Modulation of Human Tumor Suppressor Genes, Gadd45, p53 and p38 MAPK by Zinc Status in Normal Human Bronchial Epithelial Cells

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The effect of zinc status on the cell signaling transduction of tumor suppressor genes, Growth Arrest and DNA Damage inducible gene (Gadd45), p53, and p38 Mitogen Activated Protein Kinase (MAPK) were examined in Normal Human Bronchial Epithelial (NHBE) cells. Cells were cultured for one passage in different concentrations of zinc: < 0.4 μM (ZD, zinc-deficient); 4 μM (ZN, zinc normal) as normal zinc level found in most culture medium; 16 μM (ZA, zinc adequate) represented normal human plasma zinc level; and 32 μM (ZS, zinc supplementation) represented the optimal plasma zinc attainable by oral supplementation. Cell growth inhibition, up-regulation of Gadd45, p53 and p38 MAPK mRNA and protein expressions, and blockage of G2/M cell cycle progression were observed in ZS cells. The siRNA-mediated knocking down of Gadd45 was found to alleviate G2/M blockage partially in ZS cells, which indicated that the blockage is partially Gadd45 dependent. In ZS cells, the enhanced phosphorylation of p38 MAPK and p53 were abrogated after suppressing Gadd45 by siRNA, implicating that the enhanced phosphorylation of p53 and p38 MAPK were Gadd45 dependent. By using p53 transactivation inhibitor Pifithrin, the upregulated Gadd45 protein, the enhanced Gadd45 promoter activity, and the reduced level of CDK1/Cyclin B1 complex were all restored back to normal levels in ZS cell, implying that these ZS induced changes were p53 dependent. Furthermore, the ZS induced upregulation of Gadd45 expression, displacement of CDC25B from nucleus to cytoplasm, reduction of CDK1/Cyclin B1 complex level, enhancement of the activation and phosphorylation of p53, and delay of G2/M cell cycle progression were normalized by p38 MAPK dominant negative and protein inhibitor SB202190. Thus, the ZS induced changes were dependent on the activation of p38 MAPK. Our data support the involvement of a positive Gadd45, p53 and p38 MAPK feedback loop in response to stress induced by zinc supplementation. These findings demonstrate the importance of p38 MAPK and p53 in the regulation of G2/M cell cycle progression in response to the stress induced by high zinc via Gadd45 and cell cycle checkpoint regulatory proteins, including CDK1, Cyclin B1 and CDC25B.