Deficiency in the phosphatase PHLPP1 suppresses osteoclast-mediated bone resorption and enhances bone formation in mice

Abstract

Edited by Alex Toker Enhanced osteoclast-mediated bone resorption and diminished formation may promote bone loss. Pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (Phlpp1) regulates protein kinase C (PKC) and other proteins in the control of bone mass. Germline Phlpp1 deficiency reduces bone volume, but the mechanisms remain unknown. Here, we found that conditional Phlpp1 deletion in murine osteoclasts increases their numbers, but also enhances bone mass. Despite elevating osteoclasts, Phlpp1 deficiency did not increase serum markers of bone resorption, but elevated serum markers of bone formation. These results suggest that Phlpp1 suppresses osteoclast formation and production of paracrine factors controlling osteoblast activity. Phlpp1 deficiency elevated osteoclast numbers and size in ex vivo osteoclastogenesis assays, accompanied by enhanced expression of proto-oncogene C-Fms (C-Fms) and hyper-responsiveness to macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Although Phlpp1 deficiency increased TRAP ؉ cell numbers, it suppressed actinring formation and bone resorption in these assays. We observed that Phlpp1 deficiency increases activity of PKC, a PKC isoform controlling cell polarity, and that addition of a PKC pseudosubstrate restores osteoclastogenesis and bone resorption of Phlpp1-deficient osteoclasts. Moreover, Phlpp1 deficiency increased expression of the bone-coupling factor collagen triple helix repeat-containing 1 (Cthrc1). Conditioned growth medium derived from Phlpp1-deficient osteoclasts enhanced mineralization of ex vivo osteoblast cultures, an effect that was abrogated by Cthrc1 knockdown. In summary, Phlpp1 critically regulates osteoclast numbers, and Phlpp1 deficiency enhances bone mass despite higher osteoclast numbers because it apparently disrupts PKC activity, cell polarity, and bone resorption and increases secretion of bone-forming Cthrc1.

Hdac3 deletion in myeloid progenitor cells enhances bone healing in females and limits osteoclast fusion via Pmepa1

“…In future studies, the mechanism by which Pmepa1 controls osteoclast fusion will be explored. Determining if suppression of Pmepa1 also aids in coupling to bone formation, similar to other models where osteoclastogenesis is enhanced but bone resorption is suppressed, is also of interest 68 , 69 .…”

“…On Day 14, cells were lysed in 10% bleach washed 3 times with water. Pits on bone slices were stained with toluidine blue as previously described 73 . Resorption assays were performed in triplicate and repeated three times.…”

“…TRAP staining was performed as previously described 73 . Briefly, cells were fixed on cover glass with 10% neutral buffered formalin for 10 min and then washed 3 times with phosphate-buffered saline (PBS) 73 .…”

“…TRAP staining was performed as previously described 73 . Briefly, cells were fixed on cover glass with 10% neutral buffered formalin for 10 min and then washed 3 times with phosphate-buffered saline (PBS) 73 . Fixed cells were TRAP stained using the Acid Phosphatase, Leukocyte (TRAP) Kit (#387A-1KT, Sigma-Aldrich) and mounted to slides using Vectashield with DAPI (#H-1200, Vector Laboratories, Burlingame, CA) 73 .…”

“…For osteoclastogenesis experiments, three cover glasses were used per experimental condition. For each cover glass, three fields were imaged using a 10× objective 73 . Images were digitally photographed using the Zeiss AxioVert A1 and Zen software package.…”