Calponin

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J.-p. Jin - One of the best experts on this subject based on the ideXlab platform.

  • double deletion of Calponin 1 and Calponin 2 in mice decreases systemic blood pressure with blunted length tension response of aortic smooth muscle
    Journal of Molecular and Cellular Cardiology, 2019
    Co-Authors: Hanzhong Feng, Hui Wang, Katsuhito Takahashi, J.-p. Jin
    Abstract:

    Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, Calponin 1 is smooth muscle specific and Calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that Calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that Calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of Calponin 1 and Calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that Calponin 1 and Calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of Calponin 1 was found in smooth muscle of Cnn2-/- mice but not vice versa. Cnn1-/- and Cnn2-/- double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of Calponin 1 and Calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

  • deletion of Calponin 2 attenuates the development of calcific aortic valve disease in apoe mice
    Journal of Molecular and Cellular Cardiology, 2018
    Co-Authors: Olesya Plazyo, Rong Liu, Moazzem M Hossain, J.-p. Jin
    Abstract:

    Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and lacks non-surgical treatment. The pathogenesis of CAVD involves perturbation of valvular cells by mechanical stimuli, including shear stress, pressure load and leaflet stretch, of which the molecular mechanism requires further elucidation. We recently demonstrated that knockout (KO) of Cnn2 gene that encodes Calponin isoform 2, a mechanoregulated cytoskeleton protein, attenuates atherosclerosis in ApoE KO mice. Here we report that Cnn2 KO also decreased calcification of the aortic valve in ApoE KO mice, an established model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively attenuated vascular atherosclerosis that shares many pathogenic processes with CAVD, it did not reduce aortic valve calcification in ApoE KO mice. Indicating a function in the pathogenesis of CAVD, Calponin 2 participates in myofibroblast differentiation that is a leading step in the development of CAVD. The aortic valves of ApoE KO mice exhibited increased expression of Calponin 2 and smooth muscle actin (SMA), a hallmark of myofibroblasts. The expression of Calponin 2 increased during myofibroblast-like differentiation of primary sheep aortic valve interstitial cells and during the osteogenic differentiation of mouse myofibroblasts. Cnn2 KO attenuated TGFβ1-induced differentiation of myofibroblasts in culture as shown by the lower expression of SMA and less calcification than that of wild type (WT) cells. These findings present Calponin 2 as a novel molecular target for the treatment and prevention of CAVD.

  • deletion of Calponin 2 in mouse fibroblasts increases myosin ii dependent cell traction force
    Biochemistry, 2016
    Co-Authors: Moazzem M Hossain, James H C Wang, Guangyi Zhao, Moon-sook Woo, J.-p. Jin
    Abstract:

    Cell traction force (CTF) plays a critical role in controlling cell shape, permitting cell motility, and maintaining cellular homeostasis in many biological processes such as angiogenesis, development, wound healing, and cancer metastasis. Calponin is an actin filament-associated cytoskeletal protein in smooth muscles and multiple types of non-muscle cells. An established biochemical function of Calponin is the inhibition of myosin ATPase in smooth muscle cells. Vertebrates have three Calponin isoforms. Among them, Calponin 2 is expressed in epithelial cells, endothelial cells, macrophages, myoblasts, and fibroblasts and plays a role in regulating cytoskeleton activities such as cell adhesion, migration, and cytokinesis. Knockout (KO) of the gene encoding Calponin 2 (Cnn2) in mice increased cell motility, suggesting a function of Calponin 2 in modulating CTF. In this study, we examined fibroblasts isolated from Cnn2 KO and wild-type (WT) mice using CTF microscopy. Primary mouse fibroblasts were cultured o...

  • deletion of Calponin 2 in macrophages alters cytoskeleton based functions and attenuates the development of atherosclerosis
    Journal of Molecular and Cellular Cardiology, 2016
    Co-Authors: Rong Liu, J.-p. Jin
    Abstract:

    Arterial atherosclerosis is an inflammatory disease. Macrophages play a major role in the pathogenesis and progression of atherosclerotic lesions. Modulation of macrophage function is a therapeutic target for the treatment of atherosclerosis. Calponin is an actin-filament-associated regulatory protein that inhibits the activity of myosin-ATPase and dynamics of the actin cytoskeleton. Encoded by the gene Cnn2, Calponin isoform 2 is expressed at significant levels in macrophages. Deletion of Calponin 2 increases macrophage migration and phagocytosis. In the present study, we investigated the effect of deletion of Calponin 2 in macrophages on the pathogenesis and development of atherosclerosis. The results showed that macrophages isolated from Cnn2 knockout mice ingested a similar level of acetylated low-density lipoprotein (LDL) to that of wild type (WT) macrophages but the resulting foam cells had significantly less hindered velocity of migration. Systemic or myeloid cell-specific Cnn2 knockouts effectively attenuated the development of arterial atherosclerosis lesions with less macrophage infiltration in apolipoprotein E knockout mice. Consistently, Calponin 2-null macrophages produced less pro-inflammatory cytokines than that of WT macrophages, and the up-regulation of pro-inflammatory cytokines in foam cells was also attenuated by the deletion of Calponin 2. Calponin 2-null macrophages and foam cells have significantly weakened cell adhesion, indicating a role of cytoskeleton regulation in macrophage functions and inflammatory responses, and a novel therapeutic target for the treatment of arterial atherosclerosis.

  • deletion of Calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice
    American Journal of Physiology-cell Physiology, 2016
    Co-Authors: Qi Quan Huang, Moazzem M Hossain, Richard M Pope, Wen Sun, Lianping Xing, J.-p. Jin
    Abstract:

    Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of Calponin are present in vertebrates, among which Calponin 2 encoded by the C...

Kathleen G Morgan - One of the best experts on this subject based on the ideXlab platform.

  • mechanism of Calponin stabilization of cross linked actin networks
    Biophysical Journal, 2014
    Co-Authors: Kathleen G Morgan, Mikkel H Jensen, Eliza J Morris, Cynthia Gallant, David A Weitz, Jeffrey R Moore
    Abstract:

    The actin-binding protein Calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood. To investigate this underlying physical mechanism, we studied an in vitro model system of cross-linked actin using bulk rheology. Networks with basic Calponin exhibited a delayed onset of strain stiffening (10.0% without Calponin, 14.9% with Calponin) and were able to withstand a higher maximal strain before failing (35% without Calponin, 56% with Calponin). Using fluorescence microscopy to study the mechanics of single actin filaments, we found that Calponin increased the flexibility of actin filaments, evident as a decrease in persistence length from 17.6 μm without to 7.7 μm with Calponin. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, and suggest that Calponin stabilization of actin networks can be explained purely by changes in single-filament mechanics. We propose a model in which Calponin stabilizes actin networks against shear through a reduction of persistence length of individual filaments.

  • mechanism of actin network stabilization by changes in polymer flexibility by Calponin
    Biophysical Journal, 2014
    Co-Authors: Eliza J Morris, Kathleen G Morgan, Mikkel H Jensen, Cynthia Gallant, David A Weitz, Jeffrey R Moore
    Abstract:

    The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. The actin binding protein Calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood.To investigate the underlying physical mechanism, we studied an in vitro model system of crosslinked actin using bulk rheology. Networks with basic Calponin exhibited a delayed onset of strain stiffening (gamma crit) and were able to withstand higher strains (gamma max) and stresses (sigma max) before failing. Using fluorescence microscopy to study the mechanics of single actin filaments, we found that Calponin increased the flexibility of actin filaments. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, suggesting that Calponin stabilization of actin networks can be explained by changes in single filament mechanics. Comparisons to computational models indicate that a reduction of persistence length of individual actin filaments is the primary mechanism by which Calponin stabilizes actin networks against shear.Representative stress-strain curve shown below:View Large Image | View Hi-Res Image | Download PowerPoint Slide

  • compliant or stiff two differing mechanisms of actin network stabilization by Calponin and tropomyosin
    Biophysical Journal, 2013
    Co-Authors: Mikkel H Jensen, Paul C Leavis, Kathleen G Morgan, Eliza J Morris, Cynthia Gallant, David A Weitz, Philip Graceffa, Jeffrey R Moore
    Abstract:

    The cellular actin cytoskeleton plays a central role in the ability of cells to properly sense, propagate, and respond to external stresses and other mechanical stimuli. Calponin, an actin-binding protein found both in muscle and non-muscle cells, has been implicated in actin cytoskeletal organization and regulation. While currently thought to stabilize actin in the cell, the mechanisms of this stabilization are poorly understood due to the complicated nature of the living cell. Here we use a simplified in vitro model system to dissect the specific roles of individual actin binding proteins within a complex network environment.We studied the mechanical properties of actin networks in the presence of basic Calponin and smooth muscle aortic tropomyosin. These two actin-binding proteins have distinct effects on single actin filaments; while Calponin binds over subdomain 2 of actin and makes actin more flexible, tropomyosin lies along the long-pitch helix of actin and buttresses the filament. We constructed in vitro crosslinked actin networks and studied their strain behavior both macro- and microscopically using bulk rheology and active microrheology. Actin networks decorated with either Calponin or tropomyosin exhibited increased tensile strength despite the very different effects these proteins have on individual actin filaments. While Calponin-decorated actin networks exhibited delayed strain stiffening, tropomyosin-decorated actin networks underwent stress relaxation through local network failure. When the two actin-binding proteins were present together, each of these effects were apparent, and the network tensile strength were further increased.This work demonstrates two very different mechanisms by which Calponin and tropomyosin increase the tensile strength of actin networks through purely mechanical interactions.Project funded by the NIH (HL086655), and the Harvard Materials Research Science and Engineering Center (DMR-0820484).

  • h3 acidic Calponin an actin binding protein that controls extracellular signal regulated kinase 1 2 activity in nonmuscle cells
    Molecular Biology of the Cell, 2010
    Co-Authors: Sarah Appel, J.-p. Jin, Philip G Allen, Susanne Vetterkind, Kathleen G Morgan
    Abstract:

    Migration of fibroblasts is important in wound healing. Here, we demonstrate a role and a mechanism for h3/acidic Calponin (aCaP, CNN3) in REF52.2 cell motility, a fibroblast line rich in actin filaments. We show that the actin-binding protein h3/acidic Calponin associates with stress fibers in the absence of stimulation but is targeted to the cell cortex and podosome-like structures after stimulation with a phorbol ester, phorbol-12,13-dibutyrate (PDBu). By coimmunoprecipitation and colocalization, we show that extracellular signal-regulated kinase (ERK)1/2 and protein kinase C (PKC)α constitutively associate with h3/acidic Calponin and are cotargeted with h3/acidic Calponin in the presence of PDBu. This targeting can be blocked by a PKC inhibitor but does not require phosphorylation of h3/acidic Calponin at the PKC sites S175 or T184. Knockdown of h3/acidic Calponin results in a loss of PDBu-mediated ERK1/2 targeting, whereas PKCα targeting is unaffected. Caldesmon is an actin-binding protein that regulates actomyosin interactions and is a known substrate of ERK1/2. Both ERK1/2 activity and nonmuscle l-caldesmon phosphorylation are blocked by h3/acidic Calponin knockdown. Furthermore, h3/acidic Calponin knockdown inhibits REF52.2 migration in an in vitro wound healing assay. Our findings are consistent with a model whereby h3/acidic Calponin controls fibroblast migration by regulation of ERK1/2-mediated l-caldesmon phosphorylation.

  • regulation of protein kinase c by the cytoskeletal protein Calponin
    Journal of Biological Chemistry, 2000
    Co-Authors: Barbara Leinweber, Paul C Leavis, Kathleen G Morgan, Cynthia Gallant, Amadeo M Parissenti, Samudra S Gangopadhyay, Angie Kirwanrhude
    Abstract:

    Previous studies from this laboratory have shown that, upon agonist activation, Calponin co-immunoprecipitates and co-localizes with protein kinase Ce (PKCe) in vascular smooth muscle cells. In the present study we demonstrate that Calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCe and PKCα with possible involvement of C1B. The C2 region of PKCe binds to the Calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that Calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCβII, we found that Calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCα, -e, and -βII severalfold compared with control conditions. Similarly, Calponin was found to increase the amount of 32P-labeled phosphate incorporated into PKC from [γ-32P]ATP. We also observed that Calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that Calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.

Mario Gimona - One of the best experts on this subject based on the ideXlab platform.

  • unc 87 a Calponin related protein in c elegans antagonizes adf cofilin mediated actin filament dynamics
    Journal of Cell Science, 2007
    Co-Authors: Sawako Yamashiro, Mario Gimona
    Abstract:

    Stabilization of actin filaments is critical for supporting actomyosin-based contractility and for maintaining stable cellular structures. Tropomyosin is a well-characterized ubiquitous actin stabilizer that inhibits ADF/cofilin-dependent actin depolymerization. Here, we show that UNC-87, a Calponin-related Caenorhabditis elegans protein with seven Calponin-like repeats, competes with ADF/cofilin for binding to actin filaments and inhibits ADF/cofilin-dependent filament severing and depolymerization in vitro. Mutations in the unc-87 gene suppress the disorganized actin phenotype in an ADF/cofilin mutant in the C. elegans body wall muscle, supporting their antagonistic roles in regulating actin stability in vivo. UNC-87 and tropomyosin exhibit synergistic effects in stabilizing actin filaments against ADF/cofilin, and direct comparison reveals that UNC-87 effectively stabilizes actin filaments at much lower concentrations than tropomyosin. However, the in vivo functions of UNC-87 and tropomyosin appear different, suggesting their distinct roles in the regulation of actomyosin assembly and cellular contractility. Our results demonstrate that actin binding via Calponin-like repeats competes with ADF/cofilin-driven cytoskeletal turnover, and is critical for providing the spatiotemporal regulation of actin filament stability.

  • live dynamics of gfp Calponin isoform specific modulation of the actin cytoskeleton and autoregulation by c terminal sequences
    Journal of Cell Science, 2000
    Co-Authors: Claudia Danninger, Mario Gimona
    Abstract:

    The Calponin family of F-actin-, tropomyosin- and calmodulin-binding proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 Calponin), cytoskeletal organisation in non-muscle cells (h2 Calponin) and the control of neurite outgrowth (acidic Calponin). We have now investigated the effects of Calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. It is shown that h1 CaP associates with the actin stress fibers in the more central part of the cell, whereas h2 CaP localizes to the ends of stress fibres and in the motile lamellipodial protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 CaP and expression of GFP h1 CaP resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.

  • the single ch domain of Calponin is neither sufficient nor necessary for f actin binding
    Journal of Cell Science, 1998
    Co-Authors: Mario Gimona, Renu Mital
    Abstract:

    Calponins have been implicated in the regulation of actomyosin interactions in smooth muscle cells, cytoskeletal organisation in nonmuscle cells, and the control of neurite outgrowth. Domains homologous to the amino-terminal region of Calponin have been identified in a variety of actin cross-linking proteins and signal transduction molecules, and by inference these ‘Calponin homology (CH) domains’ have been assumed to participate in actin binding. We here report on the actin binding activities of the subdomains of the Calponin molecule. All three mammalian isoforms of Calponin (basic h1, neutral h2 and acidic) possess a single CH domain at their amino terminus as well as three tandem repeats proximal to the carboxyl terminus. Calponin h2 differs, however, from h1 in lacking a consensus actin-binding motif in the region 142–163, between the CH domain and the tandem repeats, which in h1 Calponin can be chemically cross-linked to actin. Despite the absence of this consensus actin-binding motif, recombinant full-length h2 Calponin co-sediments in vitro with F-actin, suggesting the presence of another binding site in the molecule. It could be shown that this binding site resides in the C-terminal tandem repeats and not in the CH domain. Thus, constructs of h2 Calponin bearing partial or complete deletions of the triple repeated sequences failed to co-localise with actin stress fibres despite the presence of a CH domain. Deletion of the acidic carboxyl terminus, beyond the repeats, increased actin binding, suggesting that the carboxy-terminal tail may modulate actin association. Results obtained from transient transfections of amino- and carboxy-terminal truncations in h1 Calponin were consistent with the established location of the actin binding motif outside and carboxy-terminal to the CH domain, and confirm that the presence of a single CH domain alone is neither sufficient nor necessary to mediate actin binding. Instead, the carboxy-terminal tandem repeats of h1 and h2 Calponin are shown to harbour a second, independent actin binding motif.

  • Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres
    FEBS Letters, 1995
    Co-Authors: Asa Jaworowski, Mario Gimona, Peter Strasser, Kurt I Anderson, Anders Arner, Martin Engstrom, Victor J Small
    Abstract:

    Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native Calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that Calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of Calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state. (Less)

  • Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells
    Journal of Cell Science, 1994
    Co-Authors: A J North, Mario Gimona, R Cross, J V Small
    Abstract:

    Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocytochemistry we have determined the distribution of Calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that Calponin occurs in the cytoskeleton, with beta-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, Calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the beta-cytoplasmic isoform of actin. It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that Calponin, together with a Ca(2+)-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.

Katsuhito Takahashi - One of the best experts on this subject based on the ideXlab platform.

  • double deletion of Calponin 1 and Calponin 2 in mice decreases systemic blood pressure with blunted length tension response of aortic smooth muscle
    Journal of Molecular and Cellular Cardiology, 2019
    Co-Authors: Hanzhong Feng, Hui Wang, Katsuhito Takahashi, J.-p. Jin
    Abstract:

    Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, Calponin 1 is smooth muscle specific and Calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that Calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that Calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of Calponin 1 and Calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that Calponin 1 and Calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of Calponin 1 was found in smooth muscle of Cnn2-/- mice but not vice versa. Cnn1-/- and Cnn2-/- double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of Calponin 1 and Calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

  • loss of smooth muscle Calponin results in impaired blood vessel maturation in the tumor host microenvironment
    Cancer Science, 2007
    Co-Authors: Hisako Yamamura, Noriko Hirano, Hidenori Koyama, Yoshiki Nishizawa, Katsuhito Takahashi
    Abstract:

    The interactions between malignant cells and the microenvironment of the local host tissue play a critical role in tumor growth, metastasis and their response to treatment modalities. We investigated the roles of smooth muscle Calponin (Cnn1, also called Calponin h1 or basic Calponin) in the development of tumor vascul ature in vivo by analyzing mutant mice lacking the Cnn1 gene. Here we show that loss of Cnn1 in host mural cells prevents maturation of tumor vasculature. In vitro studies showed that platelet-derived growth factor B-induced vascular smooth muscle migration was downregulated by the Cnn1-deficiency, and forced expression of Cnn1 restored migration. Moreover, destruction of established tumor mass by treatment with an antivascular endothelial growth factor antibody was markedly enhanced in Cnn1-deficient mice. These data, coupled with the knowledge that structural fragility of normal blood vessels is caused by loss of the Cnn1 gene, suggest that Cnn1 plays an important role in the maturation of blood vessels, and may have implications for therapeutic strategies targeting tumor vasculature for treatment of human cancers. (Cancer Sci 2007; 98: 757–763)

  • structural fragility of blood vessels and peritoneum in Calponin h1 deficient mice resulting in an increase in hematogenous metastasis and peritoneal dissemination of malignant tumor cells
    Cancer Research, 2001
    Co-Authors: Shunichiro Taniguchi, Katsuhito Takahashi, Michiko Takeoka, Takashi Ehara, Shigenari Hashimoto, Hiroto Shibuki, Nagahisa Yoshimura, Hidekazu Shigematsu, Motoya Katsuki
    Abstract:

    We have observed weak expression of Calponin h1, which stabilizes the actin filament system, in blood vessels within human malignant tumors. This observation suggested that because of a deficiency in stabilization by Calponin h1, the structure of blood vessels in malignant tumors is fragile compared with blood vessels in normal tissues. We therefore generated Calponin h1-deficient (CN−/−) mice to examine the effect of Calponin h1 on the integrity of the barrier system in blood vessels against cancer metastasis. The CN−/− mice exhibited morphological fragility of the tissues, including the uterus and blood vessels. In particular, we frequently observed bleeding into the surrounding tissue from blood vessels of the ocular fundus in CN−/− mice. In addition, mesothelial cells, which usually express Calponin h1 in normal (CN+/+) mice, were retracted in the CN−/− mice. When fluorescein was injected i.v. into mice, the CN−/− mice exhibited a greater and more rapid leakage of fluorescein from the blood vessels of the ocular fundus compared with the CN+/+ mice. In the CN−/− mice receiving i.v. inoculations of B16 melanoma cells, significantly more metastatic nodules were formed in the lung than in the CN+/+ mice. When B16 melanoma cells were injected i.p., the severity of peritonitis carcinomatosa was greater in CN−/− than in CN+/+ mice. These results indicate that Calponin h1 plays an important role in the regulation of the integrity of the blood vessels and peritoneum, which in turn is an important factor influencing the frequency of cancer metastasis. The CN−/− mice, which exhibit fragile blood vessels and peritoneum, could serve as sensitive and useful host models to investigate cancer metastasis.

  • regulation of shortening velocity by Calponin in intact contracting smooth muscles
    Biochemical and Biophysical Research Communications, 2000
    Co-Authors: Katsuhito Takahashi, Hisako Yamamura, Ryo Yoshimoto, Kenichi Fuchibe, Ayako Fujishige, Minori Mitsuisaito, Masatoshi Hori, Hiroshi Ozaki, Nobuhisa Awata
    Abstract:

    Abstract To elucidate the function of Calponin in intact contracting smooth muscle cells in vivo, we generated mice with a mutated basic Calponin (h1) locus (Yoshikawa et al., Genes Cells 3, 685–695, 1998). Crossbridge cycling rates were estimated in aortic smooth muscle by the force redevelopment following an isometric step shortening as a function of time after K+ depolarization. Evidence is presented that Calponin is involved in the inhibition of shortening velocity in the tonic phase of contraction. The phosphorylation levels of myosin regulatory light chain and cytosolic calcium concentrations were not significantly different in paired comparisons between Calponin-deficient (−/−) and wild-type (+/+) muscles at any time point after stimulation. The force-velocity relationships in vas deferens smooth muscle showed that the maximum shortening velocity of −/− muscle was significantly faster than that of +/+ muscle. There was no change in the length-force relationships in both −/− and +/+ muscles of aorta and vas deferens. The results suggest that Calponin plays a role in regulation of the crossbridge cycling and that it may be responsible for reduced shortening velocity during a maintained contraction of mammalian smooth muscle.

  • contractile properties and proteins of smooth muscles of a Calponin knockout mouse
    The Journal of Physiology, 2000
    Co-Authors: John D Matthew, Shunichiro Taniguchi, Alexander S Khromov, M Mcduffie, Avril V Somlyo, Katsuhito Takahashi
    Abstract:

    1 The role of h1-Calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic Calponin gene. These Calponin knockout (KO) mice displayed no detectable h1-Calponin in their smooth muscles. 2 The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca2+ sensitization, initiated by flash photolysis of caged GTPγS, different. 3 The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P < 0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous Calponin to approximate WT levels; the concentration dependence of exogenous Calponin slowing of Vus was proportional to its actomyosin binding in situ. 4 Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. 5 We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by Calponin. Our results show no detectable role of Calponin in the signal transduction of the Ca2+-sensitization pathways in smooth muscle.

Michele M. Castro - One of the best experts on this subject based on the ideXlab platform.

  • matrix metalloproteinase mmp 2 activation by oxidative stress decreases aortic Calponin 1 levels during hypertrophic remodeling in early hypertension
    Vascular Pharmacology, 2019
    Co-Authors: Marcela Maria Blascke De Mello, Juliana M Parente, Richard Schulz, Michele M. Castro
    Abstract:

    Abstract Hypertension is characterized by maladaptive vascular remodeling and enhanced oxidative stress in the vascular wall. Peroxynitrite may directly activate latent matrix metalloproteinase (MMP)-2 in vascular smooth muscle cells (VSMC) by its S-glutathiolation. MMP-2 may then proteolyze Calponin-1 in aortas from hypertensive animals, which stimulates VSMC proliferation and medial hypertrophy. Calponin-1 is an intracellular protein which helps to maintain VSMC in their differentiated (contractile) phenotype. The present study therefore investigated whether aortic MMP-2 activity is increased by oxidative stress in early hypertension and then contributes to hypertrophic arterial remodeling by reducing the levels of Calponin-1. Male Wistar rats were submitted to the two kidney, one clip (2 K-1C) model of hypertension or sham surgery and were treated daily with tempol (18 mg/kg/day) or its vehicle (water) by gavage from the third to seventh day post-surgery. Systolic blood pressure (SBP) was daily assessed by tail-cuff plethysmography. After one week, aortas were removed to perform morphological analysis with hematoxylin and eosin staining and to analyze reactive oxygen‑nitrogen species levels by dihydroethidium and immunohistochemistry for nitrotyrosine. MMP-2 activity was analyzed by in situ and gelatin zymography and its S-glutathiolation was analyzed by Western blot for MMP-2 of anti-glutathione immunoprecipitates. Calponin-1 levels were identified in aortas by immunofluorescence. SBP increased by approximately 50 mmHg at the first week in 2 K-1C rats which was unaffected by tempol. However, tempol ameliorated the hypertension-induced increase in arterial media-to-lumen ratio and hypertrophic remodeling. Tempol also decreased hypertension-induced aortic oxidative stress and the enhanced MMP-2 activity. S-glutathiolation may be a potential mechanism by which oxidative stress activates MMP-2 in aortas of 2 K-1C rats. Furthermore, Calponin-1 was decreased in aortas from 2 K-1C rats and tempol prevented this. In conclusion, oxidative stress may contribute to the increase in aortic MMP-2 activity, possibly by S-glutathiolation, and this may result in Calponin-1 loss and maladaptive vascular remodeling in early hypertension.

  • matrix metalloproteinase 2 activity is associated with divergent regulation of Calponin 1 in conductance and resistance arteries in hypertension induced early vascular dysfunction and remodelling
    Basic & Clinical Pharmacology & Toxicology, 2017
    Co-Authors: Juliana M Parente, Camila A Pereira, Gustavo H Oliveirapaula, Jose E Tanussantos, Rita C Tostes, Michele M. Castro
    Abstract:

    Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As Calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of Calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas Calponin-1 expression was increased in 2K-1C mesenteric arteries, Calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in Calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing Calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased Calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of Calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.

  • matrix metalloproteinase mmp 2 decreases Calponin 1 levels and contributes to arterial remodeling in early hypertension
    Biochemical Pharmacology, 2016
    Co-Authors: Vanessa A Belo, Jose E Tanussantos, Juliana M Parente, Michele M. Castro
    Abstract:

    Increased matrix metalloproteinase (MMP)-2 is implicated in the vascular remodeling of hypertension. Calponin-1 is a contractile protein, and its absence is associated with vascular smooth muscle cell (VSMC) phenotype switch, which leads to migration and remodeling. We evaluated whether increased MMP-2 activity precedes chronic vascular remodeling by decreasing Calponin-1 and inducing VSMC proliferation. Sham or two kidney-one clip (2K1C) rats were treated with doxycycline at 30mg/kg/day. Systolic blood pressure was increased in the 2K1C rats after 1 and 2weeks post-surgery, and doxycycline was effective to reduce it only at 2weeks of hypertension (p<0.05). Increased activity of MMP-2 was observed in aortas from 2K1C at 1 and 2weeks of hypertension, followed by increased VSMC proliferation, and those effects were abolished by treating 2K1C rats with doxycycline (p<0.05). Increased aortic media to lumen ratio started to emerge in 2K1C rats at 1week of hypertension, and it was established by 2weeks. MMP-2 and Calponin-1 co-localized in the cytosol of VSMC. Aortas from 2K1C rats showed a significant reduction in Calponin-1 levels at 1week of hypertension, and doxycycline prevented its loss (p<0.05). However, at 2weeks of hypertension, Calponin-1 was upregulated in 2K1C (p<0.05 vs. Sham groups). The mRNA levels of Calponin-1 were not altered in the aortas of 2K1C at 1week of hypertension. MMP-2 may contribute to the post-translational decrease in Calponin-1, thus culminating in hypertension-induced maladaptive arterial remodeling.

  • Smoothelin-B is not a target of matrix metalloproteinase (MMP)-2 in the vasculature of endotoxemic rats.
    Canadian journal of physiology and pharmacology, 2014
    Co-Authors: Priscila De Souza, Michele M. Castro, Gillian Goobie, José Eduardo Da Silva-santos, Richard Schulz
    Abstract:

    Smoothelin-B (SMTL-B) and Calponin-1 are important regulators of vascular contraction. SMTL-B contains a Calponin-homology domain and is structurally similar to cardiac troponin T. As Calponin-1 and troponin T are proteolyzed by intracellular matrix metalloproteinase (MMP)-2 in oxidative stress injury, we hypothesized that SMTL-B is also cleaved by MMP-2 and contributes to lipopolysaccharide (LPS)-induced vascular hypocontractility. Rats received ONO-4817 (an MMP inhibitor) or its vehicle, 2 h prior to being administered lipopolysaccharide (LPS). LPS-induced aorta hypocontractility to potassium chloride or phenylephrine, and reduction of Calponin-1 levels, were abolished by ONO-4817 at 6 but not 3 h after LPS. However, the level of SMTL-B was unaltered in LPS aortas and further unaffected by ONO-4817. Despite the importance of SMTL-B in vascular tone, it is not a target of MMP-2 in LPS-induced hypocontractility.

  • matrix metalloproteinase 2 proteolysis of Calponin 1 contributes to vascular hypocontractility in endotoxemic rats
    Arteriosclerosis Thrombosis and Vascular Biology, 2012
    Co-Authors: Michele M. Castro, Michael P Walsh, Jonathan Cena, Woo Jung Cho, Richard Schulz
    Abstract:

    Objective— Matrix metalloproteinase (MMP)-2 is activated in aorta during endotoxemia and plays a role in the hypocontractility to vasoconstrictors. Calponin-1 is a regulator of vascular smooth muscle tone with similarities to troponin, a cardiac myocyte protein that is cleaved by MMP-2 in myocardial oxidative stress injuries. We hypothesized that Calponin-1 may be proteolyzed by MMP-2 in endotoxemia-induced vascular hypocontractility. Methods and Results— Rats were given a nonlethal dose of bacterial lipopolysaccharide (LPS) or vehicle. Some rats were given the MMP inhibitors ONO-4817 or doxycycline. Six hours later, plasma nitrate+nitrite increased >15-fold in LPS-treated rats, an effect unchanged by doxycycline. Both ONO-4817 and doxycycline prevented LPS-induced aortic hypocontractility to phenylephrine. LPS activated MMP-2 in the aorta by S-glutathiolation. Calponin-1 levels decreased by 25% in endotoxemic aortae, which was prevented by doxycycline. Calponin-1 and MMP-2 coimmunoprecipitated and both exhibited uniform cytosolic staining in medial vascular smooth muscle cells. In vitro incubation of Calponin-1 with MMP-2 led to Calponin-1 degradation and appearance of its cleavage product. Conclusion— Calponin-1 is a target of MMP-2, which contributes to endotoxemia-induced vascular hypocontractility.

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