Abstract
Background
25(OH)D3 is important for the osteoblast differentiation of bone marrow mesenchymual stem cells (BMMSCs), BMMSCs can directly hydroxylate 25(OH)D3 to 1α,25(OH)2D3 to induce osteoblast differentiation. Our previous research with human BMMSCs showed that the cell membrane receptor megalin is required for the 25(OH)D3-DBP complex to enter cells and thereby to stimulate osteoblast differentiation. Furthermore, leptin was shown to upregulate 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.
Methods
The BMMSCs were isolated from rat bone marrow, qRT-PCR and western immunoblots were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2 and CMA activity. The osteoblast differentiation ability was evaluated by ALP activity, ALP staining, and calcium deposition.
Results
After rBMMSCs were exposed to a combination of leptin and 25(OH)D3, osteoblast differentiation was significantly enhanced, the expression of osteoblastogenic genes ALP, COL1A1, and RUNX2 by qRT-PCR were up-regulated, and ALP activity, ALP staining, and calcium deposition were also significantly increased. The quantity of 25(OH)D3 entering rBMMSCs was increased through increased megalin receptors, and ELISA confirmed that the synthesis of 1α,25(OH)2D3 was increased. Addition of a PI3K/AKT inhibitor reduced the synergistic effect of osteoblast differentiation of rBMMSCs induced by combination leptin with 25(OH)D3; the inhibited CMA activity was partially rescued by a PI3K/AKT inhibitor, and the expression of megalin was down-regulated. Up-regulation of megalin expression by leptin played a synergistic role in osteoblast differentiation of rBMMSCs induced by 25(OH)D3; leptin promoted the expression of megalin by inhibiting the activity of CMA activity.
Conclusions
These studies indicate that leptin enhanced 25(OH)D3 stimulation of osteoblast differentiation of rBMMSCs by inhibiting CMA activity to increase megalin expression, and that PI3K/AKT signaling pathway is at least partially involved in the regulation of CMA activity and megalin expression.