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

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.

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

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.

Berberine encapsulated nanoparticles stimulate osteoblast differentiation in vitro

Berberine has been known as a traditional component for treatment of intestinal-related diseases in Asian countries. Additionally, it possesses a variety of pharmacological properties, which were studied for treating tumor, diabetes, cardiovascular disease, hyperlipidemia, inflammation, bacterial and viral infections, cerebral ischemia trauma, and mental disease. Moreover, berberine has been known as an anti-osteoporotic agent by controlling both osteoclast (bone resorption cells) and osteoblast (bone-forming cells) functions. Beside the beneficial effects of berberine, it has some drawbacks that hindered its applications and resulted in low bioavailability. One of the most drawback characteristics of berberine is that it has poor watery solubility. To overcome these limits, nanotechnology has been used as the primary approach to deliver berberine in different nano-formulations. In this study, a novel berberine nanoparticle (nanoberberine, NBB) with good water dispersion was synthesized to enhance its bioavailability. The result showed that NBB was successfully developed in spherical shape and approximately 559 nm of mean size. Besides, in vitro release study revealed that berberine content release from NBB was 3 to 4 times higher than that from free berberine. Moreover, no cytotoxicity was observed for both NBB and berberine on osteoblast MC3T3-E1 cells at the tested concentrations. Additionally, alkaline phosphatase (ALP) activity, a marker for osteoblast differentiation process, was significantly higher in NBB compared to free berberine at the same test concentrations. This result indicated that NBB could be a potential biological agent for inducing bone formation. Overall, our data indicated that NBB could improve bioavailability, especially osteogenesis activity in vitro compared to free berberine.