The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester.

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Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.

The effect of in vivo-administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF) receptor activity of the rat hepatic plasma membrane was studied. TCDD causes a significant reduction in EGF binding at an early stage of toxicity (day 2) and at very low doses (1 pug/kg, single i.p., rat). This reduction appears to be due to a decline in the number of receptors. There is a good correlation between levels of decline in EGF binding and loss of body weight among TCDD-treated rats. The reduction in EGF binding occurs at a relatively low dose in the guinea pig (a very sensitive species) and at high doses in the hamster (a tolerant species). Among three mice strains, TCDD (115 ptg/kg, single i.p.) caused 98% reduction in EGF binding in the sensitive strains (C57BL/6J and CBA/J) but only a 50% reduction in the tolerant strain (AKR/J). To relate the above biochemical changes to in vivo effects, TCDD was postnatally administered (through mother's milk) to mouse neonates. The most prominent toxic manifestations were early eye opening and incisor eruption, loss in body weight gain, and retardation of hair growth. All of these symptoms have been ascribed to EGF effects. TCDD was also found to stimulate phosphorylation of the EGF receptor in the rat hepatic plasma membrane. This phosphorylation effect was observed at day 1 and persisted until the end of the test (day 10). It has long been recognized that agents causing reduction in number of EGF receptors (e.g., phorbol esters) elicit in vivo cellular responses that are similar to those caused by exposure to excess doses of growth factors. Accordingly, a hypothesis has been proposed to ascribe some of the EGF-like effects of TCDD, such as fatty infiltration of the liver and hyperplastic proliferation of gastric epithelia and epidermal cells to its action on the EGF receptor. 2,3,7, is an extremely toxic compound. The oral LD50 values in the male guinea pig and the rat are cited as 0.6 ,g/kg and 22 pug/kg, respectively (1). The action pattern of TCDD is unusual in that animals treated with a single dose steadily lose weight for several weeks, until death occurs, usually in 2-8 weeks. There are marked species differences in sensitivity (2): e.g., as compared to the above species, the Golden Syrian hamster is very tolerant to TCDD; its LD50 value is cited as >5000 pug/kg. Furthermore, the toxic manifestations also qualitatively vary from species to species. The basic biochemical cause for such toxic actions of TCDD is unknown (3).We have recently found that many plasma membrane proteins and functions of the hepatocytes from TCDD-treated rats are quantitatively different from those of untreated rats (4-6).As a follow-up of such a study, the current investigation was undertaken with the following objectives: (i) find several biochemical parameters on the plasma membrane that are severely affected by TCDD, (ii) study whether any of the changes occur at low enough doses and at very early stages in susceptible species, and only at high doses in tol...

Since many chemical tumor promoters and some oncogenes have been shown to inhibit gap junction-mediated intercellular communication, the effect of various growth factors on gap junctional intercellular communication on normal human keratinocytes was examined. In order to measure the effect of the growth factors on gap junctional communication, the scrape loading/dye transfer technique was used on human keratinocytes grown in a serum-free medium in vitro. At 24 h after treatment epidermal growth factor (10 ng/ml), transforming growth factor-beta (1 ng/ml), whole bovine pituitary extract (70 micrograms/ml) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 ng/ml) inhibited intercellular communication. Treatment of these cells with transforming growth factor-beta (1 ng/ml) induced morphological changes in some of the cells and brought about selective intercellular communication within and between the nonaltered and altered cells. Epidermal growth factor and whole bovine pituitary extract, significantly enhanced [3H]thymidine uptake and also stimulated cellular proliferation under the experimental conditions used to inhibit intercellular communication. Both transforming growth factor-beta and TPA markedly inhibited [3H]thymidine uptake and induced differentiation of some of these cells. In order to study the possible mechanism by which the growth factors might inhibit intercellular communication, the effect of the growth factors on protein kinase C activation and alterations of intracellular free calcium was investigated. The results indicated that neither protein kinase C nor an increase in [Ca2+]i were involved in the modulation of gap junctional communication by epidermal growth factor or transforming growth factor-beta. The study suggests that in the human keratinocytes inhibition of intercellular communication may be involved (i) in the action of growth factors such as epidermal growth factor during cellular proliferation and (ii) in the differentiation of primary keratinocytes by transforming growth factor-beta.