Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) ha s been implicated in pathological conditions such as inflammatory bowel dis eases and colon cancer. Recently, it has been demonstrated that inducible n itric oxide synthase (NOS II) expression and nitric oxide (NO) production a re up-regulated in these diseases as well. However, the apparent link betwe en PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we exam ined the concomitant expression of PGHS-2 and NOS II as well as the product ion of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse c olonic epithelial cells, namely YAMC (Apc(+/+)). We found that the inductio n of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopoly saccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L -NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we te sted NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGH S-2 expression and PGE2 production. This effect was abolished by hemoglobin , a NO scavenger. Using electrophoretic mobility shift assays, we found tha t both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Supe rshift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corr oborated by the immunocytochemistry data showing the redistribution of beta -catenin from the predominantly membrane localization into the cytoplasm an d nucleus after treatment with NO donors. To further explore the possible c onnection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex form ation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Ape genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YA MC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation unde r both resting conditions as well as after induction with NO. In parallel f ashion, IMCE cells ex pressed significantly higher levels of PGHS-2 mRNA an d protein, and generated more PGE2. Overall, this study suggests that NO ma y be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is s till under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formati on of nuclear beta-catenin/LEF-1 DNA complex.-Mei, J. M., Hord, N. G., Wint erstein, D. F., Donald, S. P., and Phang, J. M. Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immorta lized murine colonic epithelial cells.