368 Gut and Liver, Vol. 5, No. 3, September 2011 gens, and growth factors,16-20 and is frequently overexpressed in various tumor cells.21 The effects of COX-2 and PG-dependent signaling on HOC activation have been reported in liver cancer. The COX-2 inhibitor-induced reduction in HOC number may be due to the COX-2-dependent inhibition of Akt phosphorylation and the induction of apoptosis.22 During liver remodeling, including hepatic fibrosis and liver regeneration, several growth factors affect HOC proliferation and differentiation.1,23 Among them, hepatocyte growth factor (HGF) has a variety of activities in various cells, exerting mito- genic24 and morpho-organogenic25 effects by activating c-Met, a tyrosine kinase receptor.26 When HGF binds the c-Met recep- tor, signaling molecules such as extracellular signal-regulated kinase (ERK)27 and AKT protein kinase28 are activated. Among them, the phosphoinositide 3-kinase (PI3K)/AKT signaling path- way is dedicated to a variety of system-related biological effects of HGF, including protection against apoptosis in primary hepa- tocytes.29 Hepatic stellate cells (HSCs) influence the growth and de- velopment of HOCs. HSCs in the periportal regions increase in response to liver injury30 and produce fibronectin (FN), a com- ponent of the extracellular matrix (ECM), which is crucial for liver regeneration. CTGF on an FN-concentrated provisional matrix facilitates HOC activation, and FN is critical for HOC proliferative functions.31 A positive correlation has been report- ed between FN concentration and COX-2 expression in human cancer cells.32-34 However, the precise mechanism linking FN and COX-2 expression during liver regeneration is unclear. Herein, we explored COX-2 expression during HOC acti- vation and liver regeneration following 2-AAF/70% partial hepatectomy (PHx), and the nature of the signals that modulate COX-2 expression. The co-localization of c-Met and EpCAM with COX-2 clearly demonstrated that COX-2 is expressed in HOCs. Our results show that an increasing level of COX-2 and c-Met expression in HOCs may occur through PG signaling during liver regeneration in the 2-AAF/PHx model. Moreover, FN was expressed in HOCs. The relationships between COX-2 and proliferation-related signals during liver regeneration were also investigated. The COX-2 inhibitor NS398 decreased HOC proliferation and significantly suppressed CTGF, FN, and AKT signaling, but did not affect phosphorylated ERK 1/2 (P-ERK 1/2). Interestingly, NS398 did not impair HOC proliferation in FN-coated dishes, indicating that FN induces HOC proliferation through COX-2 signaling. COX-2 expression increased when HOCs were challenged with a COX-2 inhibitor, followed by a change to an HGF-containing medium. MATERIALS AND METHODS 1. Animals and experimental groups Male Fischer rats weighing 120 to 150 g were divided into 3 equal groups (20 rats each). All animal experiments were conducted according to protocols approved by the Animal Care and Use Committee of the Catholic University of Korea. Time-released 2-AAF (35 mg/pellet over 14 days) treatment was achieved using a product supplied by Innovative Research (Sarasota, FL, USA). The pellets were inserted subcutaneously 7 days before PHx. Three rats from each group were killed, and their livers were collected. All time points are indicated as days after PHx treatment. 2. HOC culture A Thy1-specific antibody in conjunction with magnetic- activated cell sorting (MACS) was used to isolate HOCs, with yields averaging 3×106 cells per animal on day 9. Thy1+ cells were isolated by MACS, as described previously.5 HOCs were cultured in Iscove’s MDM solution containing 10% fetal bovine serum (FBS), 5 μg/mL insulin, 100 U/mL penicillin, and 100 mg/ mL streptomycin (Life Technologies, Grand Island, NY, USA). 3. Reverse transcription-polymerase chain reaction (RT- PCR) analysis of COX-2 and prostaglandin E2 receptor mRNA expression RT-PCR analysis was conducted on total RNA isolated from tissues from normal, PHx and 2-AAF/PHx livers, as well as iso- lated HOCs, using an RNeasy Kit (Qiagen, Valencia, CA, USA). Two micrograms of RNA were used for each round of cDNA synthesis. RT was performed using a GeneAmp RNA PCR Kit and a DNA thermal cycler (Perkin Elmer, Norwalk, CT, USA), which were also used for PCR. The primers used for COX- 2, EP1, EP2, EP3, and EP4 were: 5′-AAG CCT CGG CCA GAT GCC AT-3′ forward and 5’-GTA GTA CTG TGG GAT TGA TAT C-3′ reverse (COX-2, 340 bp), 5′-ACG TGG GTC ACT ACG AGC TAC-3’ forward and 5’-GCT GTG GTT GAA GTG ATG GAT C-3′ reverse (EP1 , 301 bp), 5′-GGG TCT CCT TGC TCT TCT GTT-3′ forward and 5′-CTT TCG GGA AGA GGT TTC ATC C-3′ reverse (EP2, 392 bp), 5′-GTA TGC CAG CCA CAT GAA GAC-3′ forward and 5′-GAT GTG CCC CAT AAG CTG GAT AGC-3′ reverse (EP3, 370 bp), and 5′-GCC AAG TGT GGT GAA AGA CAT-3′ forward and 5′-CTC TCT GGC TCC CAC TAA CCT -3′ reverse (EP4, 482 bp). Briefly, mRNA was reverse-transcribed and the cDNA was subjected to 30 cycles of denaturation at 95oC for 1 minute, an- nealing at 55oC for 1 minute, and an extension period of 72oC for 1 minute. The amplified products were subjected to 1% aga- rose gel electrophoresis and stained with ethidium bromide. The mRNA levels were normalized using GAPDH as a housekeeping gene. 4. Immunohistochemical staining Double-immunofluorescence staining for Thy1.1 (BD Biosci- ences, San Jose, CA, USA), c-Met (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and EpCAM (Abcam, Cambridge, MA, USA) with COX-2 (Transduction Lab, Franklin Lakes, NJ, USA) was performed to verify the identity of the HOCs. Addition-