Despite Transcriptional and Functional Coordination, Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 Largely Reside in Distinct Lipid Microdomains in WISH Epithelial Cells

William E. Ackerman IV¹, John M. Robinson ¹ and Douglas A. Kniss ^1*

¹ Department of Obstetrics and Gynecology (Laboratory of Perinatal Research and Division of Maternal-Fetal Medicine) (WEA,DAK), Center for Biomedical Engineering (DAK), and Department of Physiology and Cell Biology (JMR), The Ohio State University, Columbus, Ohio

^* To whom correspondence should be addressed. E-mail: kniss.1{at}osu.edu.

Submitted on April 6, 2005
Accepted on 18 May 2005

	Abstract

Cytokine-induced prostaglandin (PG) E₂ synthesis requires increased expression of cyclooxygenase-2 (COX-2) in human WISH epithelial cells. Recently, an inducible downstream PGE synthase (microsomal PGE synthase-1, mPGES-1) has been implicated in this inflammatory pathway. We evaluated cooperation between COX-2 and mPGES-1 as a potential mechanism for induced PGE₂ production in WISH cells. Cytokine stimulation led to increased expression of both enzymes. Selective pharmacological inhibition of these enzymes demonstrated that induced PGE₂ release occurred through a dominant COX-2/mPGES-1 pathway. Unexpectedly, immunofluorescent microscopy revealed that the expression of these enzymes was not tightly coordinated among cells following cytokine challenge. Within cells expressing high levels of both mPGES-1 and COX-2, immunolabeling of high-resolution semithin cryosections revealed that COX-2 and mPGES-1 were largely segregated to distinct regions within continuous intracellular membranes. Using biochemical means, it was further revealed that the majority of mPGES-1 resided within detergent-insoluble membrane fractions, whereas COX-2 was found only in detergent-soluble fractions. We conclude that although mPGES-1 and COX-2 show transcriptional and functional coordination in cytokine-induced PGE₂ synthesis, complementary morphological and biochemical data suggest that a majority of intracellular mPGES-1 and COX-2 are segregated to discrete lipid microdomains in WISH epithelial cells.

Key Words: inflammation, cytokines, prostaglandin E₂, cyclooxygenase-2, microsomal prostaglandin E synthase-1, lipid microdomains, epithelial cells

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