FK506 Augments Glucocorticoid-Mediated Cyclooxygenase–2 Down-Regulation in Human Rheumatoid Synovial Fibroblasts

Abstract

SUMMARY: Prostaglandins (PG) formed by cyclooxygenase (COX) enzymes are important mediators of inflammation in rheumatoid arthritis. The contribution of the inducible COX-2 to inflammation in the rheumatoid synovium is well documented. We examined the regulation of COX-2 mRNA and protein expression in response to both glucocorticoids (GC) and FK506 using rheumatoid synovial fibroblasts. Combined treatment of FK506 and a low concentration of dexamethasone (DEX) (10 Ϫ9 M) down-regulated synovial COX-2 mRNA and protein expression. In contrast, neither FK506 nor DEX (10 Ϫ9 M) alone influenced COX-2 expression. Immunocytochemical studies showed that pretreatment with FK506 enhanced the nuclear translocation of the glucocorticoid receptor (GR) in synovial fibroblasts in the presence of low concentrations of DEX (10 Ϫ9 M). Transient transfection experiments showed that treatment of cells with FK506 enhanced the expression of glucocorticoid-responsive gene reporter in the presence of DEX (10 Ϫ9 M). NF-B is known to mediate the transcriptional activation of the COX-2 gene. Electrophoretic mobility shift assay demonstrated that DNA-binding activity of NF-B was suppressed more profoundly by FK506 plus DEX (10 Ϫ9 M) treatment with those of DEX (10 Ϫ9 M) alone in IL-1␤-stimulated synovial cells. Our results indicated that FK506-induced potentiation of GR-mediated repression of synovial COX-2 gene transcription is the result of increased translocation of GR to the nucleus and subsequent repression of NF-B transactivation. Our results also suggest that FK506 may exert anti-inflammatory effects in the rheumatoid synovium by potentiating GR-mediated signal transduction. (Lab Invest 2000, 80:135-141).R heumatoid arthritis (RA) is an inflammatory joint disease in which perpetuation of synovitis leads to articular destruction (Zvaifler and Firestein, 1994). Rheumatoid synovial fibroblasts release various inflammatory mediators (Dayer et al, 1986). Prostaglandin (PG), one of the important mediators in rheumatoid synovitis, is synthesized by cyclooxygenases (COX) (Salmon et al, 1983;Wittenberg et al, 1993). It has been demonstrated that COX-2, an inducible COX isoform, is expressed in rheumatoid synovial fibroblasts and that treatment with IL-1␤ induces the synthesis of COX-2 mRNA and protein, while glucocorticoids (GC) suppress this induction (Crofford et al, 1994).GC has been used for decades as a clinical tool to suppress inflammatory and immune responses. GC action is known to be mediated by cellular glucocorticoid receptors (GR) (Bamberger et al, 1996). The binding of GC is followed by a conformational change in GR; the hormone-receptor complex translocates to the nucleus and binds to its acceptor site in DNA (Truss and Beato, 1993). In the cytoplasm, GR associates with several heat shock proteins (Pratt, 1993;Smith and Toft, 1993). Recent studies showed that the GR complex also contains members of the immunophilin class of proteins (Tai et al, 1992). Furthermore, Yem et al (Yem et al, 1992) reported that FK506-binding protein ...

Inhibition of Farnesyltransferase Prevents Collagen-Induced Arthritis by Down-Regulation of Inflammatory Gene Expression through Suppression of p21ras-Dependent NF-κB Activation

“…Thus, the abnormal, constitutive activation of NF-B has been associated with a number of chronic inflammatory arthritides (9). Many antiarthritic drugs, including nonsteroidal anti-inflammatory drugs, inhibit the expression of proinflammatory genes, such as iNOS, COX-2, TNF-␣, and IL-1␤, through suppression of the NF-B pathway (27). This evidence suggests that the regulation of NF-B activation may be an attractive target for the prevention or treatment for arthritic diseases.…”

Differential Expression of Vascular Endothelial Growth Factor in Glucocorticoid-related Osteonecrosis of the Femoral Head

“…Basic fibroblast growth factor stimulates VEGF release through p42/p43 mitogenactivated protein (MAP) kinase in osteoblasts and negatively regulates it by p38 MAP kinase [28]. Harada et al [10,11] reported PGE2, which is synthesized by COX-2 [17], increased VEGF mRNA levels in osteoblastic RCT-3 cells and in rat calvaria-derived osteoblast-enriched cells. Migita et al [17] examined the downregulation of COX-2 in response to glucocorticoids in human rheumatoid synovial fibroblasts and concluded glucocorticoids could reduce VEGF levels by downregulating COX-2 with subsequent reduction of PGE2.…”

“…Harada et al [10,11] reported PGE2, which is synthesized by COX-2 [17], increased VEGF mRNA levels in osteoblastic RCT-3 cells and in rat calvaria-derived osteoblast-enriched cells. Migita et al [17] examined the downregulation of COX-2 in response to glucocorticoids in human rheumatoid synovial fibroblasts and concluded glucocorticoids could reduce VEGF levels by downregulating COX-2 with subsequent reduction of PGE2. Under pathologic conditions, VEGF is strongly expressed during the time course of fracture healing [21].…”

Glucocorticoids for Human Skin: New Aspects of the Mechanism of Action

“…Moreover, combined treatment with tretinoin should reduce skin atrophy [76,77] , and ointment preparations containing a glucocorticoid plus a vitamin D analogue may improve psoriasis therapy by reducing side effects [78] . Studies in rheumatoid synovial [54] and murine skin fi broblasts [79] also indicate a synergistic effect of tacrolimus and low-dose dexamethasone which may translate into clinical improvement. Due to the still measurable potential for skin atrophy of glucocorticoids and the lower potency and the risk of photocarcinogenesis with tacrolimus and pimecrolimus [80] , a combination therapy that is safe and effective in longterm treatment of severe cases would be welcome.…”

“…Moreover, glucocorticoid response is infl uenced by the cross-talk of nuclear receptor signalling pathways, since the liganded GR does not only form a homodimer, but also heterodimers with the other nuclear receptors and can compete with those for co-factors [18] . Also receptor-associated immunophilins may induce a cross-talk with other signalling cascades as to be derived from studies in synovial fibroblasts [54] and the lung adenocarcinoma-derived A549 cell [8] stimulated with dexamethasone and FK506.…”