RESUMO
Collagen VI is a main extracellular matrix protein whose mutation is linked to myopathic diseases. In myoblasts and other cell types, collagen VI gene transcription peaks during cell-cycle exit that precedes differentiation, upon serum withdrawal or confluence. To get insight into this transcriptional regulation, we characterized a growth arrest responsive region (GARR) in the Col6a1 promoter responsible for this effect. In this work, we identify sterol regulatory element binding protein (SREBP) as a GARR binding protein and provide evidence that SREBP contributes to induction of Col6a1 transcription in serum free conditions. Furthermore, our data unveil a previously unexpected link between extracellular matrix production and LDL signaling.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Colágeno Tipo VI/biossíntese , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição , Transcrição Gênica , Animais , Northern Blotting , Núcleo Celular/metabolismo , Colágeno Tipo VI/genética , Meios de Cultura Livres de Soro/farmacologia , DNA Complementar/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Glutationa Transferase/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Células Musculares/metabolismo , Células NIH 3T3 , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.
