Skip to Content
Merck
  • E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation.

The Journal of biological chemistry (2015-11-07)
Yogesh Kumar, Isha Kapoor, Kainat Khan, Gatha Thacker, Mohd Parvez Khan, Nidhi Shukla, Jitendra Kumar Kanaujiya, Sabyasachi Sanyal, Naibedya Chattopadhyay, Arun Kumar Trivedi
ABSTRACT

Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3β are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7α. Fbw7α through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3β was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7α or GSK3β was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7α-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7α restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7α in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7α, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7α negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3β-dependent manner and thus provides a plausible explanation for GSK3β-mediated bone loss as described before.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-RUNX2 antibody produced in mouse, clone 1D8, purified immunoglobulin, buffered aqueous solution
Sigma-Aldrich
Anti-hFBXW7 antibody , Mouse monoclonal, clone FB407, purified from hybridoma cell culture
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Triton X-100, laboratory grade