IADR Abstract Archives
An actin binding protein PPP1r18 regulates actin ring in osteoclasts through Protein phosphatase binding
Objectives: Bone metabolic disorders like osteoporosis or periodontitis will be taken in case of accentuation of osteoclast activity. Thus, to understand how osteoclasts resorb bone is important and will expand therapeutic approach to bone metabolic disorders, fractures and surgery for implantation, orthodontic treatment, and so on. Osteoclasts are differentiated from hematopoietic stem cells, attached to bone matrix, and then resorb bone matrix. A tyrosine kinase c-Src deficient mice show osteopetrosis because osteoclasts are disable to attachment to bone matrix and hardly resorb bone matrix. c-Src organizes actin accumulation and subsequent actin ring, a unique actin structure in osteoclasts important in attachment. However molecular mechanisms how c-Src regulates actin ring formation in osteoclasts are not fully understood. To reveal the molecular mechanisms how c-Src regulates actin ring formation in osteoclasts, we performed mass spectrum analysis.
Methods: We overexpressed constitutively activated c-Src for actin accumulation. Cells were lysed and analyzed by mass spectrum analysis after immunoprecipitation of c-Src to identify c-Src binding protein. Osteoclasts were differentiated from mouse spleen cells. Expression and localization of PPP1r18 was examined by western blotting analysis and immunofluorescence.
Results: PPP1r18 was identified as c-Src binding protein by mass spectrum analysis. PPP1r18 was expressed and localized in actin ring with c-Src of osteoclasts. Moreover localization of PPP1r18 in c-Src deficient osteoclasts were changed from general cytosol to actin ring constructed by c-Src overexpression with adenovirus system. These results suggest that PPP1r18 bind to c-Src and involved in actin ring formation. To reveal the role of PPP1r18 in osteoclasts, we overexpressed PPP1r18 in osteoclasts. Overexpression of PPP1r18 inhibited maturation, actin ring formation and bone resorption. However, mutation of protein phosphatase 1 (PP1) binding domain of PPP1r18 did not inhibit maturation, actin ring formation and bone resorption.
Conclusions: These results suggest that PPP1r18 negatively regulates actin ring formation and bone resorption through PP1 binding.
Methods: We overexpressed constitutively activated c-Src for actin accumulation. Cells were lysed and analyzed by mass spectrum analysis after immunoprecipitation of c-Src to identify c-Src binding protein. Osteoclasts were differentiated from mouse spleen cells. Expression and localization of PPP1r18 was examined by western blotting analysis and immunofluorescence.
Results: PPP1r18 was identified as c-Src binding protein by mass spectrum analysis. PPP1r18 was expressed and localized in actin ring with c-Src of osteoclasts. Moreover localization of PPP1r18 in c-Src deficient osteoclasts were changed from general cytosol to actin ring constructed by c-Src overexpression with adenovirus system. These results suggest that PPP1r18 bind to c-Src and involved in actin ring formation. To reveal the role of PPP1r18 in osteoclasts, we overexpressed PPP1r18 in osteoclasts. Overexpression of PPP1r18 inhibited maturation, actin ring formation and bone resorption. However, mutation of protein phosphatase 1 (PP1) binding domain of PPP1r18 did not inhibit maturation, actin ring formation and bone resorption.
Conclusions: These results suggest that PPP1r18 negatively regulates actin ring formation and bone resorption through PP1 binding.