Osp94 (osmotic stress protein of 94 kDa) is known to be up-regulated by hypertonic and heat-shock stresses in mouse renal inner medullary collecting duct (mIMCD3) cells. To investigate the molecular mechanism of transcriptional regulation of the Osp94 gene under these stresses, we cloned and characterized the 5´-flanking region of the gene. Sequence analysis of the proximal 4 kb 5´-flanking region revealed a TATA-less G/C-rich promoter region containing a cluster of Sp1 sites. We also identified upstream sequence motifs similar to the consensus TonE/ORE (tonicity-response element/osmotic response element) as well as the consensus HSE (heat-shock element). Luciferase activities in cells transfected with reporter constructs containing a TonE/ORE-like element (Osp94-TonE; 5´-TGGAAAGGACCAG-3´) and HSE enhanced reporter gene expression under hypertonic stress and heat-shock stress respectively. Electrophoretic gel mobility-shift assay showed a slowly migrating band binding to the Osp94-TonE probe, probably representing binding of TonEBP (TonE binding protein) to this enhancer element. Furthermore, treatment of mIMCD3 cells with MAPK (mitogen-activated protein kinase) inhibitors (SB203580, PD98059, U0126 and SP600125) and a proteasome inhibitor (MG132) suppressed the increase in Osp94 gene expression caused by hypertonic NaCl. These results indicate that the 5´-flanking region of Osp94 gene contains a hypertonicity sensitive cis-acting element, Osp94-TonE, which is distinct from a functional HSE. Furthermore, the MAPK and proteasome systems appear to be, at least in part, involved in hypertonic-stressmediated regulation of Osp94 through Osp94-TonE.

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