Signal transduction of gap junctional genes, connexin32, connexin43 in human hepatocarcinogenesis

Abstract

Aim: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis.

Methods: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot.

Results: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein.

Conclusion: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.

Figure 1

Special indicator Fluo-3 AM was loading at 37 °C for 90 min in hepatocarcinoma cell lines in order to examine localization and concentration of intracellular calcium in MRC-1024 laser scanning confocal microscope imaging system. (A). In normal hepatocyte cell line QZG, stronger signal of calcium with lower peak released. (B). Concentration of intracellular calcium in HHCC was 108.37 nmol/L.

Figure 2

Special indicator Fluo-3 AM was loading at 37 °C for 90 min in hepatocarcinoma cell lines in order to examine localization and concentration of intracellular calcium in MRC-1024 laser scanning confocal microscope imaging system. (A). In hepatocarcinoma cell line HHCC, slight signal of calcium with lower peak was observed. (B). Concentration of intracellular calcium in HHCC was 35.13 nmol/L.

Figure 3

Special indicator Fluo-3 AM was loading at 37 °C for 90 min in hepatocarcinoma cell lines in order to examine localizatio and concentration of intracellular calcium in MRC-1024 laser scanning confocal microscope imaging system. (A). Loading by Fluo-3 AM, dark signal of calcium and lower peak was also absent in hepatocarcinoma cell line SMMC-7721. (B). Concentration of intracellular calcium was 47.08 nmol/L.

Figure 4

Western blot analysis of phosphorylation on tyrosine of CX32, CX43 proteins in various cells lines. Cells were harvested and lysed, equal amount of cell lystates were resolved of SDS-PAGE, transferred to NC membranes, and then probed with anti-phophorylation tyrosine mAb 4G10. (A). Expressions of CX32 proteins in cell lines with special anti-CX32 mAb. CX32 showed high immunoblot signal only in normal hepatocyte cell line QZG with very slight signal in hepatocarcinoma cell lines HHCC, SMMC-7721. (B). Expressions of CX43 proteins in cell lines with special anti-CX43 mAb. CX43 apperared in both QZG and SMMC-7721 cells but no in HHCC. (C). Phosphorylation on tyrosine of CX43 with special anti-phosphrylation tyrosine 4G10, unphosphorylation appeared in QZG cells even they showed high level expression of CX32, CX43. (D). Phosphorylated tyrosine of CX43 protein was detected in SMMC-7721 cells.