Second, analysis of the osteoblast cell line H1-127-30 generated from the double knockout mice.32 The and expression, and abolished their induction by ectopic OSX (Figure 3c). osteoblast phenotype and was proven to be important in the maintenance of bone homeostasis, because its postnatal deletion causes loss of bone mass and bone defects.5, 6 Several studies also found that mutations or SNPs are related to osteoporosis and and are mandatory for the development of the skeleton. Moreover, both cooperatively regulate the expression of key genes in bone biology forming a transcriptional complex.9 OSX also acts as a necessary cofactor for DLX family of transcription factors.10 Furthermore, these transcription factors are subjected to fine EMD638683 R-Form tuning by posttranscriptional regulation. For instance, MAP kinases phosphorylate DLX5, RUNX2 and OSX, leading to their activation.11, 12, 13 These studies highlighted the complexity of the transcription factor network, which controls the osteoblast differentiation process and bone development. Maturation of MSCs to the osteoblastic phenotype is usually a multi-step process that requires cell expansion, differentiation and survival. The tumour suppressor p53 is considered a grasp regulator of proliferation and apoptosis. p53 activity helps to eliminate EMD638683 R-Form damaged cells, preventing tumorigenesis.14 Furthermore, p53 has been linked to cell differentiation in a variety of cell types, such as neurons, muscular cells and osteoblasts.15, 16, 17 Surprisingly, despite the key cellular functions of p53, knockout mice did not show major developmental defects. However, detailed studies exhibited skeletal abnormalities in some animals, such as upper incisor fusion and craniofacial and limb malformations.18 knockout mice are also characterized by a denser skeleton than their wild-type littermates and the deletion overexpress and osteogenic genes through an unknown mechanism.17 Previous studies suggested that deletion allows overactivation of the BMP pathway by mechanisms that involve changes in the expression of or expression levels by an miRNA-mediated mechanism.22, 23 Therefore, although the inhibitory role of p53 in bone formation is well established, little is yet known about the molecular mechanisms by which p53 exerts this function. Moreover, an in-depth understanding of the role of p53 in bone biology could have implications in the knowledge of pathologies associated with p53 signalling network alterations. Our work focused in the identification of the molecular mechanisms by which p53 exerts a repressive effect over the osteoblast differentiation programme. We found, using either loss- or gain-of-function models, that p53 expression has a unfavorable impact on the expression of osteoblast-specific transcription factors and their targets. Our work further demonstrated that this negative role of p53 is usually impartial of p53 transcriptional activity but Rabbit polyclonal to ADAP2 instead required physical conversation between OSX and p53 at the protein level. p53 prevented OSX from binding to Sp1/GC-rich sequences and blocked OSX from interacting with DLX5 and binding to homeodomain sequences. Results p53 downregulates osteoblastic gene expression It has been previously established that p53 has an inhibitory role in osteoblast differentiation using mouse models.17, 24 There is also evidences suggesting that these phenotypes are cell autonomous, as the BM-MSCs from knockout or wild-type mice. Absence of p53 results in upregulation of important genes implicated in bone development (Physique 1a). Importantly, two transcription factors with relevant roles in bone biology, and showed a slight upregulation at the mRNA level. OSX target genes were also upregulated in knockout osteoblasts, such as EMD638683 R-Form (bone sialoprotein) or (osteocalcin).9, 26, 27 Open in a separate window Determine 1 p53 protein inhibits osteogenic differentiation transcriptional programme. (a) mRNA expression levels of primary osteoblasts from wild-type or knockout mice grown for 3 days in osteogenic differentiation medium, and (b) SaOs2-p53TetOn were treated for 24?h with doxycycline 2?nM in 1% FBS medium. indicates gene mRNA. mRNAs were measured by RT-qPCR, normalized to and expressed as relative expressionS.E.M. of at least three impartial experiments (*and expression. The upregulation of expression after induction of p53 expression could be explained by direct binding of p53 to the known p53-responsive sequences in promoter.28, 29 These results provide evidence of a p53-dependent downregulation of the expression of osteoblastic genes. EMD638683 R-Form As previous studies had identified the involvement of the osteogenic BMP pathway on p53s effects, we next focused on this signalling pathway and its modulation by p53. As previously reported, p53 deletion results in a slight upregulation of mRNA, as well as the BMP-target gene in primary osteoblasts 16, 30 (Supplementary Physique 1A, left panel). Interestingly, an.