Abstract
In previous studies we reported that stimulation of rat mesangial cells (RMC) with lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF-α) (L/T) elicits inducible nitric oxide synthase (NOS2) mRNA expression, which is inhibited by dexamethasone (DX). We have now analyzed the mechanisms responsible for this inhibitory effect. Dexamethasone had no destabilizing effect on NOS2 mRNA. Transfection of RMC with several luciferase reporter constructs from the 5' flanking regulatory region of the rat NOS2 gene established the importance of the NF-κB site in the transcriptional activation of the NOS2 gene. DNA mobility shift assays showed activation by L/T of the NF-κB complex in a time-dependent manner. Dexamethasone specifically inhibited this activation in a process dependent on the glucocorticoid receptor and with a markedly greater effect when it was added prior to L/T. Dexamethasone increased the expression of the IκB-α transcript and protein in the cytoplasm. While treatment of RMC with L/T induced the transient decrement of cytoplasmic p65 levels and its appearance in the nucleus, preincubation with DX prevented this effect. Co- immunoprecipitation and immunocytochemical studies demonstrated that IκB-α is associated with p65 in the cytoplasm of RMC after treatment with DX and L/T. These results prove that inhibition of NF-κB-mediated transcription is a crucial mechanism by which DX inhibits NOS2 expression, and that this occurs by increasing cytoplasmic IκB-α levels and sequestering the activating subunits of NF-κB in the cytoplasm. The need for previous induction of IκB-α could provide a molecular explanation for the limited efficacy of these agents in the therapy of septic shock.
Original language | English (US) |
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Pages (from-to) | 38-49 |
Number of pages | 12 |
Journal | Kidney international |
Volume | 53 |
Issue number | 1 |
DOIs | |
State | Published - 1998 |
Funding
Part of this work was presented in abstract form at the International Conference on Biochemistry and Molecular Biology of Nitric Oxide, UCLA, Los Angeles, July 1996. This work was supported by DGICYT PB-93-0044 and Biomed 2 Concerted Action BMH4-CT96-0979. M.S. and C.Z. were supported by a fellowship from Fundación Renal “Iñigo Alvárez de Toledo” and are currently at the Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, MD. We are grateful to Drs. Nancy Rice, M.T. Díaz-Meco, L. Boscáand Roussel-UCLAF for valuable reagents. The authors also thank Drs. L. Boscá and J. Rey for helpful comments.
Keywords
- Glucocorticoids
- Mesangial cells
- NF- κB
- Nitric oxide synthase
- Transcription
ASJC Scopus subject areas
- Nephrology