PARENT SESSION
Signal Transduction
377
NITROPRUSSIDE STIMULATES HUMAN MITOCHONDRIAL ACONITASE GENE EXPRESSION THROUGH THE CYCLIC AMP SIGNAL TRANSDUCTION PATHWAY IN THE HUMAN PROSTATE CARCINOMA CELLS.
Juang, Horng-Heng1, 1
ABSTRACT- The mitochondrial aconitase (mACON), an iron-requiring enzyme, have been indicated as major targets of nitric oxide in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. When treating the NO generator, sodium nitroprusside (SNP), to the human prostate carcinoma cells (PC-3), we find 0.1 mM SNP stimulates the enzymatic activity of human mACON but not the lactate dehydrogenase. However, when treating the cells with other NO generators, S-nitoso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN) in the same concentration, the enzymatic activity of mACON was block. The addition of ascorbic acid to SNP resulted in a distinct decrease in mACON enzymatic activity to a level comparable to that seen with SNAP and SIN. The results indicate the effect of redox-related species of nitric oxide on mACON enzymatic activity. SNP treatments increase the protein translation as same as enzymatic activity of human mACON determining by the Western blotting assay suggesting a putative function of nitric oxide on the mACON gene transcription. We cloned a DNA fragment (-158 to +37) containing the promoter region and a putative cAMP response element (ACGTCA) of human mACON gene into the luciferase reporter vector (pBGL-2). In the transient expression studies both 0.1 mM SNP and dibutyryl-cAMP but not 8-bromo-cGMP increase two-fold, while cAMP-dependent protein kinase inhibitor, H-89, decrease luciferase expression. Mutation of cAMP response element to the AGAGCT using the method of site-directed mutagenesis abolished the activating effects of SNP and dibutyryl-cAMP. These results identify the CRE binding site as a promoter motif that allows nitroprusside to control human mACON gene transcription and establish the function of nitroprusside as a signaling molecular to human mACON through the cAMP signal transduction pathway.
KEY WORDS: prostate, aconitase, cyclic AMP, nitroprusside