Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway

We have established that the serum- and glucocorticold-inducible protein ki nase (Sgk) is a new component of the hyperosmotic stress response. Treatmen t of NMuMg mammary epithelial cells with the organic osmolyte, sorbitol, ca used the stable accumulation of Sgk transcripts and protein after an approx imately 4-h lag. Transient transfection of a series of sgk-CAT reporter pla smids containing either 5' deletions or continuous 6-base pair substitution s identified a hyperosmotic stress-regulated element that is GC-rich and is necessary for the sorbitol stimulation of sgk gene promoter activity. Gel shift analysis identified four major DNA-protein complexes in the hyperosmo tic stress-regulated element that, by competition with excess consensus wil d type and mutant oligonucleotides and by antibody supershifts, contains th e Spl transcription factor. Several lines of evidence suggest that the p38 MAPK signaling pathway mediates the hyperosmotic stress stimulation of sgk: gene expression. Treatment with pharmacological inhibitors of p38 MAPK or with a dominant negative form of MKK3, an upstream regulator of p38 MAPK, s ignificantly reduced or ablated the sorbitol induction of sgk promoter acti vity or protein production. Using an in vitro peptide transphosphorylation assay, sorbitol treatment activates either endogenous or exogenous Sgk that is localized to the cytoplasmic compartment. Thus, we propose that the sti mulated expression of enzymatically active Sgk after sorbitol treatment is a newly defined component of the p38 MAPK-mediated response to hyperosmotic stress.