Molecular mechanisms of hepatic apoptosis

Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis

Image_2_Potential roles of synaptotagmin family members in cancers: Recent advances and prospects.TIF

With the continuous development of bioinformatics and public database, more and more genes that play a role in cancers have been discovered. Synaptotagmins (SYTs) are abundant, evolutionarily conserved integral membrane proteins composed of a short N-terminus, a variable linker domain, a single transmembrane domain, and two C2 domains, and they constitute a family of 17 isoforms. The synaptotagmin family members are known to regulate calcium-dependent membrane fusion events. Some SYTs play roles in hormone secretion or neurotransmitter release or both, and much evidence supports SYTs as Ca2+ sensors of exocytosis. Since 5 years ago, an increasing number of studies have found that SYTs also played important roles in the occurrence and development of lung cancer, gastric cancer, colon cancer, and other cancers. Down-regulation of SYTs inhibited cell proliferation, migration, and invasion of cancer cells, but promoted cell apoptosis. Growth of peritoneal nodules is inhibited and survival is prolonged in mice administrated with siSYTs intraperitoneally. Therefore, most studies have found SYTs serve as an oncogene after overexpression and may become potential prognostic biomarkers for multiple cancers. This article provides an overview of recent studies that focus on SYT family members’ roles in cancers and highlights the advances that have been achieved.</p

Image_1_Potential roles of synaptotagmin family members in cancers: Recent advances and prospects.TIF

With the continuous development of bioinformatics and public database, more and more genes that play a role in cancers have been discovered. Synaptotagmins (SYTs) are abundant, evolutionarily conserved integral membrane proteins composed of a short N-terminus, a variable linker domain, a single transmembrane domain, and two C2 domains, and they constitute a family of 17 isoforms. The synaptotagmin family members are known to regulate calcium-dependent membrane fusion events. Some SYTs play roles in hormone secretion or neurotransmitter release or both, and much evidence supports SYTs as Ca2+ sensors of exocytosis. Since 5 years ago, an increasing number of studies have found that SYTs also played important roles in the occurrence and development of lung cancer, gastric cancer, colon cancer, and other cancers. Down-regulation of SYTs inhibited cell proliferation, migration, and invasion of cancer cells, but promoted cell apoptosis. Growth of peritoneal nodules is inhibited and survival is prolonged in mice administrated with siSYTs intraperitoneally. Therefore, most studies have found SYTs serve as an oncogene after overexpression and may become potential prognostic biomarkers for multiple cancers. This article provides an overview of recent studies that focus on SYT family members’ roles in cancers and highlights the advances that have been achieved.</p

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

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

<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

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

Primer DNA sequences of real time PCR genes.

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