Salt-inducible kinase is involved in the ACTH/cAMP-dependent protein kinase signaling in Y1 mouse adrenocortical tumor cells

The involvement of salt-inducible kinase, a recently cloned protein serine/ threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mous e adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly . Its level reached the highest point in 1-2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochr ome P450 and steroidogenic acute regulatory protein, on the other hand, beg an to rise after a few hours, reaching the highest levels after 8 h. The sa lt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treate d Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained lo w. However, Kin-7 cells, when transfected with a PKA expression vector, exp ressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducib le kinase were isolated, and the mRNA levels of steroidogenic genes in thes e cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-over expressing cells was markedly low compared with that in the parent, while t he levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogen ic acute regulatory protein-mRNAs in the former were similar to those in th e latter. The ACTH-dependent expression of cholesterol sidechain cleavage c ytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the sal t-inducible kinase-overexpressing cells was significantly repressed. The pr omoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-ch ain cleavage cytochrome P450 promoter-linked reporter gene. Addition of for skolin to the culture medium enhanced the cholesterol side-chain cleavage c ytochrome P450 promoter activity, but the forskolin-dependently activated p romoter activity was inhibited when the cells were transfected with a salt- inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that enco ded an inactive kinase. The salt-inducible kinase's inhibitory effect was a lso observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expres sing, NIH3T3 cells were used for the promoter assays. These results suggest ed that salt-inducible kinase might play an important role(s) in the cAMP-d ependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol sidechain cleavage cytochrome P450 in adrenocortical cells.