Two different domains of fibronectin stimulate osteoblast differentiation by activating distinct integrins

Abstract IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3–6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

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Synergistic Stimulation of Osteoblast Differentiation of Rat Mesenchymal Stem Cells by Leptin and 25(OH)D3 is Mediated by Inhibition of Chaperone-Mediated Autophagy

10.21203/rs.3.rs-547605/v1 ◽

2021 ◽

Author(s):

Qiting He ◽

Ruixi Qin ◽

Julie Glowacki ◽

Shuanhu Zhou ◽

Jie Shi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Osteoblast Differentiation ◽

Calcium Deposition ◽

Akt Inhibitor ◽

Qrt Pcr ◽

Alp Activity ◽

Chaperone Mediated Autophagy ◽

Stimulate Osteoblast Differentiation ◽

Stimulation Of

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.

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The Effects of Receptor Activator of NF-κB Ligand-Binding Peptides on Bone Resorption and Bone Formation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.648084 ◽

2021 ◽

Vol 9 ◽

Author(s):

Fatma Rashed ◽

Shingo Kamijyo ◽

Yuri Shimizu ◽

Yuna Hirohashi ◽

Masud Khan ◽

...

Keyword(s):

Bone Resorption ◽

Cell Surface ◽

Bone Formation ◽

Ligand Binding ◽

Osteoblast Differentiation ◽

Inhibitory Effects ◽

Reverse Signaling ◽

Receptor Activator ◽

Binding Peptides ◽

Stimulate Osteoblast Differentiation

Receptor activator of NF-κB ligand (RANKL)-binding peptides inhibit bone resorption and were recently shown to activate bone formation. The stimulatory mechanism underlying bone formation associated with these peptides was explained as RANKL-reverse signaling, wherein RANKL molecules on osteoblasts work as receptors to stimulate osteoblast differentiation. However, why RANKL-binding peptides stimulate osteoblast differentiation while osteoprotegerin (OPG), which is well known to bind to RANKL, cannot activate osteoblast differentiation has remained unclear. In this mini-review, we introduce three main issues: (1) The inhibitory effects of two RANKL-binding peptides (W9 and OP3-4) on bone resorption; (2) The stimulatory effects of the RANKL-binding peptides on osteoblast differentiation; and (3) The accumulation and membrane clustering of RANKL molecules at the cell surface of osteoblasts as a potential molecular switch stimulating osteoblast differentiation by RANKL-binding peptides.

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