Jm. Mei et al., Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells, FASEB J, 14(9), 2000, pp. 1188-1201
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.