Identification of novel molecules and pathogenic pathways in primary biliary cirrhosis: cDNA array analysis of intrahepatic differential gene expression.

Abstract

Background--Primary biliary cirrhosis (PBC) is an autoimmune disease in which the pathogenesis of progressive liver injury is poorly understood.

Aim--To provide novel insights into the pathogenesis of PBC related liver injury using cDNA array analysis, which simultaneously examines expression of many genes.

Methods--Utilising cDNA arrays of 874 genes, PBC was compared with primary sclerosing cholangitis (PSC) associated cirrhosis and non-diseased liver. Differential expression of 10 genes was confirmed by real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).

Results--Array analysis identified many differentially expressed genes that are important in inflammation, fibrosis, proliferation, signalling, apoptosis, and oxidative stress. PBC was associated with increased expression of both Th1 and Th2 type molecules of the immune response. Fibrosis related gene expression featured upregulation of connective tissue growth factor and transforming growth factor beta3. Many more apoptosis associated molecules exhibited increased expression, consistent with apoptosis being a more active and regulated process, in PSC associated cirrhosis than in PBC. Increased expression of many genes of the Wnt and notch pathways implicated these highly conserved and linked pathways in PBC pathogenesis. The observed increases in expression of c-jun, c-myc, and c-fos related antigen 1 are consistent with increased Wnt pathway activity in PBC. Differential expression of four components of the Wnt pathway, Wnt-5a, Wnt-13, FRITZ, and beta-catenin, was confirmed by quantitative RT-PCR.

(Gut 2001;49:565-576)

Conclusion--Many genes implicated in intrahepatic inflammation, fibrosis, and regeneration were upregulated in PBC cirrhosis. In particular, increased expression of a number of Drosophila homologues was seen in PBC.

Keywords: primary sclerosing cholangitis; apoptosis; fibrosis; connective tissue growth factor; Wnt; Th1/Th2; brain derived neurotrophic factor; notch

The pathogenesis of primary biliary cirrhosis (PBC) involves immune mediated injury of bile ducts and is characterised by multiple autoantibodies to mitochondrial antigens (antimitochrondrial antibodies (AMA)) Investigation into the pathogenesis of PBC has focused principally on the mitochondrial antigens, [3] AMA characterisation, [2] and the nature of the immune infiltrate around bile ducts. [1] Targeting of the biliary epithelium may be explained by abnormal expression of mitochondrial antigens on the luminal surface of biliary epithelial cells. [4] The aberrant mitochondrial antigen expression is thought to result in a breakdown of tolerance leading to immune mediated liver injury. [3] An alternative hypothesis that PBC results from an immune response initiated due to an infectious agent is under investigation. [5]

In PBC, damage to the biliary epithelium and progressive liver injury leading to cirrhosis appears to be mainly due to a cell mediated immune response. The mononuclear cell infiltrate in PBC is characterised by activated [CD4.sup.+] and [CD8.sup.+] T lymphocytes with a predominant Th1 response. [6-9] However, we have observed intrahepatic upregulation of interleukin (IL)-2 but not interferon (IFN)-[gamma] in PBC, which suggests that PBC does not universally conform to the Th1 paradigm. [10]

T cells of most patients recognise purified oxo-acid dehydrogenase mitochondrial multi-enzyme complex antigens [11] but the role of autoantibodies to these antigens is controversial. [2,12] There is some evidence of "molecular mimicry" [13] where a cross reactive antigen is targeted by an immune response initially induced by a micro-organism. [14] The presence of a cross reactive antigen is possible given that the titre of [AMA.sup.2] and the T cell response [15,16] to mitochondrial antigens do not always correlate with disease severity or even the presence of disease. Binding of AMA to purified biliary epithelial cells differs markedly for various antibodies suggesting that there are multiple epithelial antigens with epitope cross reactivity.

The immune response in PBC results in progressive bile duct damage and tissue fibrosis. Many fibrotic mediators in PBG may arise from damage to biliary epithelium with release of fibrogenic growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (FGF), transforming growth factor (TGF) beta, and platelet derived growth factor (PDGF). [19,20] These fibrotic mediators lead to activation of the hepatic stellate cell (HSC) with release of basic FGF, hepatocyte growth factor (HGF), and TGF-beta. Autocrine stimulation of the activated HSC with PDGF, basic FGF, and TGF-beta, and stimulation of the biliary epithelium with EGF and HGF help maintain the initiated fibrogenic stimulus. [21,22] Inflammatory mediators such as monocyte chemotactic factor 1 (MCP-1) are thought to stimulate the fibrotic response in PBC. [20]

Analysis of differential gene expression is an important approach to understanding liver injury pathogenesis. Classical approaches have examined mRNA expression of individual candidate genes such as growth factors (for example, connective tissue growth factor (CTGF) [23]), cytokines (for example, IL-8 [10]), and chemokines (for example, IP-10 [24]). Unfortunately, methods of detecting differential gene expression such as northern blot analysis and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) are laborious and have explored few genes. Pathogenic processes involved in liver injury, such as inflammation, proliferation, apoptosis, and fibrosis, are inter-related and increasing numbers of genes are being identified as important in each pathogenic process. Clearly, approaches that can simultaneously examine differential expression of many genes are likely to lead to significant advances in understanding pathogenic pathways involved in liver injury. Therefore, in this study we used comp lementary DNA (cDNA) array analysis to examine intrahepatic differential gene expression in PBC. This paper describes multiple novel observations of differentially expressed genes and pathways implicated in the above mentioned pathogenic processes. Interestingly, the Wnt pathway was strongly implicated in PBC pathobiology for the first time.

Methods

TISSUE AND RNA ISOLATION

Total RNA was isolated from end stage cirrhotic PBC (n=6) and end stage cirrhotic primary sclerosing cholangitis (PSC) (n=4) tissue obtained from liver explants. Non-diseased tissue was obtained from transplant donor liver biopsies (n=4) and from normal liver (distant from the tumour margin) during hepatic metastasis resection (n=4). Tissue was obtained following institutional ethics committee approval and Australian Medical Research Council guidelines. Chronic inflammatory activity and end stage cirrhosis were evident in all of the cirrhosis specimens. RNA extraction using guanidine isothiocyanate dissolution and isopropanol precipitation has been described previously. [10] Poly [A.sup.+] mRNA for array probe synthesis was isolated from pools of four individual RNA samples using oligo-[dT.sub.18] cellulose (Roche Molecular Systems Inc., Branchburg, New Jersey, USA) by standard methods. [25] The pooling of samples is a means of normalising for individual differences in array analysis. [26]

cDNA ARRAY ANALYSIS

Two nylon membrane based cDNA arrays were used: (a) ATLAS Human Gene Array 1.0 (588 genes and nine housekeeping genes) and (b) ATLAS Cytokine/Receptor Array (268 genes and nine housekeeping genes) (Clontech Laboratories, Inc., Palo Alto, California, USA) (fig 1). A list of all of the genes on these two arrays with summaries of gene functions is at www. clontech.com/atlas.

Probes from cirrhotic PBC, cirrhotic PSC, donor liver, and normal liver tissues were hybridised to both an ATLAS Cytokine Array and an ATLAS Human Gene Array. Probe synthesis was performed as recommended by the manufacturer with some modifications. During first strand cDNA synthesis, 1-2 [micro]g of poly [A.sup.+] mRNA were labelled in a 20 [micro]l reaction with 7.5 [micro]l of [[alpha].sup.32]P-dCTP (10 [micro]Ci/[micro]l, 3000 Ci/mmol; NEN Life Science Products, Inc., Boston, Massachusetts, USA) with 1 [micro]l of Superscript II RT (Gibco-BRL Gaithersburg, Maryland, USA), 4 [micro]l of 5x reaction buffer, 2 [micro] of 100 mM dithiothreitol, 0.5 [micro]l of RNase inhibitor (Promega Corp., Madison, Wisconsin, USA), 1 [micro]l of gene specific primer mix (Clontech), water, and 2 [micro]l of deoxy-nucleotides (dNTPs) (5 mM 2' deoxy-adenosine 5' triphosphate (dATP),…