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J. Biol. Chem., Vol. 279, Issue 50, 52500-52516, December 10, 2004

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The Luteinizing Hormone-releasing Hormone Inhibits the Anti-apoptotic Activity of Insulin-like Growth Factor-1 in Pituitary T3 Cells by Protein Kinase C-mediated Negative Regulation of Akt^*

Annabel Rose, Pascal Froment, Valérie Perrot, Michael J. Quon¶, Derek LeRoith||, and Joëlle Dupont**

From the Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, 37380 Nouzilly, UMR INSERM U-449/INRA 1235, RTH Laënnec Medical School, University of Lyon I, 69372 Lyon, France, ^¶Diabetes Unit, Laboratory of Clinical Investigation, NCCAM, and ^||Section on Molecular and Cellular Physiology, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, April 26, 2004 , and in revised form, September 23, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the T3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in T3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) -specific inhibitor, and by a dominant negative form of PKC. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKC-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue T3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Luteinizing hormone-releasing hormone (LHRH)¹ is a hypothalamic decapeptide, which plays a crucial role in normal reproductive function. In the pituitary, LHRH-I (mammalian LHRH) stimulates the synthesis and the release of the gonadotropins, LH and FSH, and promotes cell proliferation and apoptosis in pituitary cells (1, 2). The effects of LHRH-I are mediated by a cell surface receptor (LHRH-R) belonging to the G protein-coupled receptor superfamily (3, 4). LHRH-I binding to its cognate receptor leads to interaction of the receptor with heterotrimeric G proteins, including G_q/11. These G proteins in turn activate phospholipase C, leading to the production of diacylglycerol and the subsequent activation of protein kinase C (PKC) (5–9). Activation of PKC then leads to activation of downstream protein kinases belonging to the MAPK family (9–15). Activation of LHRH-R also triggers calcium influx, resulting in an increase in intracellular calcium (16) and cAMP levels (17, 18) and leading to the activation of other protein kinases, such as the c-Jun N-terminal kinase (19, 20). This LHRH-R-mediated pathway acts independently of the PKC and MAPK signaling pathways (21). Thus, multiple signal transduction pathways may mediate LHRH-I action in the pituitary gland.

Like LHRH-I, IGF-1 is a mitogen in pituitary cells (22–24) and an anti-apoptotic factor in several cell lines (25, 26). IGF-1 activates the intrinsic protein-tyrosine kinase activity of the IGF-1 receptor (IGF-1R) (27). This activation results in auto-phosphorylation of the receptor and phosphorylation of various intracellular substrates, including several insulin receptor substrate (IRS) proteins (IRS-1 to -4) and the Src homology collagen protein (28). Tyrosine-phosphorylated IRS-1 is a multisite docking protein for numerous Src homology 2 domain-containing proteins, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and the adapter protein, Grb2 (29, 30). Downstream effectors of PI3K include the serine/threonine protein kinases, Akt/PKB and p70/S6 (31). The binding of the IRS and Src homology collagen proteins to Grb2 and its associated guanine nucleotide exchange protein, mSos, leads to the activation of the Ras-Raf-MAPK pathway (27). The role of the different pathways activated by IGF-1 during proliferative and anti-apoptotic responses is cell type-specific. For example, in human intestinal smooth muscle cells (32, 33), IGF-1 induces growth by activating both the PI3K and MAPK-ERK1/2 pathways. In contrast, only the PI3K pathway is essential for IGF-1 signaling in proliferating MCF-7 cell lines (derived from human breast tumor) (34).

IGF-1 and IGF-1R are both expressed in pituitary cells (35). Like LHRH-I, IGF-1 treatment increases LH secretion by male rat pituitary cells (36). Moreover, IGF-1 enhances the LH response to LHRH-I in pituitary cells from male rats (37), pigs (38), fish (39), and sheep (40). IGF-1 also regulates growth in pituitary gonadotrope cells (41). LHRH-I agonists inhibit cell proliferation in prostate cancer cells by interfering with some of the cellular mechanisms required for the mitogenic activity of IGF-1 (42). The degree of interaction between LHRH-I and IGF-1 signaling pathways in the pituitary gland remains unclear. In this study we have investigated these interactions by using T3–1 gonadotrope cells derived from murine pituitary tumor cells. These cells produce the glycoprotein hormone -subunit, the LHRH-R and SF-1. However, these cells do not produce LH, as they were derived from cells at an early stage of development during which LH was not produced. Although these cells do not express the , LH, and FSH subunits (43, 44), they proliferate and express the LHRH receptor. Thus, T3 cells provide a good model system for determining whether interactions exist between the LHRH-I and IGF-1 signaling pathways.

In this study, we characterized interactions taking place between the IGF-1R and LHRH-R signaling pathways during cell proliferation and apoptosis in T3 cells. We demonstrate for the first time that LHRH-I treatment enhances IGF-1-induced, MAPK-ERK1/2 and PKC signaling pathway-mediated cell proliferation, whereas it strongly diminishes the anti-apoptotic effect of IGF-1 through inhibition of PKC-mediated Akt activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—[-³²P]dCTP (6000 Ci/mmol) and [-³²P]ATP (6000 Ci/mmol) were obtained from PerkinElmer Life Sciences. Recombinant human IGF-1 was purchased from R & D (Minneapolis, MN). Recombinant human LHRH-I, buserelin, and the LHRH antagonist, trifluoroacetate, were purchased from Sigma. Recombinant human EGF was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, and trypsin were purchased from Invitrogen. PI3K-specific inhibitor (LY294002) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. MEK1/2-specific inhibitor (U0126), p38 MAPK-specific inhibitor (SB202190), PKC-specific inhibitor (GF109203X), PKC-specific inhibitor (Rottlerin), PKC-specific inhibitor (Safingol), EGF receptor tyrosine kinase-specific inhibitor (AG1470), and the calcium chelator, BAPTA, were obtained from Calbiochem. The JNK1/2 MAPK-specific inhibitor SP600125 was purchased from Biomol%20Research%20Laboratories">Biomol Research Laboratories (Plymouth Meeting, MA). All inhibitor stock solutions were prepared in MeSO. These stock solutions were prepared so that the concentration of MeSO added to the culture medium was below 0.1%.

Rabbit polyclonal antibodies against phospho-Bad (phospho-Ser-136), Bad, phospho-Akt (phospho-Ser-473), Akt, phospho-ERK1/2 (phospho-Thr-202/Tyr-204), phospho-p38 (phospho-Thr-180/Tyr-182), phospho-JNK1/2 (phospho-Thr-183/Tyr-185), phospho-GSK3/ (phospho-Ser-21/9), phospho-FKHR (phospho-Ser-256), phospho-Elk-1 (phospho-Ser-383), and cleaved caspase-3 were purchased from New England Biolabs (Beverly, MA). Rabbit polyclonal antibodies to EGFR (1005), phospho-EGFR (Tyr-1173), ERK2 (C14), p38 (C20), JNK2 (N18), IGF-1R (C20), and FAK (C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to rabbit IRS-1, IRS-2, and the p85 regulatory subunit of mouse PI3K (p85) were purchased from Upstate Biotechnology, Inc. Monoclonal anti-actin (clone AC) and monoclonal anti-phosphotyrosine (PY20) antibodies were obtained from Sigma and Transduction Laboratories (Lexington, KY), respectively. All antibodies were used at 1:1000 dilution.

Preparation of Rat Anterior Pituitary Cells and Cell Cultures—Rat pituitary cells were prepared from 5-week-old female Wistar rats. Freshly removed anterior pituitary glands were washed twice in B1 buffer (0.25% bovine serum albumin, 0.01% deoxyribonuclease, 137 mM NaCl, 5 mM KCl, 25 mM HEPES, 0.6 mM NaH₂PO₄, 0.7 mM Na₂HPO₄ 12H₂O, and 6 mM glucose) before being incubated with type I collagenase (4 mg/ml) and grade II dispase (2 mg/ml) in buffer B1 at 37 °C for 30 min. Cells were centrifuged for 4 min at 200 x g and then incubated with 0.8% type V neuraminidase in B1 buffer for 10 min at 37 °C. After centrifugation (200 x g, 4 min), dispersed cells were cultured in DMEM without red phenol and supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 µg/ml), fungizone (1.25 µg/ml), and L-glutamine (200 mM).

Mouse gonadotrope T3 cell lines and LT2 cells were provided by Dr. P. Mellon (La Jolla, CA). MCF-7, MDA-MB-231, OVCAR, and SKOV cells were obtained from the ATCC (Manassas, VA). All cell lines were routinely cultured in a humidified atmosphere of 95% air and 5% CO₂ in DMEM supplemented with 10% heat-inactivated FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml L-glutamine.

Immunoprecipitation and Immunoblotting—Cell lysates were homogenized on ice in lysis buffer A (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40) supplemented with various protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) and phosphatase inhibitors (100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2 mM sodium orthovanadate). Lysates were incubated on ice for 30 min and then centrifuged at 12,000 x g for 20 min at 4 °C. The protein concentration in the resulting supernatants was then determined using the BCA protein assay. Extracts containing 200 µg of protein were incubated with IGF-1R, IRS-1, or IRS-2 (1:1000 dilution) for 16 h at 4 °C. Immune complexes were precipitated by incubation with protein G-agarose for 1 h at 4 °C as described previously (45). Immunoprecipitates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked by incubation for 1 h at room temperature with 5% nonfat milk dissolved in Tris-buffered saline supplemented with 0.1% Tween 20 and then probed with the appropriate antibodies. After extensive washing, immune complexes were detected using horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and an enhanced chemiluminescence (ECL) detection system. The radiographs were scanned, and the optical density of each band was measured using the MacBas version 2.52 software (Fuji PhotoFilm). Western blotting was performed without immunoprecipitation using cell extracts containing 50 µg of protein.

PI3K Assay—PI3K activity was determined as described previously (45). Cell lysates were prepared on ice in extraction buffer B (20 mM Tris (pH 7.5), 137 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 150 mM Na₃VO₄, 1% Nonidet P-40, 10% glycerol (v/v), 2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin in phosphate-buffered saline (PBS)). Cell lysates were cleared by centrifugation for 35 min at 40,000 x g at 4 °C. IRS-1, IRS-2, IGF-1R, or p85 were immunoprecipitated by incubating aliquots (containing 200 µg of total protein) of each of the supernatants with the appropriate antibodies (1:1000) overnight at 4 °C. Immunoprecipitates were collected using protein G-agarose beads and washed as follows: once in PBS supplemented with 1% Nonidet P-40 and 100 µM Na₃VO₄; twice in a buffer containing 100 mM Tris-HCl (pH 7.5), 500 mM LiCl₂, 100 µM Na₃VO₄; and finally once in a buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 100 µM Na₃VO₄. Precipitates were then resuspended in 40 µl of a buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM EDTA. MnCl₂ (10 µl of a 100 mM stock solution) and phosphatidylinositol (20 µg) were then added to each sample. PI3K assays were initiated by adding 10 µl of a 440 µM ATP solution containing 30 µCi of [-³²P]ATP. Reactions were performed at room temperature and allowed to proceed for 10 min. Reactions were stopped by adding 20 µl of HCl (8N) and 160 µl of CHCl₃/CH₃OH (1:1). The reaction mixtures were then centrifuged at 3,000 x g for 4 min at 4 °C. The resulting organic phase was collected and subjected to silica gel TLC. TLC plates were developed by incubation in CHCl₃/CH₃OH/H₂O/NH₄OH (120:94:22.6:4) and then left to dry. Radioactivity was quantified using a PhosphorImager (Storm, Amersham Biosciences).

Akt and ERK1/2 MAPK Activity Assays—Akt and ERK1/2 MAPK kinase activities were measured using the appropriate assay kits (Cell Signaling Technology, Beverly, MA). T3 cells were stimulated as described in the figure legends and lysed using cell lysis buffer. Cell lysates containing 200 µg of total protein were immunoprecipitated by incubation with immobilized Akt or phospho-ERK1/2 monoclonal antibodies, with gentle rocking for 3 h at 4 °C. After two washes with cell lysis buffer followed by two washes with kinase buffer (25 mM Tris (pH 7.5), 5 mM -glycerophosphate, 2 mM dithiothreitol, 0.1 mM Na₃VO₄,10 mM MgCl₂), the immunoprecipitates were resuspended in 40 µl of kinase buffer supplemented with 200 µM ATP and 1 µg of GSK-3 or Elk-1 fusion proteins. Samples were incubated for 30 min at 30 °C. The reaction was terminated by adding 20 µl of 3x SDS loading buffer. Samples were analyzed by probing with the phospho-GSK-3/ (Ser-21/9) or phospho-Elk-1 (Ser-383) antibodies.

Cell Proliferation Assay—For growth studies, T3 cells were seeded in 96-well plates (10,000 cells per well) in DMEM supplemented with 10% of FBS. After 1 day, cells were transferred to serum-free medium and allowed to grow for 24 h before being incubated with various stimulants (see figure legends for details). Growth was analyzed using the MTT assay as described previously (45).

Flow Cytometry and Cell Cycle Analysis—T3 cultures, in which 75% of the cells were confluent, were washed and transferred to serum-free DMEM and allowed to grow for 24 h. Cells were subjected to various treatments (see figure legends for details). After treatment with IGF-1 (10 nM) and/or LHRH (10 nM) for 18 h, cells were trypsinized, centrifuged, and washed twice with PBS. Cell pellets were resuspended in citrate buffer (250 mM sucrose, 40 mM trisodium citrate (pH 7.6), 5% Me₂SO) and stored at -70 °C. Nuclei were obtained by incubating the cells first in 300 µl of solution A (3.4 mM trisodium citrate (pH 7.6), 1 mM Tris, 3 mM spermine tetrahydrochloride, 0.2% Nonidet P-40, 100 µg/ml trypsin) for 5 min at room temperature and then in 300 µl of solution B (3.4 mM trisodium citrate (pH 7.6), 1 mM Tris, 3 mM spermine tetrahydrochloride, 0.2% Nonidet P-40, 100 µg/ml ribonuclease A, and 500 µg/ml trypsin inhibitor) for 5 min at room temperature. Nuclei were stained with 30 µl of propidium iodide solution (1 mg/ml), and DNA staining was analyzed by flow cytometry using a FACSCalibur system with the CellQuest software. Cell cycle analysis was performed using the ModfitLT^TM software (version 2).

Cell Death Assays—Apoptotic cells were detected by using terminal dUTP nick-end labeling assay with ApopTag Plus Peroxidase, according to the manufacturer's instructions (Intergen Co., Purchase, NY). One thousand cells were counted, and the values obtained were used to calculate the percentage of labeled cells.

Transient Transfection of MCF-7 and T3 Cells—MCF-7 and T3 cells were plated out the day before transfection. Cells were transiently transfected using the LipofectAMINE transfection reagent (Invitrogen) according to the manufacturer's instructions. Cells were transfected with 1 µg of each DNA construct (mouse LHRH-R construct for MCF-7 cells and PKC DN or focal adhesion kinase dominant negative for T3 cells) or 1 µg of empty pcDNA3.1 vector (negative control). Twenty four hours after transfection, cells were transferred to serum-free DMEM and allowed to grow for 16 h before being stimulated with various ligands, see figure legends for details. Cells were then washed with ice-cold PBS, and total protein was extracted.

Statistics—Data were analyzed by one-way analysis of variance using the Statview 5.0 software. Values are expressed as means ± S.E. Values of p < 0.05 were considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
IGF-1R Signaling Pathways in T3 Gonadotrope Cells—We first determined whether T3 cells expressed the IGF-1 receptor (Fig. 1). Immunoblot analysis using an antibody recognizing the -subunit of the IGF-1R revealed that T3 cells did express the IGF-1R. NWTb3 (NIH-3T3 overexpressing the human IGF-1R) (46) and the human breast cancer cell line, MCF-7, were used as positive controls (Fig. 1). We then investigated the functionality of the IGF-1 receptor in T3 cells. Serum-starved cells were exposed to 10 nM IGF-1 for different times (Fig. 2). We found that IGF-1 induced a rapid and substantial increase in tyrosine phosphorylation of the IGF-1R -subunit (15-fold increase after 1 min of treatment) and of IRS-1 (5-fold increase after 1 min of treatment). Tyrosine phosphorylation levels then declined over the next 30 min. IGF-1R is involved in two main signaling pathways: the PI3K/Akt pathway and the MAPK (ERK1/2, p38, and JNK1/2) pathway (28). Thus, we tested whether these kinases were activated in response to treatment of T3 cells with IGF-1. IGF-1 stimulated PI3K activity (data not shown) and resulted in a 5-fold increase in the levels of phosphorylated Ser-473 and Thr-308 Akt after 1 min of treatment. The level of phosphorylated Akt increased with the length of the treatment, reaching a maximum of 20-fold after 10 min (Fig. 2C). IGF-1 also stimulated phosphorylation of two downstream targets of Akt: the transcription factor FKHR and the kinase GSK3 / (Fig. 2D). Although the MAPKs, ERK1/2, p38, and JNK1/2, were present in the T3 extracts, we detected only a small increase in the level of phosphorylation of these kinases in response to IGF-1 (Fig. 2E). Thus, our results indicate that T3 cells are equipped with a functional IGF-1 receptor.

View larger version (44K): [in this window] [in a new window]   FIG. 1. IGF-1R expression in T3 cells. Cells (T3, MCF-7, and NWTb3) were lysed and protein extracts were subjected to SDS-PAGE. Proteins were probed using the IGF-1R antibody. Sample loading was evaluated by reprobing each membrane with an -actin antibody. All samples contained equal amounts of protein.

View larger version (51K): [in this window] [in a new window]   FIG. 2. IGF-1 signaling pathways in T3 cells. Time course of IGF-1-stimulated IGF-1R (A), IRS-1 (B), Akt (C), GSK3/ and FKHR (D), and MAPKs (ERK1/2, p38, and JNK1/2) (E) activation in T3 cells. T3 cells were incubated in serum-free DMEM overnight before treatment with 10 nM IGF-1 for 0, 0.5, 1, 10, 30, or 60 min. Whole cell lysates were separated by SDS-PAGE and immunoblotted with different antibodies (phosphotyrosine, phospho-MAPK (ERK1/2, p38, and JNK1/2), phospho-Akt Ser and Thr, phospho-GSK3/, and phospho-FKHR). The blots were then stripped and reblotted with appropriate antibodies to determine total proteins loading. The blots are typical examples of an experiment that was replicated three to four times.

View larger version (36K): [in this window] [in a new window]   FIG. 3. LH-RH type I activates MAPK family and PI3K in T3 cells. A, time course of LHRH type I-mediated activation of ERK1/2, p38, and JNK1/2. T3 cells were incubated in serum-free DMEM overnight and then treated with LHRH-I (10 nM) for the different periods of time indicated. Whole cell lysates were subjected to SDS-PAGE, and proteins were detected using the appropriate antibodies (phospho-ERK1/2, phospho-p38, and phospho-JNK1/2). Sample loading was assessed by stripping and reprobing the membranes with antibodies to ERK1/2, p38, and JNK1/2. The blots shown are representative of those obtained from three or four independent experiments. B, PhosphorImager analysis of 32P-labeled inositol 1,3,4,5-triphosphate binding to p85, the regulatory subunit of the PI3K, in LHRH-I-treated T3 cells. The results shown are representative of those obtained from three independent experiments. Values are the mean ± S.E. C, a comparison of LHRH-I-mediated and IGF-1-mediated activation of Akt phosphorylation in T3 cells.

View larger version (23K): [in this window] [in a new window]   FIG. 4. A, additive effect of IGF-I and LHRH-1 on cell growth in T3 cells. T3 cells were maintained for 3 days in serum-free medium in the absence or presence of IGF-1 (10 nM), LHRH-I (10 nM), or a combination of these hormones. Cell number was determined indirectly by MTT assays. Values are the mean cell number (±S.E.) from three independent experiments. Five measurements were performed per experiment for each condition. B, determination of the intracellular signaling pathways involved in IGF-1- and LHRH-I-induced T3 cell proliferation. Serum-starved T3 cells were preincubated for 1 h with the following inhibitors: 50 µM LY294002, 1 µM U0126, 50 µM SB202190, 50 µM SP600125, or 10 nM GF109203X and then incubated in the absence or presence of IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both stimulants for 18 h. The percentage of cells in S phase was determined after each treatment. The error bars represent the mean ± S.E. from three independent experiments. * indicates p < 0.05 (significant difference between the effect of IGF-1 alone, LHRH-1 alone, or IGF-1 and LHRH-1, and the effect of these treatments plus the various inhibitors).

Effects of LHRH-I on IGF-1 Receptor Signaling in T3 Gonadotrope Cells—We then investigated whether any interactions took place between the various components of IGF-1 and LHRH-I receptor signaling cascades. T3 cells were starved overnight and then exposed to IGF-1 (10 nM), LHRH-I (10 nM), or both stimulants for 5 min. PI3K activity and the phosphorylation state of IRS-1, IRS-2, and the IGF-1R were then determined (Fig. 5A). Treatment with IGF-1 (10 nM, 5 min) resulted in phosphorylation of the IGF-1R subunit (Fig. 5A, panel 1), IRS-1 (Fig. 5A, panel 2), and IRS-2 (Fig. 5A, panel 3), whereas treatment with LHRH-I alone (10 nM, 5 min) did not promote phosphorylation of these proteins. However, the levels of IRS1 phosphorylation and PI3K activity were higher in cells cotreated with IGF-1 and LHRH-I than in cells treated with IGF-1 alone (Fig. 5A, panel 4). Most surprisingly, cotreatment with IGF-1 and LHRH-I did not have a synergistic or additive effect on IGF-1R and IRS-2 phosphorylation (Fig. 5A, panels 1 and 2).

View larger version (28K): [in this window] [in a new window]   FIG. 5. Effect of IGF-1 and LHRH-I on IRS-1-associated PI3K activity and the phosphorylation of IRS-1, IRS-2, and the IGF-1R subunit. A, serum-starved T3 cells were stimulated with either IGF-1 (10 nM), LHRH-I (10 nM), or both agents for 5 min. Cells were lysed, and tyrosine phosphorylation of IRS-1, IRS-2, and the IGF-IR was determined. IGF-1R (panel 1), IRS-1 (panel 2), and IRS-2 (panel 3) were immunoprecipitated (IP) from whole cell lysates. Samples were then subjected to Western blotting with antibodies recognizing phosphotyrosine (PY20, panels 1–3). IGF-1R, IRS-1, and IRS-2 levels were evaluated by reprobing the membranes with IGF-1R, IRS-1, and IRS-2 antibodies. The results presented are representative of those obtained from three independent experiments. Typical results from the PhosphorImager analysis of 32P-labeled inositol 1,3,4,5-triphosphate binding to IRS-1 are shown in panel 4. B, role of the EGFR tyrosine kinase in the LHRH-I-mediated increase in IGF-1-induced IRS-1 phosphorylation and IRS-associated PI3K activity. Panels 1 and 2, serum-starved T3 cells were either left untreated or preincubated with AG1478 (10 µM) for 1 h before stimulation with either IGF-1 (10 nM), LHRH-I (10 nM), or both agents for 5 min. IRS-1 phosphorylation and IRS-1-associated PI3K activity were determined as described above for A. Panel 3, LHRH-I-mediated activation of the EGFR tyrosine kinase. Serum-starved T3 cells were stimulated with either EGF (10 nM) or LHRH-I (10 nM). Cells were lysed, and the levels of EGFR tyrosine phosphorylation were determined. EGFR was immunoprecipitated from whole cell lysates. Samples were then subjected to Western blotting with antibodies to phosphotyrosine. EGFR levels were determined by reprobing the membranes with EGFR antibodies. The results presented are representative of three independent experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Role of the p125 FAK in the LHRH-I effect on the PI3K activity associated to IRS-1. A, coimmunoprecipitation of IRS-1 with the tyrosine-phosphorylated EGF receptor. Serum-starved T3 cells were stimulated with IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents for 5 min. Cells were lysed, and IRS-1 was immunoprecipitated from whole cell lysates. Samples were then subjected to Western blotting with antibodies to the tyrosine (residue 1173)-phosphorylated EGFR. IRS-1 levels were evaluated by reprobing with IRS-1. The results shown are representative of those obtained with three independent experiments. B, role of calcium and the EGFR in the effect of LHRH-I on IRS-1-associated PI3K activity. Effect of AG1478 (10 µM) and the calcium chelator, BAPTA (10 µM), on LHRH-I-induced IRS-1-associated PI3K activity. Cells were incubated in the presence or absence of the inhibitors for 1 h and then stimulated with LHRH (10 nM) for 5 min. Cells were lysed, and IRS-1-associated PI3K activity was determined as described in Fig. 5A. C, LHRH-I activation of p125 FAK (right panel). Serum-starved T3 cells were either left untreated or stimulated with LHRH-I (10 nM) for 1, 5, or 30 min. Cells were lysed, and the level of FAK tyrosine phosphorylation was determined. FAK was immunoprecipitated (IP) from whole cell lysates. Samples were then subjected to Western blotting with antibodies to phosphotyrosine. FAK levels were evaluated by reprobing the membrane with an FAK antibody. The results shown are representative of those obtained in three independent experiments. Left panel, effect of a FAK dominant negative construct on LHRH-I-induced IRS-1-associated PI3K activity. T3 cells were transiently transfected with either a dominant negative of FAK (FAK DN) construct or the empty vector (negative control). Serum-starved T3 cell transformants were stimulated for 5 min with IGF-1 (10 nM), LHRH-I (10 nM), or with a combination of both agents. Cells were lysed, and the level of IRS-1-associated PI3K activity was determined as described in Fig. 5A. Values (mean ± S.E., n = 3) are presented fold differences between stimulated and unstimulated cells.

We also examined the effects of IGF-1 and LHRH-I on the activation of several MAP kinases (ERK1/2, p38, and JNK1/2, Fig. 7) and Akt (Fig. 8). As shown in Fig. 7A, The levels of ERK1/2 phosphorylation were higher in T3 cells treated with LHRH-I for 5 min than in unstimulated cells. In contrast, IGF-1 had only a small effect on ERK1/2 phosphorylation. Treatment with both IGF-1 and LHRH-I had an additive effect on ERK1/2 phosphorylation (Fig. 7A). This additive effect was confirmed by measuring MAPK activity using Elk-1 as a substrate (Fig. 7B). Cotreatment had a similar effect on MEK1/2 phosphorylation (Fig. 7C). However, treatment with both LHRH-I and IGF-1 did not have an additive effect on p38 and JNK1/2 phosphorylation (Fig. 7, D and E).

View larger version (39K): [in this window] [in a new window]   FIG. 7. Additive effect of IGF-1 and LHRH-I on activation of the ERK1/2 MAPK in T3 cells. Serum-starved T3 cells were stimulated for 5 min with different concentrations (1, 10. or 100 nM) of LHRH-I or IGF-1 (10 nM) or a combination of both agents. A, ERK1/2 phosphorylation. Cells were lysed and proteins separated by SDS-PAGE. Proteins were detected using antibodies to phospho-ERK1/2 and ERK1. The ERK1 cross-reacts to a small extent with ERK2. The values (mean ± S.E., n = 3) are expressed as fold differences between stimulated and unstimulated cells. B, ERK1/2 activity. The activity of the ERK1/2 MAPK was measured by using the Elk-1 fusion protein as a substrate. The levels of phosphorylated Elk-1 were evaluated by SDS-PAGE, transferred to a nitrocellulose membrane, and then probed with a phospho-Elk-1 antibody. C, MEK1/2 phosphorylation. Whole cell lysates were subjected to SDS-PAGE, and then proteins were detected using antibodies to phospho-MEK1/2 and MEK1/2. The results shown are representative of those obtained in three independent experiments. D and E, MAPK p38 and phosphorylation of the p38 JNK1/2 MAPKs. Whole cell lysates were subjected to SDS-PAGE, and proteins were probed with antibodies to phospho-p38 and p38 (E) or with antibodies to phospho-JNK1/2 and JNK1/2 (F). The results shown are representative of those obtained with three independent experiments. F and G, signaling pathways involved in LHRH-I induced the MAPK ERK1/2 phosphorylation. Serum-starved T3 cells werepreincubated in the presence or absence of AG1478 (10 µM, F) or GF109203X (10 nM, G) for 1 h prior to stimulation with IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents for 5 min. Cells were lysed and proteins separated by SDS-PAGE. Proteins were detected by probing with antibodies to phospho-ERK1/2 and ERK1. Values (mean ± S.E., n = 3) are expressed as fold differences between stimulated and unstimulated cells. IP, immunoprecipitation.

View larger version (35K): [in this window] [in a new window]   FIG. 8. LHRH-I-mediated inhibition of IGF-1-induced Akt activation in T3 cells. Serum-starved T3 cells were stimulated with different concentrations (0.1, 1, or 10 nM) of LHRH, IGF-1 (10 nM), or a combination of both for 5 min. A, Akt Ser-473 phosphorylation. Cells were lysed and proteins separated by SDS-PAGE. Proteins were detected by probing with antibodies to phospho-Akt (Ser-473) and Akt. Values (mean ± S.E., n = 3) are expressed as fold differences between stimulated and unstimulated cells. B, effect of an LHRH-I antagonist. Serum-starved T3 cells were stimulated for 5 min with IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents in the absence or presence of a LHRH-I antagonist. Whole cell lysates were subjected to SDS-PAGE and then probed with antibodies to phospho-Akt (Ser-473) and Akt. The results shown are representative of three independent experiments. C, Akt activity. Akt activity was measured using the GSK-3 fusion protein as an artificial substrate. The substrate was by SDS-PAGE and transferred to nitrocellulose membrane. The protein was detected by probing with a phospho-GSK3/ (Ser-21/9) antibody. D, effect of IGF-1 concentration on LHRH-I-mediated inhibition. Serum-starved T3 cells were stimulated for 5 minwith different concentrations (0.1–1000 nM) of IGF-1, LHRH-I alone (10 nM), or a combination of both agents. Western blotting was performed as described for A and B. E, effect of the time of stimulation on the inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation. Serum-starved T3 cells were stimulated with IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents for different lengths of time (0.5, 1, 10, and 60 min). Western blotting was performed as described for A and B. The results shown are representative of those obtained for three independent experiments.

Phosphorylation of Akt, one of the downstream targets of PI3K, was also investigated. We found that IGF-1 induced phosphorylation of the Ser-473 residue of Akt. Unlike PI3K activity, Akt phosphorylation was unaffected by treatment with LHRH-I (Fig. 8A). However, LHRH-I treatment inhibited IGF-1-induced stimulation of Akt phosphorylation. LHRH-I also had an inhibitory effect on IGF-1-induced phosphorylation of the Thr-308 residue of Akt (data not shown). The inhibitory effects of LHRH-I were abolished by treatment of the cells with an LHRH-I antagonist (Fig. 8B). We also measured Akt kinase activity using a GSK-3 fusion protein as a substrate. We found that LHRH-I strongly inhibited IGF-1-induced phosphorylation of the Ser-21/9 residue of GSK3 (Fig. 8C). The inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation was observed at all concentrations of IGF-1 tested. In addition, the inhibitory effect of LHRH-I was very rapid, and 30 s of incubation with LHRH-I was sufficient to reduce IGF-1-induced Akt phosphorylation by 2-fold (data not shown). The inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation was still observed 1 h after treatment (Fig. 8E). Buserelin, an agonist of LHRH-I, also inhibited IGF-1-induced Akt phosphorylation (data not shown). We also investigated the effect of different types of LHRH on the IGF-1 response. We found that LHRH type II (chicken LHRH), but not LHRH type III (Lamprey LHRH), mimicked LHRH type I inhibition of IGF-1-induced Akt phosphorylation (data not shown).

To determine whether the inhibitory effect of LHRH on Akt phosphorylation was cell-specific, we treated different cell lines with IGF-1, LHRH-I, or a combination of both agents for either 5 min or 1 h. The following cell lines were tested: two ovarian (OVCAR and SKOV) and two mammary cancer cell lines (MCF-7 and MDA-MB-231) expressing both the LHRH-I and IGF-1 receptors (50, 51), the gonadotrope T3 and LT2 cell lines, and a rat primary pituitary cell line. LHRH-I only led to down-regulation of IGF-I-induced Akt phosphorylation in the gonadotrope cell lines (supplemental Fig. 1). This effect was more potent in T3 cells than in LT2 or rat primary pituitary cells. Our finding that LHRH-I did not have an inhibitory effect in ovarian and mammary cancer cell lines may be explained by the fact that these cells line express relatively low levels of the LHRH-I receptor. To check this hypothesis, we transiently transfected MCF-7 cells with either an empty vector (control) or a mouse LHRH-I receptor construct (R-LHRH+/+). Transfectants were then treated with LHRH-I, IGF-1, or a combination of the two agents for 5 min. LHRH-I significantly inhibited IGF-1-induced Akt phosphorylation in R-LHRH+/+ cells but not in MCF-7 control cells (data not shown). Thus, the inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation is dependent on the amount of LHRH-I receptor expressed by the cell.

Involvement of PKC in the LHRH-I Inhibition of IGF-1-induced Akt Phosphorylation—A panel of pharmacological inhibitors and activators was used to identify the signaling pathways involved in LHRH-I inhibition of IGF-1-induced Akt phosphorylation. Serum-starved cells were pretreated for 1 h with the following: 1 µM okadaic acid (a potent type I and 2A protein phosphatase-specific inhibitor), MAPK ERK1/2, p38, and JNK1/2 inhibitors (U0126 (1 µM), SB202190 (50 µM), and SP600125 (50 µM)), 10 µM ionomycin (an inducer of maximal calcium influx), and the PKC inhibitor GF109203X (10 nM). Cells were then incubated with IGF-1, LHRH-I, or a combination of the two agents for 5 min. LHRH-I inhibition of IGF-1-induced Akt phosphorylation was not affected by okadaic acid, the MAPK inhibitors, or ionomycin (data not shown). However, the inhibitory effect of LHRH-I was abolished by treatment with the PKC inhibitor, suggesting that the PKC signaling pathway is involved in LHRH-I-mediated inhibition (Fig. 9A). In agreement with this result, we demonstrated that PMA (1 µM), a PKC activator, mimicked the inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation (Fig. 9B), an effect that was reversed by adding the PKC inhibitor (Fig. 9B). To investigate which PKC was involved, we investigated the effect of adding PKC (Safingol, 40 µM) and PKC-specific (Rottlerin, 5 µM) inhibitors to the cell cultures. We found that the PKC inhibitor completely abolished LHRH-I inhibition of IGF-1-stimulated Akt phosphorylation, whereas the PKC inhibitor had no effect (Fig. 9, C and D). Together these results suggest that PKC is involved in the inhibitory effect of LHRH-I on IGF-1-mediated Akt activation.

View larger version (35K): [in this window] [in a new window]   FIG. 9. Role of the PKC signaling pathway in the LHRH-I-mediated inhibition of IGF-1-induced Akt activation in T3 cells. Serum-starved T3 cells were preincubated for 1 h with the following inhibitors or activators: GF109203X (10 nM, A and B), PMA (1 µM, B), Safingol (40 µM, C), and Rottlerin (5 µM, D). Cells were then incubated in the absence or presence of IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents for 5 min. Whole cell lysates were subjected to SDS-PAGE, and proteins were detected with antibodies to phospho-Akt (Ser-473) and Akt. Values (mean ± S.E., n = 3) are expressed as fold differences between stimulated and unstimulated cells.

View larger version (23K): [in this window] [in a new window]   FIG. 10. A dominant negative PKC construct abolished the inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation in T3 cells. T3 cells were transiently transfected with either the dominant negative PKC (PKC DN) construct or the empty vector (negative control). Serum-starved T3 cells were stimulated for 5 min by treatment with IGF-1 (10 nM), LHRH-I (10 nM), or a combination of both agents. Cells were lysed, and proteins were separated by SDS-PAGE. Proteins were then detected using antibodies to phospho-Akt (Ser-473) and Akt. Values (mean ± S.E., n = 3) are expressed as fold differences between stimulated and unstimulated cells.

View larger version (22K): [in this window] [in a new window]   FIG. 11. LHRH inhibits the anti-apoptotic effect of IGF-1 through the inhibition of Akt and the activation of PKCa. Cells were plated on Lab-Tek (A) or 100-mm dishes (B) containing normal culture medium. After 18 h, the medium was changed to serum-free DMEM for 48 h. Cells were then incubated with IGF-1, LHRH-I, or a combination of both agents for 24 h. For treatment with the inhibitors, GF109203X (10 nM) and Safingol (40 µM), cells were preincubated for 1 h prior to the addition of IGF-1 and/or LHRH-I. A, cells were fixed and apoptotic nuclei were identified by terminal dUTP nick-end labeling staining. The percentage of labeled cells ± S.E. was determined by counting 1,000 nuclei. Means were compared by analysis of variance. B, cells were lysed and proteins were separated by SDS-PAGE. The levels of Ser-136-phosphorylated Bad, Bad, phospho-Akt, and Akt were analyzed by probing with the appropriate antibodies.

We then investigated the mechanisms involved LHRH-I inhibition of the anti-apoptotic effect of IGF-1 in T3 cells. Growth factors may play a role in protecting cells from apoptosis. Phosphorylation and inactivation of the pro-apoptotic Bcl2 family member, Bad, may be involved in this process (55). Akt has been shown to promote cell survival by a mechanism involving the phosphorylation of the Ser-136 residue of Bad (56). We examined the phosphorylation status of Bad by using an antibody recognizing phosphorylated Ser-136. We found that phosphorylation of Bad was IGF-1-dependent and that the level of Bad phosphorylation correlated with that of Akt phosphorylation (Fig. 11B). Pretreatment with LY294002 inhibited IGF-1-induced phosphorylation of the Ser-136 residue of Bad, supporting the idea that Akt is necessary for Bad activation (data not shown). To confirm whether Bad phosphorylation had an anti-apoptotic effect, we determined the level of cleaved caspase-3. We found that IGF-1 treatment prevented caspase-3 cleavage. This inhibition of caspase-3 cleavage correlated with an up-regulation of Bad phosphorylation. We also found that serum starvation for 48 h promoted caspase-3 cleavage and down-regulated Bad phosphorylation. Serum starvation for 48 h followed by IGF-1 treatment for 24 h led to up-regulation of phosphorylation of the Ser-136 residue Bad, whereas caspase-3 cleavage was down-regulated (Fig. 11B). Cotreatment with LHRH-I and IGF-1 led to down-regulation of Bad and Akt phosphorylation and up-regulation of caspase-3 cleavage. These effects were abolished by adding GF109203X and Safingol (Fig. 11B). These data indicate that LHRH-I inhibits the anti-apoptotic effect of IGF-1 by inhibiting PKC-mediated Akt and Bad phosphorylation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We showed that in the pituitary gonadotrope T3 cells, IGF-1 stimulates cell proliferation mainly via the PI3K and MAPK ERK1/2 pathways. The effect of IGF-1 on cell proliferation in T3 cells is consistent with results described in several previous reports. In humans (57), mice (24), and rats, IGF-1 is synthesized in the pituitary in vivo (35). The IGF-1R is expressed in rat and mouse pituitary glands, and IGF-1 has been shown to stimulate the proliferation of pituitary cells (24). In addition, IGF-1 leads to an increase in basal and LHRH-stimulated release of LH from rat anterior pituitary explants and dispersed cells in vitro (36, 37, 58, 59). IGF-1 increases FSH content and LHRH-stimulated FSH release in Coho salmon pituitary cells in vitro (60). IGF-1 also stimulates LHRH release in vivo (61) and LHRH gene expression in immortalized cell lines (62, 63) and in peripubertal mice (64). Although previous studies have indicated that interactions between the IGF-1 and LHRH signaling pathways do take place in pituitary gonadotrope cells, the underlying mechanisms remain to be established. We used the T3 gonadotrope-derived cell line to investigate these interactions. T3 cells express high levels of LHRH receptors exhibiting binding characteristics similar to those found in normal mouse and rat pituitary glands (7, 65).

We demonstrated that LHRH, like IGF-1 stimulates proliferation of T3 cells. However, this stimulation is mediated by different signaling pathways. We found that LHRH uses both the MAPK ERK1/2 and the PKC signaling pathways. The effects of LHRH on cell proliferation are cell-specific. LHRH agonists (66, 67) and an LHRH antagonist (68) have been shown to have an antiproliferative effect on the human androgen-independent prostate cell lines, DU-145 and PC-3. In DU-145 prostate cancer cells, the antiproliferative effects of LHRH agonists are mediated through negative regulation of the EGFR, which is inactivated through phosphorylation by PKC (69). By ng the same cell line, Marelli et al. (42) also showed hat an LHRH agonist inhibits IGF-1-induced cell proliferation by interfering with some of the cellular mechanisms mediating the IGF-1-induced stimulation. The LHRH agonist Zoladex strongly reduces the level of IGF-1R expression and tyrosine phosphorylation (42). In our study, we found that LHRH promoted cell growth. This result is consistent with those found in previous reports showing that LHRH induces cell proliferation in pituitary gonadotrope cells (1, 22). Moreover, recent studies using primary mouse pituitary cells and the gonadotrope-derived LT2 cell line have shown that addition of a LHRH agonist increased the number of cells at S phase and decreased the number of cells at G₁ phase after 2 and 4 days of culture (71). In addition, in primary rat pituitary cell cultures (a gonadotrope-enriched population), LHRH is a mitogenic factor for both LH and FSH gonadotrope cells (1, 22). Thus, LHRH appears to be a positive regulator of pituitary gonadotrope cell proliferation. However, this effect is dependent on cell type.

Our results suggest that activation of the ERK1/2 MAPK is dependent on PKC. This hypothesis was confirmed by pretreating T3 cells with GF109203X. Indeed, we found that ERK1/2 phosphorylation was 50% lower in pretreated cells than in controls (data not shown). Previous studies have reported that the ERK (11, 12), JNK (71), and p38 MAPK (21) cascade is activated in a PKC-dependent manner by LHRH in T3 cells. Benard et al. (72) showed that LHRH-mediated activation of ERK involves two distinct signaling pathways, which converge at the step involving Raf-1. The main pathway involves direct activation of Raf-1 by PKC. This step is partially dependent on a second pathway involving dynamin-dependent Ras activation downstream of Src (72). Our results were consistent with these observations. Moreover, we showed that the EGF receptor is not involved in the activation of ERK1/2 MAPK in response to LHRH-I. This result is consistent with those obtained by Benard et al. (72). In addition, we demonstrated that LHRH-I and IGF-1 have an additive effect on cell proliferation. The ERK1/2 MAPK signaling pathway is partially responsible for IGF-1-induced cell proliferation in T3 cells. Thus, the additive effect of LHRH and IGF-1 on cell proliferation is probably the result of the additive effect of these two agents on ERK1/2 phosphorylation and activity. We found that LHRH-I enhances IGF-1-induced IRS-1 phosphorylation and IRS-1-associated PI3K activity without affecting IGF-1-induced IGF-1R tyrosine phosphorylation. Furthermore, we showed that LHRH-I transactivates the EGF receptor, which phosphorylates IRS-1. Other tyrosine kinase-like platelet-derived growth factor receptors have also been reported to be involved in the transmission of signals from G protein-coupled receptors (73). For example, Grosse and co-workers. (74, 75) showed that LHRH-I treatment resulted in a rapid increase in MAPK ERK activity in T3 gonadotrope cells. This activity was enhanced by the EGFR-specific tyrosine kinase inhibitor AG1478 (74, 75). We showed that the EGFR only played a minor role, if any, in LHRH-I activation of IRS-1-associated PI3K activity. However, we found that LHRH-I promoted phosphorylation of the focal adhesion kinase (p125 FAK or FAK), which is known to bind the p85, the regulatory subunit of PI3K.

We showed, in T3 cells and to a lesser extent in LT2 cells and primary rat pituitary cells, that LHRH-I strongly inhibited the IGF-1-induced phosphorylation of the Ser-473 and Thr-308 residues and thus the activity of Akt. A wide variety experimental approaches have shown that Akt is activated via at least two pathways, one of which is PI3K-dependent and the other is PI3K-independent (76–80). Stress, okadaic acid, and cAMP regulate the activation of Akt independently of PI3K. Our results support the idea that LHRH-I inhibits IGF-1-induced Akt activation via a PI3K-independent pathway. Indeed, LHRH-1 has a synergistic effect on IGF-1-induced IRS-1-associated PI3K activity. Moreover, both PDK1/2 phosphorylation and IGF-1R- and IRS-2-associated PI3K activity were up-regulated in response to IGF-1. Both of these responses were also slightly up-regulated following LHRH-1 treatment (data not shown). We hypothesize that the inhibitory effect of LHRH-I on IGF-1-induced Akt phosphorylation is a result of LHRH-mediated activation of a phosphatase that dephosphorylates both the Ser and Thr residues of Akt. Protein phosphatases are known to play a critical role in the regulation of the PI3K/Akt cascade. For example, granulocyte-macrophage colony-stimulating factor-induced inhibition of Akt/PKB by ceramide in human erythroleukemic TF-1 cells (81) and insulin-induced inhibition by palmitate in C2C12 skeletal muscle cells (82) both lead to PI3K-independent Ser-473 dephosphorylation by phosphatases. Inhibitors of protein phosphatases, such as okadaic acid for PP1 and PP2A, FK506 for PP2B, and sodium orthovanadate for protein-tyrosine phosphatase, are useful tools for studying the significance of protein phosphorylation. We found that none of these inhibitors blocked LHRH inhibition of IGF-1-induced Akt phosphorylation, suggesting that phosphatases, such as PP2A/B and PP1, were not involved in this process (data not shown for FK506 and sodium orthovanadate).

However, we showed that the conventional isoform-specific PKC inhibitor (GF109203X) did block the inhibitory effect of LHRH-1 on IGF-1-induced Akt activation. Several recent reports have shown that some PKCs inhibit Akt activity. For example, Wen et al. (82) used a nonselective PKC inhibitor to demonstrate that PKC negatively regulates Akt in a human airway epithelial cell culture (A549). The PKC family consists of at least 12 members divided into three groups as follows: 1) the conventional PKCs (, I, II, and ); 2) the novel PKCs (, , , and ); and 3) the atypical PKCs ( and /). As all of these PKC isoforms are expressed in T3 cells (84, 85), it was necessary to establish whether all or just one of these isoforms was involved in the LHRH-1 inhibition of IGF-1-induced Akt activation. We investigated the roles of the different isoforms using a variety of isoform-specific PKC inhibitors. Our data revealed that PKC plays a major role in LHRH-1 inhibition of IGF-1-induced Akt activation.

The inhibition of Ser/Thr phosphorylation of Akt through a PKC pathway can be mediated by either a PI3K-dependent or PI3K-independent pathway depending on the ligand and cell lineage analyzed. For example, the involvement of PKC in insulin-stimulated responses has been demonstrated by studies of various steps in the signaling cascade, including those analyzing the effect of the binding of substrates like IRS-1 and PI3K to the receptor (23, 86, 87). In contrast, our results indicated that the inhibition of Akt activity probably occurs upstream of PKC, possibly involving PI3K activity. Doornbos et al. (23) reported that PKC is a negative regulator of Akt/PKB activity and that this regulation is PI3K-independent. The PH domain of Akt has been proposed to be involved in protein-protein interactions, such as binding of PKC and the / subunits of heterotrimeric G proteins (47). Konishi et al. (70) used glutathione S-transferase fusion proteins containing the three different Akt PH domains to demonstrate that PKC and - bind to the PH domain of Akt1, -2, and -3 and that PKC binds to the PH domain of Akt1 and -2 in vitro. We found that GF10923X did not prevent LHRH from enhancing IGF-1-induced IRS-1 phosphorylation or the association of this phosphorylated form with PI3K (data not shown). We hypothesize that PMA and LHRH-mediated inhibition of IGF-1-induced Akt stimulation is mediated, at least in part, by a direct interaction between PKC and Akt. It is possible that PKC-mediated phosphorylation of Akt leads to a change in the conformation the protein, which subsequently inactivates the kinase and blocks further phosphorylation of Akt by PDK1/2.

In conclusion, our results provide strong evidence that LHRH-I regulates several steps of the IGF-1R signaling pathway in the gonadotrope cell line, T3. LHRH-I enhances IGF-1-induced IRS-1 tyrosine phosphorylation and promotes the association of this protein with PI3K. In contrast, LHRH-I strongly inhibits Akt activation through PKC (Fig. 12). This LHRH-I-mediated inhibition of IGF-1-induced Akt phosphorylation strongly inhibits the anti-apoptotic effect IGF-1. Our study provides a novel insight into the mechanisms involved in LHRH and IGF-1 signaling during growth and proliferation of gonadotrope cells.

View larger version (24K): [in this window] [in a new window]   FIG. 12. Schematic representation of a cross-talk between IGF-1R, LHRH-R, and EGFR in T3 cells. IGF-1 activates the PI3K/Akt pathway but only weakly the MAPK pathway (ERK1/2, p38, and JNK1/2), whereas LHRH-I activates mainly the MAPK pathway. LHRH-I interacts with several components of the IGF-1R signaling cascade different. First, it stimulates IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated PI3K activity by facilitating transactivation of the EGFR. LHRH-I also has an inhibitory effect on IGF-1-induced PKC-mediated Akt phosphorylation, preventing Bad phosphorylation in the T3 gonadotrope cell line and thus leading to inhibition of the anti-apoptotic effects of IGF-1.

	FOOTNOTES

The on-line version of this article (available at http://www.jbc.org) contains Fig. 1.

^** To whom correspondence should be addressed: Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Nouzilly 37380, France. Tel.: 33-2-47-42-79-64; Fax: 33-2-47-42-77-43; E-mail: jdupont{at}tours.inra.fr.

¹ The abbreviations used are: LHRH, luteinizing hormone-releasing hormone; LHRH-R, LHRH receptor; JNK, c-Jun N-terminal kinase; PI3K, phosphatidylinositol 3-kinase; IGF-1, insulin-like growth factor-1; IGF-1R, IGF-1 receptor; LH, Luteinizing hormone; FSH, follicle-stimulating hormone; EGF, epidermal growth factor; EGFR, EGF receptor; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PH, pleckstrin homology; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; IRS, insulin receptor substrate; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; FAK, focal adhesion kinase.

	ACKNOWLEDGMENTS

 
We thank Dr. Michel Blanc for the rat primary pituitary culture and Dr. Alain Caraty for the LHRH-II. We also thank Dr. Jean Simon for critical reading of the manuscript.

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