title: Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and Sgk expression during the transition of granulosa cells to luteal cells.
abstract: The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/ hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RIIalpha/beta subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RIIalpha/beta was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24-48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.
authors.affiliation: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: I J
authors.initials: IJ
authors.lastname: Gonzalez-Robayna
authors.forename: T N
authors.initials: TN
authors.lastname: Alliston
authors.forename: P
authors.initials: P
authors.lastname: Buse
authors.forename: G L
authors.initials: GL
authors.lastname: Firestone
authors.forename: J S
authors.initials: JS
authors.lastname: Richards
mesh_terms: D000818:Animals; D001141:Aromatase / Q000235:genetics*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D015870:Gene Expression; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D017874:Immediate-Early Proteins; D008184:Luteal Cells / Q000201:enzymology* / Q000648:ultrastructure; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D001141:Aromatase; D005576:Colforsin; D017362:Cyclic AMP Response Element-Binding Protein; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005640:Follicle Stimulating Hormone; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010726:Phosphodiesterase Inhibitors; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D000099184:Serum-Glucocorticoid Regulated Kinases