Bae SH, et al: Hepatic Oval Cell Proliferation via COX-2 Signaling 373 Fig. 6. Analysis of the proliferation of hepatic oval cell (HOC) grown on fibronectin (FN)-coated culture dishes by BrdU staining. (A) BrdU-labeled nuclei in HOCs. Control, non-FN-coated dish in the absence of NS398; FN, FN-coated dish in the absence of NS398; NS398, non-FN coated dish with NS398; FN/NS398, FN-coated dish with NS398 (*p<0.05) (original magnification, ×200). (B) Quantification of proliferating HOCs by BrdU staining. Fig. 7. Hepatic growth factor (HGF) induces cyclooxygenase (COX)- 2 expression in hepatic oval cells (HOCs) treated with NS398. HOCs were cultured in 100 μM NS398 for 24 hours and transferred to medium containing 5 ng/mL HGF. A representative Western blot is shown. The time-dependent increase in COX-2 expression following HGF exposure is apparent. ulation of ECM proteins is important for HOC proliferation dur- ing liver regeneration induced by 2-AAF/PHx.12,31 However, the underlying mechanism linking the COX-2-induced PG signaling pathway with HOC-mediated liver regeneration is unclear. We determined that COX-2 expression occurred in the HOC activation model, in which 2AAF pellets were implanted into animals to inhibit hepatocyte proliferation. Normal liver tissue did not express COX-2, and COX-2 and EP1-4 were expressed and upregulated in liver tissue obtained from the 2AAF/PHx model and from isolated HOCs. EpCAM is a cell surface marker for HOCs.44 Immunofluores- cence microscopy showed that COX-2-expressing cells were stained with the HOC markers albumin (albeit weakly), AFP, FN, and c-Met, but not with EpCAM or α-SMA. Therefore, the sort- ed Thy1+ cells were HOCs, contrary to the opinion that EpCAM+ cells are representative of the HOC population. An increase in c-Met and COX-2 expression was observed over the course of regeneration, suggesting that the PG signal- ing pathway plays a role in HOC activation and proliferation. Previous studies reported that selective COX-2 inhibitors re- duced cancer growth in carcinogen-treated rodents, inhibited various cancer cell lines,45,46 and delayed wound healing by re- ducing inflammation-induced epithelial cell proliferation.47 We found that NS398 suppressed HOC proliferation and decreased CTGF, FN, and AKT expression during liver regeneration. In contrast, the proliferation of HOCs cultured on FN-coated dishes was not inhibited by COX-2 inhibitor treatment. These results indicate that COX-2 and ECM proteins might be involved in HOC activation through PG-dependent and -independent sig- naling pathways. The physical condition of the normal subendothelial ECM is essential for maintaining different functions in all liver cells. We found that NS398 decreased COX-2, CTGF, FN, and AKT expression, but not ERK expression, and that it suppressed HOC proliferation. However, NS398-mediated inhibition was obvi- ated by the presence of FN on the culture dishes. These results suggest that COX-2 is critical for HOC proliferation during liver regeneration, and that the interaction between ECM proteins and COX-2 is essential for liver regeneration after liver injury. HGF is a multifunctional cytokine with mitogenic, morpho- genic, and tumor-suppressing activities, which are present on the majority of epithelial cells, including HOCs.48,49 Despite this