mRNA-selective translation induced by FSH in primary Sertoli cells

Abstract

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far.

Fig. 1.

Short FSH stimulation enhances protein neosynthesis as a function of Sertoli cell density. Cells were seeded at increasing density, as indicated, before stimulation with 100 ng/ml FSH along with ³⁵S-labeled methionine and cysteine. Ninety minutes later, cells were harvested and radiolabeled proteins were quantitated as trichloroacetic precipitable counts per minute. Results were expressed as means ± sem of triplicates. Statistical difference was calculated using two-way ANOVA with a Bonferroni posttest to compare control vs. FSH-stimulated cells. *, P < 0.05.

Fig. 2.

FSH stimulation enhances the assembly of polysomes in Sertoli cells. Sertoli cells were treated with FSH for 90 min and cell lysates were separated on a sucrose gradient. A, An experiment done with control Sertoli cells. B, FSH-stimulated cells have been fractionated. Upper panel, Representative absorbance profile at OD = 254 nm, of each collected fraction of RNA separated by velocity sedimentation through a 10–45% sucrose gradient. The positions of the 40 S and 60 S ribosomal subunits, 80 S single ribosomes and polysomes containing two to nine ribosomes are indicated. The nonpolysomal fractions also contain free RNA. Lower panel, Electrophoresis in a 1% agarose gel of an aliquot of RNA isolated from each of the 20 fractions. The 28 S and 18 S rRNA, and the tRNA are visible upon staining and UV illumination. C, Quantification of the band intensity in each fraction by scanning densitometry of the agarose gels. The histogram represents the ratio of intensity in the polysome vs. 80 S fraction, and the results are expressed as the means ± sem of five independent experiments. Statistical difference was calculated using a two-tailed unpaired t test to compare control vs. FSH-stimulated cells. P = 0.0503.

Fig. 3.

FSH increases the recruitment of mTOR to the mRNA 5′cap. A, Sertoli cells were treated with FSH for 60 min before lysis. Cap-binding proteins were precipitated using m⁷GTP-sepharose-coated beads and analyzed by Western blot to detect the recruitment of mTOR. WCL, Whole cell lysate. B, Densitometry analysis of Western blots probed with antibodies raised against mTOR normalized by the eIF4E amount. Values are expressed as means ± sem of three independent experiments. Statistical difference was calculated using a two-tailed unpaired t test in control vs. FSH-stimulated cells. ***, P < 0.005. C, ³⁵S-labeled methionine and cysteine incorporation. Cells were pretreated for 10 min with PP242 (100 μm) before FSH stimulation for 90 min. The radioactivity was counted as in Fig. 1. Results were expressed as means ± sem of triplicates. Statistical difference was calculated using one-way ANOVA with a Bonferroni posttest to compare unstimulated vs. FSH-stimulated cells and control vs. PP242-treated cells. **, P < 0.01; ***, P < 0.005.

Fig. 4.

FSH increases the functional recruitment of p70S6K to the mRNA 5′cap. A, Cap-binding proteins were precipitated from lysates of Sertoli cells treated with FSH for the indicated period of time using m⁷GTP-sepharose-coated beads and analyzed by Western blot to detect the recruitment of p70S6K. C, Western blot analysis of eIF4B phosphorylation on Ser 422 in FSH-stimulated Sertoli cells. Membranes were reprobed with an anti-GAPDH antibody to monitor gel loading. B and D, Densitometry analysis of Western blots probed with antibodies raised against p70S6K normalized by the eIF4E amount or raised against phosphorylated Ser422 of eIF4B, respectively. Values are expressed as means ± sem of three independent experiments. Statistical difference was calculated using one-way ANOVA with a Bonferroni posttest in control vs. FSH-stimulated cells. *, P < 0.05 (B and D).

Fig. 5.

FSH stimulation leads to the recruitment of eIF4G to the mRNA 5′cap, without apparent displacement of 4E-BP1. Sertoli cells were stimulated with FSH for 10 to 60 min before lysis. Cap-binding proteins were precipitated using m⁷GTP-sepharose-coated beads and analyzed by Western blot to detect the recruitment of eIF4G and eIF4E (A) and 4E-BP1 and eIF-4E (C), as indicated. B, Quantification of the eIF4G signal normalized by the eIF4E amount in three independent experiments. Values are expressed as means ± sem. Statistical difference was calculated using a two-tailed unpaired t test in control vs. FSH-stimulated cells. *, P < 0.05. C. Here is shown one representative experiment of five.

Fig. 6.

Analysis of 4E-BP1 phosphorylation profile in Sertoli cells. Lysates were prepared from cells stimulated in time course of FSH stimulation. A, The phosphorylation of 4E-BP1 was analyzed by Western blot using a specific antibody raised against phospho-Ser65. B, The densitometry analysis of three independent kinetics, and the results are expressed as mean values ± sem. C, Representative Western blot using a specific antibody raised against phosphorylated Thr70. D, Densitometry analysis of six independent kinetics. Membranes were reprobed with an anti-GAPDH antibody to monitor gel loading. Values are expressed as means ± sem. Statistical difference was calculated using one-way ANOVA with a Bonferroni posttest in control vs. FSH-stimulated cells. *, P < 0.05.

Fig. 7.

Selective recruitment of mRNA to the polysomes in FSH-stimulated Sertoli cells. The presence of the tPA (A), c-fos (B), VEGF (C), and PTEN (D) mRNA was assayed by qRT-PCR in the polysomal fractions. Results are expressed as the mean relative expression level relative to GAPDH expression ± sem in three to five independent experiments. Each qRT-PCR reaction was done twice. Statistical difference was calculated using one-way ANOVA with a Bonferroni posttest in control vs. FSH-stimulated cells. ** P < 0.01; ***, P < 0.005.

Fig. 8.

The level of c-fos protein is enhanced after as little as 90 min of FSH stimulation. A, The expression of c-fos was analyzed by Western blot. Membranes were reprobed with an anti-GAPDH antibody to monitor gel loading. B, The densitometry analysis of three independent kinetics, and the results are expressed as mean values ± sem. Statistical difference was calculated using two-tailed unpaired t test in control vs. FSH-stimulated cells. *, P < 0.05.

Fig. 9.

Hypothetical scheme of the regulation of the mRNA translational machinery by FSH-induced signaling. The Cell Designer software (http://celldesigner.org) (46) has been used to formalize the topology of the signaling network. The legend is as follows: stimulatory catalyzes are indicated by a line ended in a circle. Dashed lines mean indirect reactions. The active molecular species are surrounded by black dots. P, Phosphorylation, with the position of the phosphorylated residue being indicated when known. Complexes are surrounded by a box. Double arrows indicate reversible reactions. FSH triggers cap-dependent translation by enhancing p70S6K, mTOR, and eIF4G recruitment to the cap. p70S6K activation by FSH has been reported elsewhere (15).