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Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells
C H Clegg1, M S Abrahamsen, J L Degen
1Department of Pharmacology, University of Washington, Seattle 98195.
Abstract:
Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.
Insights
Mouse Y1 adrenal cells lacking cAMP-dependent protein kinase (cA-PK) showed repressed steroidogenesis and gene expression. Restoring cA-PK activity reversed these effects, highlighting its crucial role in adrenal cell function.
Area of Science:
- Molecular Endocrinology
- Cellular Signaling
- Gene Regulation
Background:
- cAMP-dependent protein kinase (cA-PK) is a key regulator of cellular processes.
- Adrenal tumor Y1 cells are a model for studying steroidogenesis and gene expression.
Purpose of the Study:
- To investigate the role of cA-PK in regulating basal and stimulated steroidogenesis and gene expression in Y1 cells.
- To determine if restoring cA-PK activity can rescue repressed cellular functions.
Main Methods:
- Stable transfection of Y1 cells with mutant or wild-type cA-PK subunits.
- Assays for basal and stimulated steroidogenesis.
- Measurement of cAMP-regulated gene expression (ODC, uPA, P450scc) via mRNA levels and transcription rates.
Main Results:
- Repression of basal cA-PK activity led to decreased steroidogenesis and constitutive gene expression.
- Transfection with cA-PK subunits restored basal steroidogenesis and gene expression.
- Induction of C-subunit expression restored hormone responsiveness.
- Both Cα and Cβ subunits regulated gene transcription.
Conclusions:
- cAMP-dependent protein kinase plays a dominant role in regulating basal and stimulated steroidogenesis and gene expression in Y1 adrenal cells.
- Basal cA-PK activity is essential for both basal steroidogenesis and the constitutive expression of cAMP-inducible genes.
- Restoration of cA-PK activity rescues repressed cellular functions and hormone responsiveness.