Follicle-stimulating hormone amplifies insulin-like growth factor I-mediated activation of AKT/protein kinase B signaling in immature rat Sertoli cells.
FSH and IGF-I are both important determinants of testicular development and Sertoli cell function. The present studies were performed to determine the actions of FSH and IGF-I on PI3K/AKT protein kinase signaling in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were prepared from 10-d-old rats. After 7 d in culture, Sertoli cells were treated with IGF-I, FSH, or IGF-I plus FSH. In some experiments cultures were treated with 8-bromo-cAMP (40 microM), (Bu)(2)cAMP (40 microM), or forskolin (10 microM). After treatments, cell lysates were prepared, and the activation state of AKT and cAMP response element-binding protein (CREB) was determined by Western blot analysis using phosphorylation site-specific antibodies. IGF-I had little effect on CREB phosphorylation, but rapidly increased the phosphorylation of AKT in a concentration-dependent manner. Maximal stimulatory effects of IGF-I were observed at 10-20 ng/ml. Treatment with FSH (0.9 IU/ml) or forskolin for 20 min increased CREB phosphorylation, but had little effect on AKT phosphorylation. However, FSH caused a concentration-dependent increase in IGF-I-induced AKT phosphorylation. Longer incubations (1-4 h) with FSH alone resulted in the elevation of AKT phosphorylation concomitant with an increased secretion of IGF-I and decreased production of IGF-binding protein-3, implicating endogenous IGF-I in the action of FSH on AKT phosphorylation. IGF-I- and FSH-dependent AKT phosphorylation was inhibited by LY29400 (10 microM), a PI3K inhibitor, and by IGF-binding protein 3, but not by a PKA inhibitor (H89). The present study demonstrates that immature rat Sertoli cells possess multiple protein kinase signaling cascades that are regulated by FSH. Furthermore, FSH amplifies IGF-I-mediated PI3K/AKT signaling in Sertoli cells. The results provide evidence for intracellular signaling mechanisms that may be required for the proliferation and differentiation of Sertoli cells.
Khan
Shafiq A
SA
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA. shafiq.khan@ttmc.ttuhsc.edu
Ndjountche
Lilianne
L
Pratchard
Lauren
L
Spicer
L J
LJ
Davis
John S
JS
D000818:Animals; D001141:Aromatase / Q000378:metabolism; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D005640:Follicle Stimulating Hormone / Q000494:pharmacology*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D008297:Male; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D012708:Sertoli Cells / Q000187:drug effects* / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects*
D017362:Cyclic AMP Response Element-Binding Protein; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D005640:Follicle Stimulating Hormone; D004247:DNA; D001141:Aromatase; C494918:AKT1 protein, human; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt