FHL2 Regulation On Mouse Ovary Granulosa Cell Grwoth And Mechanism

FHL2(Four and a half LIM domains protein 2),a member of FHL protein family,is essential for diverse biological functions including cell proliferation,migration,apoptosis,signal transduction and gene transcription.Previous studies demonstrated that FHL2 can regulate inhibin ? expression via interacting with NR5A1 family,but the mechanism underlying this biological processes is remained unclear.Therefore,the present study used mouse granulosa cell(mGCs)as a cell model to determine the role and functional mechanism of FHL2 in mGCs.The main methods and findings were shown as follows:(1)Expression and localization of FHL2 in mouse ovary.The expression of FHL2 was detected by employing IHC analysis.Results showed that expression of FHL2 was increased and mainly located in granulosa cells and oocytes with follicle assembly accomplishment.Western blot revealed that the expression of FHL2 increased significantly from 4 to 7 days after birth;(2)FHL2 was expressed both in the nucleus and cytoplasm of mGCs.We separated the nucleus and cytoplasm protein and applied Western Blot method,results showed that FHL2 was expressed both in the nucleus and cytoplasm,confirmed by IF assay.Most importantly,FHL2 expression was cell cycle dependent;(3)FHL2 induced the mGCs growth in vitro.MGCs were transfected with FHL2 specific siRNA and /or over-expression vector for 72 hours.CCK-8,cell counting and flow cytometry were performed to demonstrate the mGCs growth.Results indicated that knock-down of FHL2 significantly reduced cell viability(p < 0.01),blocked cell cycle(p < 0.05)and promoted apoptosis,however,over-expression of FHL2 significantly promoted the growth of mGCs(p < 0.05),improved cell viability(p < 0.01)and inhibited apoptosis(p < 0.05);(4)FHL2 regulated EGF and EGFR expression via binding with AP-1 and NF-?B transcription factors.We transfected FHL2-specific interfering RNA and found that knock-down of FHL2 significantly reduced the EGF and EGFR expression both at mRNA and protein level,whereas,over-expression of FHL2 had shown to promote the expression of EGF and EGFR.Furthermore,CHIP assay illustrated that FHL2 was the co-activator factor of AP-1 and NF-?B which directly regulated the EGF and EGFR expression;(5)EGF promoted mGCs growth which was FHL2-dependent: Results demonstrated that EGF(20 ng/ml)significantly promoted cell proliferation(p < 0.01),improved cell vitality(p < 0.01)and actuated the proportion of S phase cells(p < 0.001).However,knock-down of FHL2 expression significantly inhibited the role of EGF on mGCs growth;(6)EGF can drive FHL2 expression and to translocate into nucleus: we used EGF(20ng/ml)and specific signaling pathway inhibitor(AG1478/U1026/LY294002)to treat mGCs.Western Blot results shown that EGF can up-regulate FHL2 expression via MEK and PI3 K signaling pathway.IF result showed that EGF could induce the FHL2 expression in the nucleus.Finally,we dissected nucleus and cytoplasm protein,and Western Blot showed that FHL2 in nucleus was increased which is time-dependent.In our research,the above results indicated that FHL2 was expressed in mice ovary,and played a pivotal role in mGCs growth.EGF binds with EGFR on the cell membrane,and drived FHL2 expression in nucleus via stimulating MEK and PI3 K signaling pathway.FHL2 interacted with transcription factors AP-1 and NF-?B to regulate EGF and EGFR expression to form EGF/EGFR/FHL2 feedback loop to regulate mGCs growth.Taken together,our study demonstrated the function of FHL2 on mGCs growth and its mechanism,and provided us a further understanding on mammalian reproductive system development.This finding expands our theoretical knowledge and provide scientific basis for improving livestock fertility.