Zoledronic Acid enhances proteasome-mediated degradation of SOCS3

We have previously shown that bisphosphonates can inhibit suppressor of cytokine signaling (SOCS3) a protein expressed in macrophages, osteoclasts and osteoblasts. SOCS3 functions by preventing further signaling 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 is to understand the mechanisms by which bisphosphonates modulate the inflammatory process via their effects on SOCS3 and inflammatory cytokine production.

Methods: A murine macrophage cell line (RAW 264.7) was cultured in 6-well plates prior to treatment with control growth media alone or media containing zoledronic acid (ZA) 24 hours before subsequent exposure to LPS for 24 hours.  MG132, a 26S proteasome inhibitor, was added to cells 1 hour prior to ZA treatment (0, 30, 60 min).  Supernatants were collected for ELISA and the data was analyzed using GRAPHPAD software. Cell lysates were pooled and processed for western blot analysis to assess the changes in SOCS3 protein levels. Beta-actin was used as a loading control.

Results:   ZA pretreatment decreased constitutive expression of SOC3 and LPS-induced SOCS3 expression.  However, pretreatment with a proteasome inhibitor, MG132, prevented ZA- mediated inhibition of both SOCS2 and SOCS3 protein expression.  In addition, ZA pretreatment enhanced IL-6 production.

Conclusion: It appears that ZA regulates SOCS3 expression by enhancing its proteasome-mediated degradation.  Lowered SOCS3 expression could increase inflammatory cytokine levels in macrophages and other cell types in the presence of LPS and other inflammatory stimuli to promote the tissue destruction observed during BRONJ.