ABSTRACT
Mechanical stress is known to alter bone mass and the loss of force stimuli leads to reduction of bone mass. However, molecules involved in this phenomenon are incompletely understood. As mechanical force would affect signaling events in cells, we focused on a calcium channel, TRPV4 regarding its role in the effects of force stimuli on calcium in osteoblasts. TRPV4 expression levels were enhanced upon differentiation of osteoblasts in culture. We found that BMP-2 treatment enhanced TRPV4 gene expression in a dose dependent manner. BMP-2 effects on TRPV4 expression were suppressed by inhibitors for transcription and new protein synthesis. In these osteoblasts, a TRPV4-selective agonist, 4α-PDD, enhanced calcium signaling and the effects of 4α-PDD were enhanced in differentiated osteoblasts compared to the control cells. Fluid flow, as a mechanical stimulation, induced intracellular calcium oscillation in wild type osteoblasts. In contrast, TRPV4 deficiency suppressed calcium oscillation significantly even when the cells were subjected to fluid flow. These data suggest that TRPV4 is involved in the flow-induced calcium signaling in osteoblasts.
Subject(s)
Calcium Signaling , Cell Differentiation , Osteoblasts/cytology , Osteoblasts/metabolism , Stress, Mechanical , TRPV Cation Channels/metabolism , Animals , Bone Morphogenetic Protein 2/pharmacology , Calcium Signaling/drug effects , Calcium Signaling/genetics , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Culture Media/pharmacology , Gene Expression Regulation/drug effects , Humans , Mice , Mice, Inbred C57BL , Osteoblasts/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rheology/drug effects , TRPV Cation Channels/deficiency , TRPV Cation Channels/geneticsABSTRACT
Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration.
Subject(s)
Gene Expression Regulation, Developmental , Profilins/physiology , Alleles , Animals , Bone and Bones/metabolism , Cartilage/metabolism , Cell Movement , Cytoskeleton/metabolism , Genotype , Mesenchymal Stem Cells/cytology , Mice , Mice, Knockout , Mice, Transgenic , NIH 3T3 Cells , Osteoblasts/cytology , Osteogenesis , Profilins/metabolism , RNA, Small Interfering/metabolism , Time Factors , Transfection , X-Ray Microtomography/methodsABSTRACT
Osteoporosis causes fractures that lead to reduction in the quality of life and it is one of the most prevalent diseases as it affects approximately 10% of the population. One of the important features of osteoporosis is osteopenia. However, its etiology is not fully elucidated. Dok-1 and Dok-2 are adaptor proteins acting downstream of protein tyrosine kinases that are mainly expressed in the cells of hematopoietic lineage. Although these proteins negatively regulate immune system, their roles in bone metabolism are not understood. Here, we analyzed the effects of Dok-1 and Dok-2 double-deficiency on bone. Dok-1/2 deficiency reduced the levels of trabecular and cortical bone mass compared to wildtype. In addition, Dok-1/2 deficiency increased periosteal perimeters and endosteal perimeters of the mid shaft of long bones. Histomorphometric analysis of the bone parameters indicated that Dok-1/2 deficiency did not significantly alter the levels of bone formation parameters including mineralizing surface/bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR). In contrast, Dok-1/2 deficiency enhanced the levels of bone resorption parameters including osteoclast number (N.Oc/BS) and osteoclast surface (Oc.S/BS). Analyses of individual osteoclastic activity indicated that Dok-1/2 deficiency enhanced pit formation. Systemically, Dok-1/2 deficiency increased the levels of urinary deoxypyridinoline (Dpyr). Search for the target point of the Dok-1/2 deficiency effects on osteoclasts identified that the mutation enhanced sensitivity of osteoclast precursors to macrophage colony-stimulating factor. These data revealed that Dok-1 and Dok-2 deficiency induces osteopenia by activation of osteoclasts.
