Synergistic stimulation of osteoblast differentiation of rat mesenchymal stem cells by leptin and 25(OH)D3 is mediated by inhibition of chaperone-mediated autophagy

Abstract

Background Vitamin 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. Methods BMMSCs 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. Results The 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. Conclusions Our 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.

Role of Chaperone-Mediated Autophagy in Ageing Biology and Rejuvenation of Stem Cells

“…Accordingly, CMA and VANGL2 may represent promising targets in age-related bone loss and the development of a therapeutic strategy that acts on this axis could be useful for the treatment of osteoporosis and other diseases related to bone loss, as well as for metabolic diseases associated with bone/ marrow fat imbalance, beyond that for the various applications of regenerative medicine. At variance with the previously presented scenario, in the same year, He et al (2021) studied the differentiation capacity of rat MSCs demonstrating an increase in OB differentiation following CMA inhibition (Figure 5C). This result is inconsistent with the study of Gong et al (2021).…”

“…At variance with the previously presented scenario, in the same year, He et al (2021) studied the differentiation capacity of rat MSCs demonstrating an increase in OB differentiation following CMA inhibition ( Figure 5C ). This result is inconsistent with the study of Gong et al (2021) .…”

“…This result is inconsistent with the study of Gong et al (2021) . However, while the study by Gong et al was conducted on CMA independent of the master lysosome biogenesis regulator TFEB and of the mTORC2/PHLPP1/Akt axis that, under stress conditions such as serum starvation, induce LAMP2A assembly and consequent CMA activity ( Arias et al, 2015 ), the effects described by He et al (2021) involved treatment with Leptin, a protein known to be an inhibitor of PI3K/AKT-dependent CMA. It is also known that the MSC OB differentiation is induced by vitamin D whose active metabolite is 1α,25(OH)2D3 ( Lips and Van Schoor, 2011 ; D’ippolito et al, 2002 ).…”

“…In MSCs, megalin is among the main receptors of 1α,25(OH)2D3 ( Gao et al, 2019 ). He et al (2021) proved that leptin treatment stimulates bone differentiation by increasing megalin levels through inhibition of CMA. In particular, leptin decreased LAMP2A and Hsc70 levels and increased AKT phosphorylation.…”

Autophagy: An important target for natural products in the treatment of bone metabolic diseases

“…Downregulation of LAMP2A in BMSCs is also closely associated with impaired osteogenic differentiation during aging (Gong et al, 2021). Furthermore, leptin affects CMA-mediated expression of megalin (lipoprotein-related protein 2, a key receptor for 25(OH)D3 entry into BMSCs) by inhibiting the levels of LAMP2A and HSC70, thereby increasing the utilization of 25(OH)D3 by BMSCs and making 25(OH)D3-induced enhanced osteogenic differentiation potential of BMSCs, which also suggests that CMA is essential for the role of vitamin D in bone health (He et al, 2021).…”