Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

Abstract

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 microM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A(4) (LTA(4)) hydrolase and leukotriene C(4) (LTC(4)) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA(4) hydrolase and LTC(4) synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.

Figure 1. Summary of ROS detoxification pathways and eicosanoid biosynthetic pathways

Panel A: ROS detoxification pathways. Superoxide anion is metabolized to hydrogen peroxide by SOD. Hydrogen peroxide is then detoxified by catalase and/or various peroxidases and reductases including TrxR. In the presence of transition metals, hydrogen peroxide is converted to highly reactive hydroxyl radicals. Oxidized proteins and lipids are conjugated to glutathione by the GST enzymes to facilitate cellular elimination. Panel B: Eicosanoid biosynthetic pathways: Arachidonic acid,mobilized directly from membrane phospholipids, is oxidized to form various eicosanoids by cyclooxygenases (COX), lipoxygenases (LOX) or cytochrome P450 monooxygenases. For prostaglandin metabolism, arachidonic acid is metabolized via cyclooxygenases (COX-1 and COX-2) to produce an intermediate prostaglandin, PGH_2. Further metabolism by the prostanoid synthases, microsomal prostaglandin E₂ synthase (mPGES), prostaglandin F₂ synthase (PGFS), prostaglandin D₂ synthase (PGDS), prostaglandin I₂ synthase (PGIS) or thromboxane A₂ synthase (TXAS), forms prostaglandins (PGE₂, PGF_2α and PGD₂), prostacyclin (PGI₂) or thromboxane (TXA₂), respectively. For leukotriene biosynthesis, arachidonic acid is metabolized via 5-lipoxygenase (5-LOX), in association with the 5-LOX activating protein (FLAP), generating a series of intermediate metabolites, 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene A₄ (LTA₄). LTA₄ is further metabolized by either LTA₄ hydrolase to form leukotriene B₄ (LTB₄) or conjugated with glutathione by LTC₄ synthase to form leukotriene C₄ (LTC₄).

Figure 1. Summary of ROS detoxification pathways and eicosanoid biosynthetic pathways

Panel A: ROS detoxification pathways. Superoxide anion is metabolized to hydrogen peroxide by SOD. Hydrogen peroxide is then detoxified by catalase and/or various peroxidases and reductases including TrxR. In the presence of transition metals, hydrogen peroxide is converted to highly reactive hydroxyl radicals. Oxidized proteins and lipids are conjugated to glutathione by the GST enzymes to facilitate cellular elimination. Panel B: Eicosanoid biosynthetic pathways: Arachidonic acid,mobilized directly from membrane phospholipids, is oxidized to form various eicosanoids by cyclooxygenases (COX), lipoxygenases (LOX) or cytochrome P450 monooxygenases. For prostaglandin metabolism, arachidonic acid is metabolized via cyclooxygenases (COX-1 and COX-2) to produce an intermediate prostaglandin, PGH_2. Further metabolism by the prostanoid synthases, microsomal prostaglandin E₂ synthase (mPGES), prostaglandin F₂ synthase (PGFS), prostaglandin D₂ synthase (PGDS), prostaglandin I₂ synthase (PGIS) or thromboxane A₂ synthase (TXAS), forms prostaglandins (PGE₂, PGF_2α and PGD₂), prostacyclin (PGI₂) or thromboxane (TXA₂), respectively. For leukotriene biosynthesis, arachidonic acid is metabolized via 5-lipoxygenase (5-LOX), in association with the 5-LOX activating protein (FLAP), generating a series of intermediate metabolites, 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene A₄ (LTA₄). LTA₄ is further metabolized by either LTA₄ hydrolase to form leukotriene B₄ (LTB₄) or conjugated with glutathione by LTC₄ synthase to form leukotriene C₄ (LTC₄).

Figure 2. CEES stimulates intracellular hydrogen peroxide production and protein oxidation

Panel A: Keratinocytes were incubated with DCFH-DA (5 μM) for 15 min.. The cells were then treated with CEES (100, 300 or 1000 μM) or control and analyzed by flow cytometry. Data are presented on a three decade log scale. Panel B: Keratinocytes were treated with CEES (100, 300 or 1000 μM) or control. After 24 hr, lysates were prepared and proteins derivatized with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone-modified carbonyl groups. After separation on SDS-polyacrylamide gels, modified proteins were quantified by Western blotting using antibodies to dinitrophenylhydrazone-tagged carbonyl groups.

Figure 3. Effects of CEES on keratinocyte expression of antioxidants

Cells were treated with CEES (100, 300 or 1000 μM) or control. After 24 hr, expression of Cu,Zn-SOD, Mn-SOD, catalase, and TrxR mRNA was analyzed by real-time PCR. Data are presented as fold change in gene expression relative to control. *Significantly (p < 0.05) different from control (p < 0.05).

Figure 4. Effects of CEES on protein expression of antioxidants, eicosanoid biosynthetic enzymes and MAP kinases

Cells were treated with CEES (100-1000 μM) or control and then analyzed for protein expression by Western blotting. β-catenin was analyzed as a control for equal protein loading. Panel A: Protein expression was analyzed 24 hr after CEES treatment. Panel B: Densitometric analysis of Western blot expression normalized to β-catenin expression. Panel C: MAP kinases were analyzed 5 min after CEES treatment.

Figure 5. Effects of CEES on keratinocyte expression of glutathione S-transferases

Cells were treated with CEES (100, 300 or 1000 μM) or control. After 24 hr, expression of cytosolic and microsomal glutathione S-transferases was analyzed by real-time PCR. Data are presented as fold change in gene expression relative to control. *Significantly (p < 0.05) different from control (p < 0.05).

Figure 6. Effects of CEES on expression of prostaglandin and leukotriene biosynthetic enzymes

Cells were treated with CEES (100, 300 or 1000 μM) or control. After 24 hr, expression of eicosanoid biosynthetic enzymes was analyzed by real-time PCR. Panel A: Keratinocyte expression of prostaglandin biosynthetic enzymes. Panel B: Keratinocyte expression of leukotriene biosynthetic enzymes. Data are presented as fold change in gene expression relative to control. *Significantly (p < 0.05) different from control (p < 0.05).

Figure 7. Effects of MAP kinase inhibitors on CEES-induced expression of antioxidants and eicosanoid biosynthetic enzymes

Cells were incubated with the p38 MAP kinase inhibitor or JNK MAP kinase inhibitor or control (CTL) for 3 hr and then with CEES (100-1000 μM) or control. After 24 hr, gene expression was analyzed by real-time PCR. Data are presented as fold change in gene expression relative to control. ^aSignificantly (p < 0.05) different from control (p38 inhibitor); ^bSignificantly different (p < 0.05) from control (JNK inhibitor).