Restoration of gap‐junctional intercellular communication in a communication‐deficient rat liver cell mutant by transfection with connexin 43 cDNA

Abstract

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Localization and function of the connexin 43 gap-junction protein in normal and various oncogene-expressing rat liver epithelial cells

“…WB-F344, WB-neo, and WB-myc cells showed a PolPI/Pz Cx43 phosphorylation pattern ( Figure 4B, lanes 2, 3, and 7), whereas ras-, neu-, src-, and myc/rastransformed WB cells showed hypophosphorylated Cx43 ( Figure 4B, lanes 4-6 and 8). The WB-aB1 mutant line showed a comparable POIPI Cx43 phosphorylation pattern in both membrane-enriched fractions ( Figure 4A, lane 6) and whole-cell extracts [29]. The P2 and P i bands were not well resolved by the total protein extraction procedure; however, relative amounts of Cx43 per total cellular protein could be assessed.…”

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras‐transformed cells by 4‐Phenyl‐3‐butenoic acid

“…To quantify gap junction-mediated cell-cell communication, a fluorescence dye transfer assay was performed, modified slightly from that previously described [Jou et al, 1993]. Cells were grown to 90–100% confluence in 35mm culture dishes and treated with vehicle or drug as indicated.…”

Dual Mechanisms of Regulation of Na/H Exchanger NHE-3 by Parathyroid Hormone in Rat Kidney

“…For treatment with alkaline phosphatase, we modified a protocol described by Jou et al (48). 20 g of apical membrane was washed with reaction buffer (50 mM Tris-HCl, pH 8.5, 2 mM phenylmethylsulfonyl fluoride, 8 mM MgCl 2 , 0.1% ␤-mercaptoethanol (v/v)) three times and resuspended in 45 l of reaction buffer containing 10 units of calf-intestine alkaline phosphatase.…”