Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular differentiation

Abstract

We sought to elucidate the role of AKT in follicle-stimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-alpha, microtubule-associated protein 2D, and the PKA type IIbeta regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSH-stimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farnesyltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated up-regulation of reporter activities for LHR, inhibin-alpha, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1alpha interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers.

Fig. 1. Schematic model of the pathway leading from the FSHR to HIF-1 activation in GCs

Results support the schematic model in which FSH via cAMP stimulates the activation of PI 3-kinase/ AKT leading to the inactivation of tuberin and subsequent Rheb activation. Rheb then activates mTOR to stimulate translation by phosphorylating p70^S6k, which activates the S6 ribosomal protein. The HIF-1α protein and, thus, HIF-1 activity are consequently up-regulated via increased translation. An increase in HIF-1α leads to the induction of VEGF, inhibin-α, and LHR in GCs, leading to follicular maturation.

Fig. 2. FSH stimulation of GCs results in activation of AKT, inactivation of tuberin, and activation of p70^S6k and the S6 ribosomal protein

In panels A and B, GCs were treated with 50 ng/ml FSH for the indicated times. Western blots of total cell extracts were probed with the indicated antibodies. Phospho-specific (ph) antibodies are described under “Experimental Procedures.” AKT is used as a loading control. Results in each panel are representative of two separate experiments.

Fig. 3. FSH-stimulated inactivation of tuberin and activation of p70^S6k and the S6 ribosomal protein in GCs occurs downstream of PI 3-kinase, Rheb, and mTOR activation

In panel A, GCs were pretreated with or without 100 nM wortmannin or 10 μM H89 for 1 h and then left untreated (CON) or treated with 50 ng/ml FSH, 10 μM forskolin, or 1 mM 8-CPT-cAMP for 1 h. Results are representative of two similar experiments. In panel B, GCs were pretreated with and without 100 nM rapamycin for 15 min and then left untreated (CON) or treated with 50 ng/ml FSH for 1 h. Results are representative of three separate experiments. In panel C, GCs were pretreated with and without 10 μM FTI-277 for 18 h then left untreated (CON) or treated with 50 ng/ml FSH for 1 h. Results are representative of three separate experiments. In panel D, GCs were pretreated with or without 10 μM H89 for 1 h and then left untreated (CON) or treated with 50 ng/ml IGF-1 for 1 h. Western blots of total cell extracts were probed with the indicated antibodies. Phospho-specific (ph) antibodies are described under “Experimental Procedures.” AKT is used as a loading control for all experiments.

Fig. 4. PMSG treatment of rats results in activation of AKT, inactivation of tuberin, and activation of p70^S6k and the S6 ribosomal protein

Rats were treated for the indicated times with 25 IU of PMSG, and 40 μg of detergent-solubilized ovarian extract protein was separated by SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibodies. Phospho-specific (ph) antibodies are described under “Experimental Procedures.” AKT is used as a loading control. Results are representative of two separate experiments.

Fig. 5. Follicular differentiation markers require signaling via PI 3-kinase/AKT/mTOR pathway

In panel A, GCs were pretreated with and without 12.5 μM LY294002 for 1 h and then left untreated (CON) or treated with 50 ng/ml FSH for 1 h. In panel B, GCs were pretreated with and without 100 nM rapamycin for 15 min and then treated with 50 ng/ml FSH for 1 h. Western blots of total cell extracts were probed with the indicated antibodies. mTOR is used a loading control. Results are representative of three separate experiments. In panels C and D, GCs were transfected with promoter-Luc constructs as described under “Experimental Procedures.” GCs were transfected with LHR-α-Luc (C and E) or inhibin-α-Luc (D and F). The following day, the cells were pre-treated with or without 12.5 μM LY294002 for 1 h (C and D) or 100 nM rapamycin for 15 min (E and F) and then untreated (CON) or treated with 50 ng/ml FSH for 6 h. Results are representative of two separate experiments. The percentage of inhibition in the presence of inhibitor is calculated as stated under “Experimental Procedures” and indicated at the right of each figure. Values are expressed as a mean ± S.E. of triplicates. Student’s t test was used for compared values; ** indicates significant difference with p ≤ 0.05.

Fig. 6. FSH stimulation of GCs results in increased HIF-1α protein levels occurring by increased translation

In panel A, GCs were either untreated (CON) or treated with 50 ng/ml FSH for 4 h in the presence of either 150 μM CoCl₂ or 30 μM MG115, as indicated, to prevent degradation of HIF-1α. Western blots of total cell extracts were probed with anti-HIF-1α antibody. CREB is used as a loading control. Results are representative of three separate experiments. In panel B, GCs were treated with 50 ng/ml FSH for the indicated times. Western blots of total cell extracts were probed with anti-HIF-1β antibody. Results are representative of two separate experiments. AKT is used as a loading control. In panel C, GCs were pretreated for 1 h with 8 μM actinomycin D (ACT D) or 30 μM cycloheximide (CHX) where indicated. GCs were then left untreated (CON) or treated with 50 ng/ml FSH for 4 h in the presence or absence of 150 μM CoCl₂, as indicated. CREB is used as a loading control. Results are representative of three separate experiments.

Fig. 7. FSH stimulation of GCs leads to the induction of HRE (3)-TK-Luc and VEGF-Luc activity that is inhibited by the PI 3-kinase inhibitor LY294002

GCs were transfected with promoter-Luc constructs as described under “Experimental Procedures.” In panels A and B, GCs transfected with HRE (3)-TK-Luc (A) or VEGF-Luc (B) were left untreated (CON) or treated with 50 ng/ml FSH for 6 h. For LY294002 treatments, cells were pretreated with 12.5 μM LY294002 for 1 h prior to control (CON) or FSH treatment. The percentage of inhibition in the presence of inhibitor is calculated as stated under “Experimental Procedures” and indicated at the right of each figure. Values are expressed as the mean ± S.E. of triplicates and are representative of three separate experiments. Student’s t test was used for compared values; ** indicates significant difference with p ≤ 0.05.

Fig. 8. Effects of Rheb inhibitor FTI-277 and the mTOR inhibitor ra-pamycin on FSH activation of HRE (3)-TK-Luc and VEGF-Luc

GCs were transfected with promoter-Luc constructs as described under “Experimental Procedures.” In panels A and B, GCs transfected with HRE (3)-TK-Luc (A) or VEGF-Luc (B) were untreated (CON) or treated with 50 ng/ml FSH for 6 h. For FTI-277 treatments, cells were pretreated with 10 μM FTI-277 for 18 h prior to control (CON) or FSH treatment. In panels C and D, GCs transfected with HRE (3)-TK-Luc (C) or VEGF-Luc (D) were left untreated (CON) or treated with 50 ng/ml FSH for 6 h. For rapamycin treatments, cells were pretreated with 100 nM rapamycin for 15 min prior to FSH treatment. The percentage of inhibition in the presence of inhibitor is calculated as stated under “Experimental Procedures” and indicated at the right of each figure. Values are expressed as the mean ± S.E. of triplicates and are representative of three separate experiments. Student’s t test was used for compared values; ** indicates significant difference with p ≤ 0.05.

Fig. 9. Effects of HIF-1α dominant negative on FSH activation of VEGF-Luc, inhibin-α-Luc, and LHR-Luc

GCs were transfected with promoter-Luc constructs as described under “Experimental Procedures” with or without the expression vector for HIF-1α dominant negative. In panels A, B, and C, GCs transfected with VEGF-Luc (A), inhibin-α-Luc (B), or LHR-Luc (C), alone or in conjunction with 50 ng of HIF-1α dominant negative, were left untreated (CON) or treated with 50 ng/ml FSH for 6 h. The percentage of inhibition in the presence of dominant negative HIF-1α is calculated as stated under “Experimental Procedures” and is indicated at the right of each figure. Values are expressed as the mean ± S.E. of triplicates and are representative of three separate experiments. Student’s t test was used for compared values; ** indicates significant difference with p ≤ 0.05.