Epigenetic regulation of myogenesis by vitamin C.

The micronutrient vitamin C is essential for the maintenance of skeletal muscle health and homeostasis. The pro-myogenic effects of vitamin C have long been attributed to its role as a general antioxidant agent, as well as its role in collagen matrix synthesis and carnitine biosynthesis. Here, we show that vitamin C also functions as an epigenetic compound, facilitating chromatin landscape transitions during myogenesis through its activity as an enzymatic cofactor for histone H3 and DNA demethylation. Utilizing C2C12 myoblast cells to investigate the epigenetic effects of vitamin C on myogenesis, we observe that treatment of cells with vitamin C decreases global H3K9 methylation and increases 5-hmC levels. Furthermore, vitamin C treatment enhances myoblast marker gene expression and myotube formation during differentiation. We identify KDM7A as a histone lysine demethylase markedly upregulated during myogenesis. Accordingly, knockdown of Kdm7a prevents the pro-myogenic effects of vitamin C. Chromatin immunoprecipitation analysis showed that KDM7A occupies the promoter region of the myogenic transcription factor MyoD1 where it facilitates histone demethylation. We also confirm that the methylcytosine dioxygenases TET1 and TET2 are required for myogenic differentiation and that their loss blunts stimulation of myogenesis by vitamin C. In conclusion, our data suggest that an epigenetic mode of action plays a major role in the myogenic effects of vitamin C.

PI15, a novel secreted WNT-signaling antagonist, regulates chondrocyte differentiation.

PURPOSE/AIM OF STUDY: During the development of the vertebrate skeleton, the progressive differentiation and maturation of chondrocytes from mesenchymal progenitors is precisely coordinated by multiple secreted factors and signaling pathways. The WNT signaling pathway has been demonstrated to play a major role in chondrogenesis. However, the identification of secreted factors that fine-tune WNT activity has remained elusive. Here, in this study, we have identified PI15 (peptidase inhibitor 15, protease Inhibitor 15, SugarCrisp), a member of the CAP (cysteine rich secretory proteins, antigen 5, and pathogenesis related 1 proteins) protein superfamily, as a novel secreted WNT antagonist dynamically upregulated during chondrocyte differentiation. MATERIALS AND METHODS: ATDC5 cells, C3H10T1/2 micromass cultures and primary chondrocyte cells were used as in vitro models of chondrogenesis. PI15 levels were stably depleted or overexpressed by viral shRNA or expression vectors. Chondrogenesis was evaluated by qPCR gene expression analysis and Alcian blue staining. Protein interactions were determined by coimmunoprecipitation assays. RESULTS AND CONCLUSIONS: shRNA-mediated knockdown of PI15 in ATDC5 cells, C3H10T1/2 cells or primary chondrocytes inhibits chondrogenesis, whereas the overexpression of PI15 strongly enhances chondrogenic potential. Mechanistically, PI15 binds to the LRP6 WNT co-receptor and blocks WNT-induced LRP6 phosphorylation, thus repressing WNT-induced transcriptional activity and alleviating the inhibitory effect of WNT signaling on chondrogenesis. Altogether, our findings suggest that PI15 acts as a key regulator of chondrogenesis and unveils a mechanism through which chondrocyte-derived molecules can modulate WNT activity as differentiation proceeds, thereby creating a positive feedback loop that further drives differentiation.

Royal Jelly Enhances the Ability of Myoblast C2C12 Cells to Differentiate into Multilineage Cells.

Royal jelly (RJ) is recognized as beneficial to mammalian health. Multilineage differentiation potential is an important property of mesenchymal stem cells (MSCs). C2C12 cells have an innate ability to differentiate into myogenic cells. Like MSCs, C2C12 cells can also differentiate into osteoblast- and adipocyte-lineage cells. We recently reported that RJ enhances the myogenic differentiation of C2C12 cells. However, the effect of RJ on osteoblast or adipocyte differentiation is still unknown. Here in this study, we have examined the effect of RJ on the osteoblast and adipocyte differentiation of C2C12 cells. Protease-treated RJ was used to reduce the adverse effects caused by RJ supplementation. To induce osteoblast or adipocyte differentiation, cells were treated with bone morphogenetic proteins (BMP) or peroxisome proliferator-activated receptor γ (PPARγ) agonist, respectively. RNA-seq was used to analyze the effect of RJ on gene expression. We found that RJ stimulates osteoblast and adipocyte differentiation. RJ regulated 279 genes. RJ treatment upregulated glutathione-related genes. Glutathione, the most abundant antioxidative factor in cells, has been shown to promote osteoblast differentiation in MSC and MSC-like cells. Therefore, RJ may promote osteogenesis, at least in part, through the antioxidant effects of glutathione. RJ enhances the differentiation ability of C2C12 cells into multiple lineages, including myoblasts, osteoblasts, and adipocytes.

Inhibition of TET-mediated DNA demethylation suppresses osteoblast differentiation.

DNA methylation is an epigenetic modification critical for the regulation of chromatin structure and gene expression during development and disease. The ten-eleven translocation (TET) enzyme family catalyzes the hydroxymethylation and subsequent demethylation of DNA by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Little is known about TET protein function due to a lack of pharmacological tools to manipulate DNA hydroxymethylation levels. In this study, we examined the role of TET-mediated DNA hydroxymethylation during BMP-induced C2C12 osteoblast differentiation using a novel cytosine-based selective TET enzyme inhibitor, Bobcat339 (BC339). Treatment of C2C12 cells with BC339 increased global 5mC and decreased global 5hmC without adversely affecting cell viability, proliferation, or apoptosis. Furthermore, BC339 treatment inhibited osteoblast marker gene expression and decreased alkaline phosphatase activity during differentiation. Methylated DNA immunoprecipitation and bisulfite sequencing showed that inhibition of TET with BC339 led to increased 5mC at specific CpG-rich regions at the promoter of Sp7, a key osteoblast transcription factor. Consistent with promoter 5mC marks being associated with transcriptional repression, luciferase activity of an Sp7-promoter-reporter construct was repressed by in vitro DNA methylation or BC339. Chromatin immunoprecipitation analysis confirmed that TET2 does indeed occupy the promoter region of Sp7. Accordingly, forced overexpression of SP7 rescued the inhibition of osteogenic differentiation by BC339. In conclusion, our data suggest that TET-mediated DNA demethylation of genomic regions, including the Sp7 promoter, plays a role in the initiation of osteoblast differentiation. Furthermore, BC339 is a novel pharmacological tool for the modulation of DNA methylation dynamics for research and therapeutic applications.

Characterization of unique functionalities in c-Src domains required for osteoclast podosome belt formation.

Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.

Plectin promotes tumor formation by B16 mouse melanoma cells via regulation of Rous sarcoma oncogene activity.

BACKGROUND: Melanoma is a malignant tumor characterized by high proliferation and aggressive metastasis. To address the molecular mechanisms of the proto-oncogene, Rous sarcoma oncogene (Src), which is highly activated and promotes cell proliferation, migration, adhesion, and metastasis in melanoma. Plectin, a cytoskeletal protein, has recently been identified as a Src-binding protein that regulates Src activity in osteoclasts. Plectin is a candidate biomarker of certain tumors because of its high expression and the target of anti-tumor reagents such as ruthenium pyridinecarbothioamide. The molecular mechanisms by which plectin affects melanoma is still unclear. In this study, we examined the role of plectin in melanoma tumor formation. METHODS: We used CRISPR/Cas9 gene editing to knock-out plectin in B16 mouse melanoma cells. Protein levels of plectin and Src activity were examined by western blotting analysis. In vivo tumor formation was assessed by subcutaneous injection of B16 cells into nude mice and histological analysis performed after 2 weeks by Hematoxylin-Eosin (H&E) staining. Cell proliferation was evaluated by direct cell count, cell counting kit-8 assays, cyclin D1 mRNA expression and Ki-67 immunostaining. Cell aggregation and adhesion were examined by spheroid formation, dispase-based dissociation assay and cell adhesion assays. RESULTS: In in vivo tumor formation assays, depletion of plectin resulted in low-density tumors with large intercellular spaces. In vitro experiments revealed that plectin-deficient B16 cells exhibit reduced cell proliferation and reduced cell-to-cell adhesion. Since Src activity is reduced in plectin-deficient melanomas, we examined the relationship between plectin and Src signaling. Src overexpression in plectin knockout B16 cells rescued cell proliferation and improved cell-to-cell adhesion and cell to extracellular matrix adhesion. CONCLUSION: These results suggest that plectin plays critical roles in tumor formation by promoting cell proliferation and cell-to-cell adhesion through Src signaling activity in melanoma cells.

Tyrosine Kinase Src Is a Regulatory Factor of Bone Homeostasis.

Osteoclasts, which resorb the bone, and osteoblasts, which form the bone, are the key cells regulating bone homeostasis. Osteoporosis and other metabolic bone diseases occur when osteoclast-mediated bone resorption is increased and bone formation by osteoblasts is decreased. Analyses of tyrosine kinase Src-knockout mice revealed that Src is essential for bone resorption by osteoclasts and suppresses bone formation by osteoblasts. Src-knockout mice exhibit osteopetrosis. Therefore, Src is a potential target for osteoporosis therapy. However, Src is ubiquitously expressed in many tissues and is involved in various biological processes, such as cell proliferation, growth, and migration. Thus, it is challenging to develop effective osteoporosis therapies targeting Src. To solve this problem, it is necessary to understand the molecular mechanism of Src function in the bone. Src expression and catalytic activity are maintained at high levels in osteoclasts. The high activity of Src is essential for the attachment of osteoclasts to the bone matrix and to resorb the bone by regulating actin-related molecules. Src also inhibits the activity of Runx2, a master regulator of osteoblast differentiation, suppressing bone formation in osteoblasts. In this paper, we introduce the molecular mechanisms of Src in osteoclasts and osteoblasts to explore its potential for bone metabolic disease therapy.

Tumor necrosis factor alpha regulates myogenesis to inhibit differentiation and promote proliferation in satellite cells.

TNF-α and NF-κB signaling is involved in the wasting of skeletal muscle in various conditions, in addition to cancer cachexia. TNF-α and NF-κB signaling promotes the expression level of muscle RING finger protein 1, a ubiquitin ligase, causing muscle degradation. Several studies have indicated that of TNF-α and NF-κB signaling suppresses muscle differentiation by reducing the levels of MyoD protein. On the other hand, TNF-α and NF-κB is required for myoblast proliferation. Thus, the role of TNF-α and NF-κB signaling in the process of myogenesis and regeneration of skeletal muscle is not completely elucidated. Here, we reported that TNF-α reduced the width of single fibers of skeletal muscle in an organ culture model. TNF-α and p65 repressed the transactivation of MyoD and suppressed myoblast differentiation. In addition, TNF-α increased the number of satellite cells, and NF-κB signaling was promoted at the proliferation stage during skeletal muscle regeneration in vivo. TNF-α and NF-κB signaling regulate myogenesis to inhibit differentiation and promote proliferation in satellite cells.

p130Cas induces bone invasion by oral squamous cell carcinoma by regulating tumor epithelial-mesenchymal transition and cell proliferation.

Bone invasion is a critical factor in determining the prognosis of oral squamous cell carcinoma (OSCC) patients. Transforming growth factor ß (TGF-ß) is abundantly expressed in the bone matrix and is involved in the acquisition of aggressiveness by tumors. TGF-ß is also important to cytoskeletal changes during tumor progression. In this study, we examined the relationship between TGF-ß signaling and cytoskeletal changes during bone invasion by OSCC. Immunohistochemical staining of OSCC samples from five patients showed the expression of p130Cas (Crk-associated substrate) in the cytoplasm and phosphorylated Smad3 expression in the nucleus in OSCC cells. TGF-ß1 induced the phosphorylation of Smad3 and p130Cas, as well as epithelial-mesenchymal transition (EMT) accompanied by the downregulation of the expression of E-cadherin, a marker of epithelial cells, and the upregulation of the expression of N-cadherin, or Snail, a marker of mesenchymal cells, in human HSC-2 cells and mouse squamous cell carcinome VII (SCCVII) cells. SB431542, a specific inhibitor of Smad2/3 signaling, abrogated the TGF-ß1-induced phosphorylation of p130Cas and morphological changes. Silencing p130Cas using an short hairpin RNA (shRNA) or small interfering RNA in SCCVII cells suppressed TGF-ß1-induced cell migration, invasion, EMT and matrix metalloproteinase-9 (MMP-9) production. Compared with control SCCVII cells, SCCVII cells with silenced p130Cas strongly suppressed zygomatic and mandibular destruction in vivo by reducing the number of osteoclasts, cell proliferation and MMP-9 production. Taken together, these results showed that the expression of TGF-ß/p130Cas might be a new target for the treatment of OSCC bone invasion.

Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.

Bif-1/Endophilin B1/SH3GLB1 regulates bone homeostasis.

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 -/- ) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 -/- mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 -/- mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 -/- mice. Treatment with ß-glycerophosphate (ß-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 -/- mice. Finally, bone marrow cells from Bif-1 -/- mice showed a significantly higher colony-forming efficacy by the treatment with or without ß-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 -/- mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

A peptide that blocks the interaction of NF-κB p65 subunit with Smad4 enhances BMP2-induced osteogenesis.

Bone morphogenetic protein (BMP) potentiates bone formation through the Smad signaling pathway in vitro and in vivo. The transcription factor nuclear factor κB (NF-κB) suppresses BMP-induced osteoblast differentiation. Recently, we identified that the transactivation (TA) 2 domain of p65, a main subunit of NF-κB, interacts with the mad homology (MH) 1 domain of Smad4 to inhibit BMP signaling. Therefore, we further attempted to identify the interacting regions of these two molecules at the amino acid level. We identified a region that we term the Smad4-binding domain (SBD), an amino-terminal region of TA2 that associates with the MH1 domain of Smad4. Cell-permeable SBD peptide blocked the association of p65 with Smad4 and enhanced BMP2-induced osteoblast differentiation and mineralization without affecting the phosphorylation of Smad1/5 or the activation of NF-κB signaling. SBD peptide enhanced the binding of the BMP2-inudced phosphorylated Smad1/5 on the promoter region of inhibitor of DNA binding 1 (Id-1) compared with control peptide. Although SBD peptide did not affect BMP2-induced chondrogenesis during ectopic bone formation, the peptide enhanced BMP2-induced ectopic bone formation in subcortical bone. Thus, the SBD peptide is useful for enabling BMP2-induced bone regeneration without inhibiting NF-κB activity.

Regulation of osteoclast differentiation and actin ring formation by the cytolinker protein plectin.

Osteoclasts are cells that resorb the bone matrix and maintain bone and calcium homeostasis. An actin ring is a characteristic actin structure that is essential for bone resorption by osteoclasts. Tyrosine kinase Src deficient osteoclasts do not form actin rings; thus, Src is a key molecule for actin ring formation in osteoclasts. However, how Src regulates actin ring formation is not fully understood. We identified the cytolinker protein plectin as a Src-binding protein by immunoprecipitation and liquid chromatography tandem mass spectrometry. Plectin is a huge protein (>500 kDa) and regulates the cytoskeleton by binding to actin and tubulin. We assessed the expression and role of plectin in osteoclasts. Plectin was expressed and co-localized with Src close to the actin ring in osteoclasts. Moreover, plectin was tyrosine-phosphorylated by Src. Differentiation and actin ring formation were inhibited by downregulation of plectin. These results suggest an important role for plectin in osteoclast differentiation and actin ring formation through Src binding.

KIF22 regulates mitosis and proliferation of chondrocyte cells.

Point mutations in KIF22 have been linked to spondyloepimetaphyseal dysplasia with joint laxity, type 2 (SEMDJL2). Skeletal features of SEMDJL2 include short stature and joint laxity. Mechanisms underlying these limb abnormalities are unknown. Here in this manuscript, we have investigated the function of KIF22 in chondrocytes. Quantitative PCR and immunostaining revealed that Kif22 was highly expressed in proliferating-zone growth-plate chondrocytes. Kif22 knockdown resulted in defective mitotic spindle formation and reduced cell proliferation. Forced expression of SEMDJL-associated mutant Kif22 constructs likewise induced abnormal mitotic spindle morphology and reduced proliferation. Mice expressing a KIF22 truncation mutant had shorter growth plates and shorter tibial bones compared to wild-type mice. These results suggest that KIF22 regulates mitotic spindle formation in proliferating chondrocytes thereby linking the stunted longitudinal bone growth observed in SEMDJL2 to failures of chondrocyte division.

BMP3b regulates bone mass by inhibiting BMP signaling.

Bone morphogenetic protein 3b (BMP3b), also known as growth differentiation factor 10 (GDF10), is a non-osteogenic BMP highly expressed in the skeleton. Although in vitro studies have shown that BMP3b suppresses osteoblast differentiation, the physiological role of BMP3b in regulating bone mass in vivo remains unknown. Here, we show that BMP3b deletion in mice leads to a high bone mass phenotype via an unexpected novel mechanism involving de-repression of canonical BMP/Smad signaling. BMP3b null mice were viable, and exhibited no significant difference in body size compared to wildtype control. Trabecular bone parameters assessed by histomorphometry and µCT, revealed a significant increase in bone volume and bone mineral density. Expression of osteoblast-differentiation genes were elevated in bone tissue of BMP3b null mice, whereas expression of osteoclast-related genes remained unchanged. Consistent with this, Bmp3b was highly expressed in osteoblasts relative to osteoclast cells. Ex-vivo culture of primary bone marrow mesenchymal stem cells (BMSCs) and primary bone marrow-derived osteoclasts revealed that inactivation of BMP3b enhances osteogenesis without affecting osteoclastogenesis. Mechanistically, we found that BMP3b suppressed BMP4-induced Smad1/5 phosphorylation and inhibited the activity of a BMP4-driven Id-1 luciferase reporter. Protein-protein interaction assays revealed that BMP3b competitively interfered with the association of BMP4 and BMP type I receptors. These findings suggest that BMP3b regulates bone mass by acting as a BMP receptor antagonist. Thus, maintenance of bone mass involves antagonism of canonical BMP/Smad signaling by a member of the BMP family.

Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification.

Endochondral ossification is temporally and spatially regulated by several critical transcription factors, including Sox9, Runx2, and Runx3. Although the molecular mechanisms that control the late stages of endochondral ossification (e.g. calcification) are physiologically and pathologically important, these precise regulatory mechanisms remain unclear. Here, we demonstrate that Osterix is an essential transcription factor for endochondral ossification that functions downstream of Runx2. The global and conditional Osterix-deficient mice studied here exhibited a defect of cartilage-matrix ossification and matrix vesicle formation. Importantly, Osterix deficiencies caused the arrest of endochondral ossification at the hypertrophic stage. Microarray analysis revealed that matrix metallopeptidase 13 (MMP13) is an important target of Osterix. We also showed that there exists a physical interaction between Osterix and Runx2 and that these proteins function cooperatively to induce MMP13 during chondrocyte differentiation. Most interestingly, the introduction of MMP13 stimulated the calcification of matrices in Osterix-deficient mouse limb bud cells. Our results demonstrated that Osterix was essential to endochondral ossification and revealed that the physical and functional interaction between Osterix and Runx2 were necessary for the induction of MMP13 during endochondral ossification.

Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes.

Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2-Smad-Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPß, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.

Nobiletin, a NF-κB signaling antagonist, promotes BMP-induced bone formation.

The NF-κB family of transcription factors plays an important role in skeletal development and bone homeostasis. In osteoblast cells, NF-κB signaling has been shown to suppress survival, proliferation, and differentiation. Furthermore, pharmacological suppression of NF-κB enhances osteoblast differentiation and bone formation. Thus, NF-κB antagonists are promising candidates as anabolic agents for enhancing bone mass. In this study, we describe the mechanism by which nobiletin, an inhibitor of NF-κB activity, regulates osteoblast differentiation and mineralization. We found that in MC3T3-E1 osteoblast cells, nobiletin inhibited a TNF-α responsive NF-κB luciferase reporter and also decreased the induction of classical NF-κB target genes by TNF-α. Consistent with this, nobiletin prevented TNF-α -mediated suppression of osteogenesis and potently enhanced the differentiation and mineralization of MC3T3-E1 cells. Likewise, in an in vivo BMP2-induced ectopic bone formation assay, nobiletin markedly enhanced ossicle bone volume. Western blotting and SMAD-responsive luciferase assays also demonstrated that NF-κB suppression of BMP signaling could be inhibited by nobiletin. Thus, our data suggest that mechanistically, nobiletin prevents the endogenous repression of BMP signaling by TNF-α, thereby enhancing osteoblast activity. In conclusion, nobiletin is a novel NF-κB antagonist that may be a useful anabolic agent for bone formation.