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P Cottin

Publications and source records attributed to P Cottin.

At least 19 recordsLinked to original sources
PubMed2003

Myoblast migration is prevented by a calpain-dependent accumulation of MARCKS.

Calpains, also called calcium activated neutral cysteine proteases are presently known to play pivotal roles in physiological and biological phenomena such as signal transduction, cell spreading and motility, apoptosis, regulation of cell cycle and regulation of muscle cell differentiation. Concerning this last point, calpains have been shown to play a crucial role during the earlier myogenesis. In this study we have analyzed the involvement of calpains during an important step of myogenesis: myoblast migration. Our findings show that myoblast migration was drastically reduced when the expression of micro- and m-calpain was decreased. We have also observed that MARCKS (myristoylated alanine rich C kinase substrate), a protein localized at focal adhesion sites, was significantly accumulated when the expression levels of calpains were decreased. Also, using phorbol myristate acetate, (an activator of PKC) and plasmids carrying the full-length cDNA of MARCKS or a cDNA fragment lacking the phosphorylation site domain, we demonstrated that normal myoblast migration is dependent on MARCKS phosphorylation and localization.

Animals↗
PubMed2002

Protein kinase Calpha is a calpain target in cultured embryonic muscle cells.

Previously we isolated a micro-calpain/PKCalpha complex from skeletal muscle which suggested tight interactions between the Ca2+-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKCalpha and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture. Our results show that calpains and PKCalpha are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKCalpha content in the particulate fraction and the appearance of PKMalpha fragment. These results suggest a possible calpain dependent down-regulation process of PKCalpha in our model at the time of intense fusion. In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of PKCalpha in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKCalpha, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and micro-calpains). Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCalpha.

Animals↗
PubMed2001

Evidence for a MARCKS-PKCalpha complex in skeletal muscle.

MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a protein known to cross-link actin filament and consequently, is very important in the stabilization of the cytoskeletal structure. In addition, it has been recently demonstrated that the phosphorylation rate of this protein changes during myogenesis and that this protein is implicated in fusion events. For a better understanding of the biological function of MARCKS during myogenesis, we have undertaken to identify and purify this protein from rabbit skeletal muscle. Three chromatographic steps including an affinity calmodulin-agarose column were performed. The existence of a complex between the two proteins was confirmed by non-denaturing gel electrophoresis and immunoprecipitation. Two complexes were isolated which present an apparent molecular weight of about 600 kDa. Such interactions suggest that MARCKS is either a very good PKCalpha substrate and/or a regulator of PKC activity. These results are supported by previous studies showing preferential interactions and co-localization of PKC isozyme and MARCKS at focal adhesion sites. This is the first time that MARCKS has been purified from skeletal muscle and our data are consistent with a major role of this actin- and calmodulin-binding protein in cytoskeletal rearrangement or other functions mediated by PKalpha. Our results provide evidence for a tight and specific association of MARCKS and PKCalpha (a major conventional PKC isozyme in skeletal muscle) as indicated by the co-purification of the two proteins.

Animals↗
PubMed2001

Autolysis of mu- and m-calpain from bovine skeletal muscle.

The rate of autolysis of mu- and m-calpain from bovine skeletal muscle was measured by using densitometry of SDS polyacrylamide gels and determining the rate of disappearance of the 28 and 80 kDa subunits of the native, unautolyzed calpain molecules. Rate of autolysis of both the 28 and 80 kDa subunits of mu-calpain decreased when mu-calpain concentration decreased and when beta-casein, a good substrate for the calpains, was present. Hence, autolysis of both mu-calpain subunits is an intermolecular process at pH 7.5, 0 or 25.0 degrees C, and low ionic strength. The 78 kDa subunit formed in the first step of autolysis of m-calpain was not resolved from the 80 kDa subunit of the native, unautolyzed m-calpain by our densitometer, so autolysis of m-calpain was measured by determining rate of disappearance of the 28 kDa subunit and the 78/80 kDa complex. At Ca2+ concentrations of 1000 microM or higher, neither the m-calpain concentration nor the presence of beta-casein affected the rate of autolysis of m-calpain. Hence, m-calpain autolysis is intramolecular at Ca2+ concentrations of 1000 microM or higher and pH 7.5. At Ca2+ concentrations of 350 microM or less, the rate of m-calpain autolysis decreased with decreasing m-calpain concentration and in the presence of beta-casein. Thus, m-calpain autolysis is an intermolecular process at Ca2+ concentrations of 350 microM or less. If calpain autolysis is an intermolecular process, autolysis of a membrane-bound calpain would require selective participation of a second, cytosolic calpain, making it an inefficient process. By incubating the calpains at Ca2+ concentrations below those required for half-maximal activity, it is possible to show that unautolyzed calpains degrade a beta-casein substrate, proving that unautolyzed calpains are active proteases.

Animals↗
PubMed2000

Calpain-PKC inter-relations in mouse hippocampus: a biochemical approach.

In previous studies, we isolated and identified a mu-calpain/PKCalpha complex from rabbit skeletal muscle. Here, we have used specific purification procedures in order to study the interactions between mu-calpain and PKC in mouse hippocampus, a brain structure implicated in memory processes. We observed that mu-calpain and conventional PKCs (alpha, betaII and gamma) are co-eluted after anion exchange chromatography. In contrast to our previous results obtained on skeletal muscle, mu-calpain and PKC isoenzymes were dissociated after gel filtration chromatography. Furthermore, mu-calpain induced the proteolytic conversion of PKCalpha, betaII, and gamma into PKMalpha, betaII, and gamma with a preferential hydrolysis of PKCgamma, a specific isoenzyme of the nervous system. Although the mu-calpain/PKC interactions in the hippocampus are quite different from skeletal muscle, our results however, point out the functional importance of these inter-relations. Moreover, as PKCgamma has been involved in the biochemical events underlying learning and memory, the preferential relationship between mu-calpain and PKCgamma promotes the importance of the role that mu-calpain could play in the cellular mechanisms of memory formation.

Animals↗
PubMed2000

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse.

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.

Animals↗
PubMed1999

Hydrostatic pressure and calcium-induced dissociation of calpains.

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.

Calcium↗
PubMed1999

Degradation of protein kinase Malpha by mu-calpain in a mu-calpain-protein kinase Calpha complex.

In previous studies, we isolated and identified a mu-calpain-PKCalpha complex from rabbit skeletal muscle. At the same time we pointed out that an association between mu-calpain and PKCalpha could occur at the level of the plasma membrane of muscle cells, and that PKCalpha could thus be considered as a potential mu-calpain substrate. In the present study, using the mu-calpain-PKCalpha complex as a model, we report that mu-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 microM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed mu-calpain forms and PKCalpha hydrolysis which leads to the formation of PKMalpha; (2) in certain experimental conditions, autolyzed mu-calpain forms are able to hydrolyze PKMalpha independently of the presence of diacylglycerol.

Animals↗
PubMed1999

Potential m-calpain substrates during myoblast fusion.

Many studies have demonstrated that m-calpain was implicated in cell membrane reorganization-related phenomena during fusion via a regulation by calpastatin, the specific Ca2+-dependent proteolytic inhibitor. However, the real biological role of this protease is unclear because many targeted proteins are still unknown. Using different digestion experiments we have demonstrated that desmin, vimentin, talin, and fibronectin represent very good substrates for this proteinase capable of cleaving them in fragments which are immediately degraded by other enzymatic systems. Concerning intermediate filaments, we showed that during the phenomenon of fusion, the amount of desmin was significantly reduced while the concentration of vimentin presented a steady level. On the other hand, we have conducted biological assays on cultured myoblasts supplemented by exogenous factors such as calpain inhibitors or antisense oligonucleotides capable of stimulating or inhibiting m-calpain activity. The effect of such factors on fusion and concomitantly on the targeted substrates was analyzed and quantified. When m-calpain activity and myoblast fusion were prevented by addition of calpain inhibitors entering the cells, the amounts of desmin, talin, and fibronectin were increased, whereas the amount of vimentin was unchanged. Using antisense strategy, similar results were obtained. In addition, when the phenomenon of fusion was enhanced by preventing calpastatin synthesis, the amounts of desmin, talin, and fibronectin were significantly reduced. Taken together, these results support the hypothesis that m-calpain is involved in myoblast fusion by cleaving certain proteins identified here. This cleavage could modify membrane and cytoskeleton organization for the myoblasts to fuse.

Animals↗
PubMed1999

Pressure effects on proteolysis catalysed by calpain.

The effects of pressure on mu and m-calpain stability and specific activity have been examined. Activity and stability of these neutral calcium-dependent heterodimeric proteinases were studied using an in-house built bioreactor allowing on-line spectrophotometric monitoring with retention of pressure. Both isozymes were founded to be rather baro-sensitive with t1/2 at 1500 bar of 6 min and 11 min for mu and m-calpain respectively. Activity measurements under pressure showed a biphasic behavior for both proteinases with a slight activation for pressure up to 500 bar and 750 bar for m and mu-calpain respectively. Activation volume changes indicated that the proteolytic reaction was alternatively favored and disfavored by pressure due to catalytic step activation associated with enzyme-substrate binding step being continuously inhibited by pressure. Furthermore, autoproteolysis of calpain, a calcium dependent phenomenon was inhibited by application of pressure indicating that pressure inhibition of proteolytic activity could also be due to Ca2(+)-binding decrease under pressure. Implication of these results with catalytic mechanism of these heterodimeric proteinases is also discussed.

Animals↗
PubMed1999

Reduction of blood loss and transfusion requirement by aprotinin in posterior lumbar spine fusion.

UNLABELLED: Aprotinin reduces blood loss in many orthopedic procedures. In posterior lumbar spine fusion, blood loss results primarily from large vein bleeding and also occurs after the wound is closed. Seventy-two patients undergoing posterior lumbar spine fusion were randomly assigned to large-dose aprotinin therapy or placebo. All patients donated three units of packed red blood cells (RBCs) preoperatively. Postoperative blood loss was harvested from the surgical wound in patients undergoing two- and/or three-level fusion for reinfusion. The target hematocrit for RBC transfusion was 26% if tolerated. Total (intraoperative and 24 h postoperative) blood loss, transfusion requirements, and percentage of transfused patients per treatment group were significantly smaller in the aprotinin group than in the placebo group (1935 +/- 873 vs 2809 +/- 973 mL per patient [P = 0.007]; 42 vs 95 packed RBCs per group [P = 0.001]; 40% vs 81% per group [P = 0.02]). Hematological assessments showed an identically significant (a) intraoperative increase in both thrombin-antithrombin III complexes (TAT) and in activated factor XII (XIIa) and (b) decrease in activated factor VII (VIIa), indicating a similar significant effect on coagulation in patients of both groups (P = 0.9 for intergroup comparisons of postoperative VIIa, XIIa, and TAT). Intraoperative activation of fibrinolysis was significantly less pronounced in the aprotinin group than in the placebo group (P < 0.0001 for intergroup comparison of postoperative D-dimer levels). No adverse drug effects (circulatory disturbances, deep venous thrombosis, alteration of serum creatinine) were detected. Although administered intraoperatively, aprotinin treatment dramatically reduced intraoperative and 24-h postoperative blood loss and autologous transfusion requirements but did not change homologous transfusion in posterior lumbar spine fusion. IMPLICATIONS: In our study, aprotinin therapy significantly decreased autologous, but not homologous, transfusion requirements in posterior lumbar spine fusion.

Adult↗
PubMed1998

Calpastatin-modulation of m-calpain activity is required for myoblast fusion.

Previous studies have demonstrated a role for m-calpain in myoblast fusion. Moreover, the presence, in differentiated cells, of a highly specific endogenous inhibitor of calpain, calpastatin, has led to the hypothesis that a regulation of or a protection against m-calpain activity by calpastatin could also occur during the earlier stages of muscle cell differentiation. In order to verify this hypothesis, we have investigated, in myoblast culture, the appearance of calpastatin-mRNA and its corresponding protein. Our results provide evidence that calpastatin is already present at the earlier stages of myoblast differentiation and that a significant decrease of the levels of calpastatin mRNA and its protein precedes myoblast fusion. In addition, the induction of an artificial decrease in calpastatin level, via an appropriate antisense oligodeoxyribonucleotide methodology, leads to earlier and faster myoblast fusion. Together with previous studies, these results indicate that m-calpain and calpastatin are functionally involved in myoblast fusion. Our findings also demonstrate that an acute "hyperactivity" of m-calpain resulting from the decrease of calpastatin synthesis is necessary during the early stages of this step of differentiation.

Animals↗
PubMed1997

Myoblast fusion requires fibronectin degradation by exteriorized m-calpain.

We recently reported that when myoblasts fuse, m-calpain could be exteriorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particularly associated to extracellular matrix components. Knowing that the cell surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the phenomenon of fusion via fibronectin cleavage or degradation. Using different digestion experiments, we demonstrated that soluble purified fibronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fibronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a defined medium supplemented by exogenous factors capable of stimulating or inhibiting m-calpain activity. The effects of such factors on rat myoblast fusion and concomitantly on the targeted glycoprotein were analyzed and quantified. When m-calpain activity and the phenomenon of fusion were reduced (defined medium without insulin), the amount of the 220-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calpain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by approximately 67 and approximately 71%, respectively. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and more particularly associated to the extracellular matrix when muscle cells were initially treated by anti-m-calpain IgG. Taken together, these results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or degradation.

Animals↗
PubMed1996

Coordinate activation of lysosomal, Ca 2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle.

Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca 2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B + L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca 2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca 2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.

Adenosine Triphosphate↗
PubMed1996

Evidence for implication of muscle-specific calpain (p94) in myofibrillar integrity.

The expression and the putative function(s) of a specific muscle calcium-dependent protease were investigated during myogenesis using rat myoblast primary cultures as a model. We have shown that the levels of p94 mRNAs increase as a function of myoblast differentiation, with the greatest amount of these RNAs being present during the later stages (8th day after plating). After an antisense oligodeoxyribonucleotide treatment with p94, ultrastructural studies show dramatic perturbations in differentiated myotubes and during myofibrillogenesis, mainly involving myofibrillar stability and Z-line integrity. These results may be related to recent findings about the role of p94 gene mutations in limbgirdle muscular dystrophy type 2A.

Animals↗
PubMed1995

Desmin degradation and Ca(2+)-dependent proteolysis during myoblast fusion.

It has already been reported that, in vitro, intermediate filaments such as desmin and vimentin are very susceptible to proteolysis by calpains (Ca(2+)-activated cysteine proteinases). On the other hand, desmin and m-calpain are both present at the onset of myoblast fusion and throughout this phenomenon. Based on these observations, the aim of this study was to demonstrate, with cultured rat myoblasts, that the amount of desmin decreased significantly as multinucleated myotubes were formed. Using immunoblot analysis, it has been shown that the desmin concentration decreased 41% as myoblasts fuse. Moreover, under conditions which stimulate myoblast fusion, desmin concentration was reduced by 21% compared to the control culture. Under our experimental conditions, which lead to a reduced desmin level, the amount of m-calpain was increased about three-fold. These results suggested that m-calpain could be involved in myoblast fusion via desmin cleavage. This hypothesis was confirmed by the results obtained after calpeptin treatment. In the presence of this cell-penetrating inhibitor of calpains, desmin seems not to be degraded. Taking into account the observations obtained after different hydrolysis assays and as compared to those observed on cultured cells, it seems conceivable that m-calpain would be able to initiate desmin cleavage leading to the formation of proteolytic fragments which should be immediately degraded.

Animals↗