Expression And Mechanism Of Basic Fibroblast Growth Factor(bFGF) On The Potassium-chloride Cotransporter(KCC) In Human Lens Epithelial Cells

Objective To investigate the effect of basic fibroblast growth factor( bFGF) on the expression of potassium-chloride cotransporter isoforms KCC1, KCC3, KCC4 in huamn lens epithelial cells and explore the mechanism involved.Methods1. HLE B-3 cells at Logarithmic growth phase were incubated in the 25-cm2 plastic cell culture flask and 6 well plate overnight and the blank control group with the regular cells culture. LECs were treated by different concentrations(1,10,100?g/L) of bFGF for 6 hours and different action time(6, 12, 24, 48 hours) of bFGF for 1?g/L. The expressing levels of KCC1, KCC3, KCC4 m RNA were measured by reverse transcription-PCR.2. The expression of KCC1, KCC3, KCC4 protein stimulated by different concentrations and time of bFGF was detected by Western blot.3. Immunofluorescence was used to observe the localization of KCC1 protein in human lens epithelial cells.4. In the presence of 1?g/L bFGF, LECs were cultured in the coincubation with 10?mol/L LY294002(a specific PI3 K inhibitor), 10?mol/L U0126(a specific MAPKK inhibitor) for 12 hours respectively, the changes of the expression of KCC1 protein were examined by Western blot.Results1. There were a low KCC1, KCC3, KCC4 mRNA expression in the blank control group. Compared with the blank control group, KCC1, KCC3, KCC4 m RNA expression levels in the 1?g/L bFGF groups were significantly increased(P<0.05). But there were no significant differences between time group(P>0.05). The 1?g/L bFGF group was significantly different among various groups(P<0.05).2. Western blot showed that compared with the blank control group, KCC1, KCC3, KCC4 protein expression levels in the 1?g/L bFGF groups were significantly increased(P<0.05). And bFGF continues to increase the concentration to 100 ?g/L, the KCC1 protein expression declined(P<0.05). But there were no significant differences between time group(P>0.05). The 1?g/L bFGF group was significantly different among various groups(P<0.05).3. Fluorescence microscopy revealed immunocytochemical cytoplasm labeling of HLE-B3 cells with anti-KCC1 antibody.4. After LECs were cultured in the coincubation with LY294002 and U0126, Western blot showed that the expressions of KCC1 was significantly lowered in bFGF+LY294002 group, bFGF+U0126 group compared with the bFGF group(P<0.05), while the LY294002 group and U0126 group were no statistically significant difference compared to the control group(P>0.05). And the KCC1 protein of bFGF+LY294002 group was less than bFGF+U0126 group(P=0.002).Conclusions1. The bFGF(1?g/L) can induce the expression of KCC1, KCC3, KCC4 in human lens epithelial cells but no time relation.2. KCC1 protein mainly exists in the cytoplasm of human lens epithelial cells.3. MAPK/ERK1/2, PI3K/AKT signaling pathways mediated the expression of KCC1 stimulated by bFGF in human lens epithelial cells, PI3K/AKT signaling pathways may play more important role in this process.