dependent protein synthesis was found to depend onendogenous release of angiotensin II. Autoradiography after[³⁵S]methionine incorporation revealed stretch-dependentsynthesis of several proteins, of which SM22 and actin wereparticularly prominent. Inhibition of RhoA activity by cell-permeant C3toxin increased tissue mechanical compliance and reducedstretch-dependent extracellular signal-regulated kinase (ERK)1/2activation, growth, and synthesis of actin and SM22, suggesting a roleof the actin cytoskeleton. In contrast, inhibition of Rho-associatedkinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance butstill inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin Bconcentration-dependently reduced actin and SM22 synthesis,cytochalasin D did so at low (10⁸ M) but not at high(10⁶ M) concentration. The results show that stretchstabilizes the contractile smooth muscle phenotype. Stretch-dependentdifferentiation marker expression requires an intact cytoskeleton forstretch sensing, control of protein expression via the level ofunpolymerized G-actin, or both.

     

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.     

决定细胞形状和运动性的蛋白质 palladin

Palladin 是肌动蛋白结合蛋白家族的新成员,广泛分布于平滑肌、中枢神经系统和胚胎的各种组织中,其主要的生物功能是参与构建肌动蛋白骨架系统,并在细胞骨架的动态变化中起作用 . 在肌动蛋白细胞骨架中 palladin 与 alpha- 辅肌动蛋白共存在 . 目前发现, palladin 在决定细胞的形态和迁移或运动等过程中起关键的作用 . 在转移性癌细胞和中枢神经受损伤后的星形胶质细胞中,都有 palladin 的特殊表达 . Palladin 的表达使星形胶质细胞形成了神经胶质疤痕 .