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PubMed1999

Calponin interaction with alpha-actinin-actin: evidence for a structural role for calponin.

The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.

Actinin↗
PubMed1994

Smooth muscle calponin-caltropin interaction: effect on biological activity and stability of calponin.

Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction to occur and regulation by interaction with calcium-calmodulin. However, several investigators have been unable to find evidence of in vivo phosphorylation of calponin, and the affinity between calponin and calmodulin is not high enough to suggest that this interaction is biologically significant. In this paper, we present an alternative method of calponin regulation via calcium-caltropin and describe the calponin-caltropin complex for the first time. Caltropin, a calcium-binding protein isolated from smooth muscle, is a dimer under native conditions and interacts with calponin in a calcium-dependent fashion in the ratio of 2 mol of dimer: 1 mol of calponin. The formation of this complex can be monitored by following the fluorescence of an acrylodan label on cysteine 273 of calponin, which undergoes a 35-nm blue shift in wavelength peak from 505 to 470 nm when calponin becomes complexed with caltropin. This fluorescence change when titrated with calcium indicates that the concentration of calcium required for complex formation is approximately 10(-5) M, corresponding to the low-affinity calcium-binding sites of caltropin. This complex was further characterized by circular dichroism (CD).(ABSTRACT TRUNCATED AT 250 WORDS)

2-Naphthylamine↗
PubMed1997

3-D image reconstruction of reconstituted smooth muscle thin filaments containing calponin: visualization of interactions between F-actin and calponin.

Calponin is a putative thin filament regulatory protein of smooth muscle that inhibits actomyosin ATPase in vitro. We have used electron microscopy and three-dimensional reconstruction to elucidate the structural organization of calponin on actin and actin-tropomyosin filaments. Calponin density was clearly delineated in the reconstructions and found to occur peripherally along the long-pitch actin-helix. The main calponin mass was located over sub-domain 2 of actin, and connected axially adjacent actin monomers by binding to the "upper" and "lower" edges of sub-domains 1 of each actin. When the reconstructions were fitted to the atomic model of F-actin, calponin appeared to contact actin near the N terminus and at residues 349 to 352 close to the C terminus of sub-domain 1 on one monomer. It also touched residues 92 to 95 of sub-domain 1 on the axially neighboring actin and continued up the side of this monomer as far as residues 43 to 48 of sub-domain 2. These positions are consensus binding sites for a number of actin-associated proteins and are also near to sites of weak myosin interaction. Calponin did not appear to block strong myosin binding sites on actin. In contrast to the calponin mass which appeared monomeric in reconstructions, tropomyosin formed a continuous strand of added density along F-actin. When added to tropomyosin-containing filaments, calponin caused a shift of tropomyosin away from sub-domain 1 towards sub-domain 3 of actin, exposing strong myosin-binding sites that were previously covered by tropomyosin. This structural effect is unlike that of troponin and therefore inhibition of actomyosin ATPase by calponin and troponin cannot be strictly analogous. The location of calponin would allow it to directly compete or interact with a number of actin-binding proteins.

Actins↗
PubMed1992

Purification and characterization of calponin phosphatase from smooth muscle. Effect of dephosphorylation on calponin function.

Calponin, a thin-filament protein of smooth muscle, has been implicated in the regulation of smooth-muscle contraction, since in vitro the isolated protein inhibits the actin-activated myosin MgATPase. This inhibitory effect, and the ability of calponin to bind to actin, is lost after its phosphorylation by protein kinase C or Ca2+/calmodulin-dependent protein kinase II [Winder & Walsh (1990) J. Biol. Chem. 265, 10148-10155]. If this phosphorylation reaction is of physiological significance, there must be a protein phosphatase in smooth muscle capable of dephosphorylating calponin and restoring its inhibitory effect on the actomyosin MgATPase. We demonstrate here the presence, in chicken gizzard smooth muscle, of a single major phosphatase activity directed towards calponin. This phosphatase was purified from the soluble fraction of chicken gizzard by (NH4)2SO4 fractionation and sequential chromatography on Sephacryl S-300, DEAE-Sephacel, omega-amino-octyl-agarose and thiophosphorylated myosin 20 kDa light-chain-Sepharose columns. The purified phosphatase contained three polypeptide chains of 60, 55 and 38 kDa which were shown to be identical with the subunits of SMP-I, a smooth-muscle phosphatase capable of dephosphorylating the isolated 20 kDa light chain of myosin but not intact myosin [Pato & Adelstein (1983) J. Biol. Chem. 258, 7047-7054]. Consistent with its identity with SMP-I, calponin phosphatase was classified as a type-2A protein phosphatase. Of several potential phosphoprotein substrates examined, calponin proved to be kinetically the best, suggesting that calponin may be a physiological substrate for this phosphatase. Finally, dephosphorylation of calponin which had been phosphorylated by protein kinase C restored completely its ability to inhibit the actin-activated MgATPase of smooth-muscle myosin. These observations support the hypothesis that calponin plays a role in regulating the contractile state of smooth muscle and that this function in turn is controlled by phosphorylation-dephosphorylation.

Actins↗
PubMed1996

Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin.

Previously, we attributed the binding of F-actin to the 38-residue stretch of gizzard calponin encompassing the sequence A145-Y182 and postulated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their relative position within the actin binding domain was investigated by expressing the following fragments of mouse calponin with or without substitution or deletion of the hexapeptide V142-K147: amino acids 1-228 (CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228de1142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin including (a) expected electrophoretic mobility, (b) heat stability, (c) binding to actin, tropomyosin and calmodulin, and (d) zero-length cross-linking to actin switched by calmodulin in a calcium-dependent fashion. However, while the wild-type recombinant fragments inhibit the acto-S-1 ATPase activity to the same extent as do the parent calponin, modulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin binding capacity. The data indicate that the stretch VKYAEK is crucial for ATPase inhibition by calponin but represents only part of the actin-binding domain. These results are discussed in terms of multiple contact sites between actin and calponin.

Actins↗
PubMed1996

Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175.

alpha-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of alpha-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous alpha-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained approximately 90% of their complement of alpha-calponin, and exogenous chicken gizzard alpha-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous alpha-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca(2+)-induced contraction, with a half-maximal effect at approximately 3 microM alpha-calponin. Pretreatment of demembranated muscle strips with alpha-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of alpha-calponin was shown to be Ca(2+)-independent, since exogenous alpha-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of alpha-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of alpha-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca(2+)-induced and Ca(2+)-independent contractions of demembranated muscle strips of phosphorylated alpha-calponin and three site-specific mutants of alpha-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type alpha-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated alpha-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for alpha-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of alpha-calponin as a regulatory site of phosphorylation.

Animals↗
PubMed1992

A comparison of the effects of calponin on smooth and skeletal muscle actomyosin systems in the presence and absence of caldesmon.

Thiosphosphorylated smooth muscle myosin and skeletal muscle myosin, both of which express Ca(2+)-independent actin-activated MgATPase activity, were used to examine the functional effects of calponin and caldesmon separately and together. Separately, calponin and caldesmon inhibited the actin-activated MgATPase activities of thiophosphorylated smooth muscle myosin and skeletal muscle myosin, calponin being significantly more potent in both systems. Calponin-mediated inhibition resulted from the interaction of calponin with actin since it could be reversed by increasing the actin concentration. Caldesmon had no significant influence on the calponin-induced inhibition of the smooth muscle actomyosin ATPase, nor did calponin have a significant effect on caldesmon-induced inhibition. In the skeletal muscle system, however, caldesmon was found to override the inhibitory effect of calponin. This difference probably reflects the lower affinity of skeletal muscle actin for calponin compared with that of smooth muscle actin. Calponin inhibition of skeletal muscle actin-activated myosin MgATPase was not significantly affected by troponin/tropomyosin, suggesting that the thin filament can readily accommodate calponin in addition to the troponin complex, or that calponin may be able to displace troponin. Calponin also inhibited acto-phosphorylated smooth muscle heavy meromyosin and acto-skeletal muscle heavy meromyosin MgATPases. The most appropriate protein preparations for analysis of the regulatory effects of calponin in the actomyosin system therefore would be smooth muscle actin, tropomyosin and thiophosphorylated myosin, and for analysis of the kinetic effects of calponin on the actomyosin ATPase cycle they would be smooth muscle actin, tropomyosin and phosphorylated heavy meromyosin, due to the latter's solubility.

Actins↗
PubMed1997

Interaction of chicken gizzard smooth muscle calponin with brain microtubules.

Calponin, a major actin-, tropomyosin-, and calmodulin-binding protein in smooth muscle, interacted with tubulin, a main constituent of microtubules, in a concentration-dependent fashion in vitro. The apparent K(d) value of calponin to tubulin was calculated to be 5.2 microM with 2 mol of calponin maximally bound per 1 mol of tubulin. At low ionic strength, tubulin bound to calponin immobilized on Sepharose 4B, and the bound protein was released at about 270 mM NaCl. Chemical cross-linking experiments showed that a 1:1 molar covalent complex of calponin and tubulin was produced. The amount of calponin bound to microtubules decreased with increasing ionic strength or Ca2+ concentration. The addition of calmodulin or S100 to the mixture of calponin and microtubule proteins caused the removal of calponin from microtubules in the presence of Ca2+, but not in the presence of EGTA. Calponin-related proteins including tropomyosin, SM22, and caldesmon had little effect on the calponin binding to microtubules, whereas MAP2 inhibited the binding. Interestingly, there was little, if any, effect of mycalolide B-treated actin on the binding of calponin to microtubules. Furthermore, only about 20% of calponin-F-actin interaction was inhibited in the presence of an excess amount of tubulin (4 mol per mol of calponin), indicating that tubulin binds to calponin at a different site from that of actin. Compared with MAP2, calponin had little effect on microtubule polymerization.

Actins↗
PubMed1991

Expression of calponin in rabbit and human aortic smooth muscle cells.

Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture, calponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.

Animals↗
PubMed1993

Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.

Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide phosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM interaction was Ca(2+)-dependent: half-maximal binding of calponin to MIANS.CaM occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow fluorescence kinetic analysis demonstrated that EGTA chelation of Ca2+ from CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Calponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) x 10(6) M-1s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponin complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that calponin binds CaM with 80-fold lower affinity than myosin light-chain kinase and that calponin associates with CaM much slower than it associates with caldesmon or myosin light-chain kinase. The physiological relevance of the CaM-calponin interaction was evaluated by analysis of the effects of Ca(2+)-CaM on (i) the interaction of calponin with actin and (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca(2+)-CaM half-maximally inhibited calponin (2 microM) binding to smooth and skeletal muscle actins (9 microM) at 5.4 and 11 microM CaM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Characterization of the smooth muscle calponin and calmodulin complex.

Calponin interacts with several Ca2+ binding proteins in a Ca(2+)-dependent manner. In order to determine the possible biological relevance of these interactions in smooth muscle function, it is necessary to characterize the strength and stoichiometry of the complexes formed. The interaction between calponin and calmodulin can be monitored through an acrylodan label on a cysteine of calponin. The fluorescently labeled calponin possesses the same biological function and physical behavior in binding to calmodulin as the native calponin. This probe is very environment-sensitive and responds to the calponin-calmodulin interaction by the emission peak blue-shifting 20 nm and by the fluorescent quantum yield increasing 3.5 times at 460 nm. The stoichiometric nature of this complex has been determined using analytical ultracentrifugation and is two calmodulins to one calponin, and the interaction is Ca(2+)-sensitive with a Kd1 of < or = 0.22 microM and a Kd2 of 2.5-3.4 microM. Calmodulin is not the only protein which interacts with calponin in this manner, but rather this interaction seems to be a general feature attributable to all hydrophobic patch exposing proteins, suggesting that it may be nonspecific, occurring because of a generalized mode of interaction. Two other proteins, S-100b from bovine brain and SMCaBP-11 from smooth muscle, had stronger affinities for calponin, and in particular interaction of SMCaBP-11 with calponin may be biologically relevant. In determining the nature of calponin's interaction with these Ca2+ binding proteins, it was apparent there was no effect of Ca2+ upon calponin itself and physical studies could find no evidence that calponin interacts with calcium.

2-Naphthylamine↗
PubMed1996

Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis.

Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5 sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression.

Animals↗
PubMed1996

Acidic calponin cloned from neural cells is differentially expressed during rat brain development.

Calponin is an actin-, tropomyosin- and Ca2+ calmodulin-binding protein that inhibits in vitro the actomyosin MgATPase. Basic and acidic variants of calponin have been described to date. Although the cerebral expression of calponin remained controversial for some time, transcripts encoding acidic calponin in the adult rat brain and in cultured cerebellar cells have been reported. In the present work, we report the expression of acidic calponin mRNAs and the isolation of cDNAs encoding the full-length acidic calponin in cultured neuronal and glial cells and in adult rat brain. Sequence analysis reveals that acidic calponin in the brain is identical to that previously described in rat aortic vascular smooth muscle. In situ hybridization shows that calponin is highly expressed during ontogenesis in granule cells of the dentate gyrus of the hippocampus, in all layers of the olfactory bulb and in cerebellar granule neurons of the external and internal layers. In the adult rat brain, calponin expression decreased in these fields, but increased in choroid plexus cells. Bergmann glial cells were also labelled by a calponin probe. The reverse transcription-coupled polymerase chain reaction confirms that calponin mRNA levels are highest in the early stages of hippocampal development and that expression levels are low in adult hippocampi. The developmental expression pattern of brain acidic calponin suggests that calponin could be involved in contractile activity associated with neural cell proliferation or neuronal migration.

Acids↗
PubMed1997

Calponin distribution in human ciliary muscle and other anterior segment tissues.

PURPOSE: Calponins are a family of actin-binding proteins known to regulate aortic and tracheal smooth muscle contraction. This investigation was undertaken to assess the presence, subtype, and distribution of calponin proteins in human ciliary muscle, iris, and other anterior segment tissues as well as expression in ciliary muscle cells in vitro. METHODS: The distribution of calponin immunoreactivity was assessed in paraffin sections of human anterior segment tissue. Human ciliary muscle proteins were analyzed by polyacrylamide gel electrophoresis and Western blotting. The regulation of calponin expression was compared with alpha-sm-actin expression in preconfluent and postconfluent ciliary muscle cell cultures by immunocytochemistry. To determine total cell counts, the cultures were counter-stained with ethidium homodimer. As control specimens, expression of calponin and alpha-sm-actin also was assessed in human Tenon fibroblast cultures. RESULTS: Strong calponin immunoreactivity was present in ciliary muscle, iris dilator and sphincter muscles, and blood vessel smooth muscle. Fine immunostained strands also were observed in the scleral spur. This distribution was similar to alpha-sm-actin. Western blotting showed a single band of calponin with a molecular weight of 32 kDa. In the cultured ciliary muscle cells, calponin stained straight cable-like fibers running parallel along the long axis of the cells. Although the proportion of calponin immunoreactive cells was reduced substantially in preconfluent cultures, virtually all cells were stained in confluent primary through fourth-passage cultures. Cultured human Tenon fibroblasts did not show either calponin or alpha-sm-actin immunoreactivity. CONCLUSIONS: Calponin is expressed in human ciliary muscle, iris smooth muscles, blood vessel smooth muscle, as well as within the scleral spur. In addition, calponin is expressed by ciliary smooth muscle cells in vitro. The role of calponin in contraction of these tissues should be investigated.

Adult↗
PubMed1999

Distribution of calponin and smooth muscle myosin heavy chain in fine-needle aspiration biopsies of the breast.

The cell types that may be present in any fine-needle aspiration biopsy (FNAB) of breast include epithelial cells (EC), myoepithelial cells (MEC), bipolar stromal cells (BSC), vascular pericytes/endothelial cells (VPEC), and adipose cells (AC). The recognition of most of these benign cellular elements in aspirates of the breast is relatively straightforward, based on distinct cytomorphologic criteria. However, there is controversy regarding the recognition of MEC because BSC are often referred to as MEC by cytopathologists. It is important to identify MEC in breast aspirates, because their presence has been associated with benign epithelial proliferations. In this study we used immunocytochemical methods on archival cytology slides with antibodies specific for MEC, calponin, and smooth muscle myosin heavy chain (SMMHC), to determine the distribution of MEC in FNAB of the breast and to ascertain the distribution of MEC in in situ and invasive carcinomas. Fifteen benign FNABS of breast and corresponding tissue biopsies were obtained along with 10 malignant FNABS and corresponding excisional breast biopsies from 1989-1993. Calponin and SMMHC antibodies were used on archival alcohol-fixed Papanicolaou-stained direct smears as well as the corresponding tissue sections. The distribution and pattern of positive immunostaining with both antibodies were recorded on the benign elements and the carcinomas for both cytologic and histologic slides. Benign breast tissues demonstrated strong continuous immunostaining for calponin and SMMHC of MEC. The interlobular stromal cells as well as intralobular stromal cells showed no immunostaining with either antibody. In cytologic preparations, MEC staining with calponin and SMMHC appeared as spindle cells between epithelial cells or along the edges of the epithelial groups. The bipolar stromal cells did not stain with either antibody. The tissues with DCIS (ductal carcinoma in situ) often showed the presence of MEC with strong calponin immunostaining, but sometimes the immunostaining was discontinuous or entirely absent around markedly dilated ducts. The SMMHC antibody was invariably negative, with architectural DCIS in dilated ducts. Two cases of DCIS with prominent periductal fibrosis or inflammation were positive for calponin, but the periductal stromal cells were calponin- and SMMHC-negative. Invasive carcinoma was negative for both calponin and SMMHC, but areas of DCIS were often positive in a discontinuous pattern. In conclusion, 1) Benign cellular elements from breast tissue FNAB showed strong continuous decoration of MEC with both calponin and SMMHC. Vascular pericytes and vascular smooth muscle were positive for both antibodies, but these cells were not observed in the FNAB. Benign proliferative epithelium showed no local increase in MEC with either antibody. Bipolar stromal cells in tissue and smears did not stain for MEC antibodies. 2) BSC did not correspond morphologically to MEC, and were not decorated with calponin or SMMHC. 3) Calponin-positive MEC were commonly associated with in situ ductal lesions, although they may at times have been discontinuous or absent entirely. DCIS may be recognized in FNAB by the presence of calponin-positive MEC associated with tumor cell groups. 4) Invasive carcinomas were invariably negative for MEC with these antibodies.

Biopsy, Needle↗
PubMed1994

Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin.

Native calponin is able to bind 2 mol of calcium binding protein (CaBP) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site, and the other in the amino-terminal region of calponin. Also, the first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.

Actins↗
PubMed1999

Possible role of calponin h1 as a tumor suppressor in human uterine leiomyosarcoma.

BACKGROUND: Calponin h1, a basic actin-binding protein capable of inhibiting smooth muscle contraction, is a constitutive element of smooth muscle cells. However, in leiomyosarcoma (a type of smooth muscle neoplasm of the uterus), reduced expression of calponin h1 is observed, as we have reported previously. In this study, we sought to assess the effects (in vitro and in vivo) of increasing calponin h1 expression in leiomyosarcoma cells. METHODS: A plasmid containing a human calponin h1 complementary DNA and a bacterial neomycin-resistance gene was transfected into the human leiomyosarcoma cell lines SKN and SK-LMS-1 by electroporation. Southern blotting, reverse transcription-polymerase chain reaction analysis, western blotting, and immunohistochemistry were used to confirm DNA transfer and expression of the calponin h1 protein in neomycin-resistant clones. We characterized the morphology of calponin h1-transfected cells, and we evaluated their proliferative activity and tumorigenicity by use of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, an anchorage-independent growth assay, and a nude mouse tumorigenicity assay. RESULTS: The morphology of calponin h1-transfected cells in culture resembled that of cultured normal myometrial smooth muscle cells. With SK-LMS-1 cells, proliferation of calponin h1-transfection cells was reduced to 69% of control; with SKN cells, calponin h1 transfection reduced proliferation to 70% of control. In assays of anchorage-independent growth and in vivo tumorigenicity, both growth and tumorigenicity were statistically significantly reduced in calponin h1-transfected leiomyosarcoma cells. CONCLUSIONS: Calponin h1 may function as a tumor suppressor in leiomyosarcoma. Clinically, transfer of a calponin h1 complementary DNA into poorly differentiated leiomyosarcoma cells may be of potential therapeutic value through induction of a normal, differentiated cellular phenotype.

Animals↗
PubMed1999

Identification of the binding region of basic calponin on alpha and beta tubulins.

Calponin is a basic smooth muscle protein capable of binding to actin, calmodulin, tropomyosin, and phospholipids. We have found that the basic calponin interacted with brain tubulin under polymerized and unpolymerized conditions in vitro [Fujii, T., Hiromori, T., Hamamoto, M., and Suzuki, T. (1997) J. Biochem. 122, 344-351]. We examined the calponin-binding site on the tubulin molecule by sedimentation, limited digestion, chemical-cross linking, immunoblotting, and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometric (DE MALDI-TOF) analyses. Calponin interacts with both the alpha and beta tubulins and only slightly with the tyrosinated and acetylated form of alpha tubulin. The binding of calponin to microtubules was blocked by adding poly(L-aspartic acid) (PLAA) or MAP2. After digestion of microtubule proteins with subtilisin, the amount of calponin binding to alphabetas microtubules was reduced compared to native microtubules, but no further reduction was observed in the case of alphasbetas microtubules. The chemical cross-linked products of calponin and synthesized peptides (KDYEEVGVDSVEGE; alpha-KE) derived from the C-terminal region of alpha tubulin and (YQQYQDATADEQG; beta-YG) and (GEFEEEGEEDEA; beta-GA) from that of beta tubulin were detected by mass spectrometry. One kind of calponin-peptide complex was formed in the presence of alpha-KE or beta-YG, while five complexes (calponin:peptide = 1:1-5) were generated in the presence of beta-GA. Peptides alpha-KE and beta-GA inhibited the binding of calponin to tubulin produced by EDC in a concentration-dependent manner. These findings suggest that basic calponin interacts with both tubulin subunits and that their C-terminal regions, which also contain the binding sites of MAP2, tau, and kinesin, may be involved in calponin-binding.

Amino Acid Sequence↗