c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

Abstract

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic proteins 2 (BMP2). Mitogenactivated protein kinase (MAPK) cascades, such as extracellular signal-regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun-N-terminal kinase (JNK), the other kinase of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first showed that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3-E1 cell lines. We then showed that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss-of-function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN-JNK1). Consistently, BMP2-induced osteoblastic differentiation is reduced by JNK1 gain-offunction, such as enforced expression of a constitutively active JNK1 (CA-JNK1). Most importantly, we showed that Runx2 is required for JNK1-mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3-E1 cells to the extent similar to that achieved by overexpression of wild-type (WT) Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2-induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation. ß

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

“…Furthermore, a recent study showing that inhibition of GSK3␤ leads to attenuation of MP-induced bone loss in rats (46) further supports our finding that active GSK3␤-mediated Runx2 phosphorylation is apparently involved in its degradation by Fbw7, leading to bone loss. Although there are few studies demonstrating phosphorylation of Runx2 affecting its protein stability, none of them addressed the E3 ubiquitin ligase involved in such regulation (15,25). Our data demonstrating Runx2, GSK3␤, and Fbw7 to physically interact with each other in osteoblasts is again in agreement with previous findings that suggest physiological interaction of GSK3␤ with Runx2 is required for attenuating Runx2 functions (25).…”

“…In osteoblasts, Runx2 levels are regulated coordinately with the cell cycle machinery, apparently by E3 ubiquitin ligases (14,24). Runx2 expression in osteoblasts is also down-regulated upon its phosphorylation by GSK3␤ (25) and JNK1 (15) ligase for Runx2 based on following evidence: (a) phosphorylation of Runx2 negatively regulates its activity (13,15,25); (b) GSK3␤ negatively regulates osteogenesis by phosphorylating Runx2 and inhibiting its activity (25); (c) insulin signaling-mediatedPI3K/AKTpathwaypromotesosteogenesiswithoutphosphorylating Runx2 (26,27); and (d) there are several putative CPD motifs within Runx2 protein, and some of these CPDs also harbor GSK3␤ consensus phosphorylation motifs known to be phospho-modified by GSK3␤ (25). Here, we studied whether Fbw7␣ serves as an E3 ubiquitin ligase for Runx2.…”

“…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)(13)(14)(15); however, the identification of E3 ubiquitin ligases in such processes has remained elusive.…”

Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

“…In addition to the Smad1/5/8 transcription factors, MAPKs are also involved in BMPs osteogenic signaling transduction (11-14). Recently, we have reported that p38 and ERK1/2 MAPKs act in opposition to regulate BMP9-induced osteogenic differentiation of MSCs (15).…”

“…The classic BMPs signaling pathway operates by activation of the transcription factors Smads (2,10), and there are growing evidences that it can also act through a Smads-independent mitogen activated protein kinases (MAPKs) signaling pathway (2,11-14). Our recent study has demonstrated that p38 and ERK1/2, two subfamilies of MAPKs, play important roles in regulating BMP9-induced osteogenic differentiation of MSCs (15).…”