Stabilization of Tumor Necrosis Factor α mRNA by Chronic Ethanol

Abstract

Increased expression of tumor necrosis factor ␣ (TNF␣) in response to chronic ethanol has been implicated in the pathogenesis of alcoholic liver disease. However, the molecular mechanisms by which ethanol increases the levels of TNF␣ are not well characterized. Utilizing Kupffer cells isolated from rats fed an ethanol containing diet and a murine macrophage cell line, RAW264.7, exposed to ethanol in culture, we have demonstrated that exposure to chronic ethanol results in an enhanced expression of lipopolysaccharide (LPS)-induced TNF␣. While chronic ethanol had no effect on the rate of LPS-induced TNF␣ transcription as measured by nuclear run-on experiments, TNF␣ mRNA half-life was increased by chronic ethanol. Chronic ethanol also potentiated the activation of LPS-induced p38 mitogenactivated protein (MAP) kinase in Kupffer cells, as well as in RAW264.7 cells. Specific inhibition of p38 MAP kinase activation by SB203580 in Kupffer cells or by overexpression of dominant negative p38 MAP kinase in RAW264.7 cells blocked ethanol-mediated TNF␣ mRNA stabilization. Furthermore, using chimeric reporter constructs, we have shown that A ؉ U-rich elements in the 3-untranslated region of TNF␣ mRNA are not sufficient to impart ethanol-mediated stabilization on TNF␣ mRNA.

Chronic Ethanol Increases Lipopolysaccharide-stimulated Egr-1 Expression in RAW 264.7 Macrophages

“…There was no difference in LPS stimulation of luciferase activity when the full-length TNF␣ promoter was linked to the luciferase reporter between control and ethanol-treated cells (1.8 Ϯ 0.1 relative luciferase activity in control, compared with 1.5 Ϯ 0.4 after chronic ethanol, n ϭ 6). This is consistent with our previous finding that chronic ethanol has no net effect on total LPS-stimulated TNF␣ transcription using nuclear run-on assays (29). However, chronic ethanol changed the relative contributions of the Egr-1 and NFB site to LPS-stimulated TNF␣ promoter activity.…”

“…Interestingly, LPS-stimulated reporter activity driven by the full-length TNF␣ promoter did not differ between control and ethanol-treated cells. This is consistent with our previous observation that chronic ethanol has no net effect on total LPS-stimulated TNF␣ transcription, measured by nuclear run-on assays (29). However, from the results reported here, it is clear that the contributions of Egr-1 and NFB to LPSstimulated transcription from the TNF␣ promoter are different in control versus ethanol-treated cells.…”

“…Overexpression of dominant negative Egr-1 decreased LPS-stimulated TNF␣ transcription by 50% in control RAW 264.7 macrophages (data not shown). Furthermore, we have previously reported that inhibition of ERK1/2, either by pre-treatment with PD98059 or overexpression of dominant negative ERK1/2, has no effect on LPS-stimulated TNF␣ mRNA stability after chronic ethanol exposure (29). Taken together, these data indicate that increased Egr-1 activity was critical to the maintenance of TNF␣ transcription after chronic ethanol exposure, acting to compensate for the decrease in NFB binding and promoter activity (Fig.…”

“…Regulation occurs at transcriptional and post-transcriptional levels (3), and it is likely that ethanol acts at multiple steps in the regulation of TNF␣ production. For example, we have recently reported that chronic ethanol exposure stabilizes TNF␣ mRNA and that this stabilization contributes to increased TNF␣ production after chronic ethanol exposure (29). Although TNF␣ production is regulated at both transcriptional and post-transcriptional mechanisms, activation of TNF␣ transcription is a required first step in response to LPS (3).…”

Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease

“…KC-derived TNF␣ has been identified as an important mediator of steatosis, inflammation, and hepatocyte damage in ALD (10,11). Although the involvement of various signaling pathways such as nuclear factor-B (NF-B) and Erk and mRNA stability has been studied in KCs from ALD (8,12,13), the role of miRs is unknown in resident liver macrophages. A recent report has described the miR expression profile in a murine model of ALD (14), but the functions and physiological activity of specific miRs and their cell-specific role and expression remain to be elucidated.…”