The Mechanism Of E2-ERa Signal Regulating The Expression Of CLOCK

Circulating estrogen is modulated by circadian clock. The core molecular compoments of the circadian rhythm, clock genes and their products form the transcription-translation feedback loops, which coordinates the inner clock in bodys and external environment changes. CLOCK (circadian locomotors output cycles kaput) is one of the core transcription factors in the feedback loops. More and more evidence is suggesting that the disruption of circulating estrogen and circadian rhythm is associated with breast cancer. Howerver the exact molecular mechnism has not been clarified. Our research is aim to investigate the mechanism of E2-ERa signaling pathway regulates the expression of CLOCK by western blot, RT-PCR, luciferase reporter, and ChIP. Moreover, the roles of CLOCK protein in breast cancer cells proliferation were investigated by MTT, colony formation and three-dimensional cell culture.In our study, we found that treatment of ERa-positive breast cancer cell lines, MCF-7and T47D cells with E2resulted in increased expression of CLOCK protein in these cells, but the same treatment given to ERa-negative cell lines, MDA-MB-231and MCF10A resulted in no apparent effect on CLOCK expression. The E2-enhanced expression of CLOCK in these cells was partly reversed when the same cells were also treated with ICI182780. These data suggests that ERa regulates the level of CLOCK protein.In order to research the mechenism of the effect of E2on CLOCK protein level further, we tested the CLOCK mRNA in MCF-7cells. The E2treatment or overexpression of ERa could up-regulated the level of CLOCK mRNA in MCF-7cells, ICI182780treatment or knockdown the expression of ERa decreased the level of CLOCK mRNA. These results indicate that ERa modulates the transcription of CLOCK.Next, the promoter of CLOCK was subcloned into vector pGL3-basic generating luciferase reporter CLOCK-WT-Luc. ERa activated the expression of CLOCK-WT-Luc in a dose independent manner, but ERβ had no significant impact on the expression of CLOCK-WT-Luc compared with ERa. E2activated but ICI182780repressed the expression of CLOCK-WT-Luc. These data indicates that ERa regulates CLOCK promoter activity.A computational analysis was performed and the results indicated that potential EREs were present at CLOCK DNA sequence. The potential EREs in CLOCK promoter were mutated, luciferase reporter experiments showed that the mutation of EREs decreased the activity of reporter. ERa bounded to EREs in CLOCK promoter regions in E2dependent manner by ChIP. These data showed that ERa bounds to CLOCK promoter regions in response to E2.Finally, we found that E2promoted ERa positive cell lines MCF-7and T47D proliferation, in MCF-7cells, knocking down the expression of CLOCK by shRNA repression cells proliferation. In addition, the colony formation and three-dimensional soft-agar colony culture were used to test the effect of CLOCK on colony formation and anchor independent cell growth. Our results showed that CLOCK is required for the cell proliferation.In conclusion our study revealed the crosstalk between the expression of CLOCK and E2-ERα signal, and suggested that E2-ERα signal could positively regulate CLOCK expression. This will serve as a step forward in unraveling the complex mechanisms involved in the develpoment of breast cancer.