Objective:Id2 is a natural inhibitor of the basic helix-loop-helix(bHLH) transcription factors.Although it is well known that active Id2 prevents differentiation and promotes cell cycle progression and tumorigenesis,the molecular events that regulate Id2 activity remain to be investigated.Methods:Yeast two-hybrid,mammalian two-hybrid,GST-pulldown and immunoprecipitation(CoIP) assays were used to screen and identify novel Id2 interactors.Luciferase assays were used to detect E47-mediated transcription activity.Colony formation and BrdU incorporation assays were used to determine cellular proliferation abilities.Northorn blot,western blot and quantitative PCR methods were used to measure gene expression levels.Electrophoretic mobility shift assays(EMSAs) were performed to investigate protein/DNA binding.Results:The LIM-only protein FHL2(four-and-a-half-LIM-only protein 2) was identified to be a novel Id2 interactor.The HLH domain within Id2 is not required for its interaction with FHL2.FHL2 antagonizes the inhibitory effect of Id2 on the basic helix-loop-helix protein E47-mediated transcription.FHL2 prevents the formation of Id2-E47 heterdimer,thus releasing E47 to its target DNA and restoring its transcriptional activity.FHL2 expression was remarkably up-regulated during retinoic acid-induced differentiation of neuroblastoma cells,during which the expression of Id2 is opposite to that.Ectopic FHL2 expression in neuroblastoma cells markedly reduces the transcriptional and cell-cycle promoting functions of Id2.Conclusion:These results indicate that FHL2 is an important repressor of the oncogenic activity of Id2 in neuroblastoma cells.
Wei-dong Han Zhi-qiang Wu Ya-li Zhao Yi-ling Si Xiao-bing Fu
Objective:FHL2 was previously identified to be a novel interacting factor of Id family proteins.The aim of this study was to investigate,the effects of FHL2 on Id1-mediated transcriptional regulation activity and its oncogenic activity in human breast cancer cells.Methods:Cell transfection was performed by Superfect reagent.Id1 stably overexpressed MCF-7 cells was cloned by G418 screening.The protein level of Id1 was detected by western blot analysis.Dual relative luciferase assays were used to measure the effect of E47-mediated transcriptional activity in MCF-7 human breast cancer cells.MTT assay was used to measure cell proliferation.Transwell assay was used to measure the invasive capacity of MCF-7 cancer cells.Results:The basic helix-loop-helix(bHLH) factor E47-mediated transcription activity was markedly repressed by Id1 in MCF-7 cells.This Id1-mediated repression was effectively antagonized by FHL2 transduction.Overexpression of Id1 markedly promoted the proliferation rate and invasive capacity of MCF-7 cells;however,these effects induced by Id1 were significantly suppressed by overexpression of FHL2 in cells.Conclusion:FHL2 can inhibit the proliferation and invasiveness of human breast cancer cells by repressing the functional activity of Id1.These findings provide the basis for further investigating the functional roles of FHL2-Id1 signaling in the carcinogenesis and development of human breast cancer.
Wei-dong HanZhi-qiang WuYa-li ZhaoYi-ling SiMing-zhou GuoXiao-bing Fu
