With The Function Of The Estrogen Receptor-interacting Protein Fhl2

Estrogen receptor (ER) is one of the members of the nuclear receptor superfamily. Mammalian ER is encoded by two genes:αandβ. Both ERαand ERβcan be subdivided into six regions (A-F), which contain two transactivation function regions, called AF1 and AF2, respectively. AF1 has ligand-independent transactivation function. AF2 has ligand-dependent transactivation function. The effective transactivation of AF1 and AF2 depends on their interaction with other proteins. ERplays an important role in the development of breast cancer, and the endocrine therapy of mammary carcinoma is acquired mainly by decreasing ER transcriptional activity. Therefore, it is of great significance to discover and characterize the proteins that modulate ER activity in developing the drugs of diseases related to estrogen.Using yeast two-hybrid screen, we isolated a novel ERβ-interacting protein, FHL2 (four-and-a-half-LIM-only protein), from a human mammary library with ERβAF1(1-145aa)as a bait.To further confirm the specific interaction between FHL2 and ER both in vitro and in vivo, we performed GST pull-down and co-immunoprecipitation experiments. the results were consistent with the result of yeast two-hybrid screens. Using different deletion mutants of FHL2 and ER, we defined the regions that determine the binding behavior between FHL2 and ER. Moreover, using GST pull-down experiment, we found that FHL1 and FHL3 could also interact with ER.The functional activity experiments showed that FHL2 can inhibit the ER-responsive ERE-luc reporter gene transcription in a dose-dependent manner and such activity depends on the presence of ER, not by effect the endogenous expressions level of ER.To further understand the fuction of FHL2, we made the polyclonal antibody of FHL2 by immuning Balb/c mouse. Using the high specific polyclonal antibody we determined the distribution of FHL2 in rat tissue and breast cancer cell lines. Subsequently, the expression vectors of FHL2 siRNAs were constructed. The ability of FHL2 to inhibit the ERE-luc reporter gene transcription was decreased when human embryo kidney 293 T cells were cotransfected with FHL2 siRNAs. The same result was found in the stable transfectanted FHL2 and FHL2 siRNA T47D cell lines. FHL2 may be the corepressors of ER.Our labs had proved FHL2 interacted with Smad4, other researchers found Smad4 can inhibit E2-induced transcription. We found FHL2 and Smad4 can co-inhibit the ER-responsive ERE-luc reporter gene transcription. The results of co- immunoprecipitation experiments showed FHL2, Smad4 and ERβwere exist in a protein complex.Taken together, these results identify a new regulating factor, FHL2, which plays a role in estrogen signaling pathway through interaction with ER. Further study will help us understand its specific regulating mechanism in estrogen signaling pathway.