Cross-talk between Peroxisome Proliferator-Activated Receptor D and Cytosolic Phospholipase A 2 A/Cyclooxygenase-2/ Prostaglandin E 2 Signaling Pathways in Human Hepatocellular Carcinoma Cells Lihong Xu, Chang Han, Kyu Lim, and Tong Wu Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania Abstract Peroxisome proliferator-activated receptor D (PPARD) is a nuclear transcription factor that is recently implicated in tumorigenesis besides lipid metabolism. This study describes the cross-talk between the PPARD and prostaglandin (PG) signaling pathways that coordinately regulate human hepa- tocellular carcinoma (HCC) cell growth. Activation of PPARD by its pharmacologic ligand, GW501516, enhanced the growth of three human HCC cell lines (HuH7, HepG2, and Hep3B), whereas inhibition of PPARD by small interfering RNA prevented growth. PPARD activation up-regulates the expression of cyclooxygenase (COX)-2, a rate-limiting enzyme for PG synthesis, and tumor growth. PPARD activation or PGE 2 treatment also induced the phosphorylation of cyto- solic phospholipase A 2 A (cPLA 2 A), a key enzyme that releases arachidonic acid substrate for PG production via COX. Activation of cPLA 2 A by the calcium ionophore A23187 enhanced PPARD binding to PPARD response element (DRE) and increased PPARD reporter activity, which was blocked by the selective cPLA 2 A inhibitors. Consistent with this, addi- tion of arachidonic acid to isolated nuclear extracts enhanced the binding of PPARD to DRE in vitro , suggesting a direct role of arachidonic acid for PPARD activation in the nucleus. Thus, PPARD induces COX-2 expression and the COX-2–derived PGE 2 further activates PPARD via cPLA 2 A. Such an interaction forms a novel feed-forward growth- promoting signaling that may play a role in hepatocarcino- genesis. (Cancer Res 2006; 66(24): 11859-68) Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide (1). The pathogenesis of hepatic carcinogenesis remains incompletely understood but is believed to involve sequential events, including chronic inflammation, hepatocyte hyperplasia, dysplasia, and, ultimately, malignant transformation (2). HCC usually develops in the presence of continuous inflammation and hepatocyte regeneration in the setting of chronic hepatitis and cirrhosis, although the molecular mechanisms linking chronic inflammation to malignant transformation remain to be further defined. Several lines of evidence suggest that mediators of inflammation, such as prostaglandins (PG), are implicated in hepatic carcinogenesis (see ref. 3 for review). For example, increased cyclooxygenase (COX)-2 expression has been found in human and animal HCCs (4–10). Elevated levels of PGs, most notably PGE 2 , have also been detected in HCC (11). Overexpression of COX-2 or treatment with exogenous PGE 2 increases human HCC cell growth and invasiveness (9, 12). The COX inhibitors, nonsteroidal anti-inflammatory drugs (NSAID), inhibit the prolif- eration and induce apoptosis in cultured HCC cells and in animal models of hepatocarcinogenesis (3), although these inhibitors are known to mediate effects through both COX-dependent and COX- independent mechanisms. The first step in the formation of PGs is the liberation of arachidonic acid (5,8,11,14-eicosatetraenoic acid) from membrane- bound phospholipids, usually by the action of phospholipase enzymes, primarily phospholipase A 2 s (PLA 2 ). Although there exist multiple different isoforms of PLA 2 s in cells, it is the 85-kDa cytosolic PLA 2 a (cPLA 2 a) that most commonly supplies the arachidonic acid for PG production by COX (13, 14). Two isoforms of COXs have been identified, COX-1 and COX-2, both catalyzing the conversion of arachidonic acid into endoperoxide intermedi- ates that are ultimately converted by specific synthases to prostanoids, including PGE 2 , the most abundant PG in human neoplastic epithelial cells (15, 16). Whereas COX-1 is constitutively expressed in most cells, COX-2 is highly induced by inflammatory cytokines/chemokines, growth factors, oncogene activation, and tumor promoters, thus contributing to the enhanced PG produc- tion when these signaling pathways are activated in inflammatory and neoplastic diseases (15, 16). PGs transduce signals mainly through binding to their specific G protein–coupled receptors along the plasma membrane. Recently, eicosanoids have been shown to regulate cell functions through activation of peroxisome proliferator-activated receptors (PPAR), which belong to the superfamily of nuclear receptors that function as ligand-activated transcription factors. PPARs regulate gene expression by binding with their hetero- dimeric partner retinoid X receptor to specific peroxisome proliferator response elements. Three different PPAR subtypes have been identified: PPARa, PPARy (also termed as PPARh), and PPARg. PPARa is highly expressed in liver parenchymal cells and is implicated in lipid catabolism (17–19). PPARg is predom- inantly expressed in adipose tissue and plays an important role in adipocyte differentiation, insulin sensitization, and glucose homeostasis (20, 21). PPARh/y shows a ubiquitous expression in most tissues (18) and is implicated in fatty acid oxidation, cell differentiation, inflammation, cell motility, and cell growth (22–32). Recent studies suggest a potential role of PPARy in carcinogen- esis. For example, the expression of PPARy is increased in Requests for reprints: Tong Wu, Department of Pathology, University of Pittsburgh School of Medicine, MUH E-740, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-647-9504; Fax: 412-647-5237; E-mail: wut@upmc.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1445 www.aacrjournals.org 11859 Cancer Res 2006; 66: (24). December 15, 2006 Research Article Downloaded from http://aacrjournals.org/cancerres/article-pdf/66/24/11859/2557645/11859.pdf by guest on 19 June 2022