OSTEOPROTEGERIN, RANK AND RANKL EXPRESSION IN THE FEMORAL HEAD OF PATIENTS WITH AVN

Abstract

Introduction: Avascular necrosis (AVN) of the femoral head (FH) is a painful disorder of the hip that leads to hip collapse. The pathology of AVN involves ischemic events leading to the death of bone. Several biological substances participate in the balance between osteoclasts and osteoblasts, like osteoprotegerin, RANK and RANKL. The expression of these genes affects the maturation and function of osteoblasts and osteoclasts and determines the rate of bone remodeling. In this study, we investigate the expression of OPG, RANK and RANKL in osteonecrotic FHs derived from 44 patients with AVN.

Methods and Materials: RNA and proteins were isolated from both necrotic and normal site of FHs of 44 patients diagnosed with AVN.

Quantitative RT-PCR was performed for OPG, RANKL and RANK molecules by using the Light Cycler FastStart DNA Master Hybridization Probes kit (Roche).

Western Blotting: 22 bone tissues were run on 4–12% NuPAGE gel (Invitrogen). Anti-OPG, anti-RANKL and anti-actin antibodies were used and membranes were immersed in ECL.

Results: Quantitative RT-PCR: The mRNA levels of OPG were higher in the necrotic (median: 5.25) than the normal site (median: 4.19) of the FHs and their difference was statistically significant (p< 0.05). The expression of RANK and RANKL was significantly lower than that of OPG following a similar pattern between the necrotic and normal site. The mRNA values of RANK and RANKL were higher in the necrotic sites [necrotic median: 1.0/normal median: 0.85, necrotic median: 0.8, normal median: 0.3, respectively] than the normal, although they were not statistically significant.

Western Blotting analysis: Normal sites from all FHs showed comparable OPG protein levels (median: 0.57) which were similar to those of normal (median: 0.63). Similar pattern to that of OPG was observed also for RANKL protein expression, where the median value for RANKL/F-actin ratio was 0.49 and 0.5 in normal and necrotic sites of FHs, respectively.

Discussion: OPG, RANK and RANKL are key genes for maintaining the balance between osteoblasts and osteoclasts. Our results show marked differences in the expression of OPG between the necrotic and the normal sites of the FHs; however, mRNA levels of RANKL varied insignificantly between normal and necrotic part of FH while mRNA levels of RANK gene remain similar in both sides of FHs. In contrast, the production of OPG and RANKL at the protein level showed no remarkable divergence. This indicates that the expression and production pattern of RANK may play the key role in the maintenance of the balance between osteoblasts and osteoclasts in AVN.