phosphatidylethanol and Adenocarcinoma

Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation.

Topics: Adenocarcinoma; Alcohol Dehydrogenase; Animals; Caco-2 Cells; CCAAT-Enhancer-Binding Proteins; Cell Count; Claudin-1; Colonic Neoplasms; Endocytosis; Ethanol; Glycerophospholipids; Heat-Shock Proteins; Humans; Hyperplasia; Intestines; Membrane Proteins; Mice; Phospholipase D; Phosphoproteins; Zonula Occludens-1 Protein

Phospholipase D (PLD) is involved in the signaling by many extracellular ligands, and its regulation appears to be quite complex. We investigated the signaling pathways initiated by bradykinin (BK) or sphingosine 1-phosphate (S1P) in A549 cells to define molecular mechanisms responsible for their additive effects on PLD activity. BK and S1P each elicited a sustained increase in phosphatidic acid content through a rapid and transient activation of PLD. The two pathways demonstrated rapid homologous downregulation, but heterologous desensitization was not observed. Action of both agonists required protein kinase C (PKC) activation and Ca(2+) influx but was mediated by different heterotrimeric G proteins. In membranes, inhibition of PKCdelta by rottlerin enhanced BK activation of PLD but inhibited that by S1P. Rottlerin inhibited activation of PLD in nuclei by both BK and S1P. By in situ immunofluorescence or cell fractionation followed by immunoblotting, PLD1 was concentrated primarily in nuclei, whereas the membrane fraction contained PLD2 and PLD1. Moreover, PKCdelta specifically phosphorylated recombinant PLD2, but not PLD1. BK and S1P similarly enhanced RhoA translocation to nuclei, whereas BK was less efficacious than S1P on RhoA relocalization to membranes. Effects of both agonists on the nuclear fraction, which contains only PLD1, are compatible with a RhoA- and PKCdelta-dependent process. In membranes, which contain both PLD1 and PLD2, the stimulatory effect of S1P on PLD activity can best be explained by RhoA- and PKCdelta-dependent activation of PLD1; in contrast, the effects of BK on RhoA translocation and enhancement of BK-stimulated PLD activity by PKC inhibition are both consistent with PLD2 serving as its primary target.

Topics: Adenocarcinoma; Bradykinin; Calcium; Enzyme Activation; Glycerophospholipids; Heterotrimeric GTP-Binding Proteins; Humans; Lung Neoplasms; Lysophospholipids; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Protein Kinase C-delta; Protein Transport; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine; Subcellular Fractions; Tritium; Tumor Cells, Cultured