RESUMEN
During enchondral ossification, mesenchymal cells express genes regulating the intracellular biosynthesis of cholesterol and lipids. Here, we have investigated conditional deletion of Scap or of Insig1 and Insig2 (Scap inhibits intracellular biosynthesis and Insig proteins activate intracellular biosynthesis). Mesenchymal condensation and chondrogenesis was disrupted in mice lacking Scap in mesenchymal progenitors, whereas mice lacking the Insig genes in mesenchymal progenitors had short limbs, but normal chondrogenesis. Mice lacking Scap in chondrocytes showed severe dwarfism, with ectopic hypertrophic cells, whereas deletion of Insig genes in chondrocytes caused a mild dwarfism and shortening of the hypertrophic zone. In vitro studies showed that intracellular cholesterol in chondrocytes can derive from exogenous and endogenous sources, but that exogenous sources cannot completely overcome the phenotypic effect of Scap deficiency. Genes encoding cholesterol biosynthetic proteins are regulated by Hedgehog (Hh) signaling, and Hh signaling is also regulated by intracellular cholesterol in chondrocytes, suggesting a feedback loop in chondrocyte differentiation. Precise regulation of intracellular biosynthesis is required for chondrocyte homeostasis and long bone growth, and these data support pharmacological modulation of cholesterol biosynthesis as a therapy for select cartilage pathologies.
Asunto(s)
Desarrollo Óseo/fisiología , Colesterol/biosíntesis , Condrocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Diferenciación Celular/fisiología , Colesterol/genética , Condrocitos/citología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Células Madre Mesenquimatosas/citología , Ratones , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
Sarcomas, and the mesenchymal precursor cells from which they arise, express chondroitin sulfate proteoglycan 4 (NG2/CSPG4). However, NG2/CSPG4's function and its capacity to serve as a therapeutic target in this tumor type are unknown. Here, we used cells from human tumors and a genetically engineered autochthonous mouse model of soft-tissue sarcomas (STSs) to determine NG2/CSPG4's role in STS initiation and growth. Inhibiting NG2/CSPG4 expression in established murine and human STSs decreased tumor volume by almost two-thirds and cell proliferation rate by 50%. NG2/CSPG4 antibody immunotherapy in human sarcomas established as xenografts in mice similarly decreased tumor volume, and expression of a lentivirus blocking NG2/CSPG4 expression inhibited tumor cell proliferation and increased the latency of engraftment. Gene profiling showed that Ng2/Cspg4 deletion altered the expression of genes regulating cell proliferation and apoptosis. Surprisingly, Ng2/Cspg4 deletion at the time of tumor initiation resulted in larger tumors. Gene expression profiling indicated substantial down-regulation of insulin-like growth factor binding protein (Igfbp) genes when Ng2/Cspg4 is depleted at tumor initiation, but not when Ng2/Cspg4 is depleted after tumor initiation. Such differences may have clinical significance, as therapeutic targeting of a signaling pathway such as NG2/CSPG4 may have different effects on cell behavior with tumor progression. NG2/CSPG4 depletion has divergent effects, depending on the developmental stage of sarcoma. In established tumors, IGF signaling is active, and NG2 inhibition targets cell proliferation and apoptosis.
Asunto(s)
Antígenos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas de la Membrana/metabolismo , Proteoglicanos/metabolismo , Sarcoma/metabolismo , Sarcoma/fisiopatología , Animales , Antígenos/genética , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteoglicanos Tipo Condroitín Sulfato/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Estadificación de Neoplasias , Proteoglicanos/genética , Sarcoma/genética , Sarcoma/patologíaRESUMEN
During endochondral ossification in the vertebrate limb, multipotent mesenchymal cells first differentiate into chondroblasts (chondrogenesis) that further differentiate (via chondrocyte hypertrophy) to a terminal cellular phenotype. Dlx5 and Dlx6 are functionally redundant regulators of chondrocyte hypertrophy. We now show that Dlx5 and Dlx6 also regulate the earlier step of chondrogenesis in the limb. Limb bud mesenchymal cells from Dlx5/6(-/-) embryos show reduced chondrogenesis compared to wild-type littermates, and expression of either Dlx5 or Dlx6 stimulated differentiation of limb bud mesenchymal cells to chondroblasts. The functional overlap between Dlx5 and Dlx6 occurs despite the fact that the amino- and carboxyl-terminal domains of the encoded proteins are dissimilar. In order to reconcile the disparity between the divergent structures of Dlx5 and Dlx6 with their overlapping biological functions, we investigated the domain requirements and transcriptional activities associated with Dlx5- and Dlx6-mediated chondrogenesis. We find distinct domain requirements for the chondrogenic function of these related homeoproteins, indicating divergent molecular mechanisms of action.