Effect Of BFGF On The Expression Of ClC-3Chloride Channels In Human Lens Epithelial Cells And The Correlated Mechanism

ObjectiveTo investigate the effect of basic fibroblast growth factor (bFGF) on theexpression of ClC-3chloride channels in human lens epithelial cells and explore themechanism involved.MethodsHLE B-3cells at Logarithmic growth phase were incubated in the6or96wellplate overnight and the blank control group with the regular cells culture.LECs weretreated by1,10,100μg/L of bFGF, the expressing levels of ClC-3mRNA stimulatedby different concentrations of bFGF were measured by reverse transcription-PCR atdifferent time periods. The expression of ClC-3protein was detected by Western blotand immunofluorescence. Western blot technique was also used to detectphosphorylated ERK1/2、AKT expression in various groups. In the presence of10ug/L, LECs were cultured in the coincubation with50umol/L LY294002(a specificPI3K inhibitor)、20umol/L PD98059(a specific MAPKK inhibitor) for6hrespectively,the changes of the expression of ClC-3protein were examined byWestern blot.Results1. There were a low ClC-3mRNA expression in the blank control group. As theincrease of the bFGF concentrations（1,10ug/L）,the expression values of ClC-3mRNA were gradually elevated,which peaked at10ug/L of bFGF treatment for6hours, continue to increase the concentration to100ug/L, the mRNA expressiondeclined. Compared with the blank control group,ClC-3mRNA expression levels inthe1,10ug/L bFGF groups were significantly increased (P＜0.05). But there wereno significant differences between100ug/L bFGF group and the blank control group.2. Western blot showed that as the increase of the bFGF concentrations（1,10ug/L）,the expression values of ClC-3protein were gradually elevated,whichpeaked at10ug/L of bFGF treatment, continue to increase the concentration to100ug/L, the protein expression declined.The10ug/L bFGF group was significantlydifferent among various groups （P＜0.01).Moreover, ClC-3was found to be locatedboth in plasma membrane and cell plasma,but mainly gathered in and around thenucleus.3. The bFGF in different concentration (1,10,100ug/L)all had significant effecton increasing the expression of pERK1/2、 pAKT compared with the controlgroup(P<0.05),and AKT phosphorylation was peaking at the group treated withbFGF10ug/L while the ERK1/2phosphorylation was peaking at the group treatedwith bFGF100ug/L.The total ERK1/2、AKT changed little.4. After LECs were cultured in the coincubation with LY294002andPD98059,Western blot showed that The expressions of ClC-3was significantlylowered in bFGF+LY294002group、bFGF+PD98059group compared with thebFGF group (P<0.01),while the LY294002group and PD98059group were nostatistically significant difference compared to the control group (P>0.05).Conclusions1. bFGF（1-10ug/L） can induce the expression of ClC-3in human lensepithelial cells; ClC-3has great relation with the proliferation of human lens epithelialcells.2.MAPK-ERK1/2、PI3K/AKT signaling pathways mediated the expression ofClC-3stimulated by bFGF in human lens epithelial cells, PI3K/AKT signalingpathways may play more important role in this process.