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Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylation-deficient hepatocytes’ vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylation-deficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.

The developmentally regulated GTP binding protein 2 (DRG2) is involved in the control of cell growth and differentiation. Here, we demonstrate that DRG2 regulates microtubule dynamics in HeLa cells. Analysis of live imaging of the plus-ends of microtubules with EB1-EGFP showed that DRG2 deficiency (shDRG2) significantly reduced the growth rate of HeLa cells. Depletion of DRG2 increased 'slow and long-lived' subpopulations, but decreased 'fast and short-lived' subpopulations of microtubules. Microtubule polymerization inhibitor exhibited a reduced response in shDRG2 cells. Using immunoprecipitation, we show that DRG2 interacts with tau, which regulates microtubule polymerization. Collectively, these data demonstrate that DRG2 may aid in affecting microtubule dynamics in HeLa cells.

The levels of eIF2α phosphorylation in the livers of adult mice after 5 weeks of HFrD. Western blot analysis of liver lysates from 4-month-old Cont. and A/A Hep mice fed an RD or an HFrD for 5 wks. The * indicates non-specific bands in the Western blot. (PDF 325 kb)

Hepatocyte death and fibrosis in 13 month-old A/A Hep mice fed a regular diet (RD) or a 60% high fructose diet (HFrD) for 5 wks (a) Hematoxylin and eosin (H&E)-stained images of liver tissue sections from 13-month-old Cont. (A/A-fTg) (n = 5) and A/A Hep mice (n = 8) fed an RD. Representative images are shown. (b) TUNEL and (c) Cleaved caspase-3-stained images of liver tissue sections from 13-month-old Cont. (A/A-fTg or S/A-fTg) and A/A Hep mice fed an RD (n = 5 ~ 8 mice per group) or an HFrD (n = 7 mice per group) for 5 wks. The arrowheads indicate positive cells. Representative images are shown. (d) Sirius red-stained and (e) Masson’s trichrome-stained images of liver tissue sections from 13-month-old Cont. (A/A-fTg) (n = 5) and A/A Hep mice (n = 8) fed an RD. Representative images are shown. (PDF 5270 kb)

eIF2α phosphorylation in adult mice prevents hepatocyte death induced by a long-term HFrD. (a) Hematoxylin and eosin (H&E)-stained images of paraffin-embedded liver sections from 7-month-old Cont. and A/A Hep mice fed an RD or an HFrD for 16 wks. Inset shows a magnified view of the area outlined in the black dotted box. The image reveals a necrotic hepatocyte surrounded by leukocytes. (b) Hematoxylin and eosin (H&E)-stained images of liver tissue sections from 7-month-old A/A Liv mice fed an HFrD for 16 wks. Dashed lines delineate the area consisting of ruptured dead cells (asterisks) and dying hepatocytes with fragmented nuclei (red arrow) and atypic nuclei (arrow head). (c) Hematoxylin and eosin (H&E)-stained images of OCT-embedded liver sections from 7-month-old Cont. and A/A Hep mice fed an RD or an HFrD for 16 wks. To prepare frozen blocks, tissue was embedded in Tissue-Tek OCT compound (Sakura Finetek). OCT-embedded sections (7 μm) of frozen livers were stained with haematoxylin and eosin (H&E). Inset shows a magnified view of the area outlined in the black dotted box. The image reveals a necrotic hepatocyte surrounded by leukocytes. (d) Masson’s trichrome-stained images of liver tissue sections from 7-month-old Cont. and A/A Hep mice fed an RD for 16 wks. Representative images are shown (n = 6 mice per group). (e) TUNEL analysis and (f) Immunohistochemical staining of cleaved caspase3 of liver tissue sections from 7-month-old Cont. and A/A Hep mice fed an RD or an HFrD for 16 wks. The arrowheads indicate positive cells. Representative images are shown (n = 6 mice per group). (PDF 6082 kb)

The severity of HFrD-mediated damage is higher in middle-aged A/A Hep mice. (a) The data (serum ALT level, necrotic cell #, TUNEL positive cell #, and cleaved caspase 3 positive cell # of RD-fed adult A/A Hep mice from Fig. 3a–d and RD-fed middle-aged A/A Hep mice from Fig. 2b–e) were reused to generate the graphs. The data were statistically analyzed to evaluate the difference of measured values. Data are means ± SEM (n = 6 mice for RD-fed adult A/A Hep mice and n = 8 mice for RD-fed middle-aged A/A Hep mice). ns stands for no significant. (b) The data (serum ALT level, necrotic cell #, TUNEL positive cell #, and cleaved caspase 3 positive cell # of 16 wks HFrD-fed adult A/A Hep mice from Fig. 3a–d and 5 wks HFrD-fed middle-aged A/A Hep mice from Fig. 2b–e) were reused to generate the graphs. The data were statistically analyzed to evaluate the difference of measured values. Data are means ± SEM (n = 6 mice for 16 wks HFrD-fed adult A/A Hep mice and n = 7 mice for 5 wks HFrD-fed middle-aged A/A Hep mice); # p

A high fructose diet increases expression of proapoptotic genes in liver tissues of A/A Hep mice. (a) Quantitative real-time PCR analysis to assess the expression of selected genes (anti/proapoptotic genes) in liver tissues from 7-month-old Cont. and A/A Hep mice fed an RD or an HFrD for 16 wks. Data are means ± SEM (n = 5 ~ 6 mice per group); *p

eIF2α phosphorylation in hepatocytes is dispensable for survival of adult mice. (a) Diagram depicting the four genotypes of mice used in these experiments. S/A and A/A represent heterozygous and homozygous eIF2α Ser51Ala (*) mutation(s) in exon 2 of one eIF2α allele and both eIF2α alleles, respectively. fTg/0 represents the floxed wild type (WT) eIF2α transgene driven by the CMV enhancer and chicken β-actin promoter (Enh-Pro). The loxP sequences (black arrowheads) allow excision of the WT eIF2α floxed transgene (fTg) and expression of EGFP, an indicator of recombination. CRE Hep /0 represents the Cre recombinase transgene driven by the promoter (Alfp) of Alb1 (encoding albumin) and the enhancer of Afp (encoding alpha-fetoprotein). (b) Efficiency of deletion of the fTg in liver tissues. Results from quantitative RT-PCR analyses of transgenic and total eIF2α mRNAs are shown. Data are means ± SEM (n = 4 ~ 5 mice per group); ### p

Comparison of hepatic and serum triglyceride levels in RD and HFrD-fed mice. (a) Weight gain of Cont. and A/A Hep mice on an RD or an HFrD for 13 wks. Data are means ± SEM (n = 6 mice per group); *p