Differential regulation of cyclooxygenase-2 in nontransformed and ras-transformed intestinal epithelial cells

Abstract

To determine signaling pathways responsible for modulation of COX-2 expression in nontransformed and transformed epithelial cells, we studied a rat intestinal epithelial (RIE) cell line expressing constitutively active Ras and RhoA. Expression of COX-2 protein was higher in RIE-RhoA(63L) (four-fold) and RIE-Ras(12V) (seven-fold) cells than in parental cells. Prior work suggests that Ras hyperactivity induces the expression of transforming growth factor (TGF)beta and increases epidermal growth factor (EGF)-related peptide signaling-possible mechanisms for increased COX-2 expression. Expression of COX-2 was stimulated by TGFbeta and TGFalpha in RIE and RIE-Rho(63L) cells, but not further stimulated in RIE-Ras(12V) cells. PD153035, an inhibitor of EGF receptor tyrosine kinase, and PD98059, an inhibitor of Erk, attenuated COX-2 expression in RIE and RIE-RhoA(63L). However, the high levels of COX-2 expression in RIE-Ras(12V) cells were not inhibited by either compound. Titration with a pan-neutralizing anti-TGFbeta antibody did not decrease COX-2 in RIE-Ras(12V) cells, even with concurrent EGFR inhibition. Thus, stimulation of the EGF receptor is important in the modulation of COX-2 expression in nontransformed RIE and RIE-RhoA(63L) cells. In Ras-transformed cells, signaling by additional Ras effector pathways, perhaps the RhoA pathway, must be invoked. Identification of these pathways is critical for therapeutic manipulation of COX-2 expression.

Figure 1

Expression of COX-2 protein and determination of PGE₂ levels in intestinal epithelial cell lines. Total cellular lysates from RIE-1, RIE-Ras(12V), and RIE-RhoA(63L) cells growing in subconfluent cultures were isolated, and the basal expression of COX-2 and COX-1 was examined by Western blot analysis. The bar graph shows the relative expression of COX-2 in each cell line determined by densitometry. The graph on the right shows PGE₂ levels in serum-free cell culture medium collected from each cell line. PGE₂ levels were measured by ELISA as described in the Materials and Methods section.

Figure 2

Regulation of COX-2 expression as a function of cell density. Cultures of RIE-1, RIE-Ras(12V), and RIE-RhoA(63L) cells were grown to the point at which they just reached confluence. This time point was considered time 0. Continued postconfluent growth of cells was allowed to occur and, at 24-hour intervals thereafter, cell lysates were isolated, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a single gel, and subjected to Western blot analysis for COX-2 expression. Because RIE-Ras(12V) cells did not show downregulation of COX-2 expression 48 hours after reaching confluence, an additional 24 hours of postconfluence growth was permitted. Quantified results, obtained by densitometry, are shown in the bar graph.

Figure 3

Induction of COX-2 protein expression by TGFα and TGFβ. Rapidly growing cultures of RIE-1, RIE-Ras(12V), and RIE-RhoA(63L) cells were treated with 2 ng/ml TGFα or 2 ng/ml TGFβ 1 for the indicated time period. Total cellular lysates were collected, and COX-2 protein expression was examined by Western analysis using a COX-2-specific antibody. These experiments were performed three separate times and representative results are shown. As shown in Figures 1 and 2, the relative level of COX-2 expression is different for each cell line. In this figure, autoradiographic exposures for each cell line were varied to optimize exposure levels and results were quantified by densitometry.

Figure 4

EGFR tyrosine kinase inhibitors decrease ³H thymidine incorporation but not COX-2 levels in Ras-transformed cells. RIE-Ras(12V) cells were rapidly treated with the indicated concentration of the tyrosine kinase inhibitor, PD153035, in serum-free medium. Eighteen hours later, a 3-hour pulse of ³H thymidine (1 µCi/well) was provided, followed by measurements of thymidine incorporation into TCA-insoluble materials, as described in the Materials and Methods section. AG9, an inactive structural analogue of AG1478, was used as a control in these experiments, and identical experiments with AG1478 were performed (not shown). Results, shown in the top panel, are expressed as percent of ³H thymidine incorporation in cells treated with dimethyl sulfoxide alone. Rapidly growing cultures of RIE-1, RIE-Ras(12V), and RIE-RhoA(63L) cells were treated for 24 hours with the highly specific tyrosine kinase inhibitor, PD153035, at concentrations between 0.01 and 25 mg/ml. Note that the inhibitor concentrations varied for each cell line. Total cellular lysates were collected and COX-2 protein expression was examined by Western blot analysis using a COX-2-specific antibody. These experiments were performed three separate times and representative results are shown. As shown in Figures 1 and 2, the relative level of COX-2 expression is different for each cell line. In this figure, autoradiographic exposures for each cell line were varied to optimize exposure levels. Because of the apparent insensitivity of RIE-Ras(12V) cells to PD153035, its activity as a tyrosine kinase inhibitor was checked in separate experiments, under identical conditions, using an alternate EGFR tyrosine kinase inhibitor, EKI-758. Again, reduced levels of COX-2 are not observed (bottom right). The inset shows that at a concentration of 1 µg/ml, PD153035 markedly inhibited EGFR phosphorylation, but not COX-2 expression, as shown by the arrow. In the experiment shown in the right panel, cells were grown to near confluence and treated with a pan-neutralizing anti-TGFβ antibody for 24 hours. COX-2 protein expression in each cell line was measured by Western blot analysis.

Figure 5

Effect of Erk pathway inhibition on COX-2 expression. Rapidly growing, subconfluent RIE-1, RIE-Ras(12V), RIE-RhoA(63L), and RIE-Ras(12V)/RhoA(N19) cells were treated for 24 hours with the indicated concentration of the EGFR tyrosine kinase inhibitor, PD153035, and the MEK inhibitor, PD98059 (left panel), or with U0126, a more potent inhibitor of MEK (right panel). Total cellular lysates were isolated from each cell line and loaded onto SDS-PAGE gels. Western blot analysis was performed for COX-2 and cyclin D1 expression. This experiment was repeated three times with similar results.

Figure 6

Effect of Rho inhibition on COX-2 expression. Rapidly growing, subconfluent RIE-1, RIE-Ras(12V), and RIE-RhoA(63L) cells were treated for 24 hours with the indicated concentration of Y-27632, an inhibitor of the p160ROCK limb of the RhoA pathway. Total cellular lysates were isolated from each cell line and loaded onto SDS-PAGE gels. Western blot analysis was performed for COX-2 and cyclin D1 expression. This experiment was repeated three times with similar results.