HIF-1α facilitates osteocyte-mediated osteoclastogenesis by activating JAK2/STAT3 pathway in vitro.

Osteocytes, entrapped within the mineralized bone matrix, has been found to have numerous functions such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. Due to a specialized morphology and surrounding structure, osteocytes are more tolerant to hypoxia during osteoporosis, fracture, osteoarthritis, and orthodontic-orthognathic combination therapy. Hypoxia-inducible factor-1α (HIF-1α) is one of the master regulators of hypoxia reactions, playing an important role in bone modeling, remodeling, and homeostasis. This study aimed to investigate the pivotal functional role of HIF-1α in osteocytes initiating of bone remodeling under hypoxia. In the present study, the osteoclasts formation induced by RAW264.7 was significantly promoted in conditioned media (CM) from osteocytic MLO-Y4 exposed to hypoxia in vitro. Therefore, hypoxic MLO-Y4 cells simulated by 100 µmol/L CoCl2 or 2% O2 stably expressed HIF-1α proteins and upregulated the expression of receptor activator of nuclear factor-κB ligand (RANKL) at both the messenger RNA (mRNA) and protein level. Furthermore, with the Knockdown of HIF-1α, the expression of RANKL mRNA and protein decreased after transient transfection. In addition, the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription (STAT3) was also correlated with HIF-1α and RANKL levels under hypoxia. Then AG490, a JAK2 inhibitor, inhibited p-JAK2, p-STAT3 and RANKL expression. It was possible that AG490 disturbed the contact of HIF-1α and RANKL by JAK2/STAT3 pathway, influencing osteoclastogenesis. Our findings suggested that HIF-1α promoted the expression of RANKL by activating JAK2/STAT3 pathway in MLO-Y4 cells, and enhanced osteocyte-mediated osteoclastic differentiation in vitro.

[Mechanism of participation of osteocytes in the formation of osteoclasts under hypoxia].

OBJECTIVE: To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia. METHODS: The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected. RESULTS: DFO (100 µmol·L⁻¹) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 µmol·L⁻¹ DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01). CONCLUSIONS: Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upre-gulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.