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PubMed2000

The maximal velocity of vascular smooth muscle shortening is independent of the expression of calponin.

In smooth muscle, the phosphorylation/dephosphorylation of the 20-kDa regulatory light chain of myosin (MLC20) is known to regulate actomyosin interaction and force. However, a thin filament based regulatory system for actomyosin interaction has been suggested to exist in parallel to MLC20 phosphorylation. Calponin is a thin filament associated protein that in vitro inhibits actomyosin interaction, and has been suggested to reduce maximal shortening velocity (vmax). Using antibodies to h1- and h2-calponin, we demonstrated that calponin was present in smooth muscle from Sprague Dawley (SD) rats, while calponin was not detectable in the smooth muscle from Wistar Kyoto (WKY) rats. vmax determined from the force vs. velocity relationship at maximal Ca2+ activation was not different for either the aorta or the portal vein of SD vs. WKY rats. These results suggest that physiological levels of calponin do not contribute to a thin filament-based secondary regulation to inhibit smooth muscle contraction.

Actomyosin↗
PubMed2001

Some properties of caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation.

The interaction of caldesmon with different Ca2+-binding proteins has been analyzed, and it is supposed that one of the conformers of calmodulin might be an endogenous regulator of caldesmon. The arrangement of caldesmon and Ca2+-binding proteins within their complexes has been analyzed by different methods. The central helix of calmodulin is supposed to be located near the single Cys residue in the C-terminal domain of caldesmon. The N-terminal globular domain of calmodulin interacts with sites A and B' of caldesmon, whereas the C-terminal globular domain of calmodulin binds to site B of caldesmon. The complex of calmodulin and caldesmon is very flexible; therefore, both parallel and antiparallel orientation of polypeptide chains of the two proteins is possible in experiments with short fragments of caldesmon and calmodulin. The length, flexibility, and charge of the central helix of calmodulin play an important role in its interaction with caldesmon. Phosphorylation of caldesmon by different protein kinases in vitro has been analyzed. It was shown that phosphorylation catalyzed by casein kinase II of sites located in the N-terminal domain decreases the interaction of caldesmon with myosin and tropomyosin. Caldesmon and calponin may interact with phospholipids. The sites involved in the interaction of these actin-binding proteins with phospholipids have been mapped. It is supposed that the interaction of calponin and caldesmon with phospholipids may play a role in the formation of cytoskeleton. Calponin interacts with 90-kD heat shock protein (hsp90) that may be involved in transportation of calponin and its proper interaction with different elements of cytoskeleton. Calponin, filamin, and alpha-actinin can simultaneously interact with actin filaments. Simultaneous binding of two actin-binding proteins affects the structure of actin bundles and their mechanical properties and may be of great importance in formation of different elements of cytoskeleton.

Actins↗
PubMed1990

Effect of calponin on actin-activated myosin ATPase activity.

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.

Actins↗
PubMed1988

A novel troponin T-like protein (calponin) in vascular smooth muscle: interaction with tropomyosin paracrystals.

Calponin is a smooth muscle tropomyosin-binding protein which is antigenically related to troponin T. The fine localization of calponin and troponin T on the polar paracrystals of smooth muscle tropomyosin were studied by electron microscopy. Both calponin and troponin T bound to the filamentous tropomyosin at a site about 17 nm away from the C-terminal end of tropomyosin molecule. This calponin-binding site corresponds to the domain which is highly conserved in the amino-acid sequences among the tropomyosin isoforms from skeletal muscle and smooth muscle. The result suggests that calponin, in collaboration with tropomyosin, may play an important role in regulating contractile apparatus.

Animals↗
PubMed2006

Differential expression of smooth muscle myosin, smooth muscle actin, h-caldesmon, and calponin in the diagnosis of myofibroblastic and smooth muscle lesions of skin and soft tissue.

The diagnosis of low-grade and pseudosarcomatous spindle cell lesions of skin and soft tissue can sometimes be problematic; in particular, distinction between fibroblastic, myofibroblastic, and smooth muscle proliferations can occasionally pose difficulties on routine histologic examination. We have applied a panel of immunohistochemical markers to a series of spindle cell lesions of skin and soft tissue to assess the utility of the differential expression of smooth muscle and myofibroblastic-associated markers. Twenty-eight cases of nodular fasciitis, 42 cases of fibromatosis, and 3 cases of myofibroblastic sarcoma were stained with antibodies against smooth muscle actin (SMA), smooth muscle myosin (SMMS), calponin, and high-molecular weight caldesmon (h-caldesmon). For comparison, 12 cases of cutaneous leiomyoma and 8 cases of leiomyosarcomas involving superficial soft tissues and fascia were studied with the same panel of antibodies. Thirty-eight of 42 cases of fibromatosis were positive for SMA, 42/42 cases were positive for calponin, 39/42 cases were negative for SMMS, and all cases were negative for h-caldesmon. All cases of nodular fasciitis were positive for SMA and calponin, and all were negative for h-caldesmon and SMMS. All cases of myofibroblastic sarcoma were positive for SMA and 2/3 cases for calponin, and were negative for SMMS and h-caldesmon. All cases of cutaneous leiomyoma and leiomyosarcoma were positive for all 4 markers tested. Our results demonstrate a remarkably consistent pattern of reactivity of muscle and myofibroblastic-associated markers in lesions predominantly composed of myofibroblastic spindle cells, characterized by positive staining for SMA and calponin and absence of reactivity for SMMS and h-caldesmon. Application of this panel of stains may be of aid in the differential diagnosis of low-grade myofibroblastic lesions such as nodular fasciitis and fibromatosis from smooth muscle tumors of skin and soft tissue. This panel may additionally be of assistance in the diagnosis of myofibroblastic sarcoma.

Actins↗
PubMed1994

Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells.

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.

Animals↗
PubMed2000

Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences.

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.

Actins↗
PubMed2000

[Calponin: biological, chemical and structural properties].

Calponin distribution in smooth muscle cells and its properties in experiments in vitro are described. A comparison of these properties with those of other regulatory proteins and of proteins presumed to play this role suggest that calponin has another, yet unknown function. On the basis of existing experimental and theoretical data, an attempt has been made to quantitatively estimate the content of structural elements of the polypeptide chain and their localization. With consideration of the structural calponin domains, a general model of the secondary structure of all known calponin sequences is suggested. Based on the known roentgenographic data of the CH-domain of other proteins a possible organization of the calponin molecule hydrophobic core has been proposed.

Amino Acid Sequence↗
PubMed2001

Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors.

The calponin (basic or h1) gene, normally expressed in maturated smooth muscle cells, is aberrantly expressed in a variety of human soft tissue and bone tumors. In this study, we show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the translation initiation site. A novel conditionally replicating herpes simplex virus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trans-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bone tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synovial sarcoma, leiomyosarcoma, and osteosarcoma cells. For in vivo studies, 10 animals harboring SK-LMS-1 human leiomyosarcoma cells were randomly divided and treated twice on days 0 and 9 intraneoplastically with either 1 x 10(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with medium alone. The viral treatment group showed stable and significant inhibition of tumorigenicity with apparent cure in four of five mice by day 35. Replication of viral DNA demonstrated by PCR amplification and expression of the inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry was associated with oncolysis of d12.CALP-treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a nontreated tumor distant from the site of virus inoculation. These results indicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.

Animals↗
PubMed2002

Insight into the kinetics and the mode of the interaction between smooth muscle calponin and F-actin.

Kinetics of the smooth muscle calponin-F-actin interaction was studied by stopped-flow measurements of light scattering and fluorescence intensity of pyrene-labelled F-actin. The intensity and character of the changes in light scattering, and thus the mode of calponin binding to actin filaments leading to changes in their shape and bundling, depend on the molar ratio of the two proteins. Parallel measurements of pyrene-fluorescence quenching upon calponin binding revealed that intrinsic conformational changes in actin filaments are delayed relative to the binding process and are not markedly influenced by the mode of calponin binding. Bundling of actin filaments by calponin was not correlated with fluorescence changes and thus with alterations in the structure of actin filaments.

Actins↗
PubMed2004

Effects of caldesmon, calponin, and tropomyosin on the Mg2+-ATPase activities of smooth muscle myosin.

OBJECTIVE: To test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca2+-dependent phosphorylation (CDP), and Ca2+-independent phosphorylation (CIP) and stimulate myosin Mg2+-ATPase activities. METHODS: Mg2+-ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK. RESULTS: (1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg2+-ATPase activity was observed when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg2+-ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropomyosin, the order of relative value of Mg2+-ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls. CONCLUSIONS: The results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg2+-ATPase activity of smooth muscle myosin in Ca2+-independent manner, since Ca2+ is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.

Animals↗
PubMed1992

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.

Actins↗
PubMed1991

Co-localization of immunoreactive forms of calponin with actin cytoskeleton in platelets, fibroblasts, and vascular smooth muscle.

Calponin is an actin binding protein found in the smooth muscle cells of chicken gizzard. The localization of the protein was examined in bovine platelets, mouse fibroblasts, and the smooth muscle cells of the bovine aorta. Immunoblotting of whole platelet lysates revealed that the antibody to chicken gizzard calponin recognized two proteins with apparent molecular masses of 37 and 23 kDa in the resting state and an additional high-molecular-weight component (approximately 40 kDa) in the activated state. The localizations of calponin and caldesmon, and the correlation of their localizations with that of the actin cytoskeleton were analyzed by immunofluorescence microscopy using appropriate antibodies and rhodamine-phalloidin. In resting bovine platelets, calponin exhibited the same distribution as actin filaments, which are organized in a characteristic wheel-like structure. A similar distribution was observed with the anti-caldesmon antibody. Colocalization of calponin and actin were shown in activated platelets and along stress fibers of both fibroblasts and smooth muscle cells. These results suggest not only a cytoskeletal role associated with microfilaments but also a regulatory role of these proteins for actin-myosin interaction.

Actins↗
PubMed1993

Interaction between calponin and smooth muscle myosin.

Calponin is a thin filament-associated protein in smooth muscle that has been shown to bind actin, tropomyosin and calmodulin, and has been implicated to play a role in regulation of smooth muscle contractility. Using a centrifugation assay we found that calponin interacts with unphosphorylated filamentous smooth muscle myosin. We found that this calponin-myosin interaction is reversed by Ca(2+)-CaM, and depends on ionic strength. At 50 mM NaCl the binding constant and the stoichiometry of this interaction were estimated to be 2 x 10(6) M-1, and 1.2-2.4 calponin per myosin, respectively. We suggest that the calponin-myosin interaction could be involved in regulation of smooth muscle contractility by anchoring myosin to actin.

Animals↗
PubMed1993

Identification of the regulatory site in smooth muscle calponin that is phosphorylated by protein kinase C.

F-actin and tropomyosin inhibited the phosphorylation of calponin by protein kinase C, and the phosphorylation reduced the binding of calponin to F-actin and tropomyosin. Labeled phosphate from [gamma-32P]ATP was retained both on the chymotryptic NH2-terminal 22-kDa fragment, which contains the actin-, tropomyosin-, and calmodulin-binding regions, and on the COOH-terminal 12-kDa fragment. Fractionation of tryptic 32P-labeled peptides by high performance liquid chromatography allowed isolation of three phosphopeptides (designated T1, T2, and T3), each of which was located in three repeating amino acid motifs of calponin. Both the relative initial rates and extent of phosphorylation decreased in the order T2 > T3 > T1. Both serine and threonine residues were phosphorylated in T1 (GASQAGMTAPGTK), and only a threonine residue was phosphorylated in T2 (FASQQGMTAYGTR) and in T3 (GASQQGMTVYGLPR). As the 22-kDa fragment contained only T2, the phosphorylation site in T2 appeared to regulate the binding of calponin to F-actin and tropomyosin. The amino acid sequence of T2 indicates that protein kinase C phosphorylates Thr184. Thus Thr184 is the preferred site of phosphorylation and is functionally the most important of the sites phosphorylated by protein kinase C in smooth muscle calponin.

Actins↗
PubMed1997

[The expression of calponin in Oddi's sphincters and its actions during gallstone formation].

OBJECTIVES: To study on the expression of calponin in an animal model of gallstone disease and investigate the molecular mechanisms of gallstone formation. METHODS: After feeding a high-cholesterol diet to guinea pigs, Oddi's sphincters were disseced on day 30 and day 60 respectively. We used RT-PCR, western-blotting to evaluate expression level of calponin gene. RESULTS: Down-regulation of calponin gene expression was observed in animals with gallstone. The levels of both protein and mRNA expression for calponin on day 30 and day 60 were lower than those of control group with the level from day 60 lower than that from day 60, while myosin expressions were relatively stable. CONCLUSION: Our results indicated that the decrease of calponin could increase the pressure of sphincter of Oddi, aggravate the stasis of bile and promote the gallstone formation.

Animals↗
PubMed1997

Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart.

BACKGROUND: Coexpression of alpha- and beta-myosin heavy chain (MHC) is a characteristic of the primary myocardial tube. To establish if the smooth-muscle proteins alpha-smooth-muscle actin (alpha-SMA) and calponin, and the intermediate filament protein, desmin, contribute to the specific functional properties of these early cardiomyocytes, we studied their spatiotemporal expression pattern. METHODS: Sections of prenatal and neonatal Wistar rats were stained with antibodies against alpha- and beta-MHC, alpha-SMA, calponin, and desmin. RESULTS: The expression of alpha-SMA and calponin in embryonic cardiomyocytes increases to reach its highest level at ED14. Subsequently, these proteins gradually disappear, beginning in the interventricular septum (IVS) and followed successively by the compact myocardium of the left ventricle, the right ventricle, and the central atrium. Expression of alpha-SMA persists longer in the ventricular conduction system, making it a convenient marker for the ventricular conduction system of the fetal rat. Desmin becomes expressed one day later than alpha-SMA, but also reaches its peak at ED14, suggesting that a relatively high concentration is required to form mature sarcomeres. CONCLUSIONS: The results indicate that alpha-SMA, calponin, and desmin are involved in the myofibrillar development in rat heart. The presence of spatiotemporal differences in the expression of these proteins reveals regional differences in the developmental timing of cardiomyocyte maturation. The maturation process extends from the compact myocardium in the IVS to the left and right ventricular free walls, whereas the atrioventricular junction, the ventricular trabeculae, and developing ventricular conduction system show a relatively slow maturation. Smooth-muscle proteins may contribute to the slow shortening speed that is characteristic of the embryonic myocardium.

Actins↗
PubMed1995

Phosphorylation of calponin mediated by protein kinase C in association with contraction in porcine coronary artery.

Calponin is an actin-associated regulatory protein in smooth muscle. We report that both endothelin-1 (ET-1) and phorbol 12, 13-dibutyrate (PDBu) caused a significant increase in phosphorylation of calponin during contraction of porcine coronary artery, while high levels of KCl were ineffective. This phosphorylation was predominantly catalyzed by activation of protein kinase C(PKC). In addition, the level of phosphorylation of calponin increased closely in association with the size of the contractile force induced by PDBu. Thus, the phosphorylation of calponin in vivo by PKC might modulate in part the contraction of smooth muscle that occurs in response to ET-1 or PDBu.

Animals↗