Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer

We have recently shown that a 150‐bp Col10a1 distal promoter (?4296 to ?4147 bp) is sufficient to direct hypertrophic chondrocyte‐specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem‐repeat Runx2 binding sites within the 3′‐end of this 150‐bp region (TGTGGG‐TGTGGC, ?4187 to ?4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′‐sequence without Runx2 sites were not able to drive the cell‐specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′‐end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150‐bp promoter abolishes its capacity to drive hypertrophic chondrocyte‐specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′‐sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte‐specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis‐enhancer. This interaction is required but not sufficient for cell‐specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′‐ or 3′‐sequences of this 150‐bp promoter are needed to work with Runx2 together to mediate cell‐specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation. © 2011 American Society for Bone and Mineral Research     

The effect of growth/differentiation factor-5 (GDF-5) on genotype and phenotype in human adult mesenchymal stem cells

AIM: To evaluate the effects of GDF-5 on genotype and phenotype of human mesenchymal stem cells (hMSC). HYPOTHESIS: GDF-5 leads to up-regulation of the Type I-collagen (Col) gene without altering bone marker genes or alkaline phosphatase activity. METHODS: To test our hypothesis hMSC were treated with rmGDF-5. Using quantitative real-time PCR we analyzed mRNA for Col, Runx2 and Osterix (Osx). Furthermore, we analyzed alkaline phosphatase activity (ALP) as a phenotypical bone marker. ANOVA and post hoc statistical analyses were used to determine differences among treatments (p < 0.05). RESULTS: HMSC showed a biphasic response in both Col and Runx2 after rmGDF-5. Initial up-regulation was followed by a significant down-regulation below controls. Interestingly, the controls presented with changes for Col and Runx2 over time. There was no Osx expression in either treated hMSC or controls. No significant differences could be detected in ALP. CONCLUSION: Increased expression of Col and Runx2 might indicate differentiation towards both osteoblast and fibroblast lineage. However, no Osx expression and no change in ALP support the assumption that rmGDF-5 does not lead to an osteoblast phenotype in hMSC. Our in vitro studies confirm a possible therapeutic benefit of GDF-5 in the treatment of tendon and ligament injuries and tissue engineering approaches. Further research is necessary to prove its clinical value.     

转录激活因子4过表达在内质网过度应激所致骨质疏松中的意义

目的 观察内质网过度应激中转录激活因子4(ATF4)对骨质疏松的影响,并探讨可能的分子学机制.方法 将2对ATF4过表达杂合子小鼠进行扩增繁殖后,通过鼠尾鉴定法筛选出ATF4过表达纯合子小鼠和野生型小鼠.将12周龄的野生型小鼠和ATF4过表达纯合子小鼠分为正常野生小鼠组(对照野生组),内质网应激通路激动剂衣霉素(TM)...     

c-Jun N-terminal kinase 1 negatively regulates osteoblastic differentiation induced by BMP2 via phosphorylation of Runx2 at Ser104

Runx2 plays a crucial role in osteoblastic differentiation, which can be upregulated by bone morphogenetic proteins 2 (BMP2). Mitogen-activated protein kinase (MAPK) cascades, such as extracellular signal-regulated kinase (ERK) and p38, have been reported to be activated by BMP2 to increase Runx2 activity. The role of cjun-N-terminal kinase (JNK), the other kinase of MAPK, in osteoblastic differentiation has not been well elucidated. In this study, we first showed that JNK1 is activated by BMP2 in multipotent C2C12 and preosteoblastic MC3T3-E1 cell lines. We then showed that early and late osteoblastic differentiation, represented by ALP expression and mineralization, respectively, are significantly enhanced by JNK1 loss-of-function, such as treatment of JNK inhibitor, knockdown of JNK1 and ectopic expression of a dominant negative JNK1 (DN-JNK1). Consistently, BMP2-induced osteoblastic differentiation is reduced by JNK1 gain-of-function, such as enforced expression of a constitutively active JNK1 (CA-JNK1). Most importantly, we showed that Runx2 is required for JNK1-mediated inhibition of osteoblastic differentiation, and identified Ser104 of Runx2 is the site phosphorylated by JNK1 upon BMP2 stimulation. Finally, we found that overexpression of the mutant Runx2 (Ser104Ala) stimulates osteoblastic differentiation of C2C12 and MC3T3-E1 cells to the extent similar to that achieved by overexpression of wild-type (WT) Runx2 plus JNK inhibitor treatment. Taken together, these data indicate that JNK1 negatively regulates BMP2-induced osteoblastic differentiation through phosphorylation of Runx2 at Ser104. In addition, unraveling these mechanisms may help to develop new strategies in enhancing osteoblastic differentiation and bone formation.     

Overexpression of Smurf2 Stimulates Endochondral Ossification Through Upregulation of β‐Catenin

Ectopic expression of Smurf2 in chondrocytes and perichondrial cells accelerated endochondral ossification by stimulating chondrocyte maturation and osteoblast development through upregulation of β‐catenin in Col2a1‐Smurf2 embryos. The mechanism underlying Smurf2‐mediated morphological changes during embryonic development may provide new mechanistic insights and potential targets for prevention and treatment of human osteoarthritis. Introduction : Our recent finding that adult Col2a1‐Smurf2 mice have an osteoarthritis‐like phenotype in knee joints prompted us to examine the role of Smurf2 in the regulation of chondrocyte maturation and osteoblast differentiation during embryonic endochondral ossification. Materials and Methods : We analyzed gene expression and morphological changes in developing limbs by immunofluorescence, immunohistochemistry, Western blot, skeletal preparation, and histology. A series of markers for chondrocyte maturation and osteoblast differentiation in developing limbs were examined by in situ hybridization. Results : Ectopic overexpression of Smurf2 driven by the Col2a1 promoter was detected in chondrocytes and in the perichondrium/periosteum of 16.5 dpc transgenic limbs. Ectopic Smurf2 expression in cells of the chondrogenic lineage inhibited chondrocyte differentiation and stimulated maturation; ectopic Smurf2 in cells of the osteoblastic lineage stimulated osteoblast differentiation. Mechanistically, this could be caused by a dramatic increase in the expression of β‐catenin protein levels in the chondrocytes and perichondrial/periosteal cells of the Col2a1‐Smurf2 limbs. Conclusions : Ectopic expression of Smurf2 driven by the Col2a1 promoter accelerated the process of endochondral ossification including chondrocyte maturation and osteoblast differentiation through upregulation of β‐catenin, suggesting a possible mechanism for development of osteoarthritis seen in these mice.