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PubMedDate not supplied

Aberrant methylation and silencing of the calponin gene in human sarcoma cells.

BACKGROUND: We have recently developed a new strategy for the treatment of sarcomas by using human calponin promoter to drive replication and oncolysis of herpes simplex virus (HSV-1). In order to apply this therapy to a broad spectrum of sarcoma cells, it is essential to elucidate silencing mechanisms of the calponin promoter in a subset of sarcoma cells: MATERIALS AND METHODS: In this study, we investigated the mechanisms of calponin inactivation in human sarcoma tissues and cell lines through the analysis of CpG methylation of promoter region by bisulfite modification, because the 5'-flanking region of the human calponin gene was originally identified as a DNA fragment bound to the conserved methyl-CpG binding domain (MBD) of MeCP2. Furthermore, we examined the correlation between DNA methylation and expression of the calponin gene by treatment with the demethylating agent 5-Aza-CdR, and in vitro 5'CpG methylation in a luciferase reporter gene construct. RESULTS: Here, we have provided evidence that DNA hypermethylation of the specific 5'CpG sites in exon 1 is a mechanism accompanying decreased calponin expression in both human sarcoma tissues and cell lines. CONCLUSION: These results suggest that DNA methylation may be an important mechanism regulating calponin transcription in human sarcoma cells, and thus could potentially affect the efficacies of treatment of this malignancy utilizing the human calponin promoter.

Calcium-Binding Proteins↗
PubMed1992

Mapping of the functional domains in the amino-terminal region of calponin.

Three chymotryptic fragments accounting for almost the entire amino acid sequence of gizzard calponin (Takahashi, K., and Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288) were isolated and characterized. They encompass the segments of residues 7-144 (NH2-terminal 13-kDa peptide), 7-182 (NH2-terminal 22-kDa peptide), and 183-292 (COOH-terminal 13-kDa peptide). They arise from the sequential hydrolysis of the peptide bonds at Tyr182-Gly183 and Tyr144-Ala145 which were protected by the binding of F-actin to calponin. Only the NH2-terminal 13- and 22-kDa fragments were retained by immobilized Ca(2+)-calmodulin, but only the larger 22 kDa entity cosedimented with F-actin and inhibited, in the absence of Ca(2+)-calmodulin, the skeletal actomyosin subfragment-1 ATPase activity as the intact calponin. Since the latter peptide differs from the NH2-terminal 13-kDa fragment by a COOH-terminal 38-residue extension, this difference segment appears to contain the actin-binding domain of calponin. Zero-length cross-linked complexes of F-actin and either calponin or its 22-kDa peptide were produced. The total CNBr digest of the F-actin-calponin conjugate was fractionated over immobilized calmodulin. The EGTA-eluted pair of cross-linked actin-calponin peptides was composed of the COOH-terminal actin segment of residues 326-355 joined to the NH2-terminal calponin region of residues 52-168 which seems to contain the major determinants for F-actin and Ca(2+)-calmodulin binding.

Amino Acid Sequence↗
PubMed2000

Use of a novel marker, calponin, for myoepithelial cells in fine-needle aspirates of papillary breast lesions.

Benign and malignant papillary lesions of the breast (PBL) can be difficult to distinguish in fine-needle aspirates (FNA). This study evaluates the use of smooth muscle actin (SMA) and a new smooth muscle-specific protein, calponin, for identifying myoepithelial cells (MEC) by immunohistochemical methods in paraffin-embedded cell blocks of FNA of PBL. Formalin-fixed, paraffin-embedded cell blocks of 40 cases of PBL were stained using SMA and calponin, steam heat-induced epitope retrieval, and an avidin biotin-complex technique. Staining was evaluated in MEC, epithelial, and stromal cells. The diagnosis of benign vs. malignant papillary lesion was made by using cytomorphological criteria and the presence/absence of MEC in the cell block. These results were compared to the original cytologic and subsequent histologic diagnoses. Of 40 cases of FNA diagnosed as PBL, there were 27 intraductal papillomas (IP), 6 papillary lesions with atypical features (PLAF), and 7 papillary carcinomas (PC). In all of the IP, MEC stained both with SMA and calponin. None of the PC cases was positive for MEC with calponin, and 2 out of 7 cases were weakly positive by SMA. In 6 cases of PLAF, 2 were negative for MEC, both by SMA and calponin, and a malignant papillary lesion was confirmed by histology. The remaining 4 cases were positive for MEC with both markers and were confirmed to be benign by histology. SMA stained stromal cells strongly in all of the cases where stroma was present (18 of a total of 40 cases of PBL), while calponin stained stroma focally in only 7 cases. More than half of all cases had nuclear staining of epithelial cells with SMA; calponin did not show any nuclear staining.

Actins↗
PubMed1999

Regulatory mechanisms of calponin phosphorylation in endothelin-1-induced contraction of porcine coronary artery.

Calponin is an actin-associated protein that appears to play an auxiliary regulatory role in the contraction of smooth muscle. We report here on the mechanisms for regulation of calponin phosphorylation in the endothelin-1-induced contraction of porcine coronary artery. Treatment of strips of porcine artery with endothelin-1 increased calponin phosphorylation and contraction in a concentration-dependent manner. The time course of the phosphorylation was biphasic, with the response to endothelin-1. The extent of phosphorylation reached a maximum within 5 min of stimulation with 10(-7)M endothelin-1 and then it declined rapidly to reach a minimum at 20 min. A potent inhibitor of protein kinase C, GF109203X, inhibited both calponin phosphorylation and contraction that were induced by endothelin-1 at 5 min, without an inhibition for myosin light chain phosphorylation. Protein phosphatase inhibitor, okadaic acid, had no effect on the extent of phosphorylation at 5 min, but it significantly inhibited the subsequent decrease in calponin phosphorylation. In contrast, in PDBu-treated strips of coronary artery, okadaic acid caused a significant steady increase of the extent of calponin phosphorylation. Our results suggest that calponin phosphorylation might be regulated by protein kinase C and okadaic acid sensitive protein phosphatases, in the endothelin-1-induced contraction of porcine coronary artery.

Animals↗
PubMed1996

Immunocytochemical localization of caldesmon and calponin in chicken gizzard smooth muscle.

The distribution of caldesmon and calponin in chicken gizzard smooth muscle was investigated with immunofluorescence and immunogold electron microscopy. Immunofluorescence microscopy showed that in verapamil treated (relaxed) muscles the distributions of caldesmon and myosin appeared to be uniform throughout the cytoplasm, but clearly more textured than that of actin filaments as revealed by the distribution of tropomyosin. In shortened muscles both caldesmon and myosin became segregated, in contrast to the distribution of actin, which remained uniform. The distribution of calponin was even more textured, with no similarity to those of caldesmon or myosin. Instead, considerable overlap was observed between calponin and the cytoskeletal protein desmin and, to a lesser extent, beta-actin. By immunogold electron microscopy caldesmon appeared mostly near and around myosin filaments in both relaxed and shortened muscle. Calponin, on the other hand, was found primarily at the periphery of cytoskeletal structures in the same general region as desmin, and very often adjacent to beta-actin, which is mainly in the core. These observations indicated that caldesmon and calponin are associated with different subsets of actin filaments, caldesmon with contractile actin, while calponin with cytoskeletal actin. Thus the in situ localization of caldesmon is consistent with its proposed regulatory function. Calponin, on the other hand, is unlikely to directly regulate actomyosin interactions in these cells; instead, it may function as a bridging protein between the actin and the intermediate filament networks.

Animals↗
PubMed1990

Modulation of smooth muscle calponin by protein kinase C and calmodulin.

When smooth muscle calponin was incubated with protein kinase C, 1 mole of phosphate was incorporated per mole of calponin. The apparent Km value for calponin of the protein kinase was about 0.4 microM. The phosphorylation of calponin by protein kinase C was inhibited markedly by calmodulin in a calcium-dependent manner. Kinetic analysis of calmodulin-induced inhibition of calponin phosphorylation by protein kinase C revealed that calmodulin inhibited the phosphorylation in a noncompetitive fashion with calponin and the determined Ki value was 0.4 microM. These results suggest that interaction of calmodulin with calponin may play a regulatory role in the phosphorylation by protein kinase C and smooth muscle contraction.

Animals↗
PubMed1992

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.

Adolescent↗
PubMed1997

Adenovirus-mediated transfer of the smooth muscle cell calponin gene inhibits proliferation of smooth muscle cells and fibroblasts.

Smooth muscle cell calponin (h1 or basic isoform) is an actin-binding protein that inhibits actomyosin MgATPase activity and is abundantly expressed in differentiated smooth muscle. Western blots showed bovine tracheal (BT) smooth muscle cells in culture expressed only 2 +/- 1% (n = 8) of the amount of calponin in tissues, while NIH-3T3 fibroblasts expressed none. We tested the hypothesis that introduction of calponin to cultured BT and 3T3 cells would inhibit cytoskeletal activities associated with cell proliferation. To achieve high-efficiency expression, an adenovirus encoding the CMV-calponin construct (Adv-CaP) was generated by homologous recombination in 293 cells. With greater than 90% of BT and 3T3 cells infected with Adv-CaP, calponin expression (32 and 11 microg/mg total protein, respectively) was similar to that in smooth muscle tissues (51 microg/mg). Cells were infected with Adv-CaP for 48 h, replated at low density and proliferation rates were assessed by cell density and [3H]thymidine incorporation. Cell growth and DNA synthesis by Adv-CaP-infected cells were inhibited to one-third control values for both BT and 3T3 cells. Expressed calponin was localized primarily on stress fibers in both cell types. Calponin may act at the cytoskeletal level to retard signaling pathways that normally lead to tight coupling between cell shape and DNA synthesis.

3T3 Cells↗
PubMed2001

Mutual effects of alpha-actinin, calponin and filamin on actin binding.

The mutual effect of three actin-binding proteins (alpha-actinin, calponin and filamin) on the binding to actin was analyzed by means of differential centrifugation and electron microscopy. In the absence of actin alpha-actinin, calponin and filamin do not interact with each other. Calponin and filamin do not interfere with each other in the binding to actin bundles. Slight interference was observed in the binding of alpha-actinin and calponin to actin bundles. Higher ability of calponin to depress alpha-actinin binding can be due to the higher stoichiometry calponin/actin in the complexes formed. The largest interference was observed in the pair filamin-alpha-actinin. These proteins interfere with each other in the binding to the bundled actin filaments; however, neither of them completely displaced another protein from its complexes with actin. The structure of actin bundles formed in the presence of any one actin-binding protein was different from that observed in the presence of binary mixtures of two actin-binding proteins. In the case of calponin or its binary mixtures with alpha-actinin or filamin the total stoichiometry actin-binding protein/actin was larger than 0.5. This means that alpha-actinin, calponin and filamin may coexist on actin filaments and more than mol of any actin-binding protein is bound per two actin monomers. This may be important for formation of different elements of cytoskeleton.

Actinin↗
PubMed1995

Dephosphorylation of calponin by type 2B protein phosphatase.

Calponin is a smooth muscle-specific, thin filament-associated protein which has been implicated in the regulation of contraction via its interaction with actin and inhibition of the cross-bridge cycling rate. Calponin is phosphorylated by protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), primarily at S175, with loss of actin binding and inhibition of the actin-activated myosin MgATPase. We previously isolated calponin phosphatase from chicken gizzard smooth muscle and identified it as a type 2A protein phosphatase [Winder et al. (1992) Biochem. J. 286, 197-203]. The methods used to detect phosphatase activity in that study would additionally have detected type 1 and 2C phosphatases, but not type 2B phosphatase (Ca2+/CaM-dependent phosphatase or calcineurin). We have, therefore, examined the expression of type 2B phosphatase in smooth muscle and its ability to dephosphorylate calponin. Western blotting with polyclonal antibodies to the brain enzyme revealed the expression of type 2B phosphatase in chicken gizzard, and immunofluorescence microscopy confirmed the presence of the phosphatase in isolated smooth muscle cells (rabbit and toad stomach). The purified brain phosphatase dephosphorylated calponin (phosphorylated by PKC or CaM kinase II) in a Ca2+/CaM-dependent manner. Dephosphorylation by calcineurin restored actin-binding and actin-activated myosin MgATPase inhibition which had been reduced by PKC-catalyzed phosphorylation. We conclude that calponin dephosphorylation may be catalyzed not only by type 2A phosphatase but also by type 2B phosphatase, raising the possibility that both phosphorylation and dephosphorylation of calponin could be regulated by Ca2+/CaM.

Actins↗
PubMed1995

Interaction of calponin with actin and its functional implications.

Titration of F-actin with calponin causes the formation of two types of complexes. One, at saturation, contains a lower ratio of calponin to actin (0.5:1) and is insoluble at physiological ionic strength. The another is soluble, with a higher ratio of calponin to actin (1:1). Electron microscopy revealed that the former complex consists of paracrystalline bundles of actin filaments, whereas the latter consists of separate filaments. Ca(2+)-calmodulin causes dissociation of bundles with simultaneous increase in the number of separate calponin-containing filaments. Further increase in the calmodulin concentration results in full release of calponin from actin filaments. In motility assays, calponin, when added together with ATP to actin filaments complexed with immobilized myosin, evoked a decrease in both the number and velocity of moving actin filaments. Addition of calponin to actin filaments before their binding to myosin resulted in a formation of actin filament bundles which were dissociated by ATP.

Actins↗
PubMed1995

Expression of an acidic isoform of calponin in rat brain: western blots on one- or two-dimensional gels and immunolocalization in cultured cells.

Calponin, an actin- and Ca(2+)-calmodulin-binding protein characterized as an inhibitory factor of the smooth-muscle actomyosin activity, has also been shown to be present in some non-muscle cells. However, there is a controversy as to whether calponin is present or not in brain. Several laboratories indicate that this protein is absent in chicken or bovine brains, while Applegate et al. [Applegate, Feng, Green and Taubman (1994) J. Biol. Chem. 269, 10683-10690] have recently reported the identification of an mRNA specific for a 36 kDa non-muscle calponin analogue in homogenates of rat brains. For the first time we demonstrate, by Western blots and in situ immunofluorescence localization using monoclonal as well as affinity-purified polyclonal antibody to gizzard calponin, that a 36-37 kDa and a 35-36 kDa calponin-like proteins are expressed respectively in pig and rat brains and in rat cerebellar cultured cells. The acidic pI (5.2-5.4) of the rat brain protein revealed by isoelectric focusing is in good agreement with that of the protein coded for by the calponin isoform mRNA described by Applegate et al. and is different from that of the protein from chicken gizzard (pI 9.9). Brain calponin-like protein is different from two other Ca(2+)-calmodulin-binding proteins previously identified in brain, namely caldesmon and adducin, and from tropomyosin.

Animals↗
PubMed1996

Interaction of proteolytic fragments of calmodulin with caldesmon and calponin.

Interaction of five tryptic fragments of calmodulin with caldesmon and calponin was analysed by native gel electrophoresis. In the presence of Ca2+ intact calmodulin interacts with caldesmon and calponin with apparent Kd values equal to 0.23 and 1.3 microM respectively. The interaction was abolished in the absence of Ca2+. Two large tryptic fragments of calmodulin obtained in the presence of Ca2+ (TR1C, residues 1-77, and TR2C, residues 78-148) interact with caldesmon with apparent Kd values of 11.9 and 4.6 microM. Affinity of TR2C to calponin (Kd 3.8 microM) was comparable with that of native calmodulin and was much higher than the corresponding value for TR1C (Kd 41 microM). The short C-terminal tryptic peptide of calmodulin obtained in the presence of EGTA (TR3E, residues 107-148) interacts with caldesmon and calponin with Kd values of 23.9 and 12.1 microM, whereas the large N-terminal peptide TR1E (residues 1-106) interacts with both caldesmon and calponin with a very low affinity (Kd 60 microM). Thus although both N- and C-terminal domains of calmodulin are involved in the interaction with caldesmon and calponin, the C-terminal part of calmodulin (residues 78-148) is of special importance and has the highest contribution for caldesmon and calponin binding.

Animals↗
PubMed1998

Two distinct actin-binding sites of smooth muscle calponin.

Amino acid residues 145-163 of calponin have been proposed as a putative actin-binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201] enhanced the Ca2+-induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172-His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172-His187 inhibited the binding of calponin to F-actin in a concentration-dependent manner but not the binding of caldesmon. Gly141-Gly160, including the above-mentioned putative actin-binding site, also competed with intact calponin to the same extent as Lys172-His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141-Gly160. Lys172-His187 and other tested peptides had no effect. However, Gly141-Gly160 and Lys172-His187 reduced the fluorescence intensity of pyrene-labeled F-actin with approximately equal potency. Moreover, Lys172-His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172-His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin-binding activity. These observations suggest the direct involvement of two distinct actin-binding sites, with different regulatory functions, in the interactions of calponin with actin.

Actins↗
PubMed1997

Does calponin interact with caldesmon?

The roles of calponin and caldesmon and their interaction in regulation of smooth muscle contraction are controversial. Recently, strong binding between these two proteins has been reported (Graceffa, P., Adam, L. P., and Morgan, K. G. (1996) J. Biol. Chem. 271, 30336-30339). Results in this paper fail to confirm their data and are consistent with the concept of independent functions for calponin and caldesmon. To examine the ability of duck gizzard caldesmon to interact with calponin, three caldesmon derivatives, each containing a different sulfhydryl-specific reporter probe (6-acryloyl-2-dimethylaminonaphtalene, N-(1-pyrenyl)iodoacetamide, and N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine) attached to a single cysteine located in the C-terminal domain, were synthesized. Addition of calponin to labeled caldesmon at both low and physiological salt concentrations did not induce any changes in fluorescence intensity or maximum shift. Under the same conditions, calmodulin and tropomyosin (known to bind to the C terminus of caldesmon) produced substantial changes in these spectral parameters. Gel filtration of an equimolar caldesmon-calponin mixture on a fast protein liquid chromatography Superose-12 column revealed two base-line-separated peaks, the first containing only caldesmon and the second only calponin, thus confirming the lack of any interaction between these two proteins. Also, the addition of calponin did not change the fluorescence parameters of labeled caldesmon in complexes with F-actin and F-actin-tropomyosin.

Animals↗
PubMed1996

Molecular cloning and characterization of human non-smooth muscle calponin.

cDNA clones encoding a calponin isoform with 309 amino acids have been isolated from human heart. The deduced amino acid polypeptide (M(r) 33,697) showed a neutral isoelectric point of 7.1. The mRNA, expressed in cultured smooth muscle cells as well as in fibroblasts, vascular endothelial cells, and keratinocytes, contains a 3' untranslated region of 1.2 kilobases that includes an Alu repetitive sequence in the antisense direction. On the basis of the nucleotide sequence identity to an expressed sequence tag, HUM21ES93 [Cheng, J.-F., Boyartchuk, V., and Zhu, Y. (1994) Genomics 23, 75-84], the human neutral calponin gene is assigned to chromosome 21q11.1. The amino acid sequence indicates that this protein is the human equivalent of mouse calponin-h2 (94.8% identity) [Strasser, P., Gimona, M., Moessler, H., Herzog, M., and Small, J.V. (1993) FEBS Lett. 330, 13-18]. Three tandem repeats of 29 amino acids, a Vav-homologous region and an actin-binding sequence, originally identified in the basic calponin isoform, are conserved. There are two consensus phosphorylation sites for tyrosine kinase. An immunoreactive form of the neutral calponin appears to be localized with vinculin in the cell-to-cell junctions of cardiomyocytes. Mouse calponin-h2 is also expressed in both embryonic and adult heart. These results indicate that the human neutral calponin is a non-smooth muscle isoform, and may play a physiological role in cytoskeletal organization.

Adult↗
PubMed1995

Interaction of calponin with phospholipids.

The interaction between chicken gizzard calponin and phospholipids was examined by sedimentation assay and affinity chromatography. Calponin was sedimented with phosphatidylserine (PS) and phosphatidylinositol (PI) vesicles but not with phosphatidylcholine (PC) vesicles. The apparent Kd values of calponin to PS and PI were calculated to be 1.3 x 10(6) and 1.5 x 10(6) M-1, respectively. Domain mapping with chymotryptic digestion showed that the phospholipid-binding site resided within the N-terminal 22-kDa fragment, in which the bindings of actin, calmodulin, S100, and tropomyosin also occur. The amount of calponin bound to PS and PI vesicles decreased with increasing ionic strength or Ca2+ concentrations. The presence of MgCl2 was needed for the calponin-PS vesicle interaction. Calponin-binding proteins including actin, calmodulin, and S100 inhibited calponin binding to the phospholipid vesicles in a concentration-dependent manner, while tropomyosin had little effect on the binding. The inhibitory effects of calmodulin and S100 were found only in the presence of CaCl2. Neither caldesmon nor SM22 affected the binding.

Actins↗
PubMed1994

Calponin and smooth muscle regulation.

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.

Actins↗