Volume 132, Issue 7, Pages 2533-2541 (June 2007) Increased Gene and Protein Expression of the Novel eNOS Regulatory Protein NOSTRIN and a Variant in Alcoholic Hepatitis  Rajeshwar P. Mookerjee, Anja Wiesenthal, Ann Icking, Stephen J. Hodges, Nathan A. Davies, Kirstin Schilling, Sambit Sen, Roger Williams, Marco Novelli, Werner Müller-Esterl, Rajiv Jalan  Gastroenterology  Volume 132, Issue 7, Pages 2533-2541 (June 2007) DOI: 10.1053/j.gastro.2006.12.035 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Relative expression levels [1/CT] of eNOS, caveolin-1, NOSTRINα, and NOSTRINβ mRNA. Whole mRNA of liver sections was isolated, transcribed into cDNA, and analyzed by qRT-PCR. Values are given as relative expression 1/cycle threshold (1/CT) for normal (n = 3), alcoholic cirrhosis (AC) (n = 6), and alcoholic cirrhosis with superimposed alcoholic hepatitis (AH+C) (n = 5) specimens. (A) eNOS: AH+C patients had significantly greater eNOS mRNA (P < .01) compared with AC patients. AC patients had no significant difference in eNOS message compared with normal patients. (B) Caveolin-1: Message for caveolin-1 was also significantly higher in AH+C compared with AC patients (P < .01). No significant difference was noted in caveolin-1 mRNA between AC and normal patients. (C) NOSTRINα and (D) NOSTRINβ mRNA levels were significantly higher in AH+C patients compared with AC patients (P < .01); cirrhotic specimens had higher NOSTRINα mRNA levels compared with normal liver specimens (P < .05). Of note, NOSTRINβ mRNA was undetectable in normal liver tissue as marked on the Figure (D). Standard error of the mean is denoted by horizontal bars above each column graph, and statistical significance demonstrated according to the key: *P < .05; **P < .01; n.s., not significant. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 eNOS expression and activity in liver tissue. (A) Immunostaining of paraffin-embedded liver biopsy sections from alcoholic hepatitis (AH+C) (a) and cirrhosis alone (AC) patients (b) using monoclonal anti-human eNOS. The enzyme is present in biopsy specimens from both AH+C patients (panel A) and AC (panel B). Semiquantitative assessment of the staining intensity did not allow a clear difference to be defined in eNOS expression between AH+C and AC patients (arrows). Original magnification, ×40. (B) Graph suggests a statistically significant correlation between reduced eNOS generation and high portal pressure by demonstrating that eNOS tissue activity is inversely correlated with the severity of hepatic venous pressure gradient: r = −0.64 and P = .01. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Western blot analysis of human liver samples. (A) NOSTRINα displays a characteristic domain structure comprising an N-terminal Fes/CIP homology (FCH) domain, 2 coiled-coil domains (cc1 and cc2), and a C-terminal Src homology (SH3) domain. NOSTRINβ lacks the N-terminal, 78 amino acid residues and thus most of the FCH domain present in NOSTRINα. (B) In Western blotting, recombinantly expressed NOSTRINα and NOSTRINβ from Chinese hamster ovary (CHO) cells show molecular masses of 58 and 50 kilodaltons, respectively. (C) A limited number of liver samples were analyzed by Western blotting for expression of NOSTRINα and NOSTRINβ. Total protein of liver biopsy specimens was isolated and analyzed by SDS-PAGE and Western blotting with monoclonal anti-NOSTRIN. To estimate total protein loading, Lamp-1 is shown as a marker. NOSTRINα is detected in all liver samples analyzed, whereas expression of NOSTRINβ was restricted to patients with AC or AH+C. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Expression of NOSTRINα and NOSTRINβ in cultured hepatocytes. Hep3B cells were treated with 1 μmol/L retinoic acid (RA) or mock-treated with DMSO for up to 6 days and analyzed by (A and B) Western blotting and (C) qRT-PCR. Values given are normalized to ubiquitin C and shown as relative expression (1/CT). Both experimental setups show a clear up-regulation of NOSTRINα and NOSTRINβ expression after RA treatment. Note that B shows a blot after prolonged exposure. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 NOSTRIN localization and its interaction partners in liver tissue. (A and B) Liver samples were embedded in Tissue-Tek for cryoconservation. Twelve-micrometer sections were used for (A) H&E staining or (B) immunohistochemistry. (B) Polyclonal (a + c) and monoclonal (b + d) anti-NOSTRIN was used for staining. For control, the antibodies were preabsorbed with GST-NOSTRIN prior to staining. Bars, 50 μm. (C) Human liver was frozen, ground, and lysed. Co-immunoprecipitation was performed using rabbit anti-NOSTRIN (AS532). Western blots of co-immunoprecipitates and lysates were probed with mouse anti-eNOS, anti-NOSTRIN, and anti-caveolin-1. eNOS and caveolin-1 co-immunoprecipitate with NOSTRIN from total liver lysates. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Subcellular localization of NOSTRINα and NOSTRINβ. CHO-eNOS cells were transfected with NOSTRINα (A) or NOSTRINβ cDNA (B), fixed with methanol, and analyzed by immunofluorescence. The 2 isoforms clearly differ in their subcellular localization. Gastroenterology 2007 132, 2533-2541DOI: (10.1053/j.gastro.2006.12.035) Copyright © 2007 AGA Institute Terms and Conditions