Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

• Published in: Endocrinology (Philadelphia) 2009-02, Vol.150 (2), p.929-935
• Main Authors: Kayampilly, Pradeep P, Menon, K. M. J
• Format: Article
• Language: English
• Subjects: Adenosine kinase; Adenosine monophosphate; AKT protein; AMP; AMP-activated protein kinase; AMP-Activated Protein Kinases - antagonists & inhibitors; AMP-Activated Protein Kinases - metabolism; Animals; Biological and medical sciences; Cell activation; Cell culture; Cell Culture Techniques; Cell cycle; Cell growth; Cell proliferation; Cell Proliferation - drug effects; Cells, Cultured; Cyclin D2; Cyclin-dependent kinase inhibitor p27; Cyclin-Dependent Kinase Inhibitor p27 - genetics; Cyclin-Dependent Kinase Inhibitor p27 - metabolism; Cyclins - genetics; Cyclins - metabolism; Dose-Response Relationship, Drug; Energy balance; Enzyme Activation - drug effects; Enzyme Inhibitors - pharmacology; Female; Follicle Stimulating Hormone - pharmacology; Follicle-stimulating hormone; Fundamental and applied biological sciences. Psychology; Gene expression; Granulosa cells; Granulosa Cells - drug effects; Granulosa Cells - metabolism; Imidazole; Kinases; Oncogene Protein v-akt - metabolism; Oncogene Protein v-akt - physiology; Ovaries; Ovulation; Phenols; Phosphorylation; Protein folding; Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction - drug effects; Time Factors; Vertebrates: endocrinology
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/en.2008-1032

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway. FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.