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Background: Calcium ion (Ca²⁺) signals are required for osteoclast differentiation. Previous study showed that transient receptor potential vanilloid 5 (TRPV5) is an essential Ca²⁺ transporter in osteoclastogenesis and bone resorption. TRPV5 and TRPV6 represent two highly homologous members within the transient receptor potential (TRP) superfamily. However, the role of TRPV6 in bone metabolism is still controversial and little is known about the involvement of TRPV6 in receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Methods: In our study, gene knockout mice, RNA interference, western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, histomorphometry and measurement of serum parameters were employed to investigate the role of TRPV6 in bone homeostasis, osteoclastogenesis and bone resorption. Results: We found that TRPV6 depletion results in noticeable destruction of bone microarchitecture in TRPV6 knockout mice (TRPV6^-/-), suggesting that TRPV6 is a critical regulator in bone homeostasis. Inactivation of Trpv6 had no effect on osteoblastic bone formation. However, quantification of the TRAP staining showed a significantly increased osteoclast number and surface area in the metaphyseal area of femurs bone sections derived from TRPV6^-/- mice. In agreement with our observations from TRPV6^-/- mice, TRPV6 depletion in vitro significantly increased osteoclasts differentiation and bone resorption activity. Conclusion: Based on these results above, we can draw conclusions that TRPV6 plays an essential role in bone metabolism and is a critical regulator in osteoclasts differentiation and bone resorption.

The inhibitor effect of estrogen on osteoclasts differentiation is very important in the etiology of estrogen protecting the adult skeleton against bone loss. However, the precise molecular events underlying the effect of estrogen on osteoclasts differentiation are not known. Recent studies implicated an important role of transient receptor potential vanilloid 5 (TRPV5) in osteoclast differentiation and bone resorption. Furthermore, some studies have confirmed that estrogen is involved in the regulation of calcium ion (Ca(2+)) influx in many cells via TRPV5 channel. Therefore, we hypothesize that TRPV5 channel may be implicated in the process of estrogen-inhibited osteoclastogenesis and bone resorption. Western blot, quantitative real-time PCR, tartrate-resistant acid phosphatase (TRAP) staining, and pit formation assay were employed to investigate the role of TRPV5 in estrogen decreasing osteoclast differentiation and bone resorption. We found that the expression of TRPV5 is significantly down-regulated during estrogen deficiency-induced osteoclastogenesis. Furthermore, TRAP staining and pit formation assay showed that the depletion of TRPV5 significantly blocks the inhibitor effects of estrogen on osteoclasts differentiation and bone resorption activity. Further studies confirmed that estrogen regulates the expression of TRPV5 channel via estrogen receptor. Based on these results above, we can draw conclusion that TRPV5 may contribute to the process of estrogen-inhibited osteoclastogenesis and bone resorption activity.

Tumor necrosis factor α (TNF-α)-induced osteoclast formation have been demonstrated to play an important role in the pathogenesis of estrogen deficiency-mediated bone loss, but the exact mechanisms by which TNF-α enhanced osteoclast differentiation were not fully elucidated. The class III semaphorins members were critical to regulate bone homeostasis. Here, we identified a novel mechanism whereby TNF-α increasing Semaphorin3D expression contributes to estrogen deficiency-induced osteoporosis. In this study, we found that Semaphorin3D expression was upregulated by TNF-α during the process of RANKL-induced osteoclast differentiation. Inhibition of Semaphorin3D in pre-osteoclasts could attenuate the stimulatory effects of TNF-α on osteoclast proliferation and differentiation. Mechanistically, blocking of the Jun N-terminal kinase (JNK) signaling markedly rescued TNF-α-induced Semaphorin3D expression, suggesting that JNK signaling was involved in the regulation of Semaphorin3D expression by TNF-α. In addition, silencing of Semaphorin3D in vivo could alleviate estrogen deficiency-induced osteoporosis. Our results revealed a novel function for Semaphorin3D and suggested that increased Semaphorin3D may contribute to enhanced bone loss by increased TNF-α in estrogen deficiency-induced osteoporosis. Thus, Semaphorin3D may provide a potential therapeutic target for the treatment of estrogen-deficiency induced osteoporosis.

It is well known that glucocorticoid (GC)-induced bone loss is caused primarily by hypofunction and apoptosis of osteoblasts. However, the precise molecular events underlying the effect of GC on osteoblast apoptosis are not fully understood. Recent studies implicated an important role of E4BP4 in the regulation of osteoblast apoptosis and differentiation. Furthermore, E4BP4 is a GC-regulated gene required for GC-induced apoptosis in many cells. Therefore, we hypothesize that E4BP4 may be implicated in the process of GC-induced osteoblast apoptosis. Western blot, reverse-transcription-PCR, flow cytometry, and Hoechst 33258 staining were employed to investigate the role of E4BP4 in dexamethasone (DEX)-induced osteoblast apoptosis. We found that the expression of E4BP4 is significantly up-regulated in osteoblasts exposed to DEX. Furthermore, the depletion of E4BP4 significantly decreased DEX-induced osteoblast apoptosis. In addition, E4BP4 plays a crucial role in GC-evoked apoptosis of osteoblasts by enabling induction of Bim. On the basis of these results above, we can draw the conclusion that E4BP4 may contribute to the process of DEX-induced osteoblast apoptosis.

Objective. Nucleus pulposus (NP) and annulus fibrosus (AF) are two main components of intervertebral disc (IVD). We aimed to figure out whether NP and AF also contain stem cells and whether these stem cells share common properties with chondrocytes and/or fibroblasts in their phenotypes or whether they are completely different types of cells with different characteristics. Design. The disk cells were isolated from AF and NP tissues of the same lumbar spine of the rabbits. The properties of these disk cells were characterized by their morphology, population doubling time (PDT), stem cell marker expression, and multidifferentiation potential using tissue culture techniques, immunocytochemistry, and RT-PCR. Results. Both disk cells formed colonies in culture and expressed stem cell markers, nucleostemin, Oct-4, SSEA-4, and Stro-1, at early passages. However, after 5 passages, AFSCs became elongated and NPSCs appeared senescent. Conclusion. This study indicated that IVD contains stem cells and the characteristics of AFSCs and NPSCs are intrinsically different. The findings of this study may provide basic scientific data for understanding the properties of IVD cells and the mechanisms of lower back pain.

MG-63 human osteosarcoma cells were transfected with short hairpin RNA (shRNA) against livin and survivin using monomethoxypolyethylene glycol‑chitosan (mPEG‑CS) nanoparticles (NPs) as carriers, with the aim of evaluating the effect on cell proliferation and apoptosis. mPEG‑CS NPs sized ~100 nm were prepared by ionic crosslinking. mPEG‑CS‑livin shRNA, mPEG‑CS‑survivin shRNA and mPEG‑CS‑(livin shRNA + survivin shRNA) NPs were constructed by electrostatic adsorption at NP suspension/gene solution ratios of 3:1 to transfect MG‑63 cells. The expression levels of livin and survivin mRNA and protein were measured by reverse transcription‑polymerase chain reaction and western blotting, respectively. The inhibitory effects of downregulated livin and survivin expression on cell proliferation were measured using an MTT assay. The apoptosis‑inducing effects of livin and surivin knockdown were investigated using a Hoechst staining kit. All shRNA groups resulted in reduced expression of livin and survivin mRNA and protein in MG‑63 cells. The MTT assay and Hoechst staining indicated that simultaneous knockdown of livin and survivin genes inhibited the proliferation of MG‑63 cells and promoted their apoptosis, to a greater extent than knocking down either gene individually. The simultaneous interference mediated by mPEG‑CS NPs significantly reduced livin and survivin expression in MG‑63 cells, suppressed proliferation and facilitated apoptosis, to a greater extent than knockdown of either livin or survivin alone were. Thus the results indicate a synergistic effect of livin and survivin.