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. 2003 Dec;163(6):2555-63.
doi: 10.1016/S0002-9440(10)63610-3.

MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant-induced lung injury

Leo E Otterbein  1 , Sherrie L Otterbein, Emeka Ifedigbo, Fang Liu, Danielle E Morse, Colleen Fearns, Richard J Ulevitch, Roy Knickelbein, Richard A Flavell, Augustine M Choi
Affiliations

MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant-induced lung injury

Leo E Otterbein et al. Am J Pathol. 2003 Dec.

Abstract

The stress-inducible gene heme oxygenase (HO-1) has previously been shown to provide cytoprotection against oxidative stress. The mechanism(s) by which HO-1 provides this cytoprotection is poorly understood. We demonstrate here that carbon monoxide (CO), a byproduct released during the degradation of heme by HO, plays a major role in mediating the cytoprotection against oxidant-induced lung injury. We show in vitro that CO protects cultured epithelial cells from hyperoxic damage. By using dominant negative mutants and mice deficient in the genes for the various MAP kinases, we demonstrate that the cytoprotective effects of CO are mediated by selective activation of the MKK3/p38 beta protein MAP kinase pathway. In vivo, our experiments demonstrate that CO at a low concentration protects the lungs, extends the survival of the animals, and exerts potent anti-inflammatory effects with reduced inflammatory cell influx into the lungs and marked attenuation in the expression of pro-inflammatory cytokines.

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Figures

Figure 1.
Figure 1.
A: Effects of CO on survival following exposure to lethal hyperoxia. Mice in groups of 6 to 8 were exposed to >98% O2 or >98% O2 plus 250 ppm CO continuously and monitored for survival. Mice exposed to only O2 succumbed to O2 toxicity by 90 to 100 hours while >95% of those mice exposed in identical chambers to O2 in the presence of CO (250 ppm) were alive at 100 hours, with >50% alive at 128 hours of exposure. Data represent 20 to 25 animals per treatment group. B: Effects of CO on hyperoxia-induced PMN influx into the airways of mice. C: Effects of CO on hyperoxia-induced protein in the BAL. Mice were exposed continuously to either >98% O2 or >98% O2 plus 250 ppm CO. At 84 hours, a BAL was performed and PMN differential counts was determined. Total protein in the BAL was determined by Bradford assay. Data represent mean ± SEM of 8 to 10 mice per group. * P < 0.001 vs. air and CO. # P < 0.05 vs. CO/O2. D: Effects of CO (250 ppm) on hyperoxia-induced cytokine mRNA expression. Mice were exposed to >98% O2 in the presence or absence of CO (250 ppm). At 84 hours of exposure, the lungs were harvested and analyzed by RNase protection using the cytokine templates containing the above listed cytokines. L32 and GAPDH were used as loading controls. The blots are representative of three separate experiments in which the lungs from three mice were pooled for RNA extraction.
Figure 2.
Figure 2.
A: Hyperoxia activates the MAP kinase pathways in vivo in the lung. Mice were exposed continuously to hyperoxia and at 24, 48, and 72 hours, the lungs were removed and analyzed for the presence of phosphorylated ERK1/ERK2, JNK, p38, and MKK3/6 MAP kinases. Lanes 1 and 2, untreated air controls; 3 and 4, 24 hours hyperoxia; 5 and 6, 48 hyperoxia; 7 and 8, 72 hours hyperoxia. Total ERK1/ERK2, JNK, p38, and MKK3/6 immunoblotting were used as normalization controls. B: Effects of hyperoxia on p38 expression in vitro. A549 cells were exposed continuously to hyperoxia for 15 and 30 minutes to observe any effects on p38 phosphorylation versus untreated controls. Protein lysates were assayed by Western blot using an anti-phosphorylated p38 antibody. The membranes were then stripped and reprobed with an anti-p38 antibody to assess any differences in loading. C: Effects of CO on pan-p38 and MKK3 activation. A549 cells were exposed to CO (250 ppm) continuously for 0 and 30 minutes, and 1, 4, 8, 16, and 24 hours (lanes 1 to 7, respectively) and assayed for pan-p38 and MKK3 phosphorylation by Western blot versus untreated controls. D: A549 cells were exposed to either 5%CO2/air or 5%CO2/air/CO (250 ppm) for 24 hours and then assayed for p38α and p38β by Western blot following immunoprecipitation. Data are representative blots from three independent experiments.
Figure 3.
Figure 3.
A: Effects of CO on survival in mkk3(−/−) mice following exposure to lethal hyperoxia. Null mice and wild-type littermates were exposed to >98% O2 or >98% O2 plus 250 ppm CO continuously and monitored for survival. Mkk3(−/−) mice were hypersusceptible to O2, succumbing between 65 and 72 hours versus wild-type littermates that died between 90 and 100 hours. Furthermore, survival of the mkk3(−/−) mice could not be extended by CO (250 ppm) as observed in wild-type littermate controls. Data represent 20 to 25 animals per treatment group. B: Effects of CO (250 ppm) on hyperoxia-induced cytokine mRNA expression. Mkk3(−/−) or control littermates were exposed to >98% O2 in the presence or absence of CO (250 ppm). At 65 hours of exposure, the lungs from the mkk3(−/−) and control littermates were harvested and analyzed by RNase protection using the cytokine templates containing the cytokines listed above. L32 and GAPDH are included as loading controls. The blots are representative of three separate experiments in which the lungs from three mice were pooled for RNA extraction.
Figure 4.
Figure 4.
Effects of CO on survival of A549 cells exposed to O2. A549 cells were treated with the chemical inhibitor of p38β or SB203580 (10 μmol/L) (A) or transiently transfected with dominant negative mutants (DN) of p38β or MKK3 (1 μg/plate) (B) and then exposed to 95% O2 in the presence and absence of CO (250 ppm) for 4 days. pcDNA3 (1 μg/plate) was used as the vector control. After 4 days of exposure, cells were assessed for viability versus transfected and SB treated non-exposed cells. Results are expressed as a percentage of non-exposed cells from four replicate plates ± SD. There were no adverse effects of transfection or chemical treatment on viability in non-exposed cells. * P < 0.05 vs. O2-treated.
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