A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6.

Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1.

Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice.

One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo.

Mesenchyme-specific overexpression of nucleolar protein 66 in mice inhibits skeletal growth and bone formation.

Previous studies showed that nucleolar protein 66 (NO66), the Jumonji C-domain-containing histone demethylase for methylated histone H3K4 and H3K36 (H3K36me), negatively regulates osteoblast differentiation in vitro by inhibiting the activity of transcription factor osterix (Osx). However, whether NO66 affects mammalian skeletogenesis in vivo is not yet known. Here, we generated transgenic (TG) mice overexpressing a flag-tagged NO66 transgene driven by the Prx1 (paired related homeobox 1) promoter. We found that NO66 overexpression in Prx1-expressing mesenchymal cells inhibited skeletal growth and bone formation. The inhibitory phenotype was associated with >50% decreases in chondrocyte/osteoblast proliferation and differentiation. Moreover, we found that in bones of NO66-TG mice, expression of Igf1, Igf1 receptor (Igf1r), runt-related transcription factor 2, and Osx was significantly down-regulated (P < 0.05). Consistent with these results, we observed >50% reduction in levels of phosphorylated protein kinase B (Akt) and H3K36me3 in bones of NO66-TG mice, suggesting an inverse correlation between NO66 histone demethylase and the activity of IGF1R/Akt signaling. This correlation was further confirmed by in vitro assays of C2C12 cells with NO66 overexpression. We propose that the decrease in the IGF1R/Akt signaling pathway in mice with mesenchymal overexpression of NO66 may contribute in part to the inhibition of skeletal growth and bone formation.

Connective tissue growth factor causes EMT-like cell fate changes in vivo and in vitro.

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of chronic fibrotic diseases. However, the mechanism by which paracrine effects of CTGF control the cell fate of neighboring epithelial cells is not known. In this study, we investigated the paracrine effects of CTGF overexpressed in fibroblasts of Col1a2-CTGF transgenic mice on epithelial cells of skin and lung. The skin and lungs of Col1a2-CTGF transgenic mice were examined for phenotypic markers of epithelial activation and differentiation and stimulation of signal transduction pathways. In addition to an expansion of the dermal compartment in Col1a2-CTGF transgenic mice, the epidermis was characterized by focal hyperplasia, and basal cells stained positive for αSMA, Snail, S100A4 and Sox9, indicating that these cells had undergone a change in their genetic program. Activation of phosphorylated p38 and phosphorylated Erk1/2 was observed in the granular and cornified layers of the skin. Lung fibrosis was associated with a marked increase in cells co-expressing epithelial and mesenchymal markers in the lesional and unaffected lung tissue of Col1a2-CTGF mice. In epithelial cells treated with TGFß, CTGF-specific siRNA-mediated knockdown suppressed Snail, Sox9, S100A4 protein levels and restored E-cadherin levels. Both adenoviral expression of CTGF in epithelial cells and treatment with recombinant CTGF induced EMT-like morphological changes and expression of α-SMA. Our in vivo and in vitro data supports the notion that CTGF expression in mesenchymal cells in the skin and lungs can cause changes in the differentiation program of adjacent epithelial cells. We speculate that these changes might contribute to fibrogenesis.

E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein.

SOX9 is a transcription factor that acts as a key regulator at various stages of cartilage differentiation. There is ample evidence that intracellular SOX9 protein levels are tightly regulated both by sumoylation and by degradation through the ubiquitin-proteasome pathway. Using a proteomics approach, here we report the identification of a SOX9-binding protein, E6-AP/UBE3A, that may act as a ubiquitin ligase toward Sox9. E6-AP bound SOX9 through the region consisting mostly of its high mobility group domain in vitro. In nuclear lysates, FLAG-tagged E6-AP coprecipitated with Sox9 and its high mobility group domain. This finding was estimated using nuclear lysates from a chondrocytic cell line that endogenously expresses E6-AP and SOX9. Accordingly, ectopically expressed E6-AP and SOX9 colocalized in the nucleus. We show that E6-AP ubiquitinates SOX9 in vitro and in vivo and that SOX9 levels are enhanced after addition of the proteasome inhibitor bortezomib. Similar, siRNA knockdown of E6-AP and the E2 ligase Ubc9 increased cellular SOX9 amounts, supporting the notion that SOX9 may be ubiquitinated in hypertrophic chondrocytes by E6-AP and degraded by proteasomes. This is in accordance with the distribution of SOX9 levels, which are high in proliferating and prehypertrophic chondrocytes but low in hypertrophic chondrocytes, whereas E6-AP levels are high in hypertrophic chondrocytes and low in prehypertrophic chondrocytes. Furthermore, E6-AP-deficient mice showed SOX9 accumulation in chondrocytes and the brain. These findings support the concept that E6-AP regulates SOX9 levels in developing cartilage by acting as a ubiquitin ligase.

Structural insights into histone demethylase NO66 in interaction with osteoblast-specific transcription factor osterix and gene repression.

Osterix (Osx) is an osteoblast-specific transcriptional factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory factors during gene expression.

Regulation of the osteoblast-specific transcription factor Osterix by NO66, a Jumonji family histone demethylase.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast-specific marker genes. To better understand the control of transcriptional regulation by Osx, we identified Osx-interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)-domain containing protein, called NO66, directly interacts with Osx and inhibits Osx-mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC-dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo, and this activity was needed for the regulation of osteoblast-specific promoters. During BMP-2-induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx-target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx-target genes in osteoblasts by modulating histone methylation states.

Sp7 and Runx2 molecular complex synergistically regulate expression of target genes.

Runx2 and Sp7 transcription factors are essential for skeletogenesis. Targeted deletion of either gene results in failure of osteoblast differentiation and bone formation. Loss of bone-matrix gene expression is surprisingly similar in Sp7 and Runx2 null mice. The molecular mechanisms responsible for similar transcriptional regulation of target genes remain largely unknown. Here, we demonstrate that Runx2 and Sp7 interact physically and functionally. Both proteins are co-expressed in osteoblastic cells. We first characterized a panel of Sp7 antibodies and demonstrate that majority of the published antibodies do not recognize Sp7 protein. Co-immunoprecipitation studies revealed that endogenous Runx2 protein physically interacts with Sp7 protein. We identified that runt homology domain (RHD) of Runx2 protein is involved in physical association with Sp7. Functional consequences of Runx2-Sp7 physical interaction was then assessed by promoter-reporter assays. We selected promoters of osteocalcin (OC), a marker of mature osteoblast and fibroblast growth factor 3 (FGF3), a signaling molecule that determine the fate of embryonic ecto-mesenchyme. Runx2 and Sp7 stimulate OC-promoter activity by 3-folds in epithelial cells. However, when both proteins were co-expressed, a dose-dependent synergistic activation of 22-folds was noted. Similar pattern of synergistic activation of OC-promoter was noted in mesenchymal cell. FGF3 promoter was activated by 25 - and 30-folds with Runx2 and Sp7 respectively. Again a dose-dependent synergistic activation of 130-folds was evident when Runx2 and Sp7 were co-expressed in epithelial cells. Synergistic activation of FGF3 promoter was also noted in mesenchymal cells. Together, our data demonstrated that Runx2-Sp7 molecular complex functionally cooperate for maximal induction of cell-phenotype-restricted genes.

Transcriptional regulation of Vascular Endothelial Growth Factor (VEGF) by osteoblast-specific transcription factor Osterix (Osx) in osteoblasts.

Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

Osterix is required for cranial neural crest-derived craniofacial bone formation.

Osx plays essential roles in regulating osteoblast and chondrocyte differentiation, and bone formation during mouse skeletal development. However, many questions remain regarding the requirement for Osx in different cell lineages. In this study, we asked whether Osx is required for craniofacial bone formation derived from cranial neural crest (CNC) cells. The Osx gene was conditionally inactivated in CNC-derived cells using a Wnt1-Cre recombination system. Neural crest-specific inactivation of Osx resulted in the complete absence of intramembranous skeletal elements derived from the CNC, and CNC-derived endochondral skeletal elements were also affected by Osx inactivation. Interestingly, Osx inactivated CNC-derived cells, which were recapitulated by lacZ expression, occupied the same regions of craniofacial skeletal elements as observed for controls. However, cells lost their osteogenic ability to differentiate into functional osteoblasts by Osx inactivation. These results suggest that Osx is important for craniofacial bone formation by CNC-derived cells. This finding provides novel insights of the regulation of craniofacial development by the gene network and transcription factors, and the understanding of human diseases caused by neural crest developmental abnormalities.

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice.

The transcription factor Osterix (Osx) is required for osteoblast differentiation and bone formation during embryonic development, but it is not known whether Osx has an essential function in postnatal bone growth and in bone homeostasis. Conditional deletion of Osx at several time points postnatally revealed that Osx was essential for osteoblast differentiation and new bone formation in growing and adult bones. Additionally, inactivation of Osx in bones severely disrupted the maturation, morphology, and function of osteocytes. These findings identify Osx as having an essential role in the cell-specific genetic program of osteocytes. Interestingly, Osx inactivation also led to the massive accumulation of unresorbed calcified cartilage in a large area below the growth plate of endochondral bones. This specific area was also marked by an unanticipated almost complete lack of bone marrow cells and a marked decrease in the density and size of osteoclasts. This diminished density of osteoclasts could contribute to the lack of resorption of mineralized cartilage. In addition, we speculate that the abnormally accumulated, mainly naked cartilage represents an unfavorable substrate for osteoclasts. Our study identifies Osx as an essential multifunctional player in postnatal bone growth and homeostasis.

Osteoblast-Specific Overexpression of Nucleolar Protein NO66/RIOX1 in Mouse Embryos Leads to Osteoporosis in Adult Mice.

In previous study, we showed that nucleolar protein 66 (NO66) is a chromatin modifier and negatively regulates Osterix activity as well as mesenchymal progenitor differentiation. Genetic ablation of the NO66 (RIOX1) gene in cells of the Prx1-expressing mesenchymal lineage leads to acceleration of osteochondrogenic differentiation and a larger skeleton in adult mice, whereas mesenchyme-specific overexpression of NO66 inhibits osteochondrogenesis resulting in dwarfism and osteopenia. However, the impact of NO66 overexpression in cells of the osteoblast lineage in vivo remains largely undefined. Here, we generated osteoblast-specific transgenic mice overexpressing a FLAG-tagged NO66 transgene driven by the 2.3 kB alpha-1type I collagen (Col1a1) promoter. We found that overexpression of NO66 in cells of the osteoblast lineage did not cause overt defects in developmental bones but led to osteoporosis in the long bones of adult mice. This includes decreased bone volume (BV), bone volume density (bone volume/total volume, BV/TV), and bone mineral density (BMD) in cancellous compartment of long bones, along with the accumulation of fatty droplets in bone marrow. Ex vivo culture of the bone marrow mesenchymal stem/stromal cells (BMSCs) from adult Col1a1-NO66 transgenic mice showed an increase in adipogenesis and a decrease in osteogenesis. Taken together, these data demonstrate a crucial role for NO66 in adult bone formation and homeostasis. Our Col1a1-NO66 transgenic mice provide a novel animal model for the mechanistic and therapeutic study of NO66 in osteoporosis.

Characterization of Dkk1 gene regulation by the osteoblast-specific transcription factor Osx.

Bone formation is a developmental process involving the differentiation of mesenchymal stem cells to osteoblasts. Osterix (Osx) is an osteoblast-specific transcription factor required for bone formation and osteoblast differentiation. Previous observation that Osx inhibits Wnt signaling pathway provides a novel concept of feedback control mechanisms involved in bone formation. Wnt antagonist Dickkopf-1 (Dkk1) plays an important role on skeletal development and bone remodeling. Osx has been shown to activate the Dkk1 promoter; however, the detailed mechanism of Osx regulation on Dkk1 expression is not fully understood. In this study, quantitative real-time RT-PCR results demonstrated that Dkk1 expression was downregulated in Osx-null calvaria at two different points of E15.5 and E18.5 in mice embryos. Overexpression of Osx resulted in upregulation of Dkk1 expression in Tet-off stable C2C12 cell line. Inhibition of Osx expression by siRNA led to downregulation of Dkk1 in osteoblasts. These data suggest that Osx may target Dkk1 directly. To define minimal region of Dkk1 promoter activated by Osx, we made a series of deletion mutants of Dkk1 promoter constructs, and narrowed down the minimal region to the proximal 250bp by transient transfection assay. It was shown that two GC-rich binding sites within this minimal region of Dkk1 promoter were required for the Dkk1 promoter activation by Osx. Importantly, quantitative chromatin immunoprecipitation (ChIP) assays were performed to show that endogenous Osx associated with native Dkk1 promoter in primary osteoblasts. Taken together, these findings support our hypothesis that Dkk1 is a direct target of Osx.

Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification.

Osterix (Osx) is an essential transcription factor required for osteoblast differentiation during both intramembranous and endochondral ossification. Endochondral ossification, a process in which bone formation initiates from a cartilage intermediate, is crucial for skeletal development and growth. Osx is expressed in differentiating chondrocytes as well as osteoblasts during mouse development, but its role in chondrocytes has not been well studied. Here, the in vivo function of Osx in chondrocytes was examined in a chondrocyte-specific Osx conditional knockout model using Col2a1-Cre. Chondrocyte-specific Osx deficiency resulted in a weak and bent skeleton which was evident in newborn by radiographic analysis and skeletal preparation. To further understand the skeletal deformity of the chondrocyte-specific Osx conditional knockout, histological analysis was performed on developing long bones during embryogenesis. Hypertrophic chondrocytes were expanded, the formation of bone trabeculae and marrow cavities was remarkably delayed, and subsequent skeletal growth was reduced. The expression of several chondrocyte differentiation markers was reduced, indicating the impairment of chondrocyte differentiation and endochondral ossification in the chondrocyte-specific Osx conditional knockout. Taken together, Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes, suggesting an autonomous function of Osx in chondrocytes during endochondral ossification.

Regulation of bone formation and remodeling by G-protein-coupled receptor 48.

G-protein-coupled receptor (GPCR) 48 (Gpr48; Lgr4), a newly discovered member of the glycoprotein hormone receptor subfamily of GPCRs, is an orphan GPCR of unknown function. Using a knockout mouse model, we have characterized the essential roles of Gpr48 in bone formation and remodeling. Deletion of Gpr48 in mice results in a dramatic delay in osteoblast differentiation and mineralization, but not in chondrocyte proliferation and maturation, during embryonic bone formation. Postnatal bone remodeling is also significantly affected in Gpr48(-/-) mice, including the kinetic indices of bone formation rate, bone mineral density and osteoid formation, whereas the activity and number of osteoclasts are increased as assessed by tartrate-resistant acid phosphatase staining. Examination of the molecular mechanism of Gpr48 action in bone formation revealed that Gpr48 can activate the cAMP-PKA-CREB signaling pathway to regulate the expression level of Atf4 in osteoblasts. Furthermore, we show that Gpr48 significantly downregulates the expression levels of Atf4 target genes/proteins, such as osteocalcin (Ocn; Bglap2), bone sialoprotein (Bsp; Ibsp) and collagen. Together, our data demonstrate that Gpr48 regulates bone formation and remodeling through the cAMP-PKA-Atf4 signaling pathway.

ß-catenin is a central mediator of pro-fibrotic Wnt signaling in systemic sclerosis.

OBJECTIVES: Pathologic fibroblast activation drives fibrosis of the skin and internal organs in patients with systemic sclerosis (SSc). ß-catenin is an integral part of adherens junctions and a central component of canonical Wnt signaling. Here, the authors addressed the role of ß-catenin in fibroblasts for the development of SSc dermal fibrosis. METHODS: Nuclear accumulation of ß-catenin in fibroblasts was assessed by triple staining for ß-catenin, prolyl-4-hydroxylase-ß and 4',6-diamidino-2-phenylindole (DAPI). The expression of Wnt proteins in the skin was analysed by real-time PCR and immunohistochemistry. Mice with fibroblast-specific stabilisation or fibroblast-specific depletion were used to evaluate the role of ß-catenin in fibrosis. RESULTS: The auhors found significantly increased nuclear levels of ß-catenin in fibroblasts in SSc skin compared to fibroblasts in the skin of healthy individuals. The accumulation of ß-catenin resulted from increased expression of Wnt-1 and Wnt-10b in SSc. The authors further showed that the nuclear accumulation of ß-catenin has direct implications for the development of fibrosis: Mice with fibroblast-specific stabilisation of ß-catenin rapidly developed fibrosis within 2 weeks with dermal thickening, accumulation of collagen and differentiation of resting fibroblasts into myofibroblasts. By contrast, fibroblast-specific deletion of ß-catenin significantly reduced bleomycin-induced dermal fibrosis. CONCLUSIONS: The present study findings identify ß-catenin as a key player of fibroblast activation and tissue fibrosis in SSc. Although further translational studies are necessary to test the efficacy and tolerability of ß-catenin/Wnt inhibition in SSc, the present findings may have clinical implications, because selective inhibitors of ß-catenin/Wnt signaling have recently entered clinical trials.

NFAT and Osterix cooperatively regulate bone formation.

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

The dimerization domain of SOX9 is required for transcription activation of a chondrocyte-specific chromatin DNA template.

Mutations in SOX9, a gene essential for chondrocyte differentiation cause the human disease campomelic dysplasia (CD). To understand how SOX9 activates transcription, we characterized the DNA binding and cell-free transcription ability of wild-type SOX9 and a dimerization domain SOX9 mutant. Whereas formation of monomeric mutant SOX9-DNA complex increased linearly with increasing SOX9 concentrations, formation of a wild-type SOX9-DNA dimeric complex increased more slowly suggesting a more sigmoidal-type progression. Stability of SOX9-DNA complexes, however, was unaffected by the dimerization mutation. Both wild-type and mutant SOX9 activated transcription of a naked Col2a1 DNA template. However, after nucleosomal assembly, only wild-type and not the mutant was able to remodel chromatin and activate transcription of this template. Using a cell line, in which the Col2a1 vector was stably integrated, no differences were seen in the interactions of wild-type and mutant SOX9 with the chromatin of the Col2a1 vector using ChIP. However, the mutant was unable to activate transcription in agreement with in vitro results. We hypothesize that the SOX9 dimerization domain is necessary to remodel the Col2a1 chromatin in order to allow transcription to take place. These results further clarify the mechanism that accounts for CD in patients harboring SOX9 dimerization domain mutations.

Dermal transforming growth factor-beta responsiveness mediates wound contraction and epithelial closure.

Stromal-epithelial interactions are important during wound healing. Transforming growth factor-beta (TGF-beta) signaling at the wound site has been implicated in re-epithelization, inflammatory infiltration, wound contraction, and extracellular matrix deposition and remodeling. Ultimately, TGF-beta is central to dermal scarring. Because scarless embryonic wounds are associated with the lack of dermal TGF-beta signaling, we studied the role of TGF-beta signaling specifically in dermal fibroblasts through the development of a novel, inducible, conditional, and fibroblastic TGF-beta type II receptor knockout (Tgfbr2(dermalKO)) mouse model. Full thickness excisional wounds were studied in control and Tgfbr2(dermalKO) back skin. The Tgfbr2(dermalKO) wounds had accelerated re-epithelization and closure compared with controls, resurfacing within 4 days of healing. The loss of TGF-beta signaling in the dermis resulted in reduced collagen deposition and remodeling associated with a reduced extent of wound contraction and elevated macrophage infiltration. Tgfbr2(dermalKO) and control skin had similar numbers of myofibroblastic cells, suggesting that myofibroblastic differentiation was not responsible for reduced wound contraction. However, several mediators of cell-matrix interaction were reduced in the Tgfbr2(dermalKO) fibroblasts, including alpha1, alpha2, and beta1 integrins, and collagen gel contraction was diminished. There were associated deficiencies in actin cytoskeletal organization of vasodilator-stimulated phosphoprotein-containing lamellipodia. This study indicated that paracrine and autocrine TGF-beta dermal signaling mechanisms mediate macrophage recruitment, re-epithelization, and wound contraction.