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. 2010 Oct;53(4):655-62.
doi: 10.1016/j.jhep.2010.04.017. Epub 2010 Jun 10.

Early growth response-1 attenuates liver injury and promotes hepatoprotection after carbon tetrachloride exposure in mice

Michele T Pritchard  1 , Jessica I Cohen, Sanjoy Roychowdhury, Brian T Pratt, Laura E Nagy
Affiliations

Early growth response-1 attenuates liver injury and promotes hepatoprotection after carbon tetrachloride exposure in mice

Michele T Pritchard et al. J Hepatol. 2010 Oct.

Abstract

Background & aims: Inflammatory gene expression plays a pathological role in acute and chronic hepatic inflammation, yet, inflammation also promotes liver repair by inducing protective mechanisms to limit collateral tissue damage by priming hepatocytes for proliferation. Early growth response (Egr)-1, a transcription factor that regulates inflammatory gene expression, plays a pathological role in many animal models of acute and chronic inflammatory disease. Here, we tested the hypothesis that Egr-1 is beneficial after toxic liver injury.

Methods: Acute liver injury was induced in wild-type and egr-1-/- mice by a single injection of carbon tetrachloride (CCl(4)). Liver injury, inflammatory, and hepatoprotective gene expression and signaling events were measured 18, 48, and 72 h after CCl(4) administration.

Results: Peak liver injury was greater in egr-1-/- mice compared to wild-type mice. Enhanced injury in egr-1-/- mice was associated with reduced tumor necrosis factor (TNF)alpha mRNA and protein expression, reduced Akt phosphorylation and nuclear localization of NFkappaB-p65 in nuclei of cells in the hepatic sinusoid. Expression of inducible nitric oxide synthase and cyclooxygenase-2, TNFalpha-regulated genes that have hepatoprotective function, was attenuated in egr-1-/- mice compared to wild-type mice. Although plasma interleukin (IL)-6 protein and hepatic accumulation of IL-6, glycoprotein 130, and IL-6 receptor alpha mRNA in wild-type and egr-1-/- mice were equivalent, signal transducer and activator of transcription 3 phosphorylation was attenuated in egr-1-/- mice and associated with reduced oncostatin M expression.

Conclusions: In contrast to its role in inflammation-mediated tissue injury in other models, Egr-1 expression promotes protection in the liver after CCl(4) exposure.

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Figures

Fig. 1
Fig. 1. Absence of Egr-1 exacerbated CCl4-induced liver injury
(A) Liver histology 72h after CCl4 administration, Images are representative of n = 4 – 8 mice per group. Hematoxylin and eosin, 100X magnification. Plasma isolated from whole blood was used to determine (B) ALT and (C) AST activities. Bars are means +/− SEM, n = 4–8 mice per group.
Fig. 2
Fig. 2. Hepatocellular apoptosis was enhanced in Egr-1 deficient mice
(A) Representative 400X images of hepatic TUNEL staining from wild-type and egr-1−/− mice 72h after CCl4. DAPI staining was used to label total hepatic nuclei (not shown). (B) TUNEL quantification. The data were calculated as percent TUNEL positive cells of total DAPI positive hepatic nuclei. Bars are means +/− SEM, n = 3–4 mice per group.
Fig. 3
Fig. 3. Egr-1, TNFα, and IL-6 expression after CCl4 exposure in wild-type mice
Real-time PCR was utilized to determine mRNA accumulation of (A) Egr-1 (C) TNFα and (D) IL-6. Bars represent means +/− SEM of n = 4 – 6 mice per experimental condition. (B) Immunohistochemistry was used to localize Egr-1 protein in liver sections. Open arrowheads indicate Egr-1-positive hepatocyte nuclei, open arrows indicate Egr-1-positive nuclei in NPC-HS. Images are representative of n = 4 mice for each experimental condition
Fig. 4
Fig. 4. TNFα and IL-6 expression after CCl4 exposure
Eighteen hours after CCl4 exposure, hepatic mRNA accumulation of (A) TNFα and (C) IL-6, and plasma (B) TNFα and (D) IL-6 protein were determined by real-time PCR and ELISA, respectively. Bars represent means +/− SEM, n = 5–8 mice per group.
Fig. 5
Fig. 5. Nuclear localization of NFκB-p65 in NPC-HS, and phosphorylation of Akt and STAT3 were reduced in egr-1−/− mice after CCl4 exposure
(A) Immunohistochemistry was utilized to localize p65 in liver sections from wild-type and egr-1−/− mice. Closed arrows indicate p65-negative nuclei, while open arrows indicate p65-positive nuclei. CV = central vein; HC = hepatocyte. Outlined areas in (A) are shown enlarged below each 200X image. Images are representative of n = 4 – 8 per experimental group. Immunoblots of total hepatic protein were performed to determine expression of (B,C) phospho (Ser473)-Akt, (D,E) phospho (Tyr705)-STAT3 in livers from wild-type and egr-1−/− mice. Total (t)-Erk1/2 was used as a loading control. Representative (B) Ser473-Akt and (D) Tyr705-STAT3 immunoblots are shown. Quantification of band intensity measured by densitometry after normalization to tErk1/2 are shown in the bar graphs for (C) Ser-473 Akt and (E) Tyr705-STAT3 from n = 4–5 mice per group. Bars are means +/− SEM determined after scanning densitometry of immunoreactive bands.
Fig. 6
Fig. 6. CCl4-induced expression of hepatoprotective molecules
Eighteen hours after CCl4 exposure, hepatic (A) iNOS, (B) COX-2 (C) gp130 (D) IL-6Rα (E) OSM and (F) OSMR mRNA accumulation was determined using real-time PCR. Bars represent means +/− SEM, n = 5–8 mice per group.
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