Objective： To investigate the role of Spl as transcription factor required for transactivation of LRP16 gene by estrogen. Methods： Specific antibodies of ERα and Spl were used to precipitate the target DNA/protein complexes of MCF-7 cells at different time points after estrogen treatment （Chromatin immunoprecipitation assay）, the promoter region of LRP16 gene was amplified by semi-nested polymerase chain reaction （snPCR）. Small interfering RNA （siRNA） against Spl was transiently cotransfected with LRP16-Luc （containing the region from -213bp to -126bp of LRP16 gene promoter）in MCF-7 cells. The luciferase activities were measured by dual-luciferase assay. Results： The results of chromatin immunoprecipitation assay showed that Spl protein directly bound to the -213bp to -126bp region of LRP16 gene, and ERα could enhance the affinity of Spl to DNA. Spl-siRNA specifically decreased the transactivation of LRP16-Luc by 1713-estradio1 to 70-80%. Conclusion： The estrogen-induced transactivation of the human LRP16 gene was mediated by Spl protein. Moreover, the interactions of ERα/Sp1 functional complex with LRP16 promoter DNA were required for enhanced LRP16 gene transactivation.
LRP16 was previously identified as an estrogen-induced gene in breast cancer cells. The responsiveness of LRP16 to estrogen and its functional effects in endometrial cancer （EC） cells are still unclear. Here, we show that the mRNA level and promoter activity of the LRP16 gene were significantly increased by 17β-estradiol （E2） in estrogen receptor ot （ERα）-positive Ishikawa human EC cells. Although the growth rate of Ishikawa cells was not obviously affected by ectopic expression of LRP 16, the results of a Transwell assay showed an approximate one-third increase of the invasive capacity ofLRP 16-overexpressing cells. As a result of molecular screening, we observed that the expression of E-cadherin, an essential adhesion molecule associated with tumor metastasis, was repressed by LRP16. Further promoter analyses demonstrated that LRP 16 inhibited E-cadherin transactivation in a dose-dependent manner. However, the inhibition was abolished by estrogen deprivation, indicating that the downregulation of E-cadherin transcription by LRP16 requires ERα mediation. Chromatin immunoprecipitation analyses revealed that the binding of ERα to the E-cadherin promoter was antagonized by LRP 16, suggesting that LRP 16 could interfere with ERα-mediated transcription. These results suggest that the upregulation of LRP 16 by estrogen could be involved in invasive growth by downregulating E-cadherin in human ECs.
Yuan Guang MengWei Dong HanYa Li ZhaoKe HuangYi Ling SiZhi Qiang WuYi Ming Mu
