Increased carbonylation of the lipid phosphatase PTEN contributes to Akt2 activation in a murine model of early alcohol-induced steatosis

Abstract

The production of reactive aldehydes such as 4-hydroxynonenal (4-HNE) is a key event in the pathogenesis of alcoholic liver disease (ALD), which ranges from simple steatosis to fibrosis. The lipid phosphatase PTEN plays a central role in the regulation of lipid metabolism in the liver. In this study, the effects of chronic ethanol feeding and carbonylation on the PTEN signaling pathway were examined in a 9-week mouse feeding model for ALD. Chronic ethanol consumption resulted in altered redox homeostasis as evidenced by decreased GSH, decreased Trx1, and increased GST activity. Both PTEN expression and PTEN phosphorylation were significantly increased in the livers of ethanol-fed mice. Carbonylation of PTEN increased significantly in the ethanol-fed mice compared to pair-fed control animals, corresponding to decreased PTEN 3-phosphatase activity. Concomitantly, increased expression of Akt2 along with increased Akt phosphorylation at residues Thr(308), Thr(450), and Ser(473) was observed resulting in increased Akt2 activity in the ethanol-fed animals. Akt2 activation corresponded to a decrease in cytosolic SREBP and ChREBP. Subsequent LC/MS/MS analysis of 4-HNE-modified recombinant human PTEN identified Michael addition adducts of 4-HNE on Cys(71), Cys(136), Lys(147), Lys(223), Cys(250), Lys(254), Lys(313), Lys(327), and Lys(344). Computational-based molecular modeling analysis of 4-HNE adducted to Cys(71) near the active site and Lys(327) in the C2 domain of PTEN suggested inhibition of enzyme catalysis via either stearic hindrance of the active-site pocket or prevention of C2 domain-dependent PTEN function. We hypothesize that 4-HNE-mediated PTEN inhibition contributes to the observed activation of Akt2, suggesting a possible novel mechanism of lipid accumulation in response to increased reactive aldehyde production during chronic ethanol administration in mice.

Keywords: 4-HNE; 4-hydroxy-2-nonenal; ALD; ALT; Alcoholic liver disease; ChREBP; Free radicals; Lipid peroxidation; NASH; PI3K; PTEN; PTP; PtdIns(3,4,5)P(3); SREBP; Steatosis; Trx; TrxR; alanine aminotransferase; alcoholic liver disease; carbohydrate response element-binding protein; nonalcoholic steatohepatitis; phosphatase and tensin homolog deleted on chromosome 10; phosphatidylinositol 3,4,5-trisphosphate; phosphatidylinositol 3-kinase; protein tyrosine phosphatase; sterol-response element-binding protein; thioredoxin; thioredoxin reductase.

Figure 1. Histopathology of fixed liver tissues isolated from ethanol and control fed mice

A. Hematoxylin and eosin staining demonstrates a predominantly periportal lipid accumulation in the control and predominantly centrilobular lipid accumulation in ethanol-fed mice (Arrows/Zoom). B. Increased 4-HNE, acrolein and MDA staining in the periportal region (Zones 1–2) in liver sections from ethanol fed mice. (CV, central vein, PT, portal triad). Original Magnification 400X.

Figure 1. Histopathology of fixed liver tissues isolated from ethanol and control fed mice

A. Hematoxylin and eosin staining demonstrates a predominantly periportal lipid accumulation in the control and predominantly centrilobular lipid accumulation in ethanol-fed mice (Arrows/Zoom). B. Increased 4-HNE, acrolein and MDA staining in the periportal region (Zones 1–2) in liver sections from ethanol fed mice. (CV, central vein, PT, portal triad). Original Magnification 400X.

Figure 2. Chronic ethanol mediated changes in PTEN and Akt in mouse cytosolic liver fractions

(A) Cytosolic fractions from both ethanol fed and control groups were analyzed via Western blotting and probed for total PTEN, phospho Ser³⁸⁰ PTEN, total Akt1, total Akt2, phosphoSer⁴⁷³ Akt, phosphoThr³⁰⁸ Akt and phosphoThr⁴⁵⁰ Akt, cytosolic ChREBP and cytosolic SREBP using rabbit polyclonal antibodies (B) Quantification of the Western blots (actin normalized). (C) Using mouse liver whole cell extracts, 150µg of total protein was examined for PTEN activity using a malachite green phosphate release assay and DiC₈ PtdIns(3,4,5)P₃. Total Akt, Akt1 and Akt2 activities were determined using an in vitro Akt kinase activity assay with a synthetic GSK3αβ peptide substrate and total immunoprecipitated total Akt, Akt1 and Akt2. (D) Increased pSer⁴⁷³Akt cytosolic and nuclear staining in liver sections isolated from ethanol fed mice. Data were analyzed using paired Students t-test (respective ethanol and control group) n=5 (*p<0.05, **p<0.01).

Figure 3. Effects of chronic ethanol consumption on ChREBP nuclear localization

(A) Positive ChREBP nuclear staining was observed in the periportal region (zones 1–2) in liver sections from ethanol fed mice. (B) Digitally enhanced view of ChREBP stained nuclei. (C). Quantification of ChREBP positive nuclei per 400× field, N=3 (***p<0;001) (5 fields counted for each Con/ETOH fed animal). Original Magnification 400×.

Figure 4. Chronic ethanol induced changes in mouse cytosolic/mitochondrial liver thioredoxins

(A) Cytosolic /mitochondrial fractions from pair fed and ethanol fed groups were analyzed via Western blotting and probed for Trx1, TrxR1, Trx2 and TrxR2. (B) Quantification of the Western blots (actin/VDAC normalized). Data were analyzed using paired Students t-test (respective ethanol and control group) n=5 (*p<0.05, **p<0.01).

Figure 5. Effects of chronic ethanol consumption of carbonylation of PTEN

(A) 150 µg of cytosolic protein from ethanol or control fed mice was incubated for 2 h with 2.5 mM biotin hydrazide and purified using streptavidin pulldown. Samples were subsequently analyzed using reducing SDS-PAGE/Western blotting with rabbit polyclonal anti-PTEN. (B) Two-dimensional SDS-PAGE Western blotting of PTEN from whole cell extracts (50mM IAA treatment 30min) isolated from control and ethanol-fed mice. (C) Two-dimensional Western analysis of phosphorylated PTEN from whole cell extracts (50mM IAA 30min) isolated from control and ethanol-fed mice. (D) Two-dimensional Western analysis of PTEN from lysates isolated from HepG2 cells treated with 50µM 4-HNE (60min) followed by 50mM IAA 30 min. Data were analyzed using paired Students t-test (respective ethanol and control group) n=5 (*p<0.05)

Figure 6. 4-HNE modification of rhuPTEN using LC/MS/MS analysis

(A) 4-HNE modified peptide containing Cys71 (+158), MS/MS analysis was performed and the resulting b/y ion fragmentation confirmed peptide identity (IYNLc*AER). (B) 4-HNE modified peptide containing Lys 327 (+158), MS/MS analysis was performed and the resulting b/y ion fragmentation confirmed peptide identity (NDLDk*ANK).

Figure 7. In silico molecular modeling of 4-HNE modified Cys71 on huPTEN [60]

(A) Ribbon diagram demonstrating location of Cys⁷¹ and Cys¹²⁴ (B) Electron density map demonstrating charge polarity (blue +, red −) and relationship of 4-HNE (slight blue stick) modified Cys71(yellow) and Cys¹²⁴ (colored yellow-lower). (C) Electron density map demonstrating relative location of Cys¹²⁴ following movement into the inactive state (4-HNE light blue stick, Cys⁷¹ is colored yellow CPK, Cys¹²⁴ is colored green CPK)

Figure 8. In silico molecular modeling of 4-HNE modified Lys327 on the C2 domain of huPTEN

(A) Surface electron density map of the C2 of PTEN protein surface with 4-HNE modified Lys³²⁷(Red- negative charged residues, blue-positively charged residues, 4-HNE-stick, Lys³²⁷-yellow/magenta). Note additional hydrophobic surface formed by 4-HNE addition. (B) Ribbon diagrams demonstrating location of calcium binding regions 1,2,3 and 4-HNE modified Lys³²⁷ . (C) Ribbon diagram depicting relative location of 4-HNE-Lys327 and the Trx1 binding site. (D) Surface electron density map depicting top view of relative location of Cys211 (Yellow) and Lys327 (Magenta).

Figure 9. Model of ethanol induced carbonylation in mice

During chronic ethanol consumption, production of reactive aldehydes such as 4-HNE leads to increased carbonylation and inhibition of PTEN. The resulting decrease in PTEN activity corresponds to increased activation of Akt2 and increased steatosis.