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

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 GSK3␤ are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7␣. Fbw7␣ through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3␤ was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7␣ or GSK3␤ 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. Fbw7␣-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7␣ restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7␣ 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 Fbw7␣, 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 Fbw7␣ negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3␤-dependent manner and thus provides a plausible explanation for GSK3␤-mediated bone loss as described before.Runt domain-containing protein Runx2 is a critical regulator of the osteogenic lineage (1). Null mutation of Runx2 gene results in unmineralized skeleton in mice, and its heterozygotic loss results in human cleidocranial dysplasia (CCD) 2 phenotype (2, 3), which demonstrates the importance of Runx2 in bone biology. Runx2 induces transcription of osteopontin (OP) and osteocalcin (OC) by binding to osteoblast-specific cis-acting OSE2 elements in the promoter regions of these genes (4, 5). Runx2 expression is regulated by vitamin D 3 , estrogen, TGF-␤/ BMP2, and Wnt signaling pathways (6 -10).In comparison with transcriptional regulation, the posttranslational regulation of Runx2 in osteoblasts is relatively unexplored (11). Recent studies indicate that phosphorylation, acetylation, SUMOylation, and ubiquitination regulate Runx2 functions differently (11). The effects of phosphorylation by different kinases on the Runx2 protein stability and its subsequent function have been reported earlier (12-15); however, the identification of E3 ubiquitin ligases in such processes has remained elusive.F-box protein Fbw7 (also known as Fbwx7 and cdc4) is a component of the SCF (Skp-Cullin-F box) ubiquitin ligase complex...

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