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

ABSTRACT Increased expression of prostaglandin endoperoxide H synthase‐2 (PGHS‐2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide syn‐thase (NOS II) expression and nitric oxide (NO) production are up‐regulated in these diseases as well. However, the apparent link between PGHS‐2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS‐2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionallyimmortalized mouse colonic epithelial cells, namely YAMC (Apc+/+). We found that the induction of PGHS‐2 and generation of PGE2 in these cells by IFN‐γ and lipopolysaccharide (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 tested NO‐releasing compounds, NOR‐1 and SNAP, and found that they caused PGHS‐2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR‐1 and SNAP caused β‐catenin/LEF‐1 DNA complex formation. Super‐shift by anti‐β‐catenin antibody confirmed the presence of β‐catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic β‐catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of β‐catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS‐2 expression and β‐catenin/LEF‐1 DNA complex formation, we studied IMCE (ApcMin/+) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their β‐catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher β‐catenin/LEF‐1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS‐2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS‐2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appe