Abstract
The active form of vitamin D, 1α,25-(OH)2D3, not only promotes intestinal calcium absorption, but also regulates the formation of osteoclasts (OCs) and their capacity for bone mineral dissolution. Gal-3 is a newly discovered bone metabolic regulator involved in the proliferation, differentiation, and apoptosis of various cells. However, the role of galectin-3 (gal-3) in OC formation and the regulatory effects of 1α,25-(OH)2D3 have yet to be explored. To confirm whether gal-3 contributes to the regulatory effects of 1α,25-(OH)2D3 on osteoclastogenesis, osteoclast precursors (OCPs) were induced by macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). TRAP staining and bone resorption analyses were used to verify the formation and activation of OCs. qPCR, Western blotting, co-immunoprecipitation, and immunofluorescence assays were used to detect gene and protein expression. The regulatory effects of gal-3 in OC formation after treatment with 1α,25-(OH)2D3 were evaluated using gal-3 siRNA. The results showed that 1α,25-(OH)2D3 significantly increased gal-3 expression and inhibited OC formation and bone resorption. Expression levels of OC-related genes and proteins, matrix metalloproteinase 9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), and cathepsin K (Ctsk) were also inhibited by 1α,25-(OH)2D3. Gal-3 knockdown attenuated the inhibitory effects of 1α,25-(OH)2D3 on OC formation, activation, and gene and protein expression. In addition, gal-3 was co-localized with the vitamin D receptor (VDR). These data suggest that gal-3 contributes to the osteoclastogenesis inhibitory effect of lα,25-(OH)2D3, which is involved in bone and calcium homeostasis.
Highlights
OCs are derived from bone marrow mononuclear macrophages (BMMs) and are the only cells capable of bone resorption in the body
osteoclast precursors (OCPs), such as bone marrow cells, splenocytes, and RAW264.7 macrophages, co-cultured with stromal cells, OBs, or osteocytes in vitro can be induced into OCs by the parathyroid hormone (PTH), dexamethasone, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and 1α,25(OH)2D3, which regulate the expression of membrane-bound RANKL in OBs, stromal cells, and osteocytes [1,7]
To elucidate the effect of 1α,25-(OH)2D3 on gal-3 protein expression, 0.1, 1, and 10 nmol/L 1α,25-(OH)2D3 were added to the culture medium during OC formation induced by 25 ng/mL macrophage colony stimulating factor (M-CSF) and 50 ng/mL RANKL for 3 days. 1α,25-(OH)2D3 upregulated gal-3 protein expression in a dose-dependent manner (Figure 2)
Summary
OCs are derived from bone marrow mononuclear macrophages (BMMs) and are the only cells capable of bone resorption in the body. They degrade the bone organic and inorganic matrix and cooperate with osteoblasts (OBs) to regulate bone formation and reconstruction [1–3]. OB-lineage cells express vitamin D receptor (VDR) [14], and 1α,25-(OH)2D3 promotes OBs’ maturation and bone mineralization in vitro and in vivo via VDR and reduces the formation of unmineralized osteoids [15]. Gal-3 is a 29–35 kDa protein expressed in a variety of tissues and is a member of the β-galactosyl-binding protein family [20] It is a marker of chondrocyte and OB lineages in bone and is present in OCs and BMMs [21,22]. Our results provide insights into the mechanism underlying the regulation of osteoclastogenesis by 1α,25-(OH)2D3
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