Type II collagen is a marker of articular cartilage encoded by the COL2A1 gene. The nature of the trans factors involved in the upregulation of this gene by insulin-like growth factor-I (IGF-I) remains unclear. We found that IGF-I increased type II collagen synthesis by a transcriptional control mechanism involving a 715-bp region within the COL2A1 first-intron specific enhancer. The overproduction of L-Sox5/Sox6/Sox9 and Sp1 and decoy experiments targeting these factors demonstrated their action in concert in IGF-I trans-activation. These results were supported by the data obtained in knockdown experiments in which siRNA against Sox9/Sox6 and Sp1 prevented the IGF-I-induced increase in collagen II production. Indeed, each of these trans-activators increased the expression of others. IGF-I increased the binding of Sox9 and Sp1/Sp3 to their cis elements in the enhancer, and we provide the first evidence of Sox9 interaction with the promoter by chromatin immunoprecipitation. Interactions with COL2A1 were also observed for Sp1, p300/CBP, and Tip60. Finally, a physical interaction between Sox9, p300, Sp3, and Sp1 was detected. These data demonstrate the role of Sox9, Sp1/Sp3, and euchromatin-associated factors (p300, Tip60) in the IGF-I-induced upregulation of COL2A1, indicating possible use of this growth factor in articular cartilage engineering applications to promote repair in patients with degenerative diseases, such as osteoarthritis.
The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17-estradiol (17-E 2 ) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17-E 2 stimulates, via its receptor hER66 (human estrogen receptor 66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand independent transactivation domain (AF-1) of hER66 was mediated by 'GC' binding sites of the -266/-63 bp promoter, through physical interactions between ER, Sp1/Sp3, Sox9 and p300, as demonstrated in ChIP and Re-ChIP assays in primary and dedifferentiated cells. 17-E 2 and hER66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3 and Sox-9 siRNAs prevented hER66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hER66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17-E 2 and hER66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17-E 2 could promote chondrocyte redifferentiation. Keywords Key Messages 17-E 2 up-regulates type II collagen gene expression in articular chondrocytes. An ER66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17-E 2 action in chondrocytes. 17-E 2 might be an attractive candidate for cartilage engineering applications.2
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