Bae SH, et al: Hepatic Oval Cell Proliferation via COX-2 Signaling 371 Fig. 2. Co-localization of cyclooxy- genase (COX)-2 with thymus cell antigen1 (Thy1), c-Met, epithelial cell adhesion molecule (EpCAM), and α-smooth muscle actin markers. Double-immunofluorescence label- ing with COX-2 (red; left panels in A, D, G, J, and M) and Thy1, c-Met, EpCAM, and α-smooth muscle actin, respectively (middle panels in B, E, H, K, and N). Yellow staining on the merged pictures indicates co-local- ization of the two proteins in hepatic oval cells (HOCs). Co-localization of COX-2 (A, D, G, J, and M) and Thy1 (B and E; merged images, C and F), c-Met (H; merged image, I), EpCAM (K; merged image, L), and α-smooth muscle actin (N; merged image, O) showing colocalization. 4’,6-Diamid- ino-2-phenylindole counterstaining, blue (Original magnification: A-C, ×100; D-O, ×400). stimulates the expression of ECM genes in many biological pro- non-FN-coated dishes was suppressed significantly when HOCs cesses. Western blotting of HOCs cultured with NS398 showed were treated with NS398. Interestingly, HOC proliferation was increased levels of the ECM proteins CTGF, FN, and COX-2 (Fig. not inhibited significantly on FN-coated dishes, despite NS398 5). Moreover, AKT phosphorylation was inhibited by COX-2 sig- treatment (Fig. 6B). These observations are evidence of a posi- naling, whereas ERK 1/2 phosphorylation was not. These results tive interaction between HOCs and FN, which is critical for HOC are consistent with the expression of CTGF, FN, and phospho- proliferation. serine-specific AKT in activated HOCs via the PG pathway, and COX-2 expression increased in a time-dependent manner fol- they indicate that ERK functions independently of PG signaling. lowing a change to HGF-containing medium after NS398 expo- FN is an ECM molecule that induces cell proliferation by induc- ing COX-2 signaling and subsequent PGE2 production.33 Fur- thermore, we examined the HOC proliferation rate in FN-coated cultured dishes using BrdU staining. When HOCs were cultured on non-FN-coated dishes as a control and FN-coated dishes in sure for 24 hours (Fig. 7), suggesting a role for HGF in increased COX-2 expression during HOC proliferation. DISCUSSION the absence of NS398, dramatic proliferation was evident in We demonstrated that COX-2 is expressed and that ECM pro- the FN-coated dishes (Fig. 6A). However, the proliferation on teins were produced in activated HOCs through COX-2-induced