Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis,a common disease of the elderly population. Here, unbiased kinome RNAi screening in p...Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis,a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5(Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by si RNA, genetic deletion using the Cre-lox P system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.展开更多
Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern mole...Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping（hi MAC）, which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hi MAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hi MAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3–4-day protocol,which can be adjusted to any other cell cycle stage-dependent analysis.展开更多
基金supported by grants from the“PAKT für Forschung und Innovation2010(Leibniz Age Net:signaling pathways in age-related diseases)”German Research Foundation(DFG)Tu220/14-1,DFG(No.Ci 216/2-1)+1 种基金DFG in the framework of Collaborative Research Center CRC1149“Danger Response,Disturbance Factors and Regenerative Potential after Trauma”(No.251293561—CRC 1149,INST 40/492-1 and INST 40/492-2)Open Access funding enabled and organized by Projekt DEAL。
文摘Identification of regulators of osteoblastogenesis that can be pharmacologically targeted is a major goal in combating osteoporosis,a common disease of the elderly population. Here, unbiased kinome RNAi screening in primary murine osteoblasts identified cyclin-dependent kinase 5(Cdk5) as a suppressor of osteoblast differentiation in both murine and human preosteoblastic cells. Cdk5 knockdown by si RNA, genetic deletion using the Cre-lox P system, or inhibition with the small molecule roscovitine enhanced osteoblastogenesis in vitro. Roscovitine treatment significantly enhanced bone mass by increasing osteoblastogenesis and improved fracture healing in mice. Mechanistically, downregulation of Cdk5 expression increased Erk phosphorylation, resulting in enhanced osteoblast-specific gene expression. Notably, simultaneous Cdk5 and Erk depletion abrogated the osteoblastogenesis conferred by Cdk5 depletion alone, suggesting that Cdk5 regulates osteoblast differentiation through MAPK pathway modulation. We conclude that Cdk5 is a potential therapeutic target to treat osteoporosis and improve fracture healing.
基金supported in part by the Deutsche Forschungsgemeinschaft (DFG Grant Nos. WA2627/1-1 and WA2627/5-1) of Germany
文摘Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping（hi MAC）, which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hi MAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hi MAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3–4-day protocol,which can be adjusted to any other cell cycle stage-dependent analysis.
