Tumor Necrosis Factor-α Promotes Sustained Cyclooxygenase-2 Expression: Attenuation by Dexamethasone and NSAIDs

Abstract

Prostaglandin (PG) release is characteristic of most inflammatory diseases. The committed step in the formation of free arachidonic acid into PG products is catalyzed by cyclooxygenase (COX, prostaglandin H₂ synthase, PGHS), which exists as two genetically distinct isoforms. COX-1 is constitutively expressed and produces PGs and thromboxane A₂ during normal physiologic activities, while COX-2 is an inducible enzyme stimulated by growth factors, lipopolysaccharide, and cytokines during inflammation or cell injury. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) released into the amniotic fluid in the setting of infection have been proposed to signal amnion and decidual cells to produce PGs that may culminate in preterm labor. However, since the molecular control of this phenomenon has not been established, this study used amnion-derived WISH cells to determine if TNF-α promoted the formation of PGs through COX-2 activity. Treatment of WISH cells with TNF-α (0.1 ng/mL–100 ng/mL) caused a dose-dependent increase in COX-2 expression and the subsequent biosynthesis of PGE₂ that persisted for at least 48 hrs. In contrast, COX-1 mRNA and protein levels were unaltered by TNF-α treatment as determined by RT-PCR and immunoblot analysis, respectively. TNF-α-stimulated COX-2 expression and the subsequent formation of PGE₂ were inhibited by dexamethasone (0.1 μM). In addition, indomethacin (1 μM) and the novel COX-2-selective inhibitor, NS-398 (IC₅₀ ∼ 1.1 × 10⁻⁹ M), attenuated TNF-α-elicited PGE₂ production. Results presented here demonstrate that TNF-α elicits prolonged and regulatable induction of COX-2 in WISH cells, while COX-1 is constitutively expressed and unchanged in response to TNF-α stimulation.

Introduction

Prostaglandins (PGs) are lipid mediators of pain, fever, and other inflammatory processes as well as many physiologic and homeostatic functions. The formation of PGs and thromboxane from arachidonic acid (AA) is catalyzed by a bifunctional enzyme with cyclooxygenase and peroxidase activities, known collectively as cyclooxygenase (COX) or prostaglandin G/H synthase, (PGHS) 1, 2. Two isoforms of COX have been identified: COX-1, a constitutively expressed 70-kDa protein and COX-2, a 72–74-kDa doublet induced by endotoxin, growth factors, and proinflammatory cytokines (for review see refs. 2, 3, 4).

Tumor necrosis factor-α (TNF-α), originally described as a cytotoxic agent capable of inducing hemorrhagic necrosis of tumor cells, belongs to a class of proinflammatory cytokines that mediates cytotoxicity, inflammation, and septic shock 5, 6, 7. Pathophysiologic conditions such as preterm labor (PTL) have also been associated with TNF-α, since elevated levels of this proinflammatory cytokine have been found in the amniotic fluid and peripheral blood of women experiencing infection-mediated PTL 8, 9. The primary signal-transducing TNF-α receptor in nonhematopoietic cells (p55, TNFR I) has been localized on amnion cells [10]. In addition, stimulation of amnion cells in primary culture with TNF-α causes release of AA and increased synthesis of PGE₂, an important lipid mediator involved in the initiation and maintenance of labor 11, 12, 13. Moreover, Romero et al. [8]have shown that human decidua is a rich source of TNF-α bioactivity. Thus, while the mechanisms responsible for PTL remain unknown, it appears that the release of TNF-α in response to a pathogenic insult may promote the release of PGs that cause cervical dilation and myometrial contractions culminating in labor.

We and others have previously demonstrated that cytokines induce de novo expression of COX-2 mRNA and its cognate protein in human amnion, amnion-derived WISH cells, and decidual cells 14, 15, 16, 17. In addition, we have recently reported that epidermal growth factor (EGF) stimulates very rapid but transient onset of COX-2 expression in WISH cells [18]. Preliminary evidence in our laboratory indicated that the onset and duration of COX-2 gene expression were highly dependent upon the stimulus applied (Perkins and Kniss, unpublished observations). Therefore, in the present study we sought to determine whether COX-2 expression was induced rapidly by TNF-α in WISH cells and whether this induction was sustained or short-lived. In contrast to our earlier findings, TNF-α induced a robust and long-term increase in COX-2 mRNA synthesis that was completely abolished by co-incubation with dexamethasone (DEX).