PARENT SESSION
Signal Transduction

REGULATION OF CYCLOOXYGENASE-2 GENE EXPRESSION IN BOVINE ENDOMETRIAL CELLS.

Burns, Patrick¹, Nelson, Scott², Bruemmer, Jason¹, Escudero, Jean², Clay, Colin², ^{1 2}

ABSTRACT- Cyclooxygenase-2 (Cox-2) is a key regulatory enzyme in prostaglandin synthesis that converts arachidonic acid to prostaglandin H₂. In bovine endometrium, maximal prostaglandin F₂ synthesis is associated with increased levels of both Cox-2 mRNA and protein. The molecular mechanisms that regulate Cox-2 gene expression in this tissue are unknown. The increase in Cox-2 mRNA may be due to an increase in either gene transcription and(or) stabilization of existing message. The objective of this experiment was to determine if the increase in Cox-2 mRNA during acute prostaglandin synthesis is due to an increase in gene transcription. Bovine endometrial cells (BEND) were grown to approximately 70 to 80% confluency in 100 mm culture plates in 1:1 Ham's F12 and MEM medium supplemented with 10% fetal bovine serum and 10% heat inactivated horse serum. After reaching confluency, cells were rinsed twice with PBS and serum starved for approximately 12 to 18 hr before adding treatments. Cells were untreated (controls) or treated with phorbol 12,13 dibutyrate (PDBu; 100 ng/ml), PDBu + PD98059 (ERK1/2 inhibitor; 20 M) or PDBu + actinomycin D (an inhibitor of gene transcription; 1 g/ml) for 3 or 6 hr. PDBu stimulated an increase in Cox-2 mRNA above controls at 3 and 6 hr (P<0.05). PD98059 decreased PDBu-induced Cox-2 mRNA by approximately 50%, whereas actinomycin D totally blocked PDBu-induced Cox-2 expression. In conclusion, data from the present experiment indicate that the phorbol-induced increase in Cox-2 mRNA in BEND cells is due to an increase in gene transcription. Furthermore, activation of ERK appears to account for approximately 50% of the phorbol-stimulated increase in Cox-2 mRNA.

KEY WORDS: cyclooxygenase-2, endometrium, bovine, prostaglandin