Class I PI-3-Kinase Signaling Is Critical for Bone Formation Through Regulation of SMAD1 Activity in Osteoblasts.

Bone formation and homeostasis is carried out by osteoblasts, whose differentiation and activity are regulated by osteogenic signaling networks. A central mediator of these inputs is the lipid kinase phosphatidylinositol 3-kinase (PI3K). However, at present, there are no data on the specific role of distinct class IA PI3K isoforms in bone biology. Here, we performed osteoblast-specific deletion in mice to show that both p110α and p110ß isoforms are required for survival and differentiation and function of osteoblasts and thereby control bone formation and postnatal homeostasis. Impaired osteogenesis arises from increased GSK3 activity and a depletion of SMAD1 protein levels in PI3K-deficient osteoblasts. Accordingly, pharmacological inhibition of GSK3 activity or ectopic expression of SMAD1 or SMAD5 normalizes bone morphogenetic protein (BMP) transduction and osteoblast differentiation. Together, these results identify the PI3K-GSK3-SMAD1 axis as a central node integrating multiple signaling networks that govern bone formation and homeostasis. © 2016 American Society for Bone and Mineral Research.

Mesenchymal Stem Cells Within Gelatin/CaSO4 Scaffolds Treated Ex Vivo with Low Doses of BMP-2 and Wnt3a Increase Bone Regeneration.

The delivery of osteogenic factors is a proven therapeutic strategy to promote bone regeneration. Bone morphogenetic proteins (BMPs) constitute a family of cytokines with well-known osteogenic and bone regenerative abilities. However, clinical uses of BMPs require high doses that have been associated with complications such as osteolysis, ectopic bone formation, or hematoma formation. In the present work, we sought to improve bone tissue engineering through an approach that combines the use of bone marrow-derived mesenchymal stem cells (BMMSCs), composite scaffolds, and osteoinductive agents. We employed a composite gelatin/CaSO4 scaffold that allows for an early expansion of seeded BMMSCs, which is followed by an increased level of osteogenic differentiation after 10 days in culture. Furthermore, this scaffold enhanced bone formation by BMMSCs in a mouse model of critical-sized calvarial defect. More importantly, our results demonstrate that ex vivo pretreatment of BMMSCs with low amounts of BMP-2 (2 nM) and Wnt3a (50 ng/mL) for 24 h cooperatively increases the expression of osteogenic markers in vitro and bone regeneration in the critical-sized calvarial defect mouse model. These data provide a strong rationale for the development of an ex vivo cooperative use of BMP-2 and Wnt3a. Osteogenic factor cooperation might be applied to reduce the required amount of growth factors while obtaining higher therapeutic effects.

p38α function in osteoblasts influences adipose tissue homeostasis.

The skeleton acts as an endocrine organ that regulates energy metabolism and calcium and phosphorous homeostasis through the secretion of osteocalcin (Oc) and fibroblast growth factor 23 (FGF23). However, evidence suggests that osteoblasts secrete additional unknown factors that contribute to the endocrine function of bone. To search for these additional factors, we generated mice with a conditional osteoblast-specific deletion of p38α MAPK known to display profound defects in bone homeostasis. Herein, we show that impaired osteoblast function is associated with a strong decrease in body weight and adiposity (P < 0.01). The differences in adiposity were not associated with diminished caloric intake, but rather reflected 20% increased energy expenditure and the up-regulation of uncoupling protein-1 (Ucp1) in white adipose tissue (WAT) and brown adipose tissue (BAT) (P < 0.05). These alterations in lipid metabolism and energy expenditure were correlated with a decrease in the blood levels of neuropeptide Y (NPY) (40% lower) rather than changes in the serum levels of insulin, Oc, or FGF23. Among all Npy-expressing tissues, only bone and primary osteoblasts showed a decline in Npy expression (P < 0.01). Moreover, the intraperitoneal administration of recombinant NPY partially restored the WAT weight and adipocyte size of p38α-deficient mice (P < 0.05). Altogether, these results further suggest that, in addition to Oc, other bone-derived signals affect WAT and energy expenditure contributing to the regulation of energy metabolism.

Mitogen-activated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program.

The transcription factors Runx2 and Osx (Osterix) are required for osteoblast differentiation and bone formation. Runx2 expression occurs at early stages of osteochondroprogenitor determination, followed by Osx induction during osteoblast maturation. We demonstrate that coexpression of Osx and Runx2 leads to cooperative induction of expression of the osteogenic genes Col1a1, Fmod, and Ibsp. Functional interaction of Osx and Runx2 in the regulation of these promoters is mediated by enhancer regions with adjacent Sp1 and Runx2 DNA-binding sites. These enhancers allow formation of a cooperative transcriptional complex, mediated by the binding of Osx and Runx2 to their specific DNA promoter sequences and by the protein-protein interactions between them. We also identified the domains involved in the interaction between Osx and Runx2. These regions contain the amino acids in Osx and Runx2 known to be phosphorylated by p38 and ERK MAPKs. Inhibition of p38 and ERK kinase activities or mutation of their known phosphorylation sites in Osx or Runx2 strongly disrupts their physical interaction and cooperative transcriptional effects. Altogether, our results provide a molecular description of a mechanism for Osx and Runx2 transcriptional cooperation that is subject to further regulation by MAPK-activating signals during osteogenesis.

The p38α MAPK function in osteoprecursors is required for bone formation and bone homeostasis in adult mice.

BACKGROUND: p38 MAPK activity plays an important role in several steps of the osteoblast lineage progression through activation of osteoblast-specific transcription factors and it is also essential for the acquisition of the osteoblast phenotype in early development. Although reports indicate p38 signalling plays a role in early skeletal development, its specific contributions to adult bone remodelling are still to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated osteoblast-specific deletion of p38α to determine its significance in early skeletogenesis, as well as for bone homeostasis in adult skeleton. Early p38α deletion resulted in defective intramembranous and endochondral ossification in both calvaria and long bones. Mutant mice showed reduction of trabecular bone volume in distal femurs, associated with low trabecular thickness. In addition, knockout mice also displayed decreased femoral cortical bone volume and thickness. Deletion of p38α did not affect osteoclast function. Yet it impaired osteoblastogenesis and osteoblast maturation and activity through decreased expression of osteoblast-specific transcription factors and their targets. Furthermore, the inducible Cre system allowed us to control the onset of p38α disruption after birth by removal of doxycycline. Deletion of p38α at three or eight weeks postnatally led to significantly lower trabecular and cortical bone volume after 6 or 12 months. CONCLUSIONS: Our data demonstrates that, in addition to early skeletogenesis, p38α is essential for osteoblasts to maintain their function in mineralized adult bone, as bone anabolism should be sustained throughout life. Moreover, our data also emphasizes that clinical development of p38 inhibitors should take into account their potential bone effects.

MicroRNAs and post-transcriptional regulation of skeletal development.

MicroRNAs (miRNAs) have become integral nodes of post-transcriptional control of genes that confer cellular identity and regulate differentiation. Cell-specific signaling and transcriptional regulation in skeletal biology are extremely dynamic processes that are highly reliant on dose-dependent responses. As such, skeletal cell-determining genes are ideal targets for quantitative regulation by miRNAs. So far, large amounts of evidence have revealed a characteristic temporal miRNA signature in skeletal cell differentiation and confirmed the essential roles that numerous miRNAs play in bone development and homeostasis. In addition, microarray expression data have provided evidence for their role in several skeletal pathologies. Mouse models in which their expression is altered have provided evidence of causal links between miRNAs and bone abnormalities. Thus, a detailed understanding of the function of miRNAs and their tight relationship with bone diseases would constitute a powerful tool for early diagnosis and future therapeutic approaches.

BMP signaling in telencephalic neural cell specification and maturation.

Bone morphogenetic proteins (BMPs) make up a family of morphogens that are critical for patterning, development, and function of the central and peripheral nervous system. Their effects on neural cells are pleiotropic and highly dynamic depending on the stage of development and the local niche. Neural cells display a broad expression profile of BMP ligands, receptors, and transducer molecules. Moreover, interactions of BMP signaling with other incoming morphogens and signaling pathways are crucial for most of these processes. The key role of BMP signaling suggests that it includes many regulatory mechanisms that restrict BMP activity both temporally and spatially. BMPs affect neural cell fate specification in a dynamic fashion. Initially they inhibit proliferation of neural precursors and promote the first steps in neuronal differentiation. Later on, BMP signaling effects switch from neuronal induction to promotion of astroglial identity and inhibition of neuronal or oligodendroglial lineage commitment. Furthermore, in postmitotic cells, BMPs regulate cell survival and death, to modulate neuronal subtype specification, promote dendritic and axonal growth and induce synapse formation and stabilization. In this review, we examine the canonical and non-canonical mechanisms of BMP signal transduction. Moreover, we focus on the specific role of BMPs in the nervous system including their ability to regulate neural stem cell proliferation, self-renewal, lineage specification, and neuronal function.

MicroRNA-322 (miR-322) and its target protein Tob2 modulate Osterix (Osx) mRNA stability.

Osteogenesis depends on a coordinated network of signals and transcription factors such as Runx2 and Osterix. Recent evidence indicates that microRNAs (miRNAs) act as important post-transcriptional regulators in a large number of processes, including osteoblast differentiation. In this study, we performed miRNA expression profiling and identified miR-322, a BMP-2-down-regulated miRNA, as a regulator of osteoblast differentiation. We report miR-322 gain- and loss-of-function experiments in C2C12 and MC3T3-E1 cells and primary cultures of murine bone marrow-derived mesenchymal stem cells. We demonstrate that overexpression of miR-322 enhances BMP-2 response, increasing the expression of Osx and other osteogenic genes. Furthermore, we identify Tob2 as a target of miR-322, and we characterize the specific Tob2 3'-UTR sequence bound by miR-322 by reporter assays. We demonstrate that Tob2 is a negative regulator of osteogenesis that binds and mediates degradation of Osx mRNA. Our results demonstrate a new molecular mechanism controlling osteogenesis through the specific miR-322/Tob2 regulation of specific target mRNAs. This regulatory circuit provides a clear example of a complex miRNA-transcription factor network for fine-tuning the osteoblast differentiation program.

Antiproliferative effect of catechin in GRX cells.

The phenolic compounds present in cocoa seeds have been studied regarding health benefits, such as antioxidant and anti-inflammatory activities. Fibrosis is a wound healing response that occurs in almost all patients with chronic liver injury. A large number of cytokines and soluble intercellular mediators are related to changes in the behavior and phenotype of the hepatic stellate cell (HSC) that develop a fibrogenic and contractile phenotype leading to the development of fibrosis. The objective of this study was to assess the catechin effect in GRX liver cells in activities such as cell growth and inflammation. The GRX cells treatment with catechin induced a significant decrease in cell growth. This mechanism does not occur by apoptosis or even by autophagy because there were no alterations in expression of caspase 3 and PARP (apoptosis), and LC3 (autophagy). The expression of p27 and p53 proteins, regulators of the cell cycle, showed increased expression, while COX-2 and IL-6 mRNA showed a significant decrease in expression. This study shows that catechin decreases cell growth in GRX cells and, probably, this decrease does not occur by apoptosis or autophagy but through an anti-inflammatory effect and cell cycle arrest. Catechin also significantly decreased the production of TGF-ß by GRX cells, showing a significant antifibrotic effect.

Noncanonical BMP signaling regulates cyclooxygenase-2 transcription.

Activation of p38 MAPK has been shown to be relevant for a number of bone morphogenetic protein (BMP) physiological effects. We report here the involvement of noncanonical phosphorylated mothers against decapentaplegic (Smad) signaling in the transcriptional induction of Cox2 (Ptgs2) by BMP-2 in mesenchymal cells and organotypic calvarial cultures. We demonstrate that different regulatory elements are required for regulation of Cox2 expression by BMP-2: Runt-related transcription factor-2 and cAMP response element sites are essential, whereas a GC-rich Smad binding element is important for full responsiveness. Efficient transcriptional activation requires cooperation between transcription factors because mutation of any element results in a strong decrease of BMP-2 responsiveness. BMP-2 activation of p38 leads to increased recruitment of activating transcription factor-2, Runx2, Smad, and coactivators such as p300 at the responsive sites in the Cox2 proximal promoter. We demonstrate, by either pharmacological or genetic analysis, that maximal BMP-2 effects on Cox2 and JunB expression require the function of p38 and its downstream effector mitogen/stress-activated kinase 1. Altogether our results strongly suggest that cooperative effects between canonical and noncanonical BMP signaling allow the fine-tuning of BMP transcriptional responses on specific target genes.

Conserved regulatory motifs in osteogenic gene promoters integrate cooperative effects of canonical Wnt and BMP pathways.

Osteoblast differentiation depends on the coordinated network of evolutionary conserved transcription factors during bone formation and homeostasis. Evidence indicates that bone morphogenetic protein (BMP) and Wnt proteins regulate several steps of skeletal development. Here, we provide a molecular description of the cooperative effects of BMP and Wnt canonical pathway on the expression of the early osteogenic genes Dlx5, Msx2, and Runx2 in C2C12 cells, primary cultures of bone marrow-mesenchymal stem cells, and organotypic calvarial cultures. Coordinated regulation of these genes leads to the cooperative activation of their downstream osteogenic target gene osterix. Induction of these genes is mediated through enhancer regions with an evolutionary conserved structure encompassing both Smad and TCF/LEF1 DNA-binding sites. Formation of a cooperative complex is mediated through DNA binding of Smads and TCF4/ß-catenin to their cognate sequences, as well as protein-protein interactions between them. The formation of these cooperative transcriptional complexes results in a more efficient recruitment of coactivators such as p300. We propose that evolutionary conserved regulatory regions in specific osteogenic master genes are key integrative modules during osteogenesis.

p38 regulates expression of osteoblast-specific genes by phosphorylation of osterix.

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.