HYPOTONICITY ACTIVATES TRANSCRIPTION THROUGH ERK-DEPENDENT AND ERK-INDEPENDENT PATHWAYS IN RENAL-CELLS

Acute hypotonic shock (50% dilution of medium with sterile water, but not with isotonic NaCl) activated the extracellular signal response ki nase (ERK) mitogen-activated protein (MAP) kinases in renal medullary cells, as measured by Western analysis with a phospho-ERK-specific ant ibody and by in vitro kinase assay of epitope-tagged ERKs immunoprecip itated from stable HA-ERK transfectants. Hypotonicity also activated t he transcription factor and ERK substrate Elk-l in a partially PD-9805 9-sensitive fashion, as assessed by chimeric reporter gene assay. Cons istent with these data, hypotonic stress activated transcription of th e immediate-early gene transcription factor Egr-1 in a partially PD-98 059-sensitive fashion. Hypotonicity-inducible Egr-1 transcription was mediated in part through 5'-flanking regions containing serum response elements and in part through the minimal Egr-1 promoter. Elimination of the Ets motifs adjacent to key regulatory serum response elements i n the Egr-1 promoter diminished the effect of hypotonicity but failed to abolish it. Interestingly, hypotonicity also transiently activated p38 and c-Jun NH2-terminal kinase 1, as determined by immunoblotting w ith anti-phospho-MAP kinase antibodies. Taken together, these data str ongly suggest that hypotonicity activates immediate-early gene transcr iption in renal medullary cells via MAP kinase kinase-dependent and -i ndependent mechanisms.