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Fig. 3. Calpain activity in osteoclast lysates and intact osteoclasts, and the effect of suppressing µ-calpain expression on NO-induced calpain activity in situ in osteoclasts. (A) The main active calpain in osteoclasts is µ-calpain. A zymogram is shown as a negative image (degradation is dark) with recombinant µ-calpain and m-calpain standards, and osteoclast lysate (10 µg). The zymogram was developed in Ca²⁺-EGTA buffers with 30 µM Ca²⁺ activity. Additional zymograms (not shown) were performed under the same conditions (30 µM Ca²⁺) or at 100 µM Ca²⁺ with lysates from osteoclasts treated with NO or cGMP. These studies showed no consistent differences between lysates from stimulated and unstimulated cells. m-calpain activity was not detected even when zymograms were developed at 100 µM Ca²⁺. (B) Only small amounts of processed µ-calpain or of talin (a calpain substrate) accumulate in cGMP-activated osteoclasts, although osteoclast lysates degrade calpain substrates at constantly elevated Ca²⁺ levels. (1) Western blot of talin (10% SDS-PAGE). A minor amount of degradation fragments accumulated in osteoclasts activated by 8-pCPT-cGMP for 1 hour, compared with cells treated with the calpain inhibitor Calpeptin (another lane from the same gel). Several other conditions were tested; the difference shown is the largest seen. Talin links actin to the integrin complex and is an established target for partial proteolysis by calpain. (2) Western blot of µ-calpain (10% SDS-PAGE). Osteoclast lysates showed small amounts of partially cleaved enzyme after cGMP activation. The large subunit of µ-calpain (80 kD) did not vary measurably after treating cells with several NO or cGMP activators and inhibitors (not shown), although partially cleaved enzyme (70 kD) was increased slightly at treatment with NO donor or cGMP activating analog (compare lower bands). Osteoclast lysates (30 µg) were prepared from osteoclasts treated 1 hour with 100 µM of the cGMP activator 8-pCPT-cGMP or 50 µM of the inhibitor Rp-cGMPS. (3) MAP2 degradation by osteoclast calpain in vitro (6% SDS-PAGE; silver stain). MAP2 is a structural protein (280 kDa) that is not expressed in osteoclasts and is a sensitive and well-studied calpain substrate. (Top panel) Comparisons show 2 µg of untreated MAP2, 3 µg of osteoclast lysate (Lysate), and MAP2 incubated for 15 minutes and 1 hour in buffer containing 100 µM Ca²⁺ with 3 µg of osteoclast lysate. Lysate was made from osteoclasts pre-treated 30 minutes with 8-pCPT-cGMP. (Bottom panel) Results with lysates from osteoclasts pre-treated with 10 µg Calpeptin. (C) siRNA inhibition of µ-calpain. Five days after transfection of siRNA targeting µ-calpain, the protein was reduced 85-90% as determined by western blotting. Micrograph shows efficiency of siRNA uptake was >95% (fluorescence microscopy of Cy3-labeled siRNA). Nuclei were labeled with Hoechst dye (blue) to demonstrate that very few cells were without siRNA uptake. (D) Specificity of intra-osteoclastic proteolysis for µ-calpain. The graph shows average BOC fluorescence of 20 cells (mean ± s.e.m.) normalized to unstimulated controls, for osteoclasts transfected with either non-coding siRNA or siRNA targeting µ-calpain, and treated or not with 200 µM of the NO-donor SNP for 10 minutes. Note that the NO donor fails to stimulate significant degradation of the fluorescent calpain substrate in cells transfected with the siRNA targeting µ-calpain. The reduction in NO-dependent calpain activity was comparable to the reduction in µ-calpain protein. Photomicrographs show sample fields of cells transfected with non-coding siRNA (top panels) or µ-calpain-targeting siRNA (bottom panels) without (left) or with (right) treatment with SNP. Note that the NO donor causes significant degradation of the fluorescent calpain substrate only in the control cells (top right panel).

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