Figure 5

Schematic representation of lineage specific differentiation for MSCs in an exquisitely coordinated process with critical regulators and indicators are shown. This picture also highlights the different modes of cross-talk between TGF-β/BMP signaling and the major signaling pathways of MAPK, Wnt, Hh, Notch, and FGF in which Runx2 is a key transcriptional regulator of osteoblast differentiation and bone formation. (a) TAK1 signaling pathway regulates bone formation. Following BMP induction, MAPK pathways converge at the Runx2 and Dlx5 gene to control PMC differentiation. FGF stimulated TAK1 has been shown to increase ALP protein levels without changing Ocn and mRNA levels in the proliferation and differentiation of osteoblastic precursors. FGF may influence osteoblastogenesis at least partially through Runx2 modulation. FGF-2/EGF and BMP2 appear to be reciprocally regulated in osteoblasts, contributing to the balance of interacting signaling pathways in bone development and homeostasis. (b) BMP pathway, with their corresponding Smad proteins, and inhibitory proteins I-Smads (Smad6/7) and Smurf1 (Smad ubiquitination regulatory factor-1). Activated Smad regulates expression of transcriptional factors and transcriptional co-activators (Dlx5, Runx2 and Osx). Dlx5 the initial target of activated R-Smad which regulates Osx and Runx2/Cbfα1 to regulate osteoblast differentiation while Msx2 stimulates cell proliferation in a co-ordinatation with Shh. Ihh, Shh and BMP appear to be inter regulated in bone development. Osteoblasts specific markers, including the early osteogenic marker alkaline phosphatase (ALP), type I collagen, the late osteogenic markers osteocalcin (Oc) and osteopontin (Op), connective tissue growth factor (CTGF), inhibitor of DNA binding (Id) and CBFα1/Runx2. BMP2 exhibited increased mineralization by lime mineralization protein (LMP) and bone sialoprotein (BSP). Upon BMP9 stimulation of MSCs, CTGF was among the most up- regulated genes, especially during early stages of differentiation. Smurf1 targets type I BMP receptors and recognize bone-specific Runx2. Tob, Hox transcription factors inhibit BMP signaling as part of the negative feedback circuit. Ski onco-protein also can block BMP signaling. (c) Hedgehog-induced osteoblastogenesis occurs through Runx2. Hh binds cell surface receptor patched to relieve patched mediated suppression of Smoothened (Smo). Then Smo activate to stabilize the transcription factor Gli2, which induces transcription of Gli1 and other Hh target genes. Ihh signaling is required for early osteoblastogenesis, likely through modulation of Runx2, Osx, and ALP. And, Ihh and BMP2 appear to be reciprocally regulated in osteoblasts, contributing to the balance of interacting signaling pathways. (d) Notch signaling is important for MSC differentiation into osteoblasts. When Notch interacts with membrane bound ligands delta or jagged on the surface of neighboring cells, the Notch receptor liberating the notch intracellular domain (NICD) which binds to CSL. CSL then recruits the co-activator mastermind-like (MAML) for transcription of Hey/Hes to inhibit Runx2. In addition, NICD can interact directly with Runx2 protein to repress terminal osteoblastic differentiation. Signaling can also regulate either Smad dependent BMP signaling or Wnt-induced osteogenesis through the up-regulation of RANKL and OPG, indicating the cross-talk between osteoblasts and osteoclasts could be mediated by Notch signaling. (e) PTH binding activates PTH1R to stimulate several downstream effectors. Transcriptional factor cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts. (f) Canonical Wnt/β-catenin pathway increases bone mass through a number of mechanisms including renewal of stem cells, stimulation of preosteoblast replication, induction of osteoblastogenesis, and inhibition of osteoblast and osteocyte apoptosis. Upon intracellular accumulation of β-catenin, and enter into nucleus to interact with the transcription factor T-cell factor/lymphoid enhancer factor (TCF/LEF) for expressing of bone lineage genes such as Dlx5 and Osx. Wnt has also been linked to Runx2. Runx2 gene promoter contains a Wnt-responsive TCF regulatory element, and both β-catenin and TCF1 are recruited to the Runx2 locus. Wnt signaling and LRP5/6 coreceptor activity can be blocked by the sclerostin (SOST) and Dickkopf (Dkk), leading to a decrease in bone mass. Increasing the ratio of osteoprotegerin (OPG) to RANKL, β-catenin represses osteoclastogenesis. BMPRIA signaling upregulates Sost expression primarily through Smad-dependent signaling, while it upregulates DKK1 through Smad-dependent and non-Smad-dependent signaling. Chibby (Cby), the endogenous antagonist, interrupts the binding of β-catenin to transcriptional factors Tcf/Lef-1. (g) Sonic hedgehog (Shh) signaling is activated in osteoblasts and regulated their proliferation, differentiation, as well as osteoclast formation, via focal adhesion kinase (FAK) signaling. Shh expression is positively correlated with phosphorylated FAK Tyr to increase mesenchymal proliferation and suture mesenchyme thickness via promotion of Msx2, and similarities are present between the expression of Shh, Msx2, and BMP expression during neonatal craniofacial suture development. Shh signaling indirectly induced osteoclast differentiation by upregulating osteoblasts expression of PTHrP, which promoted receptor activator of nuclear factor kappa B ligand (RANKL) expression via PKA and its target transcription factor CREB. (h) Micro-RNAs that participate in stimulation or inhibition of osteoblast differentiation and their target genes are represented. Among these, miR-133 targets the transcription factor Runx2, a known target of BMP/Smad signaling that promotes osteoblast differentiation. Whereas, miR-218 and miR-335-5p can down regulates Dkk and Sost to play role in the period from osteocyte to bone mineralization. On the other hand, miR-135a, miR-23a, miR-133a, miR-137, miR-217 target Smad5 and Runx2 to inhibit BMP signaling.