RESUMEN
Steroid hormones including (1,25)-dihydroxyvitamin D3, estrogens, and glucocorticoids control bone development and homeostasis. We show here that the osteogenic transcription factor Runx2 controls genes involved in sterol/steroid metabolism, including Cyp11a1, Cyp39a1, Cyp51, Lss, and Dhcr7 in murine osteoprogenitor cells. Cyp11a1 (P450scc) encodes an approximately 55-kDa mitochondrial enzyme that catalyzes side-chain cleavage of cholesterol and is rate limiting for steroid hormone biosynthesis. Runx2 is coexpressed with Cyp11a1 in osteoblasts as well as nonosseous cell types (e.g. testis and breast cancer cells), suggesting a broad biological role for Runx2 in sterol/steroid metabolism. Notably, osteoblasts and breast cancer cells express an approximately 32-kDa truncated isoform of Cyp11a1 that is nonmitochondrial and localized in both the cytoplasm and the nucleus. Chromatin immunoprecipitation analyses and gel shift assays show that Runx2 binds to the Cyp11a1 gene promoter in osteoblasts, indicating that Cyp11a1 is a direct target of Runx2. Specific Cyp11a1 knockdown with short hairpin RNA increases cell proliferation, indicating that Cyp11a1 normally suppresses osteoblast proliferation. We conclude that Runx2 regulates enzymes involved in sterol/steroid-related metabolic pathways and that activation of Cyp11a1 by Runx2 may contribute to attenuation of osteoblast growth.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación Enzimológica de la Expresión Génica , Osteoblastos/enzimología , Osteogénesis/genética , Esteroles/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Mitocondrias/enzimología , Peso Molecular , Osteoblastos/citología , Regiones Promotoras Genéticas/genética , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Activación Transcripcional/genéticaRESUMEN
Runt-related transcription factor 2 (Runx2) controls lineage commitment, proliferation, and anabolic functions of osteoblasts as the subnuclear effector of multiple signaling axes (e.g. transforming growth factor-beta/BMP-SMAD, SRC/YES-YAP, and GROUCHO/TLE). Runx2 levels oscillate during the osteoblast cell cycle with maximal levels in G(1). Here we examined what functions and target genes of Runx2 control osteoblast growth. Forced expression of wild type Runx2 suppresses growth of Runx2(-/-) osteoprogenitors. Point mutants defective for binding to WW domain or SMAD proteins or the nuclear matrix retain this growth regulatory ability. Hence, key signaling pathways are dispensable for growth control by Runx2. However, mutants defective for DNA binding or C-terminal gene repression/activation functions do not block proliferation. Target gene analysis by Affymetrix expression profiling shows that the C terminus of Runx2 regulates genes involved in G protein-coupled receptor signaling (e.g. Rgs2, Rgs4, Rgs5, Rgs16, Gpr23, Gpr30, Gpr54, Gpr64, and Gna13). We further examined the function of two genes linked to cAMP signaling as follows: Gpr30 that is stimulated and Rgs2 that is down-regulated by Runx2. RNA interference of Gpr30 and forced expression of Rgs2 in each case inhibit osteoblast proliferation. Notwithstanding its growth-suppressive potential, our results surprisingly indicate that Runx2 may sensitize cAMP-related G protein-coupled receptor signaling by activating Gpr30 and repressing Rgs2 gene expression in osteoblasts to increase responsiveness to mitogenic signals.
Asunto(s)
Diferenciación Celular/fisiología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica/fisiología , Osteoblastos/metabolismo , Receptores Acoplados a Proteínas G/biosíntesis , Transducción de Señal/fisiología , Células Madre/metabolismo , Animales , Relojes Biológicos/fisiología , Ciclo Celular/fisiología , Línea Celular Transformada , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Perfilación de la Expresión Génica/métodos , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Osteoblastos/citología , Estructura Terciaria de Proteína/fisiología , Receptores Acoplados a Proteínas G/genética , Células Madre/citologíaRESUMEN
In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.