DIHYDROTESTOSTERONE DOWNREGULATES BONE RESORPTION ACTIVITY OF OSTEOCLASTS IN DOSE DEPENDENT MANNER: AN IN VITRO MODEL USING RAW 264.7 CELLS

Hnin Ei Thu,Zahid Hussain,Isa Naina Mohamed,Ahmad Nazrun Shuid

doi:10.22159/ijpps.2017v9i10.20651

Abstract

Objective: Numerous studies have evidenced the bone regulatory potential of dihydrotestosterone in androgen-deficient osteoporosis. The present study was thus aimed to explore the translational mechanism of dihydrotestosterone to down-regulate the bone resorption activity of osteoclasts using RAW 264.7 cells as in vitro model.Methods: Prior to analyze the efficacy of dihydrotestosterone (5α-DHT) to alleviate osteoclastic differentiation, their cell viability and cell proliferative ability was assessed using lactate dehydrogenase (LDH) and MTS assays. The osteoclastic differentiation capacity of dihydrotestosterone was evaluated by measuring TRAP activity and the expression of bone resorption-related proteins such as matrix metallopeptidase-9 (MMP-9), cathepsin-K, tartrate-resistant acid phosphatase (TRAP) and NFATc1. Moreover, the effects of dihydrotestosterone were also evaluated on superoxide (free radicals) generation and superoxide dismutase (SOD) activity in RANKL-induced osteoclasts.Results: Dihydrotestosterone showed no toxicity towards RAW 264.7 cells and significantly enhanced their proliferation and growth rates in a dose-dependent fashion. It was also observed that dihydrotestosterone exhibits a remarkable inhibitory effect on differentiation, maturation and activation of osteoclasts. The marked inhibition of differentiation and activation of osteoclasts caused by 5α-DHT was due to down-regulation of the expression of MMP-9, cathepsin-K, TRAP, NFATc1, generation of superoxide and up-regulation of SOD activity in the RAW 264.7 cells.Conclusion: Resulting data provided substantially in vitroevidence for the pronounced anti-osteoclastogenetic activity of dihydrotestosterone and its therapeutic value in treating osteoporosis and other bone-erosive disorders.

Highlights

IntroductionBone metabolism is a physiologic process that maintains homeostasis between the bone resorption (the activity of osteoclasts) and formation (the activity of osteoblasts)
Bone metabolism is a physiologic process that maintains homeostasis between the bone resorption and formation
RAW 264.7 cells were incubated with different concentrations of 5α-DHT and their cell viability was measured on days 1 and 5 compared to the control groups

Summary

Introduction

Bone metabolism is a physiologic process that maintains homeostasis between the bone resorption (the activity of osteoclasts) and formation (the activity of osteoblasts). Any deregulation in the homeostasis of this process may lead to bone diseases [1,2,3] including osteoporosis [4]. Osteoclasts are multinucleated cells generated from monocyte/macrophage precursor cells, and osteoclast formation requires receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The receptor activator of NF-κB (RANK) is expressed on RAW 264.7 cell surfaces and conjugates with RANKL, which is essential for osteoclastogenesis [7]. RAW 264.7 cells respond to RANKL stimulation in vitro to exhibit all characteristic features of fullydifferentiated matured osteoclast [8,9,10]. RAW 264.7 cells stimulate activation of an important transcription factor, nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), for osteoclastogenesis [11]. As a master transcription factor of osteoclastogenesis, NFATc1 regulates diverse osteoclastogenesisrelated proteins such as TRAP, cathepsin-K and MMP-9 [12,13,14]

Methods

Results

Discussion

Conclusion