Up-regulation of type II collagen gene by 17ß-estradiol in articular chondrocytes involves Sp1/3, Sox-9, and estrogen receptor α.

UNLABELLED: The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17ß-estradiol (17ß-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17ß-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), 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 hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17ß-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17ß-E2 and hERα66 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 17ß-E2 could promote chondrocyte redifferentiation. KEY MESSAGES: 17ß-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/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 17ß-E2 action in chondrocytes. 17ß-E2 might be an attractive candidate for cartilage engineering applications.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of Sp1.Sp3 ratio and of the binding activity of both factors to the COL2A1 promoter.

Type II collagen is composed of alpha1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a -63/-35 sequence that binds Sp1.Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6- and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription.