# HuR Knockdown in MLO-Y4 Osteocyte-like Cells Elevates OPG Expression and Suppresses Osteoclastogenesis In Vitro

## TL;DR

This study shows that reducing HuR in osteocyte-like cells increases OPG, which suppresses bone breakdown by osteoclasts.

## Contribution

The study reveals HuR as an indirect regulator of OPG in osteocytes, impacting bone remodeling through post-transcriptional mechanisms.

## Key findings

- HuR knockdown in MLO-Y4 cells significantly increased OPG mRNA and protein without affecting RANKL.
- HuR-deficient osteocytes reduced the osteoclastogenic environment in co-culture and conditioned-medium assays.
- HuR does not directly bind OPG mRNA, suggesting indirect regulation of OPG expression.

## Abstract

Bone remodeling is maintained through the coordinated actions of osteoblasts, osteoclasts, and osteocytes, among which osteocytes serve as major regulators of osteoclast-mediated bone resorption through the receptor activator of the nuclear factor-κB ligand (RANKL)–osteoprotegerin (OPG) signaling axis. While molecular signals regulating osteocytic RANKL-OPG expression are fairly understood, how post-transcriptional mechanisms impact osteocyte function remains poorly defined. HuR (human antigen R) encoded by Elavl1 (embryonic lethal abnormal vision-like 1), a ubiquitously expressed RNA-binding protein, is known for stabilizing AU-rich element-containing transcripts involved in inflammatory and stress responses; however, its role in osteocyte-derived bone resorption is unknown. In this study, we examined the effect of HuR loss on osteocyte–osteoclastogenesis. Short hairpin RNA (shRNA)-mediated HuR knockdown in MLO-Y4 osteocyte-like cells resulted in a significant increase in OPG mRNA and its protein expression, whereas RANKL levels remained unchanged, leading to a significantly reduced RANKL/OPG ratio. Both co-culture and conditioned-medium assays demonstrated that HuR-deficient osteocytes produced a markedly diminished osteoclastogenic environment. Actinomycin D chase experiments showed no alteration in OPG mRNA decay kinetics, and RNA immunoprecipitation (RIP)-PCR failed to detect HuR–OPG interactions, indicating that HuR regulates OPG expression through indirect mechanisms rather than mRNA binding. These findings identify HuR as an indirect regulator of osteocyte-derived OPG expression that impacts osteoclast differentiation and reveal a previously unrecognized mechanism by which HuR contributes to bone remodeling.