BMP-2 and Insulin-like Growth Factor-I Mediate Osterix (Osx) Expression in Human Mesenchymal Stem Cells via the MAPK and Protein Kinase D Signaling Pathways

Abstract

Genetic studies place the transcription factor Osterix (Osx) downstream of Runx2, but limited information is available about Osx regulation during osteoblastic differentiation. An important role for bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-I (IGF-I) on Osx expression and the requirement for p38 for the BMP-2-mediated effect was reported previously by our group. In this study, we continued to investigate the molecular mechanisms by which BMP-2 and IGF-1 regulate Osx expression during osteoblast lineage progression. IGF-I-mediated Osx expression required all three MAPK components (Erk, p38, and JNK), whereas BMP-2 required p38 and JNK signaling. As a common mediator of growth factor signaling, we also investigated the involvement of protein kinase C/D (PKC/D) signaling. BMP-2-and IGF-I-mediated Osx expression was blocked in response to a PKD inhibitor. A selective inhibitor of conventional PKCs had no effect on the BMP-2-mediated Osx expression. BMP-2 and IGF-I induced a selective phosphorylation of PKD, and PKD was required for mineralization. PKC/D and MAPK signaling also mediate Runx2 activity. Therefore, to document the implication for Runx2 in Osx regulation, we blocked Runx2 activity using a dominant negative Runx2 construct and an ubiquitination mediator for Runx2 degradation. We showed that blocking Runx2 activity inhibited the BMP-2-mediated induction of Osx. These studies implicated that multiple signaling pathways mediate Osx, a critical gene for osteoblast differentiation and bone formation. In addition to Runx2, other signaling components may be necessary to regulate Osx during osteoblast lineage progression.

Cationic Nanogel-mediated Runx2 and Osterix siRNA Delivery Decreases Mineralization in MC3T3 Cells

“…COX-2 expression stimulates prostaglandin E2 (PGE-2) expression in osteoblasts [3,27]; the PGE-2 and BMP signaling pathways converge at the transcription factor runt-related protein 2 (RUNX2) [37,38]. RUNX2 and osterix (OSX) are master regulators of osteogenesis and promote expression of alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and osteonectin (ON) [11,22,54]. These molecular cues promote the osteogenic cascade and clinical manifestation of HO.…”

Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells

“…These cells are able to differentiate along several committed phenotypes including osteogenic [1][2][3], chrondogenic [4], adipogenic [5], cardiogenic [6], and neurogenic [7,8] lineages in response to stimulation by multiple environmental factors.…”

Activation of c-Jun NH2-terminal kinase 1 increases cellular responsiveness to BMP-2 and decreases binding of inhibitory Smad6 to the type 1 BMP receptor

“…(18,21,30) In particular, the MAPK JNK is reported to be required for both late-stage spontaneous differentiation of osteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells in vitro. (20,21,23) However, in those previous studies, the points of intersection of the JNK and BMP-2 pathways were not investigated.…”

“…(17) MAPKs can be activated by osteoinductive factors such as BMPs and insulin-like growth factor 1 (IGF-1) and play an important role in cellular responses to these agents. (18)(19)(20)(21) Three structurally related MAPKs have been described in mammalian cells. (17) c-Jun NH 2 -terminal kinase (JNK) is one of these MAPKs, and it contributes to multiple cellular events such as proliferation, differentiation, and survival in response to extracellular stimuli.…”

“…(22) JNK has been reported to be required for late-stage differentiation of preosteoblasts and BMP-2-induced differentiation of preosteoblasts and pleuripotent cells. (18,20,23,24) JNK is considered a critical regulator of activating transcription factor 4, osteocalcin, and bone sialoprotein expression in spontaneously differentiating osteoblasts. (21,23) These studies suggest that JNK plays an important role in the induction of osteogenesis by BMP-2.…”