Early growth response-1 transcription factor is essential for ethanol-induced fatty liver injury in mice

Abstract

Background & aims: Early growth response-1 (Egr-1), an immediate early gene/zinc-finger transcription factor, is required for maximal stimulation of tumor necrosis factor alpha (TNF-alpha) transcription in response to lipopolysaccharide (LPS). Because chronic ethanol exposure sensitizes macrophages to LPS-stimulated TNF-alpha expression, we have investigated the role of Egr-1 in mediating increased LPS-stimulated TNF-alpha expression after chronic ethanol feeding. Furthermore, because TNF-alpha contributes to alcoholic liver injury, we tested the hypothesis that Egr-1 is required for the development of ethanol-induced fatty liver injury in wild type and egr-1 -/- mice.

Methods: Wild-type and egr-1 -/- mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks.

Results: Wild-type mice fed the ethanol diet developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. However, egr-1 -/- mice did not develop steatosis after ethanol feeding. Alanine transferase and TNF-alpha concentrations in serum were increased after ethanol feeding in wild-type but not egr-1 -/- mice. In wild-type mice, challenge with LPS increased Egr-1 messenger RNA (mRNA) and DNA binding activity in liver; this response to LPS was enhanced after chronic ethanol feeding. LPS challenge also increased hepatic TNF-alpha mRNA and serum TNF-alpha to a greater extent after ethanol feeding compared with pair-fed wild-type mice. However, chronic ethanol feeding did not enhance LPS-stimulated TNF-alpha mRNA or serum TNF-alpha in egr-1 -/- mice.

Conclusions: These data show that Egr-1 contributes to increased LPS-mediated TNF-alpha expression after chronic ethanol and that the absence of Egr-1 prevents chronic ethanol-induced fatty liver, as well as increased sensitivity to LPS.

Figure 1

Chronic ethanol feeding enhances LPS-stimulated Egr-1 expression. (A) Hepatic Egr-1 mRNA content is increased in response to LPS. Female C57BL/6 mice were fed an ethanol-containing diet over 6 weeks or pair-fed control diets. Mice were then injected intraperitoneally with 0.7 μg/g body weight LPS and livers harvested after 30, 60, or 120 minutes. Mice not treated with LPS were injected with vehicle (0.09% saline) and livers harvested after 120 minutes. Total RNA was isolated and quantity of Egr-1 mRNA determined by real-time reverse-transcriptase PCR. Values represent means ± SEM, n = 4–6 per time point, *P < .05 compared with pair-fed. (B) Nuclear Egr-1 protein quantity is increased in response to LPS. Nuclear extracts were prepared from ethanol- and pair-fed mice and relative quantity of Egr-1 and p65 protein measured by Western blot. Total ERK1/2 immunoreactivity is shown as a loading control. Figure is representative of at least 3 sets of animals. (C) Treatment with LPS stimulates Egr-1, NF-κB, and AP-1 DNA binding activity in mouse liver. Nuclear extracts were prepared from ethanol- and pair-fed mice after treatment with or without LPS for 1 hour. The binding of nuclear proteins to oligonucleotides corresponding to the Egr-1 and AP-1 binding sites in the murine TNF-α promoter, as well as consensus sequence for NF-κB binding sites, were assessed by EMSA. Figures are representative of at least 3 sets of animals.

Figure 2

Chronic ethanol feeding increases serum ALT and serum TNF-α concentrations in wild-type but not egr-1 −/− mice. Mice were allowed free access to an ethanol-containing diet or pair fed a control diet for 6 weeks. ALT activity in serum was measured enzymatically. TNF-α was measured by ELISA. Values represent mean ± standard error of the mean, n = 11 for wild-type females, 5 for egr-1 −/− females, and 3 for wild-type and egr-1 −/− males. *P < .05 compared with pair-fed controls.

Figure 3

Liver histology and triglyceride content in wild-type and egr-1 −/− mice after chronic ethanol feeding. (A) Livers from mice allowed free access to ethanol-containing diets or pair-fed control diets were perfused with saline and then fixed in formalin. Liver sections were prepared and stained with H&E. Figures are representative of at least 5 mice in each treatment group. (B) Frozen liver sections were stained with Oil Red O and counterstained with hematoxylin. Figures are representative of 3 mice in each treatment group. (C) Liver triglycerides were measured. Values represent mean ± standard error of the mean, n = 4; values with different superscripts are significantly different, P < .05.

Figure 4

Endotoxin concentrations in platelet-rich plasma after chronic ethanol feeding. Platelet-rich plasma was prepared from blood taken from the retro-orbital sinus of male ethanol- and pair-fed wild-type and egr-1 −/− mice. Endotoxin concentrations were measured using a chromogenic assay. Values represent mean ± SEM, n = 3. *P < .05 compared with pair-fed controls.

Figure 5

CYP2E1 expression is induced in livers of wild-type and egr-1 −/− mice after chronic ethanol feeding. Homogenates were prepared from livers of EtOH- and pair-fed mice and the relative expression of CYP2E1 assessed by Western blot analysis. Western blots were also probed with antibody against β-actin as a loading control (data not shown). Values represent mean ± standard error of the mean, n = 4. *P < .05 compared with pair-fed controls.

Figure 6

LPS-stimulated hepatic TNF-α mRNA and serum TNF-α concentrations. (A) Total RNA was isolated from livers from ethanol- and pair-fed wild-type and egr-1 −/− mice. Expression of TNF-α mRNA was measured by real-time PCR. Quantity of TNF-α mRNA is expressed relative to β-actin mRNA. LPS-stimulated TNF-α mRNA/β-actin mRNA ratios are expressed relative to basal ratios in wild-type mice and relative to pair-fed wild-type baseline values for egr-1 −/− mice. Values represent mean ± standard error of the mean, n = 7 for wild type and n = 3 for egr-1 −/−. *P < .05 compared with pair-fed, compared with wild type. (B) Serum TNF-α concentrations were measured by ELISA. Values represent mean ± SEM, n = 13 for wild-type and n = 6 for egr-1 −/− mice at the 2-hour time point. *P < .05 compared with pair-fed mice at the 2-hour time point.

Figure 7

(A) LPS-stimulated NF-κB DNA binding activity in livers from wild-type and egr-1 −/− mice. Nuclear extracts were prepared from livers of ethanol- and pair-fed mice after treatment with or without LPS for 30–120 minutes. The binding of nuclear proteins to an oligonucleotide corresponding to the consensus sequence for the NF-κB binding site was assessed by EMSA. Figures are representative of at least 3 sets of animals. (B) Activation of NF-κB by LPS in wild-type and egr-1 −/− mice. Postnuclear supernatants were prepared from livers of ethanol- and pair-fed mice after treatment with or without LPS for 60–120 minutes. Immunoreactivity of IκB-α and p65 were measured by Western blot analysis. Figures are representative of at least 3 sets of animals.