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PubMed1995

Calponin is not phosphorylated during contractions of porcine carotid arteries.

Calponin and caldesmon were purified from porcine carotid arteries that were preincubated with [32P]orthophosphate, and the stoichiometry of phosphorylation was measured. In resting arteries, caldesmon was phosphorylated to a level of 0.41 mol PO4/mol protein, while calponin was phosphorylated to levels < 0.01 mol PO4/mol protein. Stimulation by histamine (1 or 5 min), KCl (1, 5 or 60 min), or phorbol 12,13-dibutyrate (PDBu; 1 microM for 15 or 60 min) did not lead to measurable increases in the PO4 content of calponin. Because dephosphorylation of calponin during the purification procedure could account for these results, we also determined stoichiometries after firat denaturing endogenous phosphatases with trichloroacetic acid. In these experiments, calponin was determined to be phosphorylated to the same low levels as in the first set of experiments. Collectively, these data show that calponin is not phosphorylated to significant levels during contractions of carotid arteries under conditions where caldesmon phosphorylation is apparent. The circumstances under which calponin may be phosphorylated in intact smooth muscle, and the purpose that may be served by this potential regulatory process, remain to be determined.

Animals↗
PubMed1998

Expression of the smooth muscle calponin gene in human osteosarcoma and its possible association with prognosis.

The basic calponin gene is a smooth muscle differentiation-specific gene that encodes an actin-binding protein involved in the regulation of smooth muscle contractility. We studied the expression of the calponin gene in 8 human osteosarcoma cell lines and 17 primary human osteosarcoma tissues by RT-PCR analysis. We also analyzed mRNA expression of smooth muscle-specific genes including SM22alpha, caldesmon and alpha-actin, and for neutral and acidic calponin isoforms. The genes were expressed at various levels by osteosarcoma cell lines and tissues of diverse histological subtypes. The basic calponin protein of an expected size was detected in osteosarcoma cell lines by immunoblot analysis and was localized by immunohistochemistry in the cytoplasm of the tumor cells in osteosarcoma tissues. Survival was found to be significantly increased in patients whose tumors exhibited basic calponin expression, compared with those with no expression. Alterations in the expression of other markers examined were not correlated with prognosis. Our results suggest that the basic calponin gene product may be a novel prognostic variable in patients with osteosarcoma.

Adolescent↗
PubMed1995

Molecular cloning and gene mapping of human basic and acidic calponins.

The nucleotide and deduced amino acid sequences of human basic and acidic calponins were determined. The basic calponin cDNA from human aorta (1496 bp) contained a single open reading frame (ORF) which encodes 297 amino acids (33,169 Da). The acidic calponin cDNA from human kidney (1607 bp) contained a single ORF which encodes 329 amino acids (36,412 Da). Basic calponin mRNA was expressed in only smooth muscle tissues, but acidic calponin mRNA was expressed in non-smooth muscle tissues as well as smooth muscle tissues. Fluorescent in situ hybridization revealed that basic and acidic calponin genes localize in 19p13.1-13.2 and 1p21-22 of human chromosomes, respectively.

Amino Acid Sequence↗
PubMed1996

Expression of calponin mRNA in porcine aortic endothelial cells.

Using reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of the mRNAs for basic calponin isoforms, namely h1 and h2 forms, in the porcine aortic endothelial cells (PAECs) and aortic smooth muscle cells (PASMCs). The mRNAs for both h1 and h2 calponin isoforms were expressed in PAECs as well as in PASMCs. However, the expression of h2 isoform mRNA was more abundant than h1 isoform in PAECs, while h1 isoform was more abundant than h2 in PASMCs. These findings indicated the existence of calponin mRNA and the predominance of h2 over h1 calponin isoform in PAECs. It is suggested that h2 as well as h1, calponin isoform might have a physiological role in vascular endothelial cell. This is the first report which describes the existence of calponin h2-dominant cells.

Animals↗
PubMed2001

Calponin and h-caldesmon expression in atypical fibroxanthoma and superficial leiomyosarcoma.

To evaluate smooth muscle differentiation, myogenic markers [desmin, alpha-smooth muscle actin (SMA), and muscle-specific actin (HHF35)] have been widely used. Calponin and h-caldesmon, which are cytoskeleton-associated actin-binding proteins, have been reported to be more specific myogenic markers, especially since myofibroblasts express a small amount of h-caldesmon. Atypical fibroxanthoma (AFX) occurs in the sun-exposed skin of the elderly and follows a benign clinical course. Histologically, AFX, which is a pleomorphic spindle cell tumor and considered to be a superficial variant of malignant fibrous histiocytoma, also mimics leiomyosarcoma. AFX has been thought to differentiate along pathways with fibrohistiocytic and myofibroblastic phenotypes. AFX ( n=10), superficial leiomyosarcoma (S-LMS) ( n=17) and benign fibrous histiocytoma (BFH) ( n=17) were analyzed for myofibroblastic and smooth muscle differentiation immunohistochemically from the viewpoint of comparison. AFX and BFH showed immunoreactivities respectively for calponin (3/10, 11/17), desmin (3/10, 1/17), SMA (3/10, 13/17), and HHF35 (1/10, 5/17), but failed to express h-caldesmon (0/10, 0/17). S-LMS had a high immunoreactive rate of calponin (17/17), desmin (13/17), SMA (16/17), and HHF35 (16/17), while also expressing caldesmon (11/17). The results reveal that AFX and BFH have immunoreactivities for several myogenic markers, with myofibroblastic differentiation (calponin: +/-, h-caldesmon: -), but without the smooth muscle differentiation seen in S-LMS (calponin:+, h-caldesmon: +/-). In addition, calponin and h-caldesmon are considered to be useful markers for distinguishing AFX from S-LMS.

Adolescent↗
PubMed1992

The mechanism of inhibition of the actin-activated myosin MgATPase by calponin.

Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked acto-subfragment 1. Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model.

Actins↗
PubMed1989

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.

Animals↗
PubMed1990

Do thin filaments of smooth muscle contain calponin? A new method for the preparation.

A new method for the preparation of smooth muscle thin filaments which include calponin was established. We found that calponin readily separated from thin filaments in the presence of 10 mM ATP. By preventing thin filament extract from exposing to ATP, we obtained thin filaments which contained actin, tropomyosin, caldesmon and calponin in molar ratios of 7:0.9:0.6:0.7. We studied myosin Mg-ATPase activity by using the thin filaments in comparison with classical thin filaments prepared by the method of Marston and Smith, which contained the same amounts of caldesmon and tropomyosin as our thin filaments but lost almost all calponin. The presence of calponin reduced the Vmax value for thin filament-activated myosin Mg-ATPase activity by 33% without a significant change in Km value. These findings suggest that calponin inhibits myosin Mg-ATPase activity by modulation of a kinetic step as an integral component of smooth muscle thin filaments.

Actin Cytoskeleton↗
PubMed1990

Smooth muscle specific expression of calponin.

Calponin is an actin-, calmodulin-, and tropomyosin-binding protein that has been isolated from smooth muscle tissue. Using a monoclonal antibody specific for avian calponin, we demonstrate a differentiation-linked increase in calponin expression in embryonic chick gizzard. Cultivation of gizzard smooth muscle cells in vitro resulted in a down-regulation of calponin expression after the first 48 h that was paralleled by a loss of synthesis of metavinculin and the high molecular weight isoform of caldesmon. In early cultures of smooth muscle cells calponin was localised in the actin-containing stress fibres but labelling was restricted to the central parts of the actin cytoskeleton. Calponin expression is suggested as a potentially useful index of smooth muscle differentiation.

Animals↗
PubMed1991

Properties of calponin isolated from sheep aorta thin filaments.

Calponin, a 35 kDa actin-binding protein, was shown to be a normal component of 'native' thin filaments prepared from sheep aorta. Actin, tropomyosin, caldesmon and calponin were present in molar ratios 14:2:1:0.9. Calponin was isolated from thin filaments in yield 0.5 mg/100 mg thin filament protein. Calponin inhibited actomyosin ATPase up to 85%, half maximal at 0.2 calponin/actin. Inhibition did not depend on tropomyosin, Ca2+ or Ca2+ calmodulin. Caldesmon inhibited actomyosin with a 10-fold greater potency than calponin in the presence of tropomyosin and inhibition could be reversed by Ca2+ calmodulin under certain conditions. Calponin had no effect on caldesmon inhibition or the reversal of inhibition.

Animals↗
PubMed1991

Calponin. Developmental isoforms and a low molecular weight variant.

Two-dimensional gel analysis of basic proteins in developing human smooth muscle identifies calponin as a prominent marker of the differentiated phenotype. Adult tissue (human and mouse) typically expresses up to four calponin isoforms, three of which appear sequentially during fetal development: adult myometrial cells express the same three isoforms in primary culture in vitro and these are down-regulated, in reverse order, during the subsequent modulation of phenotype. Monospecific, polyclonal antibodies against calponin identify a lower molecular weight variant of calponin (L-calponin) that is strongly and specifically expressed in adult smooth muscles of the human urogenital tract. L-calponin is down-regulated in benign smooth muscle derived tumors (leiomyoma) and is not expressed in primary cultures of normal uterine tissue.

Animals↗
PubMed1995

A calponin peptide enhances Ca2+ sensitivity of smooth muscle contraction without affecting myosin light chain phosphorylation.

In permeabilized smooth muscle, exogenously applied calponin binds to myofibrils and reduces Ca(2+)-activated tension (Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka, T. (1994) Pflügers Arch. Eur. J. Physiol. 427, 301-308). A calponin peptide (calponin Phe173-Arg185), which inhibits the binding of calponin to actin, blocks the action of calponin and enhances the contraction induced by submaximal Ca2+ in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhances the Ca(2+)-induced contraction without a corresponding increase in the level of myosin light chain phosphorylation. These results suggest that calponin decreases the sensitivity of smooth muscle to Ca2+ at a given level of myosin light chain phosphorylation.

Actins↗
PubMed1997

Calponin and mitogen-activated protein kinase signaling in differentiated vascular smooth muscle.

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-epsilon. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-epsilon are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-epsilon to the surface membrane.

Animals↗
PubMed2000

Serum response factor-dependent regulation of the smooth muscle calponin gene.

Smooth muscle calponin is a multifunctional, thin filament-associated protein whose expression is restricted to smooth muscle cell lineages in developing and postnatal tissues. Although the physiology of smooth muscle calponin has been studied extensively, the cis-elements governing its restricted pattern of expression have yet to be identified. Here we report on smooth muscle-specific enhancer activity within the first intron of smooth muscle calponin. Sequence analysis revealed a proximal consensus intronic CArG box and two distal intronic CArG-like elements, each of which bound recombinant serum response factor (SRF) as well as immunoreactive SRF from smooth muscle nuclear extracts. Site-directed mutagenesis studies suggested that the consensus CArG box mediates much of the intronic enhancer activity; mutating all three CArG elements abolished the ability of SRF to confer enhancer activity on the smooth muscle calponin promoter. Cotransfecting a dominant-negative SRF construct attenuated smooth muscle-specific enhancer activity, and transducing smooth muscle cells with adenovirus harboring the dominant-negative SRF construct selectively reduced steady-state expression of endogenous smooth muscle calponin. These results demonstrate an important role for intronic CArG boxes and the SRF protein in the transcriptional control of smooth muscle calponin in vitro.

Animals↗
PubMed2000

Simultaneous interaction of actin with alpha-actinin and calponin.

Interaction of calponin and alpha-actinin with actin was analyzed by means of cosedimentation and electron microscopy. G-actin was polymerized in the presence of calponin, alpha-actinin, or both of these actin-binding proteins (ABPs). The single and bundled actin filaments were separated, and the stoichiometry of ABPs and actin in both types of filaments was determined. Binding of calponin to the single or bundled actin filaments was not dependent on the presence of alpha-actinin and did not displace alpha-actinin from actin. In the presence of calponin, however, less alpha-actinin was bound to the bundled actin filaments, and the binding of alpha-actinin was accompanied by a partial decrease in the calponin/actin stoichiometry in the bundles of actin filaments. Calponin had no influence on the binding of alpha-actinin to the single actin filaments. The structure of actin bundles formed in the presence of the two ABPs differed from that formed in the presence of either one singly. We conclude that calponin and alpha-actinin can coexist on actin and that nearly each actin monomer can bind one of these ABPs.

Actinin↗
PubMed2003

Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain.

Identification of myoepithelial cells using antibodies to cytoskeletal proteins, such as smooth muscle myosin heavy chain (SMM-HC) and calponin, can play an important role in distinguishing invasive carcinoma from its histologic mimics. However, antibodies to these proteins may also cross-react with stromal myofibroblasts and vascular smooth muscle cells. It has recently been demonstrated that myoepithelial cells express the nuclear protein, p63, a member of the p53 gene family. We compared the patterns of reactivity of antibodies with p63, calponin, and SMM-HC on 85 breast lesions, including 11 cases of sclerosing adenosis, 33 cases of ductal carcinoma in situ, including 10 that showed microinvasion, 6 cases of lobular carcinoma in situ, and 35 cases of infiltrating ductal carcinoma. All three antibodies were positive on the vast majority of myoepithelial cells in all cases. A small minority of cases showed focal gaps in the revealed myoepithelial cell layer, reflected in discontinuous positive immunostaining around noninvasive epithelial nests (including ductal carcinoma in situ). No case showed p63 expression by myofibroblasts or vascular smooth muscle cells, whereas myofibroblasts expressed, in 8% and 76% of cases, SMM-HC and calponin, respectively. Although no tumor cell reactivity was noted with antibodies to calponin or SMM-HC, tumor cells in 11% of cases showed at least focal p63 expression. And although antibodies to p63 offer excellent sensitivity and increased specificity for myoepithelial detection relative to antibodies to calponin and SMM-HC, they have the following diagnostic limitations: 1) they occasionally demonstrate an apparently discontinuous myoepithelial layer, particularly around ductal carcinoma in situ, and 2) they react with a small but significant subset of breast carcinoma tumor cells. p63 may represent a myoepithelial marker that can complement or replace SMM-HC and/or calponin in the analysis of difficult breast lesions.

Biomarkers, Tumor↗
PubMed2005

Suppression of cancer phenotypes through a multifunctional actin-binding protein, calponin, that attacks cancer cells and simultaneously protects the host from invasion.

Quantitative and/or qualitative alteration of actin cytoskeletal molecules, involved in the regulation of cellular dynamic functions, should be intimately related with cancer phenotypes. Based on several lines of experimental evidence from our group, and others, this report proposes a strategy to simultaneously attack cancer cells and protect the host from cancer invasion, with one molecule. Calponin h1, an actin-stabilizing protein that is also intimately related to signal transduction, is very often suppressed in vascular smooth muscle cells of malignant human tumors and in mesothelial cells by coexisting cancer cells. We generated mice deficient for calponin h1, exhibiting fragility in blood vessels and peritoneal membranes. Hematogenous cancer metastasis occurred more easily in the calponin h1-deficient mice than in wild-type mice, and the peritoneal dissemination was extremely enhanced. The fragility was rescued by the exogenous introduction of the calponin h1 gene into mesothelial cells of the peritoneum. Furthermore, calponin h1 gene transfer into several transformed cell lines resulted in a suppression of malignancy. The peritoneal dissemination of intraperitoneally-injected B16-F10 cells was suppressed by the calponin h1 gene, given to target both cancer cells and the mesothelial cells of the host. The multifunctional nature of the molecule, as a machinery player of cytoskeleton and mediator of signal transduction, probably resulted in a favorable recipient-discriminating effect on cancerous and normal cells. Thus, we believe that if we use adequate multifunctional molecules for therapy, it is possible to simultaneously suppress cancer phenotypes and protect normal cells from the attack of cancer cells.

Animals↗
PubMed1998

Regulation of smooth muscle actin-myosin interaction and force by calponin.

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin triggered by an increase in sarcoplasmic free Ca2+ concentration ([Ca2+]i). Contraction can, however, be modulated by other signal transduction pathways, one of which involves the thin filament-associated protein calponin. The h1 (basic) isoform of calponin binds to actin with high affinity and is expressed specifically in smooth muscle at a molar ratio to actin of 1:7. Calponin inhibits (i) the actin-activated MgATPase activity of smooth muscle myosin (the cross-bridge cycling rate) via its interaction with actin, (ii) the movement of actin filaments over immobilized myosin in the in vitro motility assay, and (iii) force development or shortening velocity in permeabilized smooth muscle strips and single cells. These inhibitory effects of calponin can be alleviated by protein kinase C (PKC)-catalysed phosphorylation and restored following dephosphorylation by a type 2A phosphatase. Three physiological roles of calponin can be considered based on its in vitro functional properties: (i) maintenance of relaxation at resting [Ca2+]i, (ii) energy conservation during prolonged contractions, and (iii) Ca(2+)-independent contraction mediated by phosphorylation of calponin by PKC epsilon, a Ca(2+)-independent isoenzyme of PKC.

Actins↗