Hydrogen peroxide as a potential mediator of the transcriptional regulation of heparan sulphate biosynthesis in keratinocytes.

Ionizing radiation is one of the types of oxidative stress that has a number of damaging effects on cutaneous tissues. One of the histological features of radiation-induced cutaneous fibrosis is the accumulation of extracellular matrix (ECM) components, including heparan sulfate proteoglycan (HSPG), which are required for the repair of tissue damage, and operate by interacting with a variety of growth factors. In this study, we established a model of human HaCaT keratinocytes overexpressing anti-oxidative enzyme genes to elucidate the mechanism of oxidative stress leading to the accumulation of HSPG and the role of its accumulation. Catalase overexpression induced an increase in anti-HS antibody (10E4) epitope expression in these cells. Western blotting showed that the smeared bands of HSPG were obviously shifted to a higher molecular weight in the catalase transfectants due to glycosylation. After heparitinase I treatment, the core proteins of HSPG were expressed in the catalase transfectants to almost the same extent as in the control cells. In addition, the transcript levels of all the enzymes required for the synthesis of the heparan sulfate chain were estimated in the catalase transfectant clones. The levels of five enzyme transcripts - xylosyltransferase-II (XT-II), EXTL2, D-glucuronyl C5-epimerase (GLCE), HS2-O-sulfotransferase (HS2ST), and HS6-O-sulfotransferase (HS6ST) - were significantly increased in the transfectants. Moreover, hydrogen peroxide was found to down-regulate the levels of these enzymes. By contrast, siRNA-mediated repression of catalase decreased 10E4 epitope expression, the transcript level of HS2ST1, and the growth rate of HaCaT cells. These findings suggested that peroxide-mediated transcriptional regulation of HS metabolism-related genes modified the HS chains in the HaCaT keratinocytes

Role of catalase in monocytic differentiation of U937 cells by TPA: hydrogen peroxide as a second messenger

Human promonocytic cell line U937 cells can be induced to differentiate into macrophages by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment induced the expression of the monocytic differentiation markers CD11b and CD36, with concomitant morphological changes. Moreover, TPA enhanced reactive oxygen species (ROS) generation in these cells, and phagocytic ability was also stimulated during differentiation. The antioxidant agent N-acetyl-L-cysteine inhibited the TPA-induced differentiation of U937 cells. TPA treatment decreased the expression level of catalase, which catalyzes the decomposition of hydrogen peroxide (H(2)O(2)) to H(2)O and O(2). In contrast, TPA increased the level of manganese superoxide dismutase, which catalyzes the dismutation of superoxide into H(2)O(2) and O(2) without affecting the levels of copper-zinc superoxide dismutase or glutathione peroxidase 1, which removes H(2)O(2) using glutathione as substrate. Treatment of U937 cells with catalase inhibited the enhancement of ROS generation induced by TPA, and blocked the TPA-induced differentiation of U937 cells. Human promyelocytic cell line HL60 cells were also induced to differentiate into macrophages by TPA. However, HP100-1 cells, its variant cell line overexpressing catalase, were resistant to TPA-induced differentiation. Our results suggest that catalase inhibits monocytic differentiation by TPA; the decrease in catalase level and the accumulation of H(2)O(2) are significant events for monocyte/macrophage differentiation by TPA