Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

Abstract

4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86-98 fold within 6h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2-/- mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress.

Keywords: Antioxidants; Caveolae; Heme oxygenase-1; Nrf2; Skin; Stress proteins.

Figure 1. Metabolism of 4-HNE in PAM 212 keratinocytes

Cells were treated with 30 or 100 μM 4-HNE for increasing periods of time up to 120 min. For analysis of 4-HNE, cells were washed and 4-HNE extracted as described in the Materials and Methods. Panel A. Representative HPLC tracing of 4-HNE extracted from cells 15 min and 120 min post treatment with 100 μM 4-HNE. Panel B. Time course of 4-HNE accumulation and degradation in cells treated with 100 μM 4-HNE. Panel C. Time-course of 4-HNE metabolism in S9 fractions of mouse keratinocytes. S9 fractions of PAM 212 cells were incubated with 100 μM 4-HNE in the absence and presence of 1 mM NADH or NADPH. At the indicated times, 4-HNE was extracted and quantified by HPLC. Panel D. Time-dependent formation of 4-HNE-protein adducts in intact cells treated with control, 30, or 100 μM 4-HNE. For this analysis, treated cells were lysed and analyzed by Western blotting using a monoclonal antibody to 4-HNE. Arrowheads show the appearance of modified proteins with molecular weights of 43 and 75 kDa.

Figure 2. Effects of 4-HNE on HO-1 expression in keratinocytes

PAM 212 cells (panels A and B) or primary cultures of mouse keratinocytes (panels C and D) were treated with increasing concentrations of 4-HNE for 6 h (panels A and C) or with 30 μM 4-HNE for increasing periods of time (panels B and D). Cells were then analyzed for HO-1 mRNA and protein expression by real time PCR and Western blotting, respectively. In each panel, the upper figures show HO-1 mRNA expression, while the lower panels show Western blots for HO-1 protein expression. Expression of β-actin is shown in each of the western blots as a control. Data for mRNA are expressed as fold increase relative to control. Bars represent the mean ± SE (n = 3).

Figure 3. Role of MAP kinase and PI3K/Akt signaling in 4-HNE-induced HO-1 expression in keratinocytes

Upper panel. Effects of 4-HNE on expression of MAP kinases and PI3K/Akt. PAM 212 keratinocytes were treated with vehicle control or 30 μM 4-HNE for 15, 30 or 60 min. Cell lysates were prepared and analyzed for total and phosphorylated p38, JNK, Erk1/2, or PI3K/Akt by western blotting. Center panel. Effects of MAP kinase and PI3K/Akt inhibitors on 4-HNE-induced HO-1 mRNA expression. PAM 212 cells and primary mouse keratinocytes were pre-incubated for 3 h with inhibitors of p38 kinase (SB203580, 10 μM), JNK (SP600125, 20 μM), Erk1/2 (PD98059, 10 μM) or PI3K (wortmannin, 0.1 μM) and then with 30 μM 4-HNE for additional 6 h. Expression of HO-1 mRNA was analyzed by real-time PCR and is expressed as fold-increase relative to vehicle control. Each bar represents the mean ± SE (n = 3). *Significantly different from control (p < 0.05). Lower panel. Effects of MAP kinase and PI3K/Akt inhibitors on 4-HNE-induced HO-1 protein expression in PAM 212 keratinocytes. Total cell lysates were prepared from control and treated cells and analyzed for HO-1 protein expression by Western blotting.

Figure 4. Localization of 4-HNE-induced HO-1 in caveolae

Upper panel. Distribution of HO-1 in caveolar fractions (CF) and non-caveolar fractions (NCF) of PAM 212 cells. Keratinocytes were treated with 0, 10 or 30 μM 4-HNE. After 6 h, caveolar and non-caveolar fractions were prepared and analyzed for HO-1 and caveolin-1 (Cav-1) protein expression by western blotting. Center panel. Effects of kinase inhibitors on expression of HO-1 in caveolar and non-caveolar fractions of PAM 212 cells. Keratinocytes were pre-incubated with MAP kinase and PI3K inhibitors and then 4-HNE (0, 3, 10 or 30 μM) as described in the legend to Figure 3. After an additional 6 h, caveolar and non-caveolar fractions were prepared and analyzed for HO-1 protein by western blotting. Lower panel. Effects of cholesterol depletion on HO-1 expression. Cells were pretreated with 5 mM methyl-β-cyclodextrin (MbCD) or control (C) for 3 h and then with 4-HNE for 6 h. Total cell lysates were then prepared and analyzed for HO-1 protein expression by western blotting.

Figure 5. Role of Nrf2 in 4-HNE-induced HO-1 expression in mouse keratinocytes

Keratinocytes were incubated with control or 4-HNE and analyzed for Nrf2 or HO-1 expression. Upper panel. Effects of 4-HNE on nuclear localization of Nrf2. PAM 212 cells were treated with 30 μM 4-HNE for 0, 1, 3 and 6 h or 0, 10, 30 and 100 μM 4-HNE for 3 h. Nuclear and cytoplasmic fractions of the cells were then prepared and Nrf2 expression analyzed by western blotting. Lower panel. Effects of Nrf2 expression on 4-HNE-induced HO-1 expression. Primary keratinocytes from wild type and Nrf2−/− mice were treated with 0, 10, 30 and 100 μM 4-HNE for 6 h and expression of HO-1 mRNA (upper panel) and protein (lower panel) analyzed by real-time PCR and Western blotting, respectively, as described in the Materials and Methods. HO-1 mRNA expression is presented as fold-increase relative to control. Each bar represents the mean ± SE (n = 3).