Junping Wei

Osteopontin inhibits expression of cytochrome c oxidase in RAW 264.7 murine macrophages.

Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two CAM molecules: alpha(v)beta(3)-integrin and CD44. OPN initiates a number of signal transduction pathways that control cell survival, proliferation, and migration. In this study, utilizing RAW 264.7 murine macrophages, we demonstrate that expression of the mitochondrial protein, CCOI, is significantly decreased in the setting of OPN stimulation. This effect is blocked by the CD44 competitive ligand, hylauronate; GRGDSP, a hexapeptide that blocks OPN-integrin binding, had no effect. CCOI mRNA and transcription were significantly decreased in the presence of OPN; CCOI mRNA half-life was unaltered by OPN. Additional mitochondrial run-on studies, which included genes from L-strand and H-strand, suggest that OPN terminates transcription of the distal H-strand. CCO enzyme activity was also significantly decreased by OPN. Our results indicate that OPN inhibits CCOI expression as the result of a novel CD44-dependent transcriptional regulatory mechanism of the mitochondrial H strand.

Endotoxin-Stimulated Nitric Oxide Production Inhibits Expression of Cytochrome c Oxidase in ANA-1 Murine Macrophages

In endotoxin (LPS)-mediated states of sepsis, inducible NO synthase expression and NO production are associated with molecular regulatory functions that determine the host inflammatory response. NO inhibits cellular respiration and mitochondrial electron transport by inhibition of cytochrome c oxidase (CcO) activity. CcO is the terminal complex of the mitochondrial respiratory chain, responsible for 90% of cellular oxygen consumption and essential for cellular energy production. Subunit 1 (CcO I) is considered to be the most critical of the 13 CcO component subunits. In this regard little is known of the effect of NO on the transcriptional program for CcO expression. In ANA-1 murine macrophages, LPS-mediated NO synthesis decreases CcO enzyme activity, CcO I protein expression, and CcO I steady mRNA levels. Mitochondrial run-on analysis demonstrates unaltered CcO I mitochondrial gene transcription. Half-life analysis indicates that CcO I mRNA stability is significantly decreased in the presence of LPS-mediated NO synthesis. In this study using LPS-stimulated ANA-1 murine macrophages, we demonstrate that expression of the mitochondrial gene product, CcO I, is significantly decreased as the result of a unique and previously uncharacterized, NO-dependent post-transcriptional regulatory mechanism.

Peroxide-mediated chromatin remodelling of a nuclear factor kappa B site in the mouse inducible nitric oxide synthase promoter

Hepatocyte expression of iNOS (inducible nitric oxide synthase) and synthesis of nitric oxide convey protective antioxidant functions in models of sepsis, shock and reperfusion. However, the underlying redox-sensitive mechanisms that regulate hepatocyte expression of iNOS and its antioxidant functions are largely unknown. Activity of the transcription factor NF-kappaB (nuclear factor kappaB) is known to be redox-modulated. In this regard, the mouse hepatocyte iNOS promoter has NF-kappaB-binding sites at nt -1044 to -1034 and at nt -114 to -104, which are considered to be critical for iNOS expression in response to pro-inflammatory cytokine stimulation. The relative contribution of these two NF-kappaB-binding sites in the mouse iNOS promoter to hepatocyte iNOS promoter activity in the context of oxidative stress has not been characterized previously. In addition, although the cis - and trans -regulatory factors controlling mouse hepatocyte iNOS expression have been well-characterized, the local changes in chromatin structure that accompany activation of iNOS gene transcription have not been considered. In the present study, we demonstrate that (1) in the absence of exogenous oxidative stress, the NF-kappaB site at nt -114 is inactive and (2) peroxide-mediated oxidative stress induces hyperacetylation and enhanced accessibility of the restriction enzyme to this NF-kappaB region. Our results suggest that chromatin structural changes activate this NF-kappaB site and increase interleukin-1beta-stimulated iNOS expression in the presence of oxidative stress.