Mitogen-activated protein kinases regulate HO-1 gene transcription after ischemia-reperfusion lung injury

Abstract

Lung ischemia-reperfusion (I-R) is an important model of oxidant-mediated acute lung and vascular injury. Heme oxygenase-1 (HO-1) is a cytoprotective gene that is markedly induced by lung I-R injury. HO-1 mRNA is increased in mouse lung after 30 min of lung hilar clamping (ischemia) followed by 2-6 h of unclamping (reperfusion) compared with control mice. In a variety of vascular cell types, HO-1 mRNA is induced after 24 h of anoxia followed by 30 min-1 h of reoxygenation (A-R). Transfection studies reveal that the promoter and 5'-distal enhancer E1 are necessary and sufficient for increased HO-1 gene transcription after A-R. Immunoblotting studies show all three subfamilies of MAPKs (ERK, JNK, and p38) are activated by 15 min of reperfusion. We also demonstrate that HO-1 gene transcription after A-R involves ERK, JNK, and p38 MAPK pathways. Together, our data show that I-R not only induces HO-1 gene expression in mouse lungs and vascular cells but that gene transcription occurs via the promoter and E1 enhancer and involves upstream MAPK pathways.

Carbon Monoxide Modulates Fas/Fas Ligand, Caspases, and Bcl-2 Family Proteins via the p38α Mitogen-activated Protein Kinase Pathway during Ischemia-Reperfusion Lung Injury

“…Cells were incubated for 6 h with DNA mixtures containing serum-free medium, Fu-GENE 6 reagent (Roche Applied Science), and wild type or dominant negative mutant plasmids. After incubation, cells were cultured for an additional 16 h in complete medium and then exposed to A-R in the presence or absence of 15 ppm CO. We have demonstrated the transfection efficiency of PAECs to exceed 80% using pEGFP transfections and by examining the cells under phase-contrast and fluorescence microscopy as described previously (15).…”

“…1A we show a time course of when p38 activation occurs in the presence and absence of CO in PAECs. CO increases phospho-p38 levels during anoxia after 8 h, but p38 activation is maximal at 24 h. Of note, in the absence of CO, pulmonary artery endothelial cell death is maximal after 24 h of anoxia and is maintained during 30 min to 8 h of reoxygenation (15). Therefore, in subsequent assays we use 24 h of anoxia and 24 h of anoxia followed by 1 h of reoxygenation as the time points of interest in PAECs.…”

Carbon Monoxide Differentially Modulates STAT1 and STAT3 and Inhibits Apoptosis via a Phosphatidylinositol 3-Kinase/Akt and p38 Kinase-dependent STAT3 Pathway during Anoxia-Reoxygenation Injury

“…To determine whether PI3K/Akt was upstream of p38 MAPK, we blocked the PI3K/Akt pathway with LY294002, a compound that acts as a competitive inhibitor of the ATP binding site of PI3K and has been shown to cause specific inhibition (23,24). We blocked the p38 MAPK signaling pathway with SB203580 as described previously (16). LY294002 pretreatment significantly inhibited CO-mediated activation of both Akt and p38 MAPK (Fig.…”

“…Endothelial Cell A-R CO Exposure-Rat pulmonary artery endothelial cells (PAEC) have been described previously (16) and were exposed to A-R in the presence or absence of 15 ppm of CO according to our previous methods (4).…”

“…After incubation, cells were cultured for an additional 16 h in complete medium and then exposed to A-R in the presence or absence of 15 ppm. Our past studies have demonstrated the transfection efficiency of PAEC to exceed 80% using pEGFP transfections (16).…”