Growth Hormone Mediates Its Protective Effect in Hepatic Apoptosis through Hnf6

<div><p>Background and Aims</p><p>Growth hormone (GH) not only supports hepatic metabolism but also protects against hepatocyte cell death. <i>Hnf6</i> (or <i>Oc1)</i> belonging to the Onecut family of hepatocyte transcription factors known to regulate differentiated hepatic function, is a GH-responsive gene. We evaluate if GH mediates Hnf6 activity to attenuate hepatic apoptotic injury.</p><p>Methods</p><p>We used an animal model of hepatic apoptosis by bile duct ligation (BDL) with <i>Hnf6</i> -/- (KO) mice in which hepatic <i>Hnf6</i> was conditionally inactivated. GH was administered to adult wild type WT and KO mice for the 7 days of BDL to enhance Hnf6 expression. <i>In vitro</i>, primary hepatocytes derived from KO and WT liver were treated with LPS and hepatocyte apoptosis was assessed with and without GH treatment.</p><p>Results</p><p>In WT mice, GH treatment enhanced Hnf6 expression during BDL, inhibited Caspase -3, -8 and -9 responses and diminished hepatic apoptotic and fibrotic injury. GH-mediated upregulation of Hnf6 expression and parallel suppression of apoptosis and fibrosis in WT BDL liver were abrogated in KO mice. LPS activated apoptosis and suppressed Hnf6 expression in primary hepatocytes. GH/LPS co-treatment enhanced Hnf6 expression with corresponding attenuation of apoptosis in WT-derived hepatocytes, but not in KO hepatocytes. ChiP-on-ChiP and electromobility shift assays of KO and WT liver nuclear extracts identified <i>Ciap1</i> (or <i>Birc2</i>) as an Hnf6-bound target gene. Ciap1 expression patterns closely follow Hnf6 expression in the liver and in hepatocytes.</p><p>Conclusion</p><p>GH broad protective actions on hepatocytes during liver injury are effected through Hnf6, with Hnf6 transcriptional activation of <i>Ciap1</i> as an underlying molecular mediator.</p></div

Primer DNA sequences of real time PCR genes.

<p>Primer DNA sequences of real time PCR genes.</p

Mouse <i>Ciap1</i> promoter DNA sequences containing Hnf6 binding sites.

<p>Mouse <i>Ciap1</i> promoter DNA sequences containing Hnf6 binding sites.</p

Apoptosis in LPS-treated WT and KO primary hepatocytes.

<p>Representative western blot of Hnf6, Ciap1, Procaspase-3, Cleaved Caspase-3, and b-Actin expression of total proteins from primary hepatocytes in WT and KO mice (n = 3–5) after treatment for 32 hours with 1 ug/ml of LPS, with or without GH cotreatment.</p

Iap gene and protein gene expression in GH-treated BDL liver.

<p>(A) Hepatic <i>Ciap1</i> expression in SH or BDL WT and BDL liver with PBS (□) or GH (■) treatment, with levels relative to SH WT liver and significant p values. (B) Representative western blot gel of SH or BDL WT and KO liver, and graph of quantitated signal from GH-treated WT and KO BDL liver with significant p value. (C-F) <i>Ciap2</i>, <i>Xiap</i>, <i>Survivin</i>, <i>Bcl2</i> expression in SH or BDL WT and KO liver with PBS (□) or GH (■) treatment, with levels relative to SH WT liver.</p

TUNEL assay and Caspase expression in WT and KO liver.

<p>(A) Representative TUNEL-labeled liver micrographs from Sham (SH) WT or KO mice without (WT, KO) or with GH treatment (WTGH and KOGH); and Bile duct ligation (BDL) WT or KO mice without (WTBDL, KOBDL) or with GH treatment (GHWTBDL and GHKOBDL) (n = 8–10/group). The arrows identify TUNEL + hepatocytes. (B) Bar graph of % TUNEL (+) hepatocytes/100x high power field from WT or KO SH or BDL liver treated with PBS (□) or GH (■) and significant p value. (C-E) <i>Caspase-3</i> (C), <i>-8</i> (D) and <i>-9</i> (E) in WT or KO liver treated with PBS (□) or GH (■) and significant p values. Gene levels were calculated relative to SH WT liver. (F) Representative western blots of Procaspase-8, -9, -3, cleaved proteins and b-Actin in SH or BDL WT and KO liver and the corresponding bar graphs of caspase staining intensity in BDL samples with significant p values.</p

Downstream signaling response to GH treatment.

<p>(A) Representative western blot of phosphorylated Stat5 and b-Actin of liver nuclear proteins from Sham (SH) or BDL WT and KO liver treated with PBS or GH. (B) <i>Igf1</i> expression in WT SH and KO SH as well as WT BDL and KO BDL liver treated with PBS (□) or GH (■), with levels relative to SH KO PBS liver and significant p values. (C) <i>Hnf</i>6 expression in SH or BDL WT and KO liver treated with PBS (□) or GH (■), with levels relative to SH KO liver and significant p values. (D) Representative western blot of Hnf6 nuclear protein extracts from SH or BDL WT and KO liver treated with PBS or GH. (E) Baseline expression levels of Iap (<i>Ciap1</i>, <i>Ciap2</i>, <i>Xiap</i>, <i>Livin</i>, <i>Naip</i>, <i>Apollen and Survivin</i>) family of genes from WT and KO liver with levels shown relative to KO liver and significant p values. (F) Baseline expression levels of Bcl2 (<i>Bcl2</i>, <i>Bad</i>, <i>Bax</i>, <i>Bcl-x</i>, <i>Bak1</i>) families of genes from WT and KO liver with levels shown relative to KO liver and significant p values.</p

Hepatic cholestasis and fibrosis in WT and KO mice.

<p>(A) Serum Alkaline Phosphatase levels (a marker of cholestasis) in SH or BDL WT and KO mice treated with PBS (□) or GH (■) and significant p values. (B) Representative micrographs of α-Sma immunostaining of SH or BDL WT and KO liver. (C) Bar graph of α-Sma immunostaining of SH or BDL WT and KO liver treated with PBS (□) or GH (■) with intensity relative to SH WT and significant p value. (D) <i>Ctgf</i> expression in SH or BDL WT and KO mice treated with PBS (□) or GH (■) with levels relative to Sham WT and significant p values. (E) <i>Tgfb2R</i> expression in SH or BDL WT and KO mice treated with PBS (□) or GH (■) with levels relative to SH WT and significant p values.</p

<i>Ciap1</i> promoter occupancy by Hnf6.

<p>(A) <i>Ciap1-</i> and <i>Xiap</i>-precipitated promoter fragments bound by mock, control IgG or anti-Hnf6 antibody were amplified by real time PCR and significant p value is shown. The gel insert shows precipitated anti-Hnf6 antibody treated Hnf6-DNA complex in WT vs KO nuclear extracts. Relative <i>Ciap1</i> promoter amplification is graphed relative to the corresponding WT mock data. (B) Electromobility shift assay EMSA of <i>in vitro</i> synthesized Hnf6 protein in the absence (Negative) or in the presence of 3 <i>Ciap1</i> promoter fragments with arrow showing the Oligonucleotide-Hnf6 protein precipitated complex. The Digoxin-labeled Oligonucleotide could be inhibited with DIG Oligo Competitor. (C) EMSA of nuclear extracts from KO and WT liver against <i>Ciap1</i> promoter oligonucleotide 1, with the arrow showing the location of the DNA-protein complex.</p