Mu-calpain is functionally required for alpha-processing of Alzheimer's beta-amyloid precursor protein

Alzheimer's beta-amyloid precursor protein (APP) is normally processed by an unidentified alpha-secretase. A unique feature of this protease is its high sensitivity to phorbol esters, yet the mechanism involved is unclear. We have previously reported that phorbol 12,13-dibutyrate (PDBu) activates calpain, a Ca2+-dependent protease, and PDBu-induced release of APPs (secreted APP) is sensitive to calpain inhibitors, suggesting that calpain is involved in APP alpha-processing. In the present study, we found that PDBu markedly promoted the expression of both mu- and m-calpains in cultured fibroblasts. Dose-response and time course studies revealed that mu-calpain was more sensitive to PDBu than m-calpain and the temporal course of the mu-calpain change coincides better with that of APPs release. Moreover, the stimulatory effect of PDBu on mu-calpain was selectively blocked by mu-calpain-specific siRNA (small interference RNA) and the blockage was accompanied by a concomitant decrease in APPs release. In contrast, m-calpain siRNA did not affect APPs release significantly. Measurement of amyloid beta protein (Abeta) release in the mu-calpain siRNA-treated cells indicated that Abeta40 and Abeta42 levels inversely changed in relation to APPs, and the changes in Abeta42 were more prominent than in Abeta40. Together, these data suggest that calpain, particularly mu-calpain, is a potential candidate for alpha-secretase in the regulated APP alpha-processing, and that changes in this protease can affect the outcome of the overall APP processing.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.     

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.     

Effect of mu-calpain on m-calpain

The free Ca(2+) concentrations required for half-maximal proteolytic activity of m-calpain are in the range of 400-800 microM and are much higher than the 50-500 nM free Ca(2+) concentrations that exist in living cells. Consequently, a number of studies have attempted to find mechanisms that would lower the Ca(2+) concentration required for proteolytic activity of m-calpain. Although autolysis lowers the Ca(2+) concentration required for proteolytic activity of m-calpain, 90-400 microM Ca(2+) is required for a half-maximal rate of autolysis of m-calpain, even in the presence of phospholipid. It has been suggested that mu-calpain, which has a lower Ca(2+) requirement than m-calpain, might proteolyze m-calpain and reduce its Ca(2+) requirement to a level that would allow it to be active at physiological Ca(2+) concentrations. We have incubated m-calpain with mu-calpain for 60 min at a ratio of 1:50 mu-calpain:m-calpain, in the presence of 50 microM free Ca(2+); this Ca(2+) concentration is high enough for more than half-maximal activity of mu-calpain, but does not activate m-calpain. Under these conditions, mu-calpain caused no detectable proteolytic degradation of the m-calpain polypeptide and did not change the Ca(2+) concentration required for proteolytic activity of m-calpain. mu-Calpain also did not degrade the m-calpain polypeptide at 1000 microM Ca(2+), which is a Ca(2+) concentration high enough to completely activate m-calpain. It seems unlikely that mu-calpain could act as an "activator" of m-calpain in living cells. Because m-calpain rapidly degrades itself (autolyzes) at 1000 microM Ca(2+) and because the subsite specificities of mu- and m-calpain are very similar if not identical, failure of mu-calpain to rapidly degrade m-calpain at 1000 microM Ca(2+) suggests a unique role of autolysis in calpain function.     

Functional dissection of human protease mu-calpain in cell migration using RNAi

Calpains are a family of calcium-dependent cysteine proteases involved in a variety of cellular functions. Two isoforms, m-calpain and mu-calpain, have been implicated in cell migration. However, since conventional inhibitors used for the studies of the functions of these enzymes lack specificity, the individual physiological function and biochemical mechanism of these two isoforms, especially mu-calpain, are not clear. In contrast, RNA interference has the potential to allow a sequence-specific destruction of target RNA for functional assay of gene of interest. In the present study, we found that small interfering RNAs-mediated knockdown of mu-calpain expression in MCF-7 cells that do not express m-Calpain led to a reduction of cell migration. This isoform-specific function of mu-calpain was further confirmed by the rescue experiment as overexpression of mu-calpain but not m-calpain could restore the cell migration rate. Knockdown of mu-calpain also altered cell morphology with increased filopodial projections and a highly elongated tail that seemed to prevent cell spreading and migration with reduced rear detachment ability. Furthermore, knockdown of mu-calpain decreased the proteolytic products of filamin and talin, which were specifically rescued by overexpression of mu-calpain but not m-calpain, suggesting that their proteolysis could be one of the key mechanisms by which mu-calpain regulates cell migration.     

Studies on Calpain Expression during Differentiation of Rat Satellite Cells in Primary Cultures in the Presence of Heparin or a Mimic Compound

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.     

The effect of early posthatch starvation on calpain mRNA levels

The calpain system is a family of calcium activated proteases that degrade myofibrillar protein. Male broiler chickens (Ross) were provided a standard starter diet top-dressed with Oasis((R)) nutritional supplement (fed; Novus International, St. Louis, MO, USA), or they were not provided any feed (starved) for the first 3 days posthatch. Subsequently, the standard starter diet was provided to all chickens between 3 and 7 days posthatch. RNA was extracted from the Pectoralis thoracicus, and skeletal muscle-specific n-calpain-1 (p94) calpain, mu-calpain, and m-calpain expression was evaluated using quantitative Northern analysis. Early posthatch starvation did not (P>0.05) affect calpain mRNA levels on each day examined. Similarly, there were no (P>0.05) changes in mu-calpain or m-calpain mRNA levels between 0 and 7 days posthatch in fed birds. However, p94 calpain mRNA levels were significantly (P<0.05) lower at 7 days posthatch compared to 0 or 2 days posthatch. Therefore, in the early posthatch chicken, it appears that the calpain system may not be affected by the presence of oral nutrition, and that there is an age-related downregulation of p94 calpain mRNA expression.     

Disruption of the mouse mu-calpain gene reveals an essential role in platelet function

Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu-calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m-calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu-calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu-calpain-deficient mice are viable and fertile. The complete deficiency of mu-calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alphaIIbbeta3 integrin, talin, and ABP-280 (filamin). However, mu-calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alphaIIbbeta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu-calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.     

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.     

CD147 overexpression on synoviocytes in rheumatoid arthritis enhances matrix metalloproteinase production and invasiveness of synoviocytes

Macrophage-like synoviocytes and fibroblast-like synoviocytes (FLS) are known as the most active cells of rheumatoid arthritis (RA) and are close to the articular cartilage in a position enabling them to invade the cartilage. Macrophage-like synoviocytes and FLS expression of matrix metalloproteinases (MMPs) and their interaction has aroused great interest. The present article studied the expression of CD147, also called extracellular matrix metalloproteinase inducer, on monocytes/macrophages and FLS from RA patients and its potential role in enhancing MMPs and the invasiveness of synoviocytes. Expression of CD147 on FLS derived from RA patients and from osteoarthritis patients, and expression of CD147 on monocytes/macrophages from rheumatic synovial fluid and healthy peripheral blood were analyzed by flow cytometry. The levels of CD147, MMP-2 and MMP-9 mRNA in FLS were detected by RT-PCR. The role of CD147 in MMP production and the cells' invasiveness in vitro were studied by the co-culture of FLS with the human THP-1 cell line or monocytes/macrophages, by gel zymography and by invasion assay. The results showed that the expression of CD147 was higher on RA FLS than on osteoarthritis FLS and was higher on monocytes/macrophages from rheumatic synovial fluid than on monocytes/macrophages from healthy peripheral blood. RT-PCR showed that the expressions of CD147, MMP-2 and MMP-9 mRNA was higher in RA FLS than in osteoarthritis FLS. A significantly elevated secretion and activation of MMP-2 and MMP-9 were observed in RA FLS co-cultured with differentiated THP-1 cells or RA synovial monocytes/macrophages, compared with those co-cultured with undifferentiated THP-1 cells or healthy control peripheral blood monocytes. Invasion assays showed an increased number of invading cells in the co-cultured RA FLS with differentiated THP-1 cells or RA synovial monocytes/macrophages. CD147 antagonistic peptide inhibited the MMP production and the invasive potential. Our studies demonstrated that the CD147 overexpression on monocytes/macrophages and FLS in RA patients may be responsible for the enhanced MMP secretion and activation and for the invasiveness of synoviocytes. These findings suggest that CD147 may be one of the important factors in progressive joint destruction of RA and that CD147 may be a potential therapeutic target in RA treatment.     

Calpain is required for MMP-2 and u-PA expression in SV40 large T-antigen-immortalized cells

The absence of both mu- and m-calpain activity, caused by disruption of the capn4 gene in mice, retarded migration, and disrupted the cytoskeleton, both in primary capn4(-/-) embryonic fibroblasts (mEF) and in capn4(-/-) mEF immortalized with SV40 large T-antigen (TAg). These results are thought to reflect the role of calpain in integrin signaling to the cytoskeleton. The integrins are also involved, together with matrix metalloproteinases (MMP) and plasminogen activators (PA), in cellular invasion. This study therefore aimed to establish whether links exist between the calpain, MMP, and PA systems, using both primary and TAg-immortalized capn4(+/+) and capn4(-/-) embryonic fibroblasts. Both Matrigel invasion, and expression of MMP-2 and u-PA activities, correlated with calpain expression in TAg-containing cells, but not in primary cells. MMP-2 mRNA synthesis also correlated with calpain expression in the presence of TAg, but u-PA mRNA synthesis was not so correlated. The results suggest that calpain acquires new regulatory roles in the presence of TAg. Calpain is also required for v-Src-mediated transformation. It appears that calpain may have previously unsuspected roles in oncogenic transformation.     

Involvement of mu- and m-calpains and protein kinase C isoforms in L8 myoblast differentiation

The objectives were to investigate the roles of different calpains and protein kinase C (PKC) isoforms in muscle differentiation. Concentrations of mu- and m-calpain increased significantly whereas PKCalpha and delta declined significantly during L8 myoblast differentiation. Both mu-calpain and m-calpain antisense oligonucleotides inhibited myotube formation and creatine kinase activity during L8 myoblast differentiation. These results implied that both mu- and m-calpain were involved in L8 myoblast differentiation. To investigate the involvement of calpain in regulation of PKC concentrations, mu-calpain antisense oligonucleotides were added to L8 myoblasts. PKCalpha remained unchanged and PKCdelta declined. By adding m-calpain antisense oligonucleotides instead, PKCalpha level remained unchanged and PKCdelta concentrations increased significantly during differentiation. These results suggest that PKCalpha, but not PKCdelta, is the substrate for mu-calpain and PKCalpha and delta are the substrates for the m-calpain. In addition, more phosphorylated myogenin was found in day 2 antisense oligonucleotides treated L8 cells. It is concluded that the decline of PKCalpha mediated by m- and mu-calpain is essential for L8 myoblast differentiation. The decline of PKC during myoblast differentiation may cause hypo-phosphorylation of myogenin, which in turn activates muscle-specific genes during myogenesis.     

Roles of mu-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system

The goal of this work was to characterize the roles of mu-calpain in skeletal muscle protein degradation. Three approaches were developed to alter mu-calpain activity in rat myotubes. These included over-expression of antisense mu-calpain (mu-AS), dominant negative mu-calpain (mu-DN) and the antisense 30-kDa calpain subunit (30-AS). Constructs were expressed in rat L8 myotubes, and their effects on protein degradation and on concentrations of intact and/or degraded fodrin, desmin and tropomyosin were examined. An ecdysone-inducible expression system, in which we replaced a constitutively active CMV promoter with a skeletal muscle-specific alpha-actin promoter, was used to drive expression. Cell lines were evaluated by expression of the gene-of-interest following addition of ponasterone A (PA; ecdysone analog) to culture medium. Changes in calpain activity were assessed by evaluating fodrin degradation. 30-AS, which should alter both mu- and m-calpain activities, increased intact fodrin concentration. mu-DN and mu-AS reduced fodrin degradation products. mu-DN reduced total protein degradation by 7.9% (P<0.01) at 24 h and by 10.6% (P<0.01) at 48 h. mu-AS reduced total protein degradation by 6.4% at 24 h (P<0.05). 30-AS reduced total protein degradation by 13.4% (P<0.05) and 7.3% (P<0.05) following 24 and 48 h of PA administration, respectively. We assessed effects of mu-DN, mu-AS and 30-AS on concentrations of desmin and tropomyosin. Inhibition of calpains stabilized desmin, but had no effect on tropomyosin. These data indicate that fodrin and desmin are mu-calpain substrates and that mu-calpain accounts for a small proportion of total protein degradation in muscle cells. Tropomyosin is not degraded by calpain in muscle cells.     

The calpain system in human placenta

The calpain system is involved in a number of human pathologies ranging from the muscular dystrophies to Alzheimer's disease. It is important, therefore, to be able to obtain and to characterize both mu-calpain and m-calpain from human tissue. Although human mu-calpain can be conveniently obtained from either erythrocytes or platelets, no readily available source of human m-calpain has been described. Human placenta extracts contain both mu-calpain and m-calpain in nearly equal proportions and in significant quantities (3-4 mg mu-calpain and 4-5 mg m-calpain/1000 g placenta tissue). Placenta also contains calpastatin that elutes off ion-exchange columns over a wide range of KCl concentrations completely masking the mu-calpain activity eluting off these columns and even partly overlapping m-calpain elution. Placenta mu-calpain requires 50-70 microM Ca2+ and placenta m-calpain requires 450-460 microM Ca2+ for half-maximal proteolytic activity. Western analysis of washed placenta tissue shows that placenta contains both mu- and m-calpain, although some of the mu-calpain in whole placenta extracts likely originates from the erythrocytes that are abundant in the highly vascularized placenta. Placenta calpastatin could not be purified with conventional methods. The most prominent form of calpastatin in Western analyses of placenta obtained as soon as possible after birth was approximately 48-51 kDa; partly purified preparations of placenta calpastatin also contained 48-51 and 70 kDa polypeptides. Human placenta extracts likely contain two different calpastatin isoforms, a 48-51 kDa "placenta calpastatin" and a 70 kDa erythrocyte calpastatin.     

The calpastatin-derived calpain inhibitor CP1B reduces mRNA expression of matrix metalloproteinase-2 and -9 and invasion by leukemic THP-1 cells

The ubiquitous proteases mu- and m-calpain are Ca(2+)-dependent cysteine endopeptidases. Besides involvement in a variety of physio(patho)logical processes, recent studies suggest a pivotal role of calpains in differentiation of hematopoietic cells and tumor cell invasion. However, the precise actions of calpains and their endogenous inhibitor, calpastatin, in these processes are only partially understood. Here we have studied the role of the calpain/calpastatin system in the invasion of leukemic cells under basal and differentiation-stimulating conditions. To further differentiate the human leukaemic cell line THP-1 (monocytic), the cells were treated for 24 hours with the differentiation-stimulating reagents phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO). Macrophage- and granulocyte-like differentiation was confirmed by induction of vimentin expression as well as by microscopic and fluorescence-assisted cytometric analysis. Extracellular matrix (ECM) invasion of both the basal and differentiation-stimulated cells in a Matrigel assay was inhibited by pre-incubation of the cells with the specific calpain inhibitor CP1B for 24 hours. Inhibition of invasiveness correlated with decreased mRNA expression and secretion of the matrix metalloproteinases MMP-2 and MMP-9. In contrast, addition of CP1B only during the invasion process did neither influence transmigration nor MMP release. This is the first report showing that the calpain/calpastatin system mediates MMP-mRNA expression of the leukemic THP-1 cells and as a consequence their invasiveness.     

Effect of Ca2+ on binding of the calpains to calpastatin

Autolyzed mu-calpain, unautolyzed mu-calpain, autolyzed m-calpain, and unautolyzed m-calpain (mu-calpain is the micromolar Ca2+-requiring proteinase, m-calpain is the millimolar Ca2+-requiring proteinase) were passed through a calpastatin-affinity column at different free Ca2+ concentrations, and binding of the calpains to calpastatin was compared with proteolytic activity of that calpain at each Ca2+ concentration. Unautolyzed m-calpain, autolyzed m-calpain, and autolyzed mu-calpain required less Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Unautolyzed mu-calpain, however, required slightly more Ca2+ for half-maximal binding to calpastatin than for half-maximal activity. Half-maximal binding of oxidatively inactivated mu- or m-calpain to calpastatin required approximately the same Ca2+ concentrations as half-maximal binding of unautolyzed mu- or m-calpain, respectively, to calpastatin. Binding of unautolyzed m-calpain and autolyzed mu-calpain to calpastatin occurred over a wide range of Ca2+ concentrations, and it seems likely that two or more Ca2+-binding sites with different Ca2+-binding constants are involved in binding of the calpains to calpastatin. Proteolytic activity occurs at different Ca2+ concentrations than calpastatin binding, suggesting a second set of Ca2+-binding sites associated with proteolytic activity. Third and fourth sets of Ca2+-binding sites may be involved in autolysis and in binding to phosphatidylinositol or cell membranes; these four Ca2+-dependent properties of the calpains may require the eight potential Ca2+-binding sites that amino acid sequences predict are present in the calpain molecules.     

Calpain is required for normal osteoclast function and is down-regulated by calcitonin

Osteoclast motility is thought to depend on rapid podosome assembly and disassembly. Both mu-calpain and m-calpain, which promote the formation and disassembly of focal adhesions, were observed in the podosome belt of osteoclasts. Calpain inhibitors disrupted the podosome belt, blocked the constitutive cleavage of the calpain substrates filamin A, talin, and Pyk2, which are enriched in the podosome belt, induced osteoclast retraction, and reduced osteoclast motility and bone resorption. The motility and resorbing activity of mu-calpain(-/-) osteoclast-like cells were also reduced, indicating that mu-calpain is required for normal osteoclast activity. Histomorphometric analysis of tibias from mu-calpain(-/-) mice revealed increased osteoclast numbers and decreased trabecular bone volume that was apparent at 10 weeks but not at 5 weeks of age. In vitro studies suggested that the increased osteoclast number in the mu-calpain(-/-) bones resulted from increased osteoclast survival, not increased osteoclast formation. Calcitonin disrupted the podosome ring, induced osteoclast retraction, and reduced osteoclast motility and bone resorption in a manner similar to the effects of calpain inhibitors and had no further effect on these parameters when added to osteoclasts pretreated with calpain inhibitors. Calcitonin inhibited the constitutive cleavage of a fluorogenic calpain substrate and transiently blocked the constitutive cleavage of filamin A, talin, and Pyk2 by a protein kinase C-dependent mechanism, demonstrating that calcitonin induces the inhibition of calpain in osteoclasts. These results indicate that calpain activity is required for normal osteoclast activity and suggest that calcitonin inhibits osteoclast bone resorbing activity in part by down-regulating calpain activity.     

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.     

Calpain specificity and expression in chicken tissues

We have compared ubiquitous calpains in chicken (Gallus gallus), turkey (Meleagris gallopavo) and mammals. In chicken, we studied their distribution in different tissues. The calpain activity was determined by casein zymography, a technique avoiding any prior sample purification, thus limiting any autolysis and denaturation reactions. Our results show that two ubiquitous calpains are present in chicken: (1) a mu-calpain having a greater calcium sensitivity and a lower electrophoretic mobility than the mammalian one, (2) a mu/m-calpain, named like this by Sorimachi et al. [Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S., Suzuki, K., 1995. Identification of a third ubiquitous calpain species-chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta, 1261, 381-93.], having a calcium sensitivity intermediate between that of the two mammalian mu-calpain and the m-calpain. Tissue distribution of the two chicken isozymes vary and mu/m-calpain predominates, whereas mu-calpain levels are very low in some tissues, unlike in mammalian tissues. The characteristics of mu/m-calpain and its preponderance in all organs suggest that it may play a different role in chicken than in mammals.