Abstract
BackgroundVitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D3 to biologically active 1α,25(OH)2D3 in BMMSCs promotes osteoblastogenesis in an autocrine/paracrine manner. Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA.MethodsBMMSCs were isolated from rat bone marrow (4-week-old male SD rats); qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit.ResultsThe combination of leptin and 25(OH)D3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor.ConclusionsOur data reveal the mechanism by which leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D’s role in skeletal health.
Highlights
The maintenance of bone mass in the human body results from the dynamic balance of bone resorption and bone formation, osteoclasts absorb calcified bone matrix, and osteoblasts synthesize new bone matrix
To test whether leptin has a synergistic effect on osteoblast differentiation of rat bone marrow mesenchymal stem cells (rBMMSCs) with 25(OH)D3, we measured mRNA expression level of osteoblast genes (ALP, Collagen type I (COL1A1), Runt-related transcription factor 2 (RUNX2), OSP, OSX), 12 h, 3, and 5 days showed leptin enhanced the protein expression of rBMMSCs megalin; c relative quantitative analysis of megalin IF staining; d 25(OH)D3 had no effect on the relative mRNA expression of megalin with or without leptin in osteoblast differentiation medium (ODM) after 8 h; e IF showed 25(OH)
The results showed that leptin or 25(OH)D3 used alone had no significant effect on osteoblast differentiation of rBMMSCs at the experimental concentrations, but osteoblast differentiation was significantly enhanced when a combination of two agents at 12 h, 3, and 5 days, synergistic effect of leptin and 25(OH)D3 was gradually increased with the extension of time
Summary
The maintenance of bone mass in the human body results from the dynamic balance of bone resorption and bone formation, osteoclasts absorb calcified bone matrix, and osteoblasts synthesize new bone matrix. Bone marrow mesenchymal stem cells (BMMSCs) have multilineage differentiation potential to osteoblasts, adipocytes, and chondrocytes in suitable culture environments, proliferate in vitro [4, 5]. BMMSCs are the precursors of osteoblasts, but the osteoblast differentiation of BMMSCs involves a series of complex mechanisms, which still need to be studied [7]. Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. we reported that leptin upregulates megalin in those cells. We tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA
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