Xin M. Wang

Gene expression profiling of alcoholic liver disease in the baboon (Papio hamadryas) and human liver

The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis.

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell–extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9‐overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8‐overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein‐transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg‐Gly‐Asp integrin‐binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell–extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced β‐catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell–extracellular matrix interactions, and thus may regulate tissue remodeling.

Reversible Inactivation of Human Dipeptidyl Peptidases 8 and 9 by Oxidation

Hydrogen peroxide (H2O2) can act as an intracellular messenger by oxidizing sulfhydryl groups in cysteines that can be oxidized at neutral pH. The oxidizing agents H2O2 and pyrroloquinoline quinone and the large thiol reagents N-ethylmaleimide and 4-(hydroxymercuri) benzoate each inhibited dipeptidyl peptidase (DP) activity in the intracellular DPIV-related proteins DP8 and DP9 at pH 7.5. In contrast, these treatments did not alter activity in DPIV and fibroblast activation protein. Peptidase inhibition was completely reversed by 2-mercaptoethanol or reduced glutathione. Alkylation of DP8 by the small thiol reagent iodoacetamide prevented inhibition by H2O2, N-ethylmaleimide or pyrroloquinoline qui- none. Two cysteines were reactive per peptidase monomer. We exploited these properties to highly purify DP8 by thiol affinity chromatography. Homology modelling of DP8 and DP9 was consistent with the proposal that the mechanism in- volves decreased protein flexibility caused by intramolecular disulfide bonding. These novel data show that DP8 and DP9 are reversibly inactivated by oxidants at neutral pH and suggest that DP8 and DP9 are H2O2 sensing proteins.

Regulation of dipeptidyl peptidase 8 and 9 expression in activated lymphocytes and injured liver

AIM To investigate the expression of dipeptidyl peptidase (DPP) 8 and DPP9 in lymphocytes and various models of liver fibrosis. METHODS DPP8 and DPP9 expression were measured in mouse splenic CD4⁺ T-cells, CD8⁺ T-cells and B-cells (B220⁺), human lymphoma cell lines and mouse splenocytes stimulated with pokeweed mitogen (PWM) or lipopolysaccharide (LPS), and in dithiothreitol (DTT) and mitomycin-C treated Raji cells. DPP8 and DPP9 expression were measured in epidermal growth factor (EGF) treated Huh7 hepatoma cells, in fibrotic liver samples from mice treated with carbon tetrachloride (CCl₄) and from multidrug resistance gene 2 (Mdr2/Abcb4) gene knockout (gko) mice with biliary fibrosis, and in human end stage primary biliary cirrhosis (PBC). RESULTS All three lymphocyte subsets expressed DPP8 and DPP9 mRNA. DPP8 and DPP9 expression were upregulated in both PWM and LPS stimulated mouse splenocytes and in both Jurkat T- and Raji B-cell lines. DPP8 and DPP9 were downregulated in DTT treated and upregulated in mitomycin-C treated Raji cells. DPP9-transfected Raji cells exhibited more annexin V⁺ cells and associated apoptosis. DPP8 and DPP9 mRNA were upregulated in CCl₄ induced fibrotic livers but not in the lymphocytes isolated from such livers, while DPP9 was upregulated in EGF stimulated Huh7 cells. In contrast, intrahepatic DPP8 and DPP9 mRNA expression levels were low in the Mdr2 gko mouse and in human PBC compared to non-diseased livers. CONCLUSION These expression patterns point to biological roles for DPP8 and DPP9 in lymphocyte activation and apoptosis and in hepatocytes during liver disease pathogenesis.

Intrahepatic expression of collagen and fibroblast activation protein (FAP) in hepatitis C virus infection.

Mark D. Gorrell, Xin M. Wang, Miriam T. Levy, Eleanor Kable, George Marinos , Guy Cox and Geoffrey W. McCaughan 1. A. W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, Centenary Institute of Cancer Medicine and Cell Biology and the University of Sydney, NSW Australia. 2. Electron Microscope Unit, University of Sydney. 3. Gastroenterology Department, Prince of Wales Hospital, Sydney

Structure and Function in Dipeptidyl Peptidase IV and Related Proteins

Potential therapeutic applications of DPIV inhibitors have fuelled interest in understanding the biological roles of DPIV and its relatives. Such efforts are confounded by the ubiquitous expression of DPIV, inhibitor selectivity questions and the variety of identified substrates. DPIV is not essential, but is such a useful enzyme that all animal species express it. The enzyme activity’s ancient and primary function is probably nutritional, providing more complete proteolysis of food and recycled proteins. This function is unnecessary in well-fed humans. The development of selective inhibitors of proteolytic activity and identification of ligand binding activities in this gene family would lead to rapid advances in understanding the biology of the POP gene family.

Discoidin Domain Receptor 1: Isoform Expression and Potential Functions in Cirrhotic Human Liver

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and is activated by collagens. Transcriptional profiling of cirrhosis in human liver using a DNA array and quantitative PCR detected elevated mRNA expression of DDR1 compared with that in nondiseased liver. The present study characterized DDR1 expression in cirrhotic and nondiseased human liver and examined the cellular effects of DDR1 expression. mRNA expression of all five isoforms of DDR1 was detected in human liver, whereas DDR1a demonstrated differential expression in liver with hepatitis C virus and primary biliary cirrhosis compared with nondiseased liver. In addition, immunoblot analysis detected shed fragments of DDR1 more readily in cirrhotic liver than in nondiseased liver. Inasmuch as DDR1 is subject to protease-mediated cleavage after prolonged interaction with collagen, this differential expression may indicate more intense activation of DDR1 protein in cirrhotic compared with nondiseased liver. In situ hybridization and immunofluorescence localized intense DDR1 mRNA and protein expression to epithelial cells including hepatocytes at the portal-parenchymal interface and the luminal aspect of the biliary epithelium. Overexpression of DDR1a altered hepatocyte behavior including increased adhesion and less migration on extracelular matrix substrates. DDR1a regulated extracellular expression of matrix metalloproteinases 1 and 2. These data elucidate DDR1 function pertinent to cirrhosis and indicate the importance of epithelial cell-collagen interactions in chronic liver injury.

Extra-Enzymatic Roles of DPIV and FAP in Cell Adhesion and Migration on Collagen and Fibronectin

FAP and DPIV overexpression in the HEK293 cell line reduced cell adhesion, migration and invasion on ECM components, increased proliferation and promoted apoptosis, independently of enzyme activity. These observations may aid an understanding of the roles of FAP and DPIV in tissue remodeling.

The pro-fibrotic role of dipeptidyl peptidase 4 in carbon tetrachloride-induced experimental liver injury

Liver fibrosis is a progressive pathological process involving inflammation and extracellular matrix deposition. Dipeptidyl peptidase 4 (DPP4), also known as CD26, is a cell surface glycoprotein and serine protease. DPP4 binds to fibronectin, can inactivate specific chemokines, incretin hormone and neuropeptides, and influences cell adhesion and migration. Such properties suggest a pro‐fibrotic role for this peptidase but this hypothesis needs in vivo examination. Experimental liver injury was induced with carbon tetrachloride (CCl4) in DPP4 gene knockout (gko) mice. DPP4 gko had less liver fibrosis and inflammation and fewer B cell clusters than wild type mice in the fibrosis model. DPP4 inhibitor‐treated mice also developed less liver fibrosis. DNA microarray and PCR showed that many immunoglobulin (Ig) genes and some metabolism‐associated transcripts were differentially expressed in the gko strain compared with wild type. CCl4‐treated DPP4 gko livers had more IgM+ and IgG+ intrahepatic lymphocytes, and fewer CD4+, IgD+ and CD21+ intrahepatic lymphocytes. These data suggest that DPP4 is pro‐fibrotic in CCl4‐induced liver fibrosis and that the mechanisms of DPP4 pro‐fibrotic action include energy metabolism, B cells, NK cells and CD4+ cells.

DP8 and DP9 have extra-enzymatic roles in cell adhesion, migration and apoptosis

The biological significance of the new DPIV family members DP8 and DP9 is unknown. In order to obtain correlations between cell behaviors and peptidase expression levels, DP8 and DP9 overexpression in transfected cells was quantified by expressing green fluorescent protein fusion proteins. We found that, like DPIV and FAP, cells overexpressing DP8 and DP9 exhibit behavioral changes in the presence of ECM components. We demonstrated that these effects were independent of enzyme activity, and of the RGD motif that occurs in DP9. This study is the first indication of some similarities as well as differences between DP8, DP9, DPIV and FAP in their cell biological roles.