
<ArticleTitle><prot>Salt-inducible kinase</prot> is involved in the <prot>ACTH</prot>/<prot>cAMP-dependent protein kinase</prot> signaling in Y1 mouse adrenocortical tumor cells.</ArticleTitle>
<AbstractText>The involvement of <prot>salt-inducible kinase</prot>, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by <prot>ACTH</prot>, the cellular content of <prot>salt-inducible kinase</prot> 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 <prot>cholesterol side-chain cleavage cytochrome P450</prot> and <prot>steroidogenic acute regulatory protein</prot>, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The <prot>salt-inducible kinase</prot> mRNA level in <prot>ACTH</prot>-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed <prot>salt-inducible kinase</prot> mRNA. Y1 cells that overexpressed <prot>salt-inducible kinase</prot> were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of <prot>cholesterol side-chain cleavage cytochrome P450</prot> mRNA in the <prot>salt-inducible kinase</prot>-overexpressing cells was markedly low compared with that in the parent, while the levels of <prot>Ad4BP/steroidogenic factor-1</prot>-, <prot>ACTH receptor</prot>-, and <prot>steroidogenic acute regulatory protein</prot>-mRNAs in the former were similar to those in the latter. The <prot>ACTH</prot>-dependent expression of <prot>cholesterol side-chain cleavage cytochrome P450</prot>- and <prot>steroidogenic acute regulatory protein</prot>-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the <prot>cholesterol side-chain cleavage cytochrome P450</prot> gene was assayed by using Y1 cells transfected with a human <prot>cholesterol side-chain cleavage cytochrome P450</prot> promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the <prot>cholesterol side-chain cleavage cytochrome P450</prot> promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a <prot>salt-inducible kinase</prot> expression vector. This inhibition did not occur when the cells were transfected with a <prot>salt-inducible kinase</prot> (K56M) vector that encoded an inactive kinase. The <prot>salt-inducible kinase</prot>'s inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/<prot>steroidogenic factor-1</prot> -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that <prot>salt-inducible kinase</prot> might play an important role(s) in the cAMP-dependent, but Ad4BP/<prot>steroidogenic factor-1</prot>-independent, gene expression of <prot>cholesterol side-chain cleavage cytochrome P450</prot> in adrenocortical cells.</AbstractText>
