Enhanced Gadd45 expression and delayed G2/M progression are p53-dependent in zinc-supplemented human bronchial epithelial cells.

Zinc is an essential nutrient for humans; however, this study demonstrated for the first time that an elevated zinc status, created by culturing cells at optimal plasma zinc concentration attainable by oral zinc supplementation, is cytotoxic for normal human bronchial epithelial (NHBE) cells. p53 plays a central role in the modulation of cell signal transduction in response to the stress from DNA damage, hypoxia and oncogene activation. The present study was designed to determine whether the previously reported increased Gadd45 expression and delayed G2/M cell cycle progression in zinc-supplemented NHBE cells is p53-dependent, and to decipher the mechanisms responsible for the regulation of Gadd45 expressions by p53, and elucidate the Gadd45 functions in impaired cell growth and cell cycle progression in NHBE cells. Cells were cultured for one passage in different concentrations of zinc: <0.4 micromol/L (ZD) as severe zinc-deficient; 4 micromol/L (ZN) as normal zinc level in culture medium; 16 micromol/L (ZA) as normal human plasma zinc level; and 32 micromol/L (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth and upregulation of p53 mRNA and protein expression were observed in ZS cells. Most importantly, ZS treatment also enhanced Gadd45 nuclear protein level and promoter activity, decreased CDK1-Cyclin B1 level and delayed G2/M cell cycle progression. These changes were normalized to those observed in ZN by treating ZS cells with Pifitherin, an inhibitor of p53 transactivation activity. Thus, our findings support the p53 dependency of the Gadd45-CDK1/Cyclin B1-G2/M cell cycle progression pathway in ZS NHBE cells.

Zinc-induced G2/M blockage is p53 and p21 dependent in normal human bronchial epithelial cells.

The involvement of p53 and p21 signal pathway in the G2/M cell cycle progression of zinc-supplemented normal human bronchial epithelial (NHBE) cells was examined using the small interferring RNA (siRNA) approach. Cells were cultured for one passage in a different concentration of zinc: <0.4 microM (ZD) as zinc deficient; 4 microM as normal zinc level (ZN) in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 microM (ZS) as the high end of plasma zinc attainable by oral supplementation. Nuclear p21 protein and mRNA levels as well as promoter activity in ZS cells, but not in ZD cells, were markedly elevated to almost twofold compared with ZN control cells. G2/M blockage in ZS cells was coupled with the observation of elevated p21 gene expression. In ZS cells, the abrogation of p21 protein induction by the transfection of p21 siRNA was shown to alleviate the G2/M blockage, demonstrating the positive linkage of p21 elevation and G2/M blockage. Abolishment of the increase in p53 protein in ZS cells with transfection of p53 siRNA normalized the elevated p21 protein to a similar level as in ZN control cells, which demonstrated that the p21 induction is p53 dependent. Furthermore, the normalization of p53 protein by siRNA treatment in ZS cells alleviated cell growth depression and G2/M blockage, which demonstrated that p53 was involved in the high zinc status-induced G2/M blockage and growth depression. Thus high zinc status in NHBE cells upregulates p53 expression which in turn elevates p21 that eventually induces G2/M blockage.

Suppression of Gadd45 alleviates the G2/M blockage and the enhanced phosphorylation of p53 and p38 in zinc supplemented normal human bronchial epithelial cells.

An adequate zinc status is essential for optimal cellular functions and growth. Yet, excessive zinc supplementation can be cytotoxic and can impair cell growth. Gadd45 plays a vital role as cellular stress sensor in the modulation of cell signal transduction in response to stress. The present study was designed to determine the influence of zinc status on Gadd45 expression and cell cycle progression in zinc deficient and supplemented normal human bronchial epithelial (NHBE) cells, and to decipher the molecular mechanism(s) exerted by the suppression of Gadd45 expression on cell growth and cell cycle progression in this cell type. Cells were cultured for one passage in different concentration of zinc: <0.4 muM (ZD) as severe zinc deficient; 4 muM as normal zinc level in culture medium; 16 microM (ZA) as normal human plasma zinc level; and 32 muM (ZS) as the high end of plasma zinc attainable by oral supplementation. Inhibition of cell growth, upregulation of Gadd45 mRNA and protein expression, and blockage of G2/M cell cycle progression were observed in ZS cells. In contrast, little or no changes in these parameters were seen in ZD cells. The siRNA-mediated knocking down of Gadd45 was found to relieve G2/M blockage in ZS cells, which indicated that the blockage was Gadd45 dependent. Moreover, the enhanced phosphorylation of p38 and p53 (ser15) in ZS cells was normalized after suppression of Gadd45 by siRNA, implicating that the enhanced phosphorylation of these proteins was Gadd45 dependent. Thus, we demonstrated for the first time that an elevated zinc status modulated signal transduction to produce a delay at G2/M during cell cycle progression in NHBE cells.

Zinc deficiency depresses p21 gene expression: inhibition of cell cycle progression is independent of the decrease in p21 protein level in HepG2 cells.

The influence of zinc status on p21 gene expression was examined in human hepatoblastoma (HepG2) cells. Cells were cultured for one passage in a basal medium depleted of zinc to induce severely zinc-deficient (ZD) cells or in basal medium supplemented with 0.4, 4.0, 16, or 32 microM zinc to represent mild zinc deficiency (ZD0.4), the amount of zinc in most normal media (ZN), the normal human plasma zinc level (zinc-adequate; ZA), or the high end of plasma zinc attainable by oral supplementation (ZS), respectively. In ZD and ZD0.4 cells, the nuclear p21 protein level, mRNA abundance, and promoter activity were reduced to 40, 70, and 65%, respectively, of ZN cells. However, p21 protein and mRNA levels, as well as p21 promoter activity, were not altered in ZA and ZS cells compared with ZN cells. Moreover, the amounts of acetylated histone-4 associated with the proximal and distal p21 promoter regions, as a measure of p21 promoter accessibility, were decreased in ZD (73 and 64%, respectively) and ZD0.4 (82 and 77%, respectively) cells compared with ZN cells (100 and 100%, respectively). Thus multiple lines of evidence indicate that the transcriptional process of p21 is downregulated by depressed zinc status in HepG2 cells. Furthermore, the transfection of 5 microg of plasmid cytomegalovirus-p21 plasmid, which constitutively expressed p21, was able to normalize the reduction in p21 protein level and cyclin D1-cdk4 complex activity but not the inhibition of cell growth and G1/S cell cycle progression in ZD cells.