RESUMO
BMP2 signaling and RUNX2 regulatory pathways converge for transcriptional control of bone formation in vivo. SMAD proteins are recruited to RUNX2 regulatory complexes via an overlapping nuclear matrix targeting signal/Smad interacting domain sequence (391-432) in Runx2. To establish the contribution of RUNX2-SMAD interaction to osteoblastogenesis, we characterized a number of point mutants. Only a triple mutation of amino acids 426-428 (HTY-AAA) results in loss of RUNX2 interactions with either BMP2- or TGF-beta- responsive SMADs and fails to integrate the BMP2/TGF-beta signal on target gene promoters. In a Runx2 null cell reconstitution assay, the HTY mutant did not activate the program of osteoblast differentiation (alkaline phosphatase, collagen type 1, osteopontin, bone sialoprotein and osteocalcin) in response to BMP2 signaling. Thus, subnuclear targeting function and formation of a RUNX2-SMAD osteogenic complex are functionally inseparable. Taken together, these studies provide direct evidence that RUNX2 is essential for execution and completion of BMP2 signaling for osteoblast differentiation.
Assuntos
Aminoácidos/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Diferenciação Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/química , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/citologia , Proteínas Smad/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Sinais de Localização Nuclear/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/metabolismoRESUMO
Bone-specific transcription of the osteocalcin (OC) gene is regulated principally by the Runx2 transcription factor and is further stimulated in response to 1alpha,25-dihydroxyvitamin D3 via its specific receptor (VDR). The rat OC gene promoter contains three recognition sites for Runx2 (sites A, B, and C). Mutation of sites A and B, which flank the 1alpha,25-dihydroxyvitamin D3-responsive element (VDRE), abolishes 1alpha,25-dihydroxyvitamin D3-dependent enhancement of OC transcription, indicating a tight functional relationship between the VDR and Runx2 factors. In contrast to most of the members of the nuclear receptor family, VDR possesses a very short N-terminal A/B domain, which has led to the suggestion that its N-terminal region does not contribute to transcriptional enhancement. Here, we have combined transient-overexpression, coimmunoprecipitation, in situ colocalization, chromatin immunoprecipitation, and glutathione S-transferase pull-down analyses to demonstrate that in osteoblastic cells expressing OC, VDR interacts directly with Runx2 bound to site B, which is located immediately adjacent to the VDRE. This interaction contributes significantly to 1alpha,25-dihydroxyvitamin D3-dependent enhancement of the OC promoter and requires a region located C terminal to the runt homology DNA binding domain of Runx2 and the N-terminal region of VDR. Together, our results indicate that Runx2 plays a key role in the 1alpha,25-dihydroxyvitamin D3-dependent stimulation of the OC promoter in osteoblastic cells by further stabilizing the interaction of the VDR with the VDRE. These studies demonstrate a novel mechanism for combinatorial control of bone tissue-specific gene expression. This mechanism involves the intersection of two major pathways: Runx2, a "master" transcriptional regulator of osteoblast differentiation, and 1alpha,25-dihydroxyvitamin D3, a hormone that promotes expression of genes associated with these terminally differentiated bone cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Osteoblastos/fisiologia , Osteocalcina/genética , Osteocalcina/metabolismo , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Elemento de Resposta à Vitamina D , Animais , Sítios de Ligação , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/genética , Genes Reporter , Substâncias Macromoleculares , Osteoblastos/citologia , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/genética , Transcrição Gênica , Regulação para CimaRESUMO
Multiple dimensions of epigenetic control contribute to regulation of gene expression that governs bone biology and pathology. Once confined to DNA methylation and a limited number of post-translational modifications of histone proteins, the definition of epigenetic mechanisms is expanding to include contributions of non-coding RNAs and mitotic bookmarking, a mechanism for retaining phenotype identity during cell proliferation. Together these different levels of epigenetic control of physiological processes and their perturbations that are associated with compromised gene expression during the onset and progression of disease, have contributed to an unprecedented understanding of the activities (operation) of the genomic landscape. Here, we address general concepts that explain the contribution of epigenetic control to the dynamic regulation of gene expression during eukaryotic transcription. This article is part of a Special Issue entitled Epigenetics and Bone.