Anti-CAPN1 (aa 468-481) polyclonal antibody

In skeletal remodeling, osteoclasts degrade bone, detach and move to new locations. Mechanical stretch and estrogen regulate osteoclast motility via nitric oxide ( NO). We have found previously that NO stimulates guanylyl cyclase, activating the cGMP-dependent protein kinase 1 (PKG1), reversibly terminating osteoclast matrix degradation and attachment, and initiating motility. The PKG1 substrate vasodilator-stimulated protein (VASP), a membrane-attachment-related protein found in complexes with the integrin alpha v beta 3 in adherent osteoclasts, was also required for motility. Here, we studied downstream mechanisms by which the NO-dependent pathway mediates osteoclast relocation. We found that NO-stimulated motility is dependent on activation of the Ca2+-activated proteinase mu-calpain. RNA interference (RNAi) showed that NO-dependent activation of mu-calpain also requires PKG1 and VASP. Inhibition of Src kinases, which are involved in the regulation of adhesion complexes, also abolished NO-stimulated calpain activity. Pharmacological inhibition and RNAi showed that calpain activation in this process is mediated by the inositol ( 1,4,5)-trisphosphate receptor 1 [Ins( 1,4,5) P(3)R1] Ca2+ channel. We conclude that NO-induced motility in osteoclasts requires regulated Ca2+ release, which activates mu-calpain. This occurs via the Ins( 1,4,5) P(3)R1.

Amyloid-peptides (A beta), which can aggregate oligomers or fibrils into neurons, play a critical role in Alzheimer's disease (AD). Calpain 1 (CAPN1) is one of the Ca2+ regulated proteases, involved in cell death, apoptosis, and motility. As an important signal transduction pathway, it has been reported to be closely related to muscle atrophy, myoblast fusion, diabetes mellitus, and neurodegenerative diseases, including Alzheimer's disease. Our previous study investigated the effect of CAPN1 RNAi on neurotoxicity and apoptosis in primary neurons induced by A beta 25-35. The primary neurons were cultured and separated into different groups pretreated with A beta 25-35 (10 mu M). Three groups were treated with CAPN1 shRNA, while one group was not. Along with CAPN1 shRNA, P25 (2 mu g/mL) and rapamycin (20 ng/mL) were added to the culture. The results showed that CAPN1 RNAi enhanced the formation of autophagosomes in cells, as observed by confocal microscopy. Parallelly, CAPN1 RNAi enhanced the cell viability, as observed by Cell Counting Kit-8 (CCK8) assay, and decreased the apoptotic rate, as observed by flow cytometry. In addition, CAPN1 RNAi decreased the mRNA level of CAPN1 and downregulated the expression levels of CDK5, GSK3 beta, and p-tau in primary neurons induced by A beta 25-35. All these results indicated the effect of CAPN1 RNAi against A beta-induced neurotoxicity. Our results suggested that CAPN1 RNAi exhibits a neuroprotective effect on the treatment of AD and provided a pharmacological basis for its clinical treatment.