绝经后妇女雌激素剥夺与骨关节炎(OA)之间存在联系,这表明17β-雌二醇(17β-E2)可能是软骨稳态的调节剂。这里,我们证明17β-E2刺激,通过其受体人雌激素受体α66(hERα66),II型胶原在分化和去分化(反映OA表型)关节软骨细胞中的表达。hERα66的非配体依赖性反式激活域(AF-1)对II型胶原基因(COL2A1)的反式激活是由-266/-63-bp启动子的“GC”结合位点介导的,通过ERα之间的物理相互作用,Sp1/Sp3,Sox9和p300,如在原代和去分化细胞中的染色质免疫沉淀(ChIP)和Re-染色质免疫沉淀(Re-ChIP)测定中所示。17β-E2和hERα66增加了Sp1/Sp3和Sox-9对COL2A1启动子和增强子区域的DNA结合活性。此外,Sp1,Sp3和Sox-9小干扰RNA(siRNA)阻止了hERα66诱导的COL2A1反式激活,表明这些因子及其各自的顺式区域是hERα66介导的COL2A1上调所必需的。我们的结果强调了基因组途径,17β-E2和hERα66通过该途径调节Sp1/Sp3异聚体结合活性,并同时参与分别参与软骨细胞分化状态和COL2A1转录激活的必需因子Sox-9和p300的募集。因此,这些新发现对于OA中软骨的组织工程具有吸引力,17β-E2能促进软骨细胞再分化。
结论:17β-E2上调关节软骨细胞中II型胶原基因的表达。ERa66/Sp1/Sp3/Sox-9/p300蛋白复合物介导这种刺激作用。这种异聚复合物在体内与Col2a1启动子和增强子相互作用并结合。我们的发现强调了软骨细胞中17β-E2作用的新调节机制。17β-E2可能是软骨工程应用的有吸引力的候选者。
The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17β-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. 17β-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 17β-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 17β-E2 could promote chondrocyte redifferentiation.
CONCLUSIONS: 17β-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 17β-E2 action in chondrocytes. 17β-E2 might be an attractive candidate for cartilage engineering applications.