Elevated Nrf-2 responses are insufficient to mitigate protein carbonylation in hepatospecific PTEN deletion mice

<div><p>Objective</p><p>In the liver, a contributing factor in the pathogenesis of non-alcoholic fatty liver disease (NASH) is oxidative stress, which leads to the accumulation of highly reactive electrophilic α/β unsaturated aldehydes. The objective of this study was to determine the impact of NASH on protein carbonylation and antioxidant responses in a murine model.</p><p>Methods</p><p>Liver-specific phosphatase and tensin homolog (PTEN)-deletion mice (PTEN^LKO) or control littermates were fed a standard chow diet for 45–55 weeks followed by analysis for liver injury, oxidative stress and inflammation.</p><p>Results</p><p>Histology and Picrosirius red-staining of collagen deposition within the extracellular matrix revealed extensive steatosis and fibrosis in the PTEN^LKO mice but no steatosis or fibrosis in controls. Increased steatosis and fibrosis corresponded with significant increases in inflammation. PTEN-deficient livers showed significantly increased cell-specific oxidative damage, as detected by 4-hydroxy-2-nonenal (4-HNE) and acrolein staining. Elevated staining correlated with an increase in nuclear DNA repair foci (γH2A.X) and cellular proliferation index (Ki67) within zones 1 and 3, indicating oxidative damage was zonally restricted and was associated with increased DNA damage and cell proliferation. Immunoblots showed that total levels of antioxidant response proteins induced by nuclear factor erythroid-2-like-2 (Nrf2), including GSTμ, GSTπ and CBR1/3, but not HO-1, were elevated in PTEN^LKO as compared to controls, and IHC showed this response also occurred only in zones 1 and 3. Furthermore, an analysis of autophagy markers revealed significant elevation of p62 and LC3II expression. Mass spectrometric (MS) analysis identified significantly more carbonylated proteins in whole cell extracts prepared from PTEN^LKO mice (966) as compared to controls (809). Pathway analyses of identified proteins did not uncover specific pathways that were preferentially carbonylated in PTEN^LKO livers but, did reveal specific strongly increased carbonylation of thioredoxin reductase and of glutathione-<i>S</i>-transferases (GST) M6, O1, and O2.</p><p>Conclusions</p><p>Results show that disruption of PTEN resulted in steatohepatitis, fibrosis and caused hepatic induction of the Nrf2-dependent antioxidant system at least in part due to elevation of p62. This response was both cell-type and zone specific. However, these responses were insufficient to mitigate the accumulation of products of lipid peroxidation.</p></div

Impact of PTEN^LKO on expression of antioxidant responses.

<p>Western immunoblotting analysis of GCLC, GSTμ, GSTπ, CBR1/3, HO-1, Prdx5 and Gpx1 in liver lysates prepared from control and PTEN^LKO mice. All exposures were normalized using GAPDH expression. Data are means +/- SEM, n = 6 per genotype.</p

Basic pathology in PTEN^LKO livers.

<p>Panels A, E. Histology of livers from control or PTEN^LKO mice. Panels B, F. Bright-field images of PSR-staining. Panels C, G. Polarized light exposure of PSR-staining. Panels D, H. Cytokeratin 7 staining. Representative images are shown; n = at least 3 mice per genotype (CV, central vein, PT, portal triad).</p

Impact of PTEN^LKO on DNA double-strand break repair and proliferation.

<p>Tissue sections isolated from control and PTEN^LKO mice were immunostained using antibodies directed against γH2A.X and Ki67. N = at least 3 mice per genotype. Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198139#pone.0198139.g001" target="_blank">Fig 1</a>.</p

Impact of PTEN^LKO on protein carbonylation.

<p>Tissue sections isolated from control and PTEN^LKO mice were immunostained using antibodies directed against 4-HNE or acrolein, as indicated. A. 100X magnification, B. 400X magnification of PTEN^LKO livers. Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198139#pone.0198139.g001" target="_blank">Fig 1</a>.</p

Inflammatory infiltrates in PTEN^LKO livers.

<p>Liver sections from control and PTEN^LKO mice were immunostained using antibodies directed against MPO (panels A, E); F4/80 (panels B, F); CD3 (panels C, G); or B220 (panels D, H). Representative images are shown; n = at least 3 mice per genotype. Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198139#pone.0198139.g001" target="_blank">Fig 1</a>.</p

VENN analysis of carbonylated proteins in control and PTEN^LKO livers.

<p>VENN analysis of carbonylated proteins in control and PTEN^LKO livers.</p

Impact of PTEN^LKO on expression of markers of autophagy.

<p>Western immunoblotting analysis of p62, LC3I and LC3II in liver lysates prepared from control and PTEN^LKO mice. All exposures were normalized using GAPDH expression. Data are means +/- SEM, n = 6 per genotype.</p

Differentiation of hematopoietic lineages in spleen of <i>Zfp148</i>-deficient mice.

<p>Flow cytometric analysis of spleen from P14 pups (<i>n</i> = 3) and adult mice, ages 3-6 months; wild type (<i>n</i> = 6) and <i>Zfp148</i><b>^<i>gt/gt</i></b> (<i>n</i> = 5). (A) Percentage of CD4- or CD8-positive T-cells, CD4-positive T-cells and CD8-positive T-cells among lymphocytes. (B) Representative cytograms from adult spleens. (C) Percentage of CD69-positive cells among lymphocytes. (D) Representative cytograms from adult spleens. (E) CD62L low expressing cells among lymphocytes. (F) Percentage of B220-positive cells among lymphocytes in P14 and adult spleen. (G) Representative cytograms from adult spleens. Lymphocytes were gated based on side scatter (SSC). Values are expressed as mean ± SEM. Significance levels: *P<0.05, **P<0.01.</p

Differentiation of hematopoietic lineages in thymus and bone marrow of <i>Zfp148</i>-deficient mice.

<p>Flow cytometric analysis of bone marrow (A–D) and thymocytes (E–H) from wild type and <i>Zfp148</i>^<i>gt/gt</i> mice. Graphs and representative cytograms show percentage of (A, B) Mac1-, B220- and (C, D) CD4 and/or CD8-positive cells among leukocytes in bone marrow from adult mice, ages 3-6 months; wild type (<i>n</i> = 6) and <i>Zfp148</i><b>^<i>gt/gt</i></b> (<i>n</i> = 5). (E–H) Percentage of (E) double negative (DN) CD4/CD8, (F) double positive (DP) CD4/CD8, (G) CD4 single positive (SP) and CD8 SP cells among thymocytes from P14 pups (left, <i>n</i> = 3) and adult mice (right, <i>n</i> = 5). (H) Representative cytograms from adult thymus. Values are expressed as mean ± SEM. Significance levels: *P<0.05, **P<0.01.</p