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

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.

Interleukin-11 and interleukin-6 protect cultured human endothelial cells from H2O2-induced cell death.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with 100% oxygen. Previous studies have determined that both interleukin (IL)-6 and IL-11 are protective in oxygen toxicity. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene (-) animals, and both IL-6 and IL-11 markedly diminished this cell death response. However, the mechanism(s) by which these cytokines protect cells from death is unclear. In the present study, we characterized the effects of H2O2 on subconfluent human umbilical vein endothelial cell (HUVEC) and human pulmonary microvascular endothelial cell (HPMEC) cultures. We found that preincubation of HUVEC cultures with either IL-6 or IL-11 diminished H2O2 (1.0 mM)-induced cell death. Similar effects were noted with HPMEC showing that this effect is not HUVEC-specific. The protective effects of both IL-6 and IL-11 were not associated with any changes in antioxidants and were decreased by approximately 80% in the presence of U0126, a specific inhibitor of MEK-1-dependent pathways. The cytoprotective effects of IL-11 and IL-6 were also completely eliminated in STAT3 dominant-negative transduced HUVEC cultures. These studies demonstrate that IL-6 and IL-11 both confer cytoprotective effects that diminish oxidant-mediated endothelial cell injury. They also demonstrate that this protection is mediated, at least in part, by a STAT3 and MEK-1-dependent specific signal transduction pathway(s).

Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.

Cytosolic Ca(2+) (Ca(i)(2+)) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca(2+) waves, Ca(2+) oscillations, and other subcellular Ca(2+) signaling patterns, but little is known about the subcellular organization of Ca(2+) signaling in cholangiocytes. Therefore, we examined Ca(2+) signaling and the subcellular distribution of Ca(2+) release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca(2+) signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP(3)R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP(3)R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP(3)R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca(2+) via InsP(3) in cholangiocytes, induced Ca(2+) oscillations in both cholangiocytes and NRC cells. Acetylcholine (ACh) induced apical-to-basal Ca(2+) waves. In conclusion, Ca(2+) signaling in cholangiocytes occurs as polarized Ca(2+) waves that begin in the region of the type III InsP(3)R. Differential subcellular localization of InsP(3)R isoforms may be an important molecular mechanism for the formation of Ca(2+) waves and oscillations in cholangiocytes. Because Ca(i)(2+) is in part responsible for regulating ductular secretion, these findings also may have implications for the molecular basis of cholestatic disorders.