| Introduction: It was previously shown that bisphosphonate compounds reduce the levels of suppressor of cytokine signaling 3 (SOCS3), a protein expressed in macrophages, osteoclasts, osteoblasts, etc. SOCS3 prevents further production of inflammatory cytokines such as interleukin 6 (Il-6) which can cause osteoclast activation and T-cell differentiation. Regulation of these phenomena may be important in understanding why bisphosphonate-related osteonecrosis of the jaw (BRONJ) occurs in patients using these medications. Objective: The purpose of this study was to identify the mechanism by which bisphosphonates modulate the inflammatory process via their effects on SOCS3 and inflammatory cytokine production. We hypothesized that zoledronic acid inducing proteosomal degradation of SOCS3 to regulate its levels. Methods: A murine macrophage cell line (RAW 264.7) was used because of their involvement in the inflammatory response in BRONJ. The cells were cultured with media containing zoledronic acid (ZA) or carrier to evaluate its capacity to repress SOCS3 and promote Il-6 production, by western blot and ELISA assays, respectively. Cells were also treated with lipopolysaccharide (LPS) as a positive inflammatory control. To determine the mechanism inducing SOCS3 degradation upon ZA treatment, we added the proteosomal inhibitor MG132 to the cultured cells 1 hour prior to ZA treatment for 0, 30, and 60 minutes. Co-Immunocoprecipitation (Co-IP) was conducted to determine whether SOCS3 ubiquitination was the mechanism used to promote SOCS3 proteasomal degradation. Results: ZA pretreatment decreased constitutive expression of SOCS3 and LPS-induced SOCS3 expression. Decreased SOCS3 was accompanied by increased Il-6 production. However, pretreatment with the proteasome inhibitor MG132 prevented ZA-mediated decrease of SOCS3 protein levels. Co-IP results revealed an increase in the co-precipitate of SOCS3 and ubiquitin following ZA treatment. Conclusion: ZA regulates SOCS3 levels by enhancing its proteasome-mediated degradation via ubiquitination. Lowered SOCS3 levels increase inflammatory cytokine levels in macrophages in the presence of LPS inflammatory stimulus. This inflammatory response appears to promote the tissue destruction observed during BRONJ. |
