INVOLVEMENT OF TRANSCRIPTIONAL MECHANISMS IN THE INHIBITION OF NOS2 EXPRESSION BY DEXAMETHASONE IN RAT MESANGIAL CELLS

In previous studies we reported that stimulation of rat mesangial cell s (RMC) with lipopolysaccharide (LPS) + tumor necrosis factor alpha (T NF-alpha) (L/T) elicits inducible nitric oxide synthase (NOS2) mRNA ex pression, which is inhibited by dexamethasone (DX). We have now analyz ed the mechanisms responsible for this inhibitory effect. Dexamethason e had no destabilizing effect on NOS2 mRNA. Transfection of RMC with s everal luciferase reporter constructs from the 5' flanking regulatory region of the rat NOS2 gene established the importance of the NF-kappa B site in the transcriptional activation of the NOS2 gene. DNA mobili ty shift assays showed activation by L/T of the NF-kappa B complex in a time-dependent manner. Dexamethasone specifically inhibited this act ivation in a process dependent on the glucocorticoid receptor and with a markedly greater effect when it was added prior to L/T. Dexamethaso ne increased the expression of the I kappa B-alpha transcript and prot ein in the cytoplasm. While treatment of RMC with L/T induced the tran sient decrement of cytoplasmic p65 levels and its appearance in the nu cleus, preincubation with DX prevented this effect. Co-immunoprecipita tion and immunocytochemical studies demonstrated that I kappa B-alpha is associated with p65 in the cytoplasm of RMC after treatment with DX and L/T. These results prove that inhibition of NF-kappa B-mediated t ranscription is a crucial mechanism by which DX inhibits NOS2 expressi on, and that this occurs by increasing cytoplasmic I kappa B-alpha lev els and sequestering the activating subunits of NF-kappa B in the cyto plasm. The need for previous induction of I kappa B-alpha could provid e a molecular explanation for the limited efficacy of these agents in the therapy of septic shock.