Induction of cyclooxygenase-2 by platelet-derived growth factor (PDGF) and its inhibition by dexamethasone are independent of NF-κB/IκB transcription factors

Abstract

Glucocorticoids are potent inhibitors of cyclooxygenase-2 (prostaglandin G/H synthase-2, COX-2) expression. The focus of this work was to investigate the molecular mechanisms, by which glucocorticoids interfere with platelet-derived growth factor (PDGF)-mediated induction of COX-2 with special emphasis on the role of the transcription factors NF-kappaB/IkappaB alpha. In rat renal mesangial cells, PDGF induced a rapid and transient increase of COX-2 mRNA and protein, which reached maximal levels after 1-2 and 4 h, respectively. The in vivo half-life of COX-2 mRNA, which was estimated to be less than 1 h, was reduced by dexamethasone. Kinetic studies and COX-2 promoter activity assays indicated that dexamethasone also interfered with COX-2 transcription. Inhibition of COX-2 induction by dexamethasone was abrogated by cycloheximide, an inhibitor of translation, indicating dependence on de novo protein synthesis. As a possible mediator of dexamethasone action, the NF-kappaB/IkappaB alpha system of transcription factors was investigated. Dexamethasone doubled IkappaB alpha protein levels within 1 h and reduced complex formation of nuclear NF-kappaB proteins with DNA. Newly synthesized IkappaB alpha may thus bind to NF-kappaB and interfere with gene activation. PDGF-induced signalling, however, barely affected the NF-kappaB/IkappaB alpha system: IkappaB alpha protein remained unaltered for 30 min after treatment of mesangial cells with PDGF and was only reduced by 30% after 1 h. Concomitantly, binding of NF-kappaB proteins to DNA, detected by electrophoretic mobility shift assays, was slightly increased by 30%. Furthermore, stably transfected COX-2 promoter constructs with and without the NF-KB binding site were comparably activated by PDGF (2.5-fold increase of luciferase activity). Taken together, these data indicate that although dexamethasone interferes with the NF-kappaB/IkappaB alpha system of transcription factors, this mechanism is not essential for the inhibition of PDGF-induced COX-2 expression.

A JNK1/AP-1–Dependent, COX-2 Induction Is Implicated in 12-O-Tetradecanoylphorbol-13-Acetate–Induced Cell Transformation through Regulating Cell Cycle Progression

“…Wadleigh et al (47) has reported the similar phenomena that inhibiting endotoxin-induced NF-nB activation by expression of an inhibitor-nB a mutant does not block endotoxin-dependent COX-2 reporter activity. Our results supports the notion that the role of NF-nB in COX-2 induction may depend on cell type and stimulus (47,48).…”

Anti-inflammatory functions of glucocorticoid-induced genes

“…In some studies anti-inflammatory effects of GCs were not accompanied by changes in DNA-binding activity of NFκB as measured by electrophoretic mobility shift assay (Brostjan et al, 1996;De Bosscher et al, 1997;Hofmann et al, 1998;Nissen and Yamamoto, 2000;Ray et al, 1997;Wissink et al, 1998). However, in other cases GCs were found to inhibit NFκB activation as measured by the same assay (Eberhardt et al, 2002;Goppelt-Struebe et al, 2000;Kurata and Yamamoto, 1999;Kurokouchi et al, 2000;Ma et al, 2004;Mukaida et al, 1994;Vital et al, 2003). The latter observations are inconsistent with the transrepression model.…”

“…IκBα expression is tissue-specifically regulated, and is enhanced by GCs in several primary or transformed cell types (Almon et al, 2005;Auphan et al, 1995;Kang et al, 2006;Kurata and Yamamoto, 1999;Kurokouchi et al, 2000;Quan et al, 2000;Ramdas and Harmon, 1998;Scheinman et al, 1995a;Shames et al, 1998;Stojadinovic et al, 2006;Thiele et al, 1999). In A c c e p t e d M a n u s c r i p t A Clark Anti-inflammatory functions of glucocorticoid-induced genes Page 17 of 53 some cases GC treatment inhibited IκBα degradation, nuclear translocation of p65 or formation of nuclear NFκB complexes (Eberhardt et al, 2002;Goppelt-Struebe et al, 2000;Kurata and Yamamoto, 1999;Kurokouchi et al, 2000;Ma et al, 2004;Mukaida et al, 1994;Vital et al, 2003). This is not consistent with a model in which GCs inhibit NFκB only at a level downstream of DNA binding, although it does not conclusively prove that upregulation of IκBα is critical.…”

Negative regulation of nuclear factor-kappaB activation and function by glucocorticoids

“…In addition, the antagonism of NF-B by GCs was shown to be independent of I B induction (Heck et al 1997, De Bosscher et al 2000b, Goppelt-Struebe et al 2000. This was based on the findings that in spite of stimulation of I B synthesis by GCs, increased I B availability did not affect (De Bosscher et al 1997, 2000b or only partially affected (Crinelli et al 2000) the effects of GCs, and that the effects of GCs were resistant to cycloheximide treatment (De Bosscher et al 1997), thereby arguing against de novo induction of an NF-B inhibitor protein as a potential mechanism by which GCs antagonized NF-B.…”

“…In the light of arguments in favor of or against induction of I B synthesis as the mechanism by which GCs antagonized NF-B, it appears that stimulation of I B synthesis and thus antagonism of NF-B activity by GCs is either an independent event (Bourke & Moynagh 1999, Goppelt-Struebe et al 2000, and/or is cell type specific (Costas et al 2000, De Bosscher et al 2000b. However, the latter mechanism is questioned as contradictory effects of GCs on I B synthesis were observed in the same tissue and cell types.…”