Novel Signal Transduction Pathway for Luteinizing Hormone and Its Interaction with Insulin: Activation of Janus Kinase/Signal Transducer and Activator
Abstract'k, http://www.100md.com
The actions of LH are mediated through a single class of cell surface LH/human chorionic gonadotropin receptor, which is a member of the G protein-coupled receptor family. In the present study we showed that LH induced rapid tyrosine phosphorylation and activation of the Janus kinase 2 (JAK2) in rat ovary. Upon JAK2 activation, tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT-1), STAT-5b, insulin receptor substrate-1 (IRS-1), and Src homology and collagen homology (Shc) were detected. In addition, LH induced IRS-1/phosphoinositol 3-kinase and Shc /growth factor receptor-binding protein 2 (Grb2) associations and downstream AKT (protein kinase B, homologous to v-AKT) serine phosphorylation and ERK tyrosine phosphorylation, respectively. The simultaneous infusion of insulin and LH induced higher phosphorylation levels of JAK2, STAT5b, IRS-1, and AKT compared with each hormone alone in the whole ovary of normal rats. By immunohistochemistry we demonstrated that these late events take place in follicular cells and both external and internal theca. These results indicate a new signal transduction pathway for LH and show that there is positive cross-talk between the insulin and LH signaling pathways at the level of phosphoinositol 3-kinase/AKT pathway in this tissue.
Introductionq5, http://www.100md.com
THE MAMMALIAN LH receptor is a G protein-coupled receptor (GPCR) with a transmembrane domain composed of seven segments. Binding of the receptor by LH leads to activation of Gs, which activates the membrane-associated adenylyl cyclase, causing an increase in intracellular cAMP (1, 2, 3, 4). This nucleotide acts as a second messenger for the regulation of steroidogenic acute regulatory protein (StAR) and the cytochrome P450 enzyme system (5). Alternative signaling pathways by LH or human chorionic gonadotropin (hCG) receptor were described in the last years, including chloride ion influx, activation of the phosphoinositol pathway, and calcium ion mobilization (6, 7, 8).q5, http://www.100md.com
Insulin can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, such as low density lipoprotein (LDL) receptor, StAR, and P450 cholesterol side-chain cleavage enzyme (CYP11A) (7, 9, 10). Considerable evidence demonstrates that insulin receptor tyrosine kinase activity is essential for many, if not all, of the biological effects of insulin. In most cells this primary event leads to the subsequent tyrosyl phosphorylation of the insulin receptor substrate 1 (IRS-1) and IRS-2. Both IRS-1 and IRS-2 have been implicated as the first postreceptor step in insulin signal transmission. In animal tissues and cultured cells, phosphorylated IRS-1 and -2 can bind and activate phosphatidylinositol 3-kinase (PI 3-kinase) (11, 12, 13). A downstream substrate of PI 3-kinase activity is the serine/threonine protein kinase B or AKT (protein kinase B, homologous to v-AKT) (5). Upon insulin receptor tyrosine kinase activation and autophosphorylation, there is also recruitment of protein Shc (Src homology and collagen homology) and growth factor receptor-binding protein 2 (Grb2), leading to activation of the extracellular signal-regulated kinase (ERK) pathway (15, 16, 17).
Recently, it was demonstrated that in cultured granulosa cells, putative suppression of PI 3-kinase with wortmannin or LY294002 trigged in vitro apoptosis (18), raising the hypothesis that LH may signal through classical tyrosine kinase pathways. In addition, other hormones that act through GPCRs, including angiotensin II and vasopressin, can induce tyrosine phosphorylation of cytoplasmic proteins (19, 20, 21, 22). Angiotensin II activates Janus kinase 2 (JAK2), a member of the JAK family, and probably uses this kinase to induce several intracellular protein tyrosine phosphorylations.6w-2^, http://www.100md.com
In this study we evaluated the ability of LH to activate JAK2 and to induce the tyrosine phosphorylation of IRS-1, Shc, signal transducer and activator of transcription 1 (STAT1), and STAT5b as well as IRS-1/PI 3-kinase and Shc/Grb2 associations and the phosphorylation of AKT/PKB and ERK in rat ovary in vivo. We also assessed the possible cross-talk between the LH and insulin signaling pathways. Our data reveal a new signal transduction pathway for LH and show that there is positive cross-talk between insulin and LH signaling systems.
Materials and Methodsk63{ie, 百拇医药
Materialsk63{ie, 百拇医药
The reagents for SDS-PAGE, immunoprecipitation, and immunoblotting were obtained from sources previously cited (21, 22, 23, 24). Highly purified hCG, LH, and PRL were obtained from the NIDDK National Hormone and Pituitary Program (Torrance, CA), and human recombinant insulin (Humulin R) was purchased from Eli Lilly \|[amp ]\| Co. (Indianapolis, IN). The PI 3-kinase inhibitors (wortmannin and LY294002) were obtained from Sigma-Aldrich (St. Louis, MO), and the protein kinase A (PKA) inhibitor (H89) was purchased from Calbiochem (San Diego, CA). Anti-IR, anti-IRS-1, anti-STAT1, anti-STAT5b, anti-Shc, anti-Grb2, antiphosphotyrosine, and anti-JAK2 antibodies were purchased from Santa Cruz Technology, Inc. (Santa Cruz, CA). Anti-PI 3-kinase was from Upstate Biotechnology, Inc. (Lake Place, NY). Anti-phosphoserine-AKT, anti-phospho-ERK, anti-phospho-STAT1, anti-phospho-STAT3, and anti-phospho-STAT5b antibodies were obtained from New England Biolabs, Inc. (Beverly, MA).
Animal tissue extracts and immunoblotting%!/?, http://www.100md.com
Female Wistar rats (200–220 g) were housed with access to standard rodent chow and water ad libitum. Food was withdrawn 6 h before the experiments. All procedures with animals were conducted in accord with the principles and procedures described by NIH Guidelines for the Care and Use of Experimental Animals. The studies were conducted using animals at the diestrous phase. The rats were anesthetized with sodium thiopental (25 mg/kg, ip) and were used 10–15 min later, as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the cava vein was exposed, and 0.5 ml saline (0.9% NaCl) with or without insulin, PRL, LH/hCG, or insulin plus LH was injected at the doses indicated as bolus infusion (doses stated in the figures). The ovaries were removed at the times indicated and homogenized in ice-cold extraction buffer containing 100 mM Tris (pH 7.4), 10 mM EDTA, 1% Triton X-100, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.01 mg aprotinin/ml. Pools of two ovarian extracts from female rats at the diestrous phase were centrifuged at 15,000 rpm at 4 C for 15 min to remove insoluble material; the supernatant was then used for the assay. Protein determination was performed by the Bradford dye binding method using the reagent from Bio-Rad Laboratories, Inc. (Richmond, CA) and BSA as the standard. Two milligrams of protein from the supernatants were used for immunoprecipitation with anti-IRS-1, anti-JAK2, anti-STAT-1, anti-STAT-5b, anti-Shc, and protein A-Sepharose 6MB before Laemmli sample buffer treatment and electrophoresis in SDS-PAGE as described previously (21, 22, 23, 24). For whole tissue extracts, similarly sized aliquots (100 µg protein) were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT, anti-phospho-ERK, anti-phospho-STAT1, anti-phospho-STAT3, and anti-phospho-STAT5b antibodies. Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) (21, 22, 23, 24). To reduce nonspecific protein binding to the nitrocellulose, the filter was preincubated for 2 h at room temperature in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blots were incubated overnight at 4 C with antibodies against phosphotyrosine, the p85 subunit of PI 3-kinase, STAT-1, STAT-5b, GRB2, phosphoserine-AKT, and phospho-ERK diluted in blocking buffer with 3% nonfat dry milk, followed by washing for 30 min in blocking buffer without milk. The blots were incubated with 2 µCi [125I]protein A (30 µCi/µg) in 10 ml blocking buffer for 2 h at room temperature and then washed again for 30 min as described above. [125I]Protein A bound to the specific antibodies was detected by autoradiograph using preflashed Kodak XAR film (Eastman Kodak Co., Rochester, NY) with Cronex Lightning Plus intensifying screens (DuPont, Wilmington, DE) at -8 C for 48–72 h. Band intensities were quantitated by optical densitometry (model GS 300, Hoefer Scientific, San Francisco, CA) of the developed autoradiographs.
JAK2 in vitro tyrosine kinase activity+]?r), http://www.100md.com
JAK2 tyrosine kinase activity was measured by autophosphorylation. A low dose of LH (2 pg) was injected into the vena cava to stimulate partial JAK2 autophosphorylation. JAK2 was immunoprecipitated as described above. The resulting immune complexes were collected on protein A-Sepharose. The protein kinase activity of the immunoprecipitates was measured by incubating the immune complexes in 100 µl buffer containing 50 mM Tris (pH 7.5), 0.2 mM sodium vanadate, 0.1% Triton X-100, 3 mM MnCl2, and 15 µM cold ATP for 30 min at room temperature. The complexes were washed twice with cold buffer, then resuspended in Laemmli sample buffer and analyzed by SDS-PAGE (21, 23, 24). The incorporation of phosphate into the separated proteins was visualized by autoradiography using antiphosphotyrosine immunoblots after transfer to nitrocellulose.+]?r), http://www.100md.com
Immunohistochemistry+]?r), http://www.100md.com
Four ovaries from two diestrous female rats from each treatment group (saline, insulin, LH, and insulin plus LH infusion) were examined to determine the expression and tissue distribution of proteins participating in the insulin signaling pathway. Hydrated 5-µm sections of paraformaldehyde-fixed, paraffin-embedded tissue were stained by the avidin-peroxidase method. Sections were incubated for 30 min with 2% normal rabbit or normal mouse serum at room temperature and then were exposed for 12 h in a moisture chamber at 4 C to the primary antibodies against insulin receptor (1:80), p-STAT-1 (1:50), p-STAT-5b (1:50), p-AKT (1:40), or p-ERK (1:50). Biotinylated secondary antibodies were used in incubations for 2 h at room temperature, followed by a 1-h incubation with ready to use avidin-coupled peroxidase (Vector Laboratories, Inc., Burlingame, CA). The resulting immunocomplexes were detected with 50 mg/100 ml diaminobenzidine:4 HCl/0.01 ml/100 ml H2O2 dissolved in 5 mmol/liter Tris, pH 7.6. Analysis and photodocumentation were performed using a Microphot FXA microscope (Nikon, Milville, NY).
Preparation of perfused rat ovary)x9lljp, http://www.100md.com
Diestrous female rats underwent surgical isolation of the right ovary with connecting vasculature as described by Matousek and co-workers (25). The ovaries were placed in a perfusion system for 30 min to examine the effect of PI 3-kinase inhibitors [wortmannin (0.1 µM) and LY 294002 (50 µM)] and PKA inhibitor (H89, 10 µM) on LH-induced AKT serine phosphorylation.)x9lljp, http://www.100md.com
Results)x9lljp, http://www.100md.com
LH stimulates JAK2 tyrosine kinase activity)x9lljp, http://www.100md.com
To investigate whether LH stimulates JAK2 phosphorylation, LH was infused into the cava vein of female rats, and the ovaries were extracted and analyzed by immunoblotting. JAK2 tyrosine phosphorylation was measured by immunoprecipitation with polyclonal anti-JAK2 antibodies. The precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted with a monoclonal antiphosphotyrosine antibody (Fig. 1). Parallel experiments were performed in which the rats received an infusion of PRL, because this hormone is known to induce the tyrosine phosphorylation of JAK2 in ovary cells (26). Figure 1A shows that LH induces JAK2 tyrosine phosphorylation 3 min after this hormone infusion. As expected, the infusion of PRL also induced JAK2 tyrosine phosphorylation a little latter, although less markedly than LH (Fig. 1B). Both figures show that the tyrosine phosphorylation of JAK2 in response to LH and PRL is rapid and transient, and no change in JAK2 protein expression was observed after hormone infusion (Fig. 1, A and B, lower panel). LH-stimulated phosphorylation of JAK2 was dose dependent (Fig. 1C). Phosphorylated JAK2 was detectable after the injection of as little as 2 pg LH, and half-maximal stimulation occurred at approximately 200 pg, which, when distributed in the blood volume, may be within the physiological range for female rats.
fig.ommitteed*, 百拇医药
Figure 1. LH stimulates JAK2 tyrosine phosphorylation in the ovary of rats. Saline (0), 2 ng LH, or 200 ng PRL (Pro) were administered at the times indicated (A and B) into the vena cava as a bolus injection in normal female rats. For the dose-response analysis, the indicated doses were injected 3 min after LH infusion (C). A pool of ovarian extracts was immunoprecipitated with anti-JAK2, followed by immunoblotting with antiphosphotyrosine monoclonal antibody. The phosphorylation levels of JAK2 were quantified with an image analyzer and are shown as the fold increase from the basal value (n = 5). JAK2 tyrosine kinase activity (D) was measured by autophosphorylation in vitro in the absence (-) or presence (+) of 15 µM ATP before and after 3 min of LH (2 pg) administration. SDS-PAGE (8%) was performed, and there was an increase in JAK2 autophosphorylation after LH infusion plus the addition of ATP in vitro.*, 百拇医药
To investigate the effect of LH on JAK2 kinase activity, we performed an in vitro kinase assay. Ovaries from rats that were stimulated submaximally with LH to obtain limited tyrosine phosphorylation of JAK2 were immunoprecipitated with anti-JAK2 antibody and incubated with exogenous ATP to permit autophosphorylation in vitro. Tyrosine phosphorylation was quantified by immunoblotting using antiphosphotyrosine antibody. After the infusion of a low dose of LH in vivo and the addition of ATP in vitro, there was a clear increase in JAK2 autophosphorylation (Fig. 1D).
LH induces STAT-1 and STAT-5b tyrosine phosphorylationkn, http://www.100md.com
Although activation of the JAK-STAT pathway was originally thought to occur through cytokine receptors, it has recently been shown that the key activating event, STAT tyrosine phosphorylation, may be regulated/activated by GPCRs (27). Thus, an investigation of whether LH infusion activates the JAK-STAT signal transduction pathway in the ovary of intact rats was undertaken. We initially used coimmunoprecipitation analysis to determine whether acute iv infusion of LH would activate STAT-1, STAT-3, and STAT-5 transcription factors to form JAK2 complexes. Supernatants from a pool of ovaries from female rats that received LH infusion or saline were immunoprecipitated with anti-JAK2 antibody. The membranes containing these immune complexes were incubated with STAT-1, STAT-3, and STAT-5b. In Fig. 2A, data obtained from this approach show that after LH infusion there was an increase in JAK2/STAT-1 and JAK2/STAT-5b association, but not JAK2/STAT-3 association (data not shown).
fig.ommitteed:xcf#ch, 百拇医药
Figure 2. LH stimulates JAK2 coimmunoprecipitation with STAT-1, STAT-5b, and STAT-1, and STAT-5b phosphorylation in ovaries of normal rats in vivo. Saline (-) or 2 ng LH or PRL (Pro) was administered into the vena cava as a bolus injection in normal female rats. The ovaries were extracted 3 min after LH infusion for immunoprecipitation with anti-JAK2 antibody (A). A pool of ovarian extracts was immunoprecipitated with anti-JAK2 (A), anti-STAT-1 (B), or anti-STAT-5b (C) antibodies. The membranes containing JAK2 immune complexes were incubated with anti-STAT-1 or anti-STAT-5b. The membranes containing anti-STAT-1 or anti-STAT-5b were immunoblotted with antiphosphotyrosine antibody (B and C). Shown are representative autoradiographs from four different experiments.:xcf#ch, 百拇医药
We next investigated the tyrosine phosphorylation of STATs after LH infusion. LH not only induced increased JAK2/STAT-1 and JAK2/STAT-5b associations, but also increased the tyrosine phosphorylation of STAT-1 and STAT-5b proteins within 3–5 min after hormone injection (Fig. 2, B and C). However, the tyrosine phosphorylation of STAT-3 was not detected even in samples previously immunoprecipitated with anti-phospho-STAT-3 after LH stimulus, consistent with our previous finding that there was no detectable coimmunoprecipitation of JAK2 with STAT-3. As expected, the infusion of PRL induced STAT-5b tyrosine phosphorylation, but not STAT-1 (Fig. 2, B and C, right). STAT1 and STAT5b protein expression did not change after LH infusion (Fig. 2, B and C, lower panels).
LH stimulates IRS-1 tyrosine phosphorylation and AKT serine phosphorylationo9, 百拇医药
The rate of LH-induced IRS-1 tyrosine phosphorylation was estimated in a time-course experiment (Fig. 3A). Proteins from ovaries of rats treated with LH were immunoprecipitated with anti-IRS-1 antibody and then immunoblotted with a mouse monoclonal antiphosphotyrosine antibody. IRS-1 was maximally tyrosine-phosphorylated 3 min after the infusion of LH (Fig. 3A, upper panel), with no change in IRS-1 protein expression (Fig 3A, lower panel). Phosphorylated IRS-1 binds and activates PI 3-kinase and at least two adapter molecules, Grb2 and Nck (homologous to v-Nck) (11, 28). We evaluated the ability of LH to stimulate IRS-1/PI 3-kinase association in the rat ovary in vivo. After LH stimulation, there was an increase in IRS-1/PI 3-kinase association (Fig. 3A, middle panel) in the ovaries of intact rats, which paralleled the increase in IRS-1 tyrosine phosphorylation.o9, 百拇医药
fig.ommitteed
Figure 3. LH stimulates the IRS-1/PI 3-kinase/AKT pathway. After the injection of 2 ng LH at the indicated time, ovaries were extracted from rats. The supernatants were used for immunoprecipitation with anti-IRS-1 (A) antibodies and for whole tissue extracts (B–D). The immunoblots were performed with antiphosphotyrosine (A, upper panel), anti-PI 3-kinase (A, middle panel), anti-IRS-1 (A, lower panel), and anti-pAKT (B and C) antibodies. C, Effect of LH on AKT serine phosphorylation in ovaries extracted from prepubertal 4-wk-old rats. D, In perfused ovaries, as described in Materials and Methods, pretreatment with LY294002 (50 µmol, iv), wortmannin (100 nmol; E), or H89 (10 mM) was followed 30 min later by iv administration of LH or insulin. E and F, Effect of hCG in the phosphorylation of JAK2 (E) and IRS-1 (F) in ovaries extracts of normal rats 3 min after hormone infusion. Ovarian extracts were immunoprecipitated with anti-JAK2 or anti-IRS-1 antibodies, and immunoblottings were performed with antibody against phosphotyrosine residues. Results are representative of four experiments./m0l48, 百拇医药
To confirm that the actions of LH on tyrosine phosphorylation of JAK2 and IRS-1 are mediated by classical LH receptor, and that this receptor binds hCG as well as LH, we performed experiments using an infusion of 100 IU hCG instead of LH (4). The results demonstrated that hCG is able to induce JAK2 (Fig. 4C) and IRS-1 (Fig. 4D
fig.ommitteedf!x, 百拇医药
Figure 4. LH stimulates Shc/Grb2/ERK pathway. After 2 ng LH injection at the time indicated, ovaries were extracted from rats. The supernatants were used for immunoprecipitation with anti-Shc (A) antibodies and for whole tissue extracts (B). The immunoblots were performed with antiphosphotyrosine (A, upper panel), with anti-GRB2 (A, middle panel), with anti-Shc (A, lower panel), and with anti-pERK and anti-ERK antibodies (B). These are representative autoradiographs from four different experiments.f!x, 百拇医药
It is well established that insulin-induced IRS-1 tyrosine phosphorylation and association/activation with PI 3-kinase are accompanied by an increase in AKT serine phosphorylation (14, 29). Because LH was able to induce IRS-1 tyrosine phosphorylation and association with PI 3-kinase, we investigated the effect of LH on AKT serine phosphorylation. LH infusion induced only a 2-fold increase in AKT serine phosphorylation at 3 min, which then decreased gradually, returning to basal levels at 10 min (Fig. 3B, upper panel), without changes in AKT protein expression (Fig. 3B, lower panel). We performed similar experiments in immature rats (4 wk old), and the results demonstrated the same effect of LH inducing AKT phosphorylation, with similar a time course similar to that in mature rats, indicating that this pathway is present in animals that lack corpora lutea (Fig. 3C).
To verify whether LH is able to induce AKT phosphorylation in perfused ovaries and determine the effect of inhibitors of PI 3-kinase or PKA in this pathway, we prepared perfused ovaries as described in Materials and Methods and treated them with LH after pretreatment (30 min) with LY294002, wortmannin, or H89. We also used insulin as a control. As shown in Fig. 3D, in perfused ovaries LH induced AKT serine phosphorylation similar to that in rat ovaries in vivo, and pretreatment with LY294002 (Fig. 3D, middle panel) or wortmannin (Fig. 3D, upper panel) abolished LH-induced AKT phosphorylation. As expected insulin-induced AKT phosphorylation was also blocked by these inhibitors of PI 3-kinase. When we used H89 for 30 min before LH treatment, no effect was observed on LH-induced AKT phosphorylation, suggesting that PKA is probably not involved or has only a minor influence on this pathway (Fig. 3D, lower panel).*$;dt.%, 百拇医药
LH stimulates Shc tyrosine phosphorylation and ERK phosphorylation
LH-induced Shc tyrosine phosphorylation was observed within 3 min after hormone infusion and was maximal at 10 min (Fig. 4A, upper panel), promoting no change in Shc protein expression (Fig. 4A, lower panel). LH also increased the Shc/Grb2 association in a similar time course of LH-induced Shc tyrosine phosphorylation (Fig. 4A, middle panel). The LH-stimulated phosphorylation of Shc was dose dependent and similar to that for JAK2 (data not shown).-|, 百拇医药
Because Shc tyrosine phosphorylation leads to activation of ERK pathways through the Grb2/SOS complex, we investigated the effect of LH on ERK phosphorylation levels. LH infusion leads to an approximately 8-fold increase in ERK phosphorylation at 10 min, as determined by immunoblotting with an antiphospho-ERK antibody (Fig. 4B, upper panel) without changes in ERK protein expression.-|, 百拇医药
Effect of simultaneous administration of LH and insulin on JAK/STAT, IRS-1/PI3K/AKT, and Shc/ERK pathways-|, 百拇医药
At first we measured the tyrosine phosphorylation of JAK2 in rat ovaries that were stimulated with LH, insulin, or both hormones for 3 and 10 min. The phosphorylation of JAK2 in ovaries showed an increase of approximately 7-fold 3 min after LH infusion and of approximately 6-fold 3 min after insulin infusion. LH and insulin together showed approximately 10-fold and approximately 7-fold increases in JAK2 tyrosine phosphorylation 3 and 10 min, respectively, significantly higher than with each hormone alone (Fig. 5A).
fig.ommitteed4;t$%#(, http://www.100md.com
Figure 5. LH plus insulin enhances JAK2/STAT-5b and IRS-1/PI 3-kinase/Akt signaling pathway with no effect on Shc/GRB2/ERK pathway in the ovary of normal rats in vivo. Saline (0), 2 ng LH, 6 µg insulin, or 2 ng LH plus 6 µg insulin (LH-Ins) were administered into vena cava as a bolus injection of female normal rats. The ovaries were extracted at the indicated time, and after homogenization and centrifugation the supernatants were used for immunoprecipitation with the following antibodies against JAK2 (A), STAT-1 (B), STAT-5b (C), IRS-1 (D and E), and Shc (G and H) or for whole tissue extracts and immunoblotted with anti-pAkt (F) and with anti-pERK (I). Bands were quantitated using a densitometer. Data are the mean ± SEM for four rats for each group. *, P < 0.05 vs. LH-Ins and LH-Ins plus LH; #, P < 0.05 vs. Ins-Ins plus LH.4;t$%#(, http://www.100md.com
Figure 5B shows that LH induced maximal STAT1 tyrosine phosphorylation (~ 7-fold increase) at 10 min, and insulin induced only a mild increase in STAT1 phosphorylation. The administration of both hormones did not induce an additive effect over each other on STAT1 phosphorylation.
LH or insulin induced a moderate increase in STAT5b tyrosine phosphorylation. The infusion of both hormones induced approximately 6- and 11-fold increases in STAT5b tyrosine phosphorylation at 3 and 10 min, respectively, significantly higher than the effect of each hormone alone (Fig. 5C).1hgy, 百拇医药
Figure 5D shows that the simultaneous infusion of LH and insulin induced a higher level of IRS-1 tyrosine phosphorylation at 3 and 10 min than each hormone alone, indicating a synergistic effect of the hormones. The effects of LH, insulin, or both hormones on IRS-1/PI 3-kinase association showed were similar on IRS-1 tyrosine phosphorylation (Fig. 5E).1hgy, 百拇医药
LH induced only a mild and transitory increase in AKT serine phosphorylation. After insulin infusion, there was an increase in AKT serine phosphorylation of approximately 7-fold at 3 and 10 min. The simultaneous infusion of both hormones showed an additive effect, with a significant increase in AKT serine phosphorylation at 3 and 10 min (Fig. 5F).
Figure 5G shows that LH or insulin induced a moderate increase in Shc tyrosine phosphorylation at 3 and 10 min, but there is no additive effect after the infusion of both hormones. The effect of LH, insulin, or both hormones on Shc/Grb2 association showed a similar behavior of Shc tyrosine phosphorylation (Fig. 5H.1j9:, 百拇医药
The insulin infusion induced an approximately 7-fold increase in ERK phosphorylation at 3 and 10 min. The simultaneous infusion of both hormones showed no additive effect in ERK phosphorylation compared with that detected with insulin alone (Fig. 5I).1j9:, 百拇医药
Tissue distribution of IR and the molecular events induced by LH and insulin1j9:, 百拇医药
By performing immunohistochemical studies of rat ovary, a broad distribution of IR was detected. Impressive staining for IR was observed in interstitial cells of the stroma, in cells of the external an internal theca, and in follicular cells of the diestrous ovary (Fig. 6A). Under LH, insulin, or LH plus insulin stimulation the induction of Ser473 phosphorylation of AKT was most evident in follicular cells (Fig. 6B). For ERK, a prestimulus, constitutive pattern of tyrosine phosphorylation was detected. After LH, insulin, or LH plus insulin treatment an impressive increase in staining was induced mostly in follicular cells of the diestrous ovary (Fig. 6B). STAT1 and STAT5b tyrosine phosphorylations were also induced by LH, insulin, or LH plus insulin treatment. Again, thecal and follicular cells were the sites of highest staining (Fig. 6B). Although immunohistochemistry does not serve as a quantitative method, on a comparative basis an apparent additive effect of LH and insulin was detected on AKT and STAT5b treatment-induced phosphorylation.
fig.ommitteeduz:+s*d, http://www.100md.com
Figure 6. Immunohistochemical analysis of IR (upper panel) distribution in ovary of diestrous rats and of LH, insulin, and LH- and insulin-induced Akt, ERK, STAT1, and STAT5 phosphorylation (lower panel). IR is present in most cells of the rat ovary, including interstitial cells of the stroma, external and internal thecal cells, and follicular cells (upper panel). LH and insulin induced Ser473 Akt phosphorylation is detected mostly in follicular cells (lower panel, upper line). LH- and insulin-induced tyrosine phosphorylation of ERK is detected mostly in follicular cells (lower panel, second line). LH- and insulin-induced tyrosine phosphorylation of STAT1 is detected mostly in thecal and follicular cells (lower panel, third line), and LH- and insulin-induced tyrosine phosphorylation of STAT5b is detected mostly in thecal and follicular cells (lower panel, lower line).uz:+s*d, http://www.100md.com
Discussionuz:+s*d, http://www.100md.com
In the present study we have examined the LH action on JAK/STAT, Shc/Grb2/ERK, and IRS-1/PI 3-kinase/AKT pathways and investigated the possibility of direct interactions between LH and insulin in the ovary, focusing on some key intermediate steps of these signaling pathways. Our results show that LH induces rapid tyrosine phosphorylation and activation of JAK2 in rat ovary. Upon JAK2 activation, tyrosine phosphorylation of STAT-1, STAT-5b, IRS-1, and Shc was detected. In addition, LH induced IRS-1/PI 3-kinase and Shc/Grb2 associations and downstream AKT serine phosphorylation and ERK phosphorylation, respectively. The simultaneous infusion of insulin and LH induced higher phosphorylation levels of JAK2, STAT-5b, IRS-1, and AKT compared with each hormone alone in the ovary of normal rats. These events occurred mostly in follicular cells and external and internal theca. Thus, the results indicate a new signal transduction pathway for LH and show that there is positive cross-talk between the insulin and LH signaling pathways at the levels of JAK2/STAT-5b and PI 3-kinase/AKT pathway in this tissue.
This is the first report that LH activates the JAK-STAT signaling pathway. JAK-STAT was originally identified as a cytokine-activated intracellular signaling pathway associated with an inflammatory response, but recently the JAK-STAT pathway was shown to be activated by peptide growth factors as well as GPCRs (reviewed in Ref. 27). The molecular mechanisms by which the LH receptor, which lacks intrinsic tyrosine kinase activity, couples to tyrosine phosphorylation events are not known. Because hCG binds LH receptor and was able to induce tyrosine phosphorylation of JAK2 and IRS-1, we suggest that the effects of LH are mediated by LH receptor. Accumulating evidence suggests that the GPCRs, like cytokine and GH receptors, serve as a docking site for signaling molecules that initiate tyrosine phosphorylation cascades. In the present study the rapid tyrosine phosphorylation and association of JAK2 and STAT-1 and -5b proteins suggest that a large signaling complex is formed with the LH receptor upon LH treatment. Coimmunoprecipitation of JAK2 and STAT-1 and -5b could be due the direct association of JAK2, STAT1, and STAT5b with the LH receptor or indirect association of STAT1 and -5b with the JAK2 kinase. There are several mechanisms by which JAK2 and STATs may associate with LH receptor. One possibility is that JAK2 initially associates with the LH receptor and leads to recruitment of STAT1 and -5b proteins. A second is that LH receptor recruits STAT proteins, which serve as adapter molecules for binding JAK2. A third possibility is that both JAK and STATs associate with the receptor, and upon ligand binding to the receptor, JAK2 phosphorylates the associated STAT proteins. Further studies (mutational analysis of the LH receptor and experiments in JAK2-deficient cell lines) will be required to assess this issue.
Recent evidence indicates that ligands signaling through GPCs may mimic some well-known effects classically observed after activation of tyrosine kinase receptors by the activation of JAK2, including the tyrosine phosphorylation of IRSs, Shc, and the activation of MAPK and PI 3-kinase (19, 20, 21, 22, 30, 31, 32). Our results demonstrate that LH is able to induce tyrosine phosphorylation of IRS-1 and Shc. Tyrosine-phosphorylated IRS-1 and Shc can proceed through the Grb2/Sos and Ras pathways, leading to activation of ERK (15, 16, 17). The present study also demonstrates that LH induces ERK activation. Although distinct pathways may link membrane receptors to activation of the ERK cascade (27), our results suggest that tyrosine phosphorylation of IRS-1 and Shc is a possible pathway used by LH to induce MAPK activation. Furthermore, the present results are in accordance with recent evidence that LH or hCG time- and dose-dependently activated ERK1 and ERK2 in human granulosa-lutein cells, and that this activation is required as a regulator of progesterone synthesis (33).
PI 3-kinase also transduces proliferative signals. The major lipid product of PI 3-kinase activity is phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate has binding affinity for a sequence called the pleckstrin homology domain. Thus, pleckstrin homology domaincontaining proteins are localized to membrane-associated signaling complexes (14, 27). One target of PI 3-kinase lipid products is AKT/PKB and its upstream activator, phosphoinositide-dependent kinase. AKT/PKB activates various enzymes involved in cell growth and inhibition of apoptosis (18, 29). Previous study supported a role for active PI 3kinase/AKT signaling in maintenance of the preovulatory follicle granulosa layer and demonstrated that FSH induces a biphasic increase in AKT phosphorylation in estrogen-primed, immature, rat granulosa cells (34). Our data showing that LH is able to induce AKT serine phosphorylation in rat ovary in vivo suggest that some degree of cross-talk among cell survival pathways is clearly a possibility, but this has not been adequately addressed.
Insulin can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, StAR, P450 cholesterol side-chain cleavage enzyme (CYP11A), 17{alpha} -hydroxylase/17,20-lyase (CYP17), and LDL receptor (7, 9, 10, 35). The mechanisms underlying the foregoing bihormonal interactions have been extensively studied and involve the ability of insulin to enhance LH-stimulated ovarian cAMP accumulation with a consequent increase in the expression of StAR and CYP11A. However, in the case of CYP17, the addition of cAMP failed to fully mimic LH enhancement of insulin action in thecal cells (35).(, 百拇医药
In the present study, H89 inhibition of PKA had no effect on insulin- or LH-induced AKT serine phosphorylation, but the use of PI 3-kinase inhibitors completely abolished this pathway. On the other hand, the pharmacological block of PI 3-kinase and ERK impeded the ability of insulin to enhance LH-stimulated LDL receptor transcriptional activity (7). Taken together, these results allow for the possibility of greater regulatory complexity of the LH-insulin interaction.
Our results show additive sites for the positive cross-talk between LH and insulin. Simultaneous stimulation with both hormones led to an increase in IRS-1 tyrosine phosphorylation and serine phosphorylation of AKT compared with the effect of acute LH or insulin administration. The predominant sites of cross-talk seem to be follicular and thecal cells, which are sites of rapid growth, tissue remodeling, and metabolic requirements. Although insulin has an LH-sensitizing effect, in the present study we found that LH and insulin have no additive effects on ERK phosphorylation.c|&, 百拇医药
Intracellular interactions between different signaling systems may function as mechanisms for enhancing or counterregulating hormone action. In the case of insulin, the cross-talk with LH-mediated pathways resulted in direct interactions between insulin and LH signaling systems at the levels of JAK2 and STAT5b. Simultaneous stimulation with both hormones led to increased tyrosine phosphorylation of JAK2 and STAT5b. In contrast, no effect on STAT1 phosphorylation was observed compared with acute insulin or LH administration. These results suggest that the positive cross-talk between insulin and LH signaling was due in part to additive effects on JAK2 activation and divergence of association between JAK2 and STAT1 and -5b. Another possible reason for this difference is differential insulin/LH signal amplification. STAT5b can be activated either by insulin through the insulin receptor in a JAK-independent fashion (36, 37) or with LH receptor by LH.
In conclusion, we have provided evidence for rapid direct effects of LH administration in vivo on intracellular signaling pathways demonstrating the existence of an additional signaling pathway stimulated by LH in the rat ovary in vivo. We also observed a convergence of LH and insulin signaling at the level of MAPK without synergism. Moreover, our results indicate direct and positive cross-talk between LH and insulin at the levels of JAK2, STAT-5b, and IRS-1 tyrosine phosphorylation and AKT serine phosphorylation. This mechanism may serve to potentiate the activities of both LH and insulin pathways and to increase stimulation in physiological processes such as the regulation of steroidogenesis that are under the combined control of both hormones.u@-4!z, 百拇医药
Acknowledgmentsu@-4!z, 百拇医药
We thank Mr. L. Janeri, Mr. J. L. Santos, and Mrs. L. Silva for technical assistance, and the National Institute of Diabetes and Digestive Kidney Diseases National Hormone and Pituitary Program and A. F. Parlow for the LH and hCG.
Received July 11, 2002.z4#da\, 百拇医药
Accepted for publication October 29, 2002.z4#da\, 百拇医药
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The actions of LH are mediated through a single class of cell surface LH/human chorionic gonadotropin receptor, which is a member of the G protein-coupled receptor family. In the present study we showed that LH induced rapid tyrosine phosphorylation and activation of the Janus kinase 2 (JAK2) in rat ovary. Upon JAK2 activation, tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT-1), STAT-5b, insulin receptor substrate-1 (IRS-1), and Src homology and collagen homology (Shc) were detected. In addition, LH induced IRS-1/phosphoinositol 3-kinase and Shc /growth factor receptor-binding protein 2 (Grb2) associations and downstream AKT (protein kinase B, homologous to v-AKT) serine phosphorylation and ERK tyrosine phosphorylation, respectively. The simultaneous infusion of insulin and LH induced higher phosphorylation levels of JAK2, STAT5b, IRS-1, and AKT compared with each hormone alone in the whole ovary of normal rats. By immunohistochemistry we demonstrated that these late events take place in follicular cells and both external and internal theca. These results indicate a new signal transduction pathway for LH and show that there is positive cross-talk between the insulin and LH signaling pathways at the level of phosphoinositol 3-kinase/AKT pathway in this tissue.
Introductionq5, http://www.100md.com
THE MAMMALIAN LH receptor is a G protein-coupled receptor (GPCR) with a transmembrane domain composed of seven segments. Binding of the receptor by LH leads to activation of Gs, which activates the membrane-associated adenylyl cyclase, causing an increase in intracellular cAMP (1, 2, 3, 4). This nucleotide acts as a second messenger for the regulation of steroidogenic acute regulatory protein (StAR) and the cytochrome P450 enzyme system (5). Alternative signaling pathways by LH or human chorionic gonadotropin (hCG) receptor were described in the last years, including chloride ion influx, activation of the phosphoinositol pathway, and calcium ion mobilization (6, 7, 8).q5, http://www.100md.com
Insulin can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, such as low density lipoprotein (LDL) receptor, StAR, and P450 cholesterol side-chain cleavage enzyme (CYP11A) (7, 9, 10). Considerable evidence demonstrates that insulin receptor tyrosine kinase activity is essential for many, if not all, of the biological effects of insulin. In most cells this primary event leads to the subsequent tyrosyl phosphorylation of the insulin receptor substrate 1 (IRS-1) and IRS-2. Both IRS-1 and IRS-2 have been implicated as the first postreceptor step in insulin signal transmission. In animal tissues and cultured cells, phosphorylated IRS-1 and -2 can bind and activate phosphatidylinositol 3-kinase (PI 3-kinase) (11, 12, 13). A downstream substrate of PI 3-kinase activity is the serine/threonine protein kinase B or AKT (protein kinase B, homologous to v-AKT) (5). Upon insulin receptor tyrosine kinase activation and autophosphorylation, there is also recruitment of protein Shc (Src homology and collagen homology) and growth factor receptor-binding protein 2 (Grb2), leading to activation of the extracellular signal-regulated kinase (ERK) pathway (15, 16, 17).
Recently, it was demonstrated that in cultured granulosa cells, putative suppression of PI 3-kinase with wortmannin or LY294002 trigged in vitro apoptosis (18), raising the hypothesis that LH may signal through classical tyrosine kinase pathways. In addition, other hormones that act through GPCRs, including angiotensin II and vasopressin, can induce tyrosine phosphorylation of cytoplasmic proteins (19, 20, 21, 22). Angiotensin II activates Janus kinase 2 (JAK2), a member of the JAK family, and probably uses this kinase to induce several intracellular protein tyrosine phosphorylations.6w-2^, http://www.100md.com
In this study we evaluated the ability of LH to activate JAK2 and to induce the tyrosine phosphorylation of IRS-1, Shc, signal transducer and activator of transcription 1 (STAT1), and STAT5b as well as IRS-1/PI 3-kinase and Shc/Grb2 associations and the phosphorylation of AKT/PKB and ERK in rat ovary in vivo. We also assessed the possible cross-talk between the LH and insulin signaling pathways. Our data reveal a new signal transduction pathway for LH and show that there is positive cross-talk between insulin and LH signaling systems.
Materials and Methodsk63{ie, 百拇医药
Materialsk63{ie, 百拇医药
The reagents for SDS-PAGE, immunoprecipitation, and immunoblotting were obtained from sources previously cited (21, 22, 23, 24). Highly purified hCG, LH, and PRL were obtained from the NIDDK National Hormone and Pituitary Program (Torrance, CA), and human recombinant insulin (Humulin R) was purchased from Eli Lilly \|[amp ]\| Co. (Indianapolis, IN). The PI 3-kinase inhibitors (wortmannin and LY294002) were obtained from Sigma-Aldrich (St. Louis, MO), and the protein kinase A (PKA) inhibitor (H89) was purchased from Calbiochem (San Diego, CA). Anti-IR, anti-IRS-1, anti-STAT1, anti-STAT5b, anti-Shc, anti-Grb2, antiphosphotyrosine, and anti-JAK2 antibodies were purchased from Santa Cruz Technology, Inc. (Santa Cruz, CA). Anti-PI 3-kinase was from Upstate Biotechnology, Inc. (Lake Place, NY). Anti-phosphoserine-AKT, anti-phospho-ERK, anti-phospho-STAT1, anti-phospho-STAT3, and anti-phospho-STAT5b antibodies were obtained from New England Biolabs, Inc. (Beverly, MA).
Animal tissue extracts and immunoblotting%!/?, http://www.100md.com
Female Wistar rats (200–220 g) were housed with access to standard rodent chow and water ad libitum. Food was withdrawn 6 h before the experiments. All procedures with animals were conducted in accord with the principles and procedures described by NIH Guidelines for the Care and Use of Experimental Animals. The studies were conducted using animals at the diestrous phase. The rats were anesthetized with sodium thiopental (25 mg/kg, ip) and were used 10–15 min later, as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the cava vein was exposed, and 0.5 ml saline (0.9% NaCl) with or without insulin, PRL, LH/hCG, or insulin plus LH was injected at the doses indicated as bolus infusion (doses stated in the figures). The ovaries were removed at the times indicated and homogenized in ice-cold extraction buffer containing 100 mM Tris (pH 7.4), 10 mM EDTA, 1% Triton X-100, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM sodium vanadate, 2 mM phenylmethylsulfonylfluoride, and 0.01 mg aprotinin/ml. Pools of two ovarian extracts from female rats at the diestrous phase were centrifuged at 15,000 rpm at 4 C for 15 min to remove insoluble material; the supernatant was then used for the assay. Protein determination was performed by the Bradford dye binding method using the reagent from Bio-Rad Laboratories, Inc. (Richmond, CA) and BSA as the standard. Two milligrams of protein from the supernatants were used for immunoprecipitation with anti-IRS-1, anti-JAK2, anti-STAT-1, anti-STAT-5b, anti-Shc, and protein A-Sepharose 6MB before Laemmli sample buffer treatment and electrophoresis in SDS-PAGE as described previously (21, 22, 23, 24). For whole tissue extracts, similarly sized aliquots (100 µg protein) were subjected to SDS-PAGE and immunoblotted with anti-phospho-AKT, anti-phospho-ERK, anti-phospho-STAT1, anti-phospho-STAT3, and anti-phospho-STAT5b antibodies. Electrotransfer of proteins from the gel to nitrocellulose was performed for 90 min at 120 V (constant) (21, 22, 23, 24). To reduce nonspecific protein binding to the nitrocellulose, the filter was preincubated for 2 h at room temperature in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blots were incubated overnight at 4 C with antibodies against phosphotyrosine, the p85 subunit of PI 3-kinase, STAT-1, STAT-5b, GRB2, phosphoserine-AKT, and phospho-ERK diluted in blocking buffer with 3% nonfat dry milk, followed by washing for 30 min in blocking buffer without milk. The blots were incubated with 2 µCi [125I]protein A (30 µCi/µg) in 10 ml blocking buffer for 2 h at room temperature and then washed again for 30 min as described above. [125I]Protein A bound to the specific antibodies was detected by autoradiograph using preflashed Kodak XAR film (Eastman Kodak Co., Rochester, NY) with Cronex Lightning Plus intensifying screens (DuPont, Wilmington, DE) at -8 C for 48–72 h. Band intensities were quantitated by optical densitometry (model GS 300, Hoefer Scientific, San Francisco, CA) of the developed autoradiographs.
JAK2 in vitro tyrosine kinase activity+]?r), http://www.100md.com
JAK2 tyrosine kinase activity was measured by autophosphorylation. A low dose of LH (2 pg) was injected into the vena cava to stimulate partial JAK2 autophosphorylation. JAK2 was immunoprecipitated as described above. The resulting immune complexes were collected on protein A-Sepharose. The protein kinase activity of the immunoprecipitates was measured by incubating the immune complexes in 100 µl buffer containing 50 mM Tris (pH 7.5), 0.2 mM sodium vanadate, 0.1% Triton X-100, 3 mM MnCl2, and 15 µM cold ATP for 30 min at room temperature. The complexes were washed twice with cold buffer, then resuspended in Laemmli sample buffer and analyzed by SDS-PAGE (21, 23, 24). The incorporation of phosphate into the separated proteins was visualized by autoradiography using antiphosphotyrosine immunoblots after transfer to nitrocellulose.+]?r), http://www.100md.com
Immunohistochemistry+]?r), http://www.100md.com
Four ovaries from two diestrous female rats from each treatment group (saline, insulin, LH, and insulin plus LH infusion) were examined to determine the expression and tissue distribution of proteins participating in the insulin signaling pathway. Hydrated 5-µm sections of paraformaldehyde-fixed, paraffin-embedded tissue were stained by the avidin-peroxidase method. Sections were incubated for 30 min with 2% normal rabbit or normal mouse serum at room temperature and then were exposed for 12 h in a moisture chamber at 4 C to the primary antibodies against insulin receptor (1:80), p-STAT-1 (1:50), p-STAT-5b (1:50), p-AKT (1:40), or p-ERK (1:50). Biotinylated secondary antibodies were used in incubations for 2 h at room temperature, followed by a 1-h incubation with ready to use avidin-coupled peroxidase (Vector Laboratories, Inc., Burlingame, CA). The resulting immunocomplexes were detected with 50 mg/100 ml diaminobenzidine:4 HCl/0.01 ml/100 ml H2O2 dissolved in 5 mmol/liter Tris, pH 7.6. Analysis and photodocumentation were performed using a Microphot FXA microscope (Nikon, Milville, NY).
Preparation of perfused rat ovary)x9lljp, http://www.100md.com
Diestrous female rats underwent surgical isolation of the right ovary with connecting vasculature as described by Matousek and co-workers (25). The ovaries were placed in a perfusion system for 30 min to examine the effect of PI 3-kinase inhibitors [wortmannin (0.1 µM) and LY 294002 (50 µM)] and PKA inhibitor (H89, 10 µM) on LH-induced AKT serine phosphorylation.)x9lljp, http://www.100md.com
Results)x9lljp, http://www.100md.com
LH stimulates JAK2 tyrosine kinase activity)x9lljp, http://www.100md.com
To investigate whether LH stimulates JAK2 phosphorylation, LH was infused into the cava vein of female rats, and the ovaries were extracted and analyzed by immunoblotting. JAK2 tyrosine phosphorylation was measured by immunoprecipitation with polyclonal anti-JAK2 antibodies. The precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and then immunoblotted with a monoclonal antiphosphotyrosine antibody (Fig. 1). Parallel experiments were performed in which the rats received an infusion of PRL, because this hormone is known to induce the tyrosine phosphorylation of JAK2 in ovary cells (26). Figure 1A shows that LH induces JAK2 tyrosine phosphorylation 3 min after this hormone infusion. As expected, the infusion of PRL also induced JAK2 tyrosine phosphorylation a little latter, although less markedly than LH (Fig. 1B). Both figures show that the tyrosine phosphorylation of JAK2 in response to LH and PRL is rapid and transient, and no change in JAK2 protein expression was observed after hormone infusion (Fig. 1, A and B, lower panel). LH-stimulated phosphorylation of JAK2 was dose dependent (Fig. 1C). Phosphorylated JAK2 was detectable after the injection of as little as 2 pg LH, and half-maximal stimulation occurred at approximately 200 pg, which, when distributed in the blood volume, may be within the physiological range for female rats.
fig.ommitteed*, 百拇医药
Figure 1. LH stimulates JAK2 tyrosine phosphorylation in the ovary of rats. Saline (0), 2 ng LH, or 200 ng PRL (Pro) were administered at the times indicated (A and B) into the vena cava as a bolus injection in normal female rats. For the dose-response analysis, the indicated doses were injected 3 min after LH infusion (C). A pool of ovarian extracts was immunoprecipitated with anti-JAK2, followed by immunoblotting with antiphosphotyrosine monoclonal antibody. The phosphorylation levels of JAK2 were quantified with an image analyzer and are shown as the fold increase from the basal value (n = 5). JAK2 tyrosine kinase activity (D) was measured by autophosphorylation in vitro in the absence (-) or presence (+) of 15 µM ATP before and after 3 min of LH (2 pg) administration. SDS-PAGE (8%) was performed, and there was an increase in JAK2 autophosphorylation after LH infusion plus the addition of ATP in vitro.*, 百拇医药
To investigate the effect of LH on JAK2 kinase activity, we performed an in vitro kinase assay. Ovaries from rats that were stimulated submaximally with LH to obtain limited tyrosine phosphorylation of JAK2 were immunoprecipitated with anti-JAK2 antibody and incubated with exogenous ATP to permit autophosphorylation in vitro. Tyrosine phosphorylation was quantified by immunoblotting using antiphosphotyrosine antibody. After the infusion of a low dose of LH in vivo and the addition of ATP in vitro, there was a clear increase in JAK2 autophosphorylation (Fig. 1D).
LH induces STAT-1 and STAT-5b tyrosine phosphorylationkn, http://www.100md.com
Although activation of the JAK-STAT pathway was originally thought to occur through cytokine receptors, it has recently been shown that the key activating event, STAT tyrosine phosphorylation, may be regulated/activated by GPCRs (27). Thus, an investigation of whether LH infusion activates the JAK-STAT signal transduction pathway in the ovary of intact rats was undertaken. We initially used coimmunoprecipitation analysis to determine whether acute iv infusion of LH would activate STAT-1, STAT-3, and STAT-5 transcription factors to form JAK2 complexes. Supernatants from a pool of ovaries from female rats that received LH infusion or saline were immunoprecipitated with anti-JAK2 antibody. The membranes containing these immune complexes were incubated with STAT-1, STAT-3, and STAT-5b. In Fig. 2A, data obtained from this approach show that after LH infusion there was an increase in JAK2/STAT-1 and JAK2/STAT-5b association, but not JAK2/STAT-3 association (data not shown).
fig.ommitteed:xcf#ch, 百拇医药
Figure 2. LH stimulates JAK2 coimmunoprecipitation with STAT-1, STAT-5b, and STAT-1, and STAT-5b phosphorylation in ovaries of normal rats in vivo. Saline (-) or 2 ng LH or PRL (Pro) was administered into the vena cava as a bolus injection in normal female rats. The ovaries were extracted 3 min after LH infusion for immunoprecipitation with anti-JAK2 antibody (A). A pool of ovarian extracts was immunoprecipitated with anti-JAK2 (A), anti-STAT-1 (B), or anti-STAT-5b (C) antibodies. The membranes containing JAK2 immune complexes were incubated with anti-STAT-1 or anti-STAT-5b. The membranes containing anti-STAT-1 or anti-STAT-5b were immunoblotted with antiphosphotyrosine antibody (B and C). Shown are representative autoradiographs from four different experiments.:xcf#ch, 百拇医药
We next investigated the tyrosine phosphorylation of STATs after LH infusion. LH not only induced increased JAK2/STAT-1 and JAK2/STAT-5b associations, but also increased the tyrosine phosphorylation of STAT-1 and STAT-5b proteins within 3–5 min after hormone injection (Fig. 2, B and C). However, the tyrosine phosphorylation of STAT-3 was not detected even in samples previously immunoprecipitated with anti-phospho-STAT-3 after LH stimulus, consistent with our previous finding that there was no detectable coimmunoprecipitation of JAK2 with STAT-3. As expected, the infusion of PRL induced STAT-5b tyrosine phosphorylation, but not STAT-1 (Fig. 2, B and C, right). STAT1 and STAT5b protein expression did not change after LH infusion (Fig. 2, B and C, lower panels).
LH stimulates IRS-1 tyrosine phosphorylation and AKT serine phosphorylationo9, 百拇医药
The rate of LH-induced IRS-1 tyrosine phosphorylation was estimated in a time-course experiment (Fig. 3A). Proteins from ovaries of rats treated with LH were immunoprecipitated with anti-IRS-1 antibody and then immunoblotted with a mouse monoclonal antiphosphotyrosine antibody. IRS-1 was maximally tyrosine-phosphorylated 3 min after the infusion of LH (Fig. 3A, upper panel), with no change in IRS-1 protein expression (Fig 3A, lower panel). Phosphorylated IRS-1 binds and activates PI 3-kinase and at least two adapter molecules, Grb2 and Nck (homologous to v-Nck) (11, 28). We evaluated the ability of LH to stimulate IRS-1/PI 3-kinase association in the rat ovary in vivo. After LH stimulation, there was an increase in IRS-1/PI 3-kinase association (Fig. 3A, middle panel) in the ovaries of intact rats, which paralleled the increase in IRS-1 tyrosine phosphorylation.o9, 百拇医药
fig.ommitteed
Figure 3. LH stimulates the IRS-1/PI 3-kinase/AKT pathway. After the injection of 2 ng LH at the indicated time, ovaries were extracted from rats. The supernatants were used for immunoprecipitation with anti-IRS-1 (A) antibodies and for whole tissue extracts (B–D). The immunoblots were performed with antiphosphotyrosine (A, upper panel), anti-PI 3-kinase (A, middle panel), anti-IRS-1 (A, lower panel), and anti-pAKT (B and C) antibodies. C, Effect of LH on AKT serine phosphorylation in ovaries extracted from prepubertal 4-wk-old rats. D, In perfused ovaries, as described in Materials and Methods, pretreatment with LY294002 (50 µmol, iv), wortmannin (100 nmol; E), or H89 (10 mM) was followed 30 min later by iv administration of LH or insulin. E and F, Effect of hCG in the phosphorylation of JAK2 (E) and IRS-1 (F) in ovaries extracts of normal rats 3 min after hormone infusion. Ovarian extracts were immunoprecipitated with anti-JAK2 or anti-IRS-1 antibodies, and immunoblottings were performed with antibody against phosphotyrosine residues. Results are representative of four experiments./m0l48, 百拇医药
To confirm that the actions of LH on tyrosine phosphorylation of JAK2 and IRS-1 are mediated by classical LH receptor, and that this receptor binds hCG as well as LH, we performed experiments using an infusion of 100 IU hCG instead of LH (4). The results demonstrated that hCG is able to induce JAK2 (Fig. 4C) and IRS-1 (Fig. 4D
fig.ommitteedf!x, 百拇医药
Figure 4. LH stimulates Shc/Grb2/ERK pathway. After 2 ng LH injection at the time indicated, ovaries were extracted from rats. The supernatants were used for immunoprecipitation with anti-Shc (A) antibodies and for whole tissue extracts (B). The immunoblots were performed with antiphosphotyrosine (A, upper panel), with anti-GRB2 (A, middle panel), with anti-Shc (A, lower panel), and with anti-pERK and anti-ERK antibodies (B). These are representative autoradiographs from four different experiments.f!x, 百拇医药
It is well established that insulin-induced IRS-1 tyrosine phosphorylation and association/activation with PI 3-kinase are accompanied by an increase in AKT serine phosphorylation (14, 29). Because LH was able to induce IRS-1 tyrosine phosphorylation and association with PI 3-kinase, we investigated the effect of LH on AKT serine phosphorylation. LH infusion induced only a 2-fold increase in AKT serine phosphorylation at 3 min, which then decreased gradually, returning to basal levels at 10 min (Fig. 3B, upper panel), without changes in AKT protein expression (Fig. 3B, lower panel). We performed similar experiments in immature rats (4 wk old), and the results demonstrated the same effect of LH inducing AKT phosphorylation, with similar a time course similar to that in mature rats, indicating that this pathway is present in animals that lack corpora lutea (Fig. 3C).
To verify whether LH is able to induce AKT phosphorylation in perfused ovaries and determine the effect of inhibitors of PI 3-kinase or PKA in this pathway, we prepared perfused ovaries as described in Materials and Methods and treated them with LH after pretreatment (30 min) with LY294002, wortmannin, or H89. We also used insulin as a control. As shown in Fig. 3D, in perfused ovaries LH induced AKT serine phosphorylation similar to that in rat ovaries in vivo, and pretreatment with LY294002 (Fig. 3D, middle panel) or wortmannin (Fig. 3D, upper panel) abolished LH-induced AKT phosphorylation. As expected insulin-induced AKT phosphorylation was also blocked by these inhibitors of PI 3-kinase. When we used H89 for 30 min before LH treatment, no effect was observed on LH-induced AKT phosphorylation, suggesting that PKA is probably not involved or has only a minor influence on this pathway (Fig. 3D, lower panel).*$;dt.%, 百拇医药
LH stimulates Shc tyrosine phosphorylation and ERK phosphorylation
LH-induced Shc tyrosine phosphorylation was observed within 3 min after hormone infusion and was maximal at 10 min (Fig. 4A, upper panel), promoting no change in Shc protein expression (Fig. 4A, lower panel). LH also increased the Shc/Grb2 association in a similar time course of LH-induced Shc tyrosine phosphorylation (Fig. 4A, middle panel). The LH-stimulated phosphorylation of Shc was dose dependent and similar to that for JAK2 (data not shown).-|, 百拇医药
Because Shc tyrosine phosphorylation leads to activation of ERK pathways through the Grb2/SOS complex, we investigated the effect of LH on ERK phosphorylation levels. LH infusion leads to an approximately 8-fold increase in ERK phosphorylation at 10 min, as determined by immunoblotting with an antiphospho-ERK antibody (Fig. 4B, upper panel) without changes in ERK protein expression.-|, 百拇医药
Effect of simultaneous administration of LH and insulin on JAK/STAT, IRS-1/PI3K/AKT, and Shc/ERK pathways-|, 百拇医药
At first we measured the tyrosine phosphorylation of JAK2 in rat ovaries that were stimulated with LH, insulin, or both hormones for 3 and 10 min. The phosphorylation of JAK2 in ovaries showed an increase of approximately 7-fold 3 min after LH infusion and of approximately 6-fold 3 min after insulin infusion. LH and insulin together showed approximately 10-fold and approximately 7-fold increases in JAK2 tyrosine phosphorylation 3 and 10 min, respectively, significantly higher than with each hormone alone (Fig. 5A).
fig.ommitteed4;t$%#(, http://www.100md.com
Figure 5. LH plus insulin enhances JAK2/STAT-5b and IRS-1/PI 3-kinase/Akt signaling pathway with no effect on Shc/GRB2/ERK pathway in the ovary of normal rats in vivo. Saline (0), 2 ng LH, 6 µg insulin, or 2 ng LH plus 6 µg insulin (LH-Ins) were administered into vena cava as a bolus injection of female normal rats. The ovaries were extracted at the indicated time, and after homogenization and centrifugation the supernatants were used for immunoprecipitation with the following antibodies against JAK2 (A), STAT-1 (B), STAT-5b (C), IRS-1 (D and E), and Shc (G and H) or for whole tissue extracts and immunoblotted with anti-pAkt (F) and with anti-pERK (I). Bands were quantitated using a densitometer. Data are the mean ± SEM for four rats for each group. *, P < 0.05 vs. LH-Ins and LH-Ins plus LH; #, P < 0.05 vs. Ins-Ins plus LH.4;t$%#(, http://www.100md.com
Figure 5B shows that LH induced maximal STAT1 tyrosine phosphorylation (~ 7-fold increase) at 10 min, and insulin induced only a mild increase in STAT1 phosphorylation. The administration of both hormones did not induce an additive effect over each other on STAT1 phosphorylation.
LH or insulin induced a moderate increase in STAT5b tyrosine phosphorylation. The infusion of both hormones induced approximately 6- and 11-fold increases in STAT5b tyrosine phosphorylation at 3 and 10 min, respectively, significantly higher than the effect of each hormone alone (Fig. 5C).1hgy, 百拇医药
Figure 5D shows that the simultaneous infusion of LH and insulin induced a higher level of IRS-1 tyrosine phosphorylation at 3 and 10 min than each hormone alone, indicating a synergistic effect of the hormones. The effects of LH, insulin, or both hormones on IRS-1/PI 3-kinase association showed were similar on IRS-1 tyrosine phosphorylation (Fig. 5E).1hgy, 百拇医药
LH induced only a mild and transitory increase in AKT serine phosphorylation. After insulin infusion, there was an increase in AKT serine phosphorylation of approximately 7-fold at 3 and 10 min. The simultaneous infusion of both hormones showed an additive effect, with a significant increase in AKT serine phosphorylation at 3 and 10 min (Fig. 5F).
Figure 5G shows that LH or insulin induced a moderate increase in Shc tyrosine phosphorylation at 3 and 10 min, but there is no additive effect after the infusion of both hormones. The effect of LH, insulin, or both hormones on Shc/Grb2 association showed a similar behavior of Shc tyrosine phosphorylation (Fig. 5H.1j9:, 百拇医药
The insulin infusion induced an approximately 7-fold increase in ERK phosphorylation at 3 and 10 min. The simultaneous infusion of both hormones showed no additive effect in ERK phosphorylation compared with that detected with insulin alone (Fig. 5I).1j9:, 百拇医药
Tissue distribution of IR and the molecular events induced by LH and insulin1j9:, 百拇医药
By performing immunohistochemical studies of rat ovary, a broad distribution of IR was detected. Impressive staining for IR was observed in interstitial cells of the stroma, in cells of the external an internal theca, and in follicular cells of the diestrous ovary (Fig. 6A). Under LH, insulin, or LH plus insulin stimulation the induction of Ser473 phosphorylation of AKT was most evident in follicular cells (Fig. 6B). For ERK, a prestimulus, constitutive pattern of tyrosine phosphorylation was detected. After LH, insulin, or LH plus insulin treatment an impressive increase in staining was induced mostly in follicular cells of the diestrous ovary (Fig. 6B). STAT1 and STAT5b tyrosine phosphorylations were also induced by LH, insulin, or LH plus insulin treatment. Again, thecal and follicular cells were the sites of highest staining (Fig. 6B). Although immunohistochemistry does not serve as a quantitative method, on a comparative basis an apparent additive effect of LH and insulin was detected on AKT and STAT5b treatment-induced phosphorylation.
fig.ommitteeduz:+s*d, http://www.100md.com
Figure 6. Immunohistochemical analysis of IR (upper panel) distribution in ovary of diestrous rats and of LH, insulin, and LH- and insulin-induced Akt, ERK, STAT1, and STAT5 phosphorylation (lower panel). IR is present in most cells of the rat ovary, including interstitial cells of the stroma, external and internal thecal cells, and follicular cells (upper panel). LH and insulin induced Ser473 Akt phosphorylation is detected mostly in follicular cells (lower panel, upper line). LH- and insulin-induced tyrosine phosphorylation of ERK is detected mostly in follicular cells (lower panel, second line). LH- and insulin-induced tyrosine phosphorylation of STAT1 is detected mostly in thecal and follicular cells (lower panel, third line), and LH- and insulin-induced tyrosine phosphorylation of STAT5b is detected mostly in thecal and follicular cells (lower panel, lower line).uz:+s*d, http://www.100md.com
Discussionuz:+s*d, http://www.100md.com
In the present study we have examined the LH action on JAK/STAT, Shc/Grb2/ERK, and IRS-1/PI 3-kinase/AKT pathways and investigated the possibility of direct interactions between LH and insulin in the ovary, focusing on some key intermediate steps of these signaling pathways. Our results show that LH induces rapid tyrosine phosphorylation and activation of JAK2 in rat ovary. Upon JAK2 activation, tyrosine phosphorylation of STAT-1, STAT-5b, IRS-1, and Shc was detected. In addition, LH induced IRS-1/PI 3-kinase and Shc/Grb2 associations and downstream AKT serine phosphorylation and ERK phosphorylation, respectively. The simultaneous infusion of insulin and LH induced higher phosphorylation levels of JAK2, STAT-5b, IRS-1, and AKT compared with each hormone alone in the ovary of normal rats. These events occurred mostly in follicular cells and external and internal theca. Thus, the results indicate a new signal transduction pathway for LH and show that there is positive cross-talk between the insulin and LH signaling pathways at the levels of JAK2/STAT-5b and PI 3-kinase/AKT pathway in this tissue.
This is the first report that LH activates the JAK-STAT signaling pathway. JAK-STAT was originally identified as a cytokine-activated intracellular signaling pathway associated with an inflammatory response, but recently the JAK-STAT pathway was shown to be activated by peptide growth factors as well as GPCRs (reviewed in Ref. 27). The molecular mechanisms by which the LH receptor, which lacks intrinsic tyrosine kinase activity, couples to tyrosine phosphorylation events are not known. Because hCG binds LH receptor and was able to induce tyrosine phosphorylation of JAK2 and IRS-1, we suggest that the effects of LH are mediated by LH receptor. Accumulating evidence suggests that the GPCRs, like cytokine and GH receptors, serve as a docking site for signaling molecules that initiate tyrosine phosphorylation cascades. In the present study the rapid tyrosine phosphorylation and association of JAK2 and STAT-1 and -5b proteins suggest that a large signaling complex is formed with the LH receptor upon LH treatment. Coimmunoprecipitation of JAK2 and STAT-1 and -5b could be due the direct association of JAK2, STAT1, and STAT5b with the LH receptor or indirect association of STAT1 and -5b with the JAK2 kinase. There are several mechanisms by which JAK2 and STATs may associate with LH receptor. One possibility is that JAK2 initially associates with the LH receptor and leads to recruitment of STAT1 and -5b proteins. A second is that LH receptor recruits STAT proteins, which serve as adapter molecules for binding JAK2. A third possibility is that both JAK and STATs associate with the receptor, and upon ligand binding to the receptor, JAK2 phosphorylates the associated STAT proteins. Further studies (mutational analysis of the LH receptor and experiments in JAK2-deficient cell lines) will be required to assess this issue.
Recent evidence indicates that ligands signaling through GPCs may mimic some well-known effects classically observed after activation of tyrosine kinase receptors by the activation of JAK2, including the tyrosine phosphorylation of IRSs, Shc, and the activation of MAPK and PI 3-kinase (19, 20, 21, 22, 30, 31, 32). Our results demonstrate that LH is able to induce tyrosine phosphorylation of IRS-1 and Shc. Tyrosine-phosphorylated IRS-1 and Shc can proceed through the Grb2/Sos and Ras pathways, leading to activation of ERK (15, 16, 17). The present study also demonstrates that LH induces ERK activation. Although distinct pathways may link membrane receptors to activation of the ERK cascade (27), our results suggest that tyrosine phosphorylation of IRS-1 and Shc is a possible pathway used by LH to induce MAPK activation. Furthermore, the present results are in accordance with recent evidence that LH or hCG time- and dose-dependently activated ERK1 and ERK2 in human granulosa-lutein cells, and that this activation is required as a regulator of progesterone synthesis (33).
PI 3-kinase also transduces proliferative signals. The major lipid product of PI 3-kinase activity is phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate has binding affinity for a sequence called the pleckstrin homology domain. Thus, pleckstrin homology domaincontaining proteins are localized to membrane-associated signaling complexes (14, 27). One target of PI 3-kinase lipid products is AKT/PKB and its upstream activator, phosphoinositide-dependent kinase. AKT/PKB activates various enzymes involved in cell growth and inhibition of apoptosis (18, 29). Previous study supported a role for active PI 3kinase/AKT signaling in maintenance of the preovulatory follicle granulosa layer and demonstrated that FSH induces a biphasic increase in AKT phosphorylation in estrogen-primed, immature, rat granulosa cells (34). Our data showing that LH is able to induce AKT serine phosphorylation in rat ovary in vivo suggest that some degree of cross-talk among cell survival pathways is clearly a possibility, but this has not been adequately addressed.
Insulin can amplify gonadotropin-stimulated steroidogenesis by augmenting the expression of key sterol regulatory genes in ovarian cells, StAR, P450 cholesterol side-chain cleavage enzyme (CYP11A), 17{alpha} -hydroxylase/17,20-lyase (CYP17), and LDL receptor (7, 9, 10, 35). The mechanisms underlying the foregoing bihormonal interactions have been extensively studied and involve the ability of insulin to enhance LH-stimulated ovarian cAMP accumulation with a consequent increase in the expression of StAR and CYP11A. However, in the case of CYP17, the addition of cAMP failed to fully mimic LH enhancement of insulin action in thecal cells (35).(, 百拇医药
In the present study, H89 inhibition of PKA had no effect on insulin- or LH-induced AKT serine phosphorylation, but the use of PI 3-kinase inhibitors completely abolished this pathway. On the other hand, the pharmacological block of PI 3-kinase and ERK impeded the ability of insulin to enhance LH-stimulated LDL receptor transcriptional activity (7). Taken together, these results allow for the possibility of greater regulatory complexity of the LH-insulin interaction.
Our results show additive sites for the positive cross-talk between LH and insulin. Simultaneous stimulation with both hormones led to an increase in IRS-1 tyrosine phosphorylation and serine phosphorylation of AKT compared with the effect of acute LH or insulin administration. The predominant sites of cross-talk seem to be follicular and thecal cells, which are sites of rapid growth, tissue remodeling, and metabolic requirements. Although insulin has an LH-sensitizing effect, in the present study we found that LH and insulin have no additive effects on ERK phosphorylation.c|&, 百拇医药
Intracellular interactions between different signaling systems may function as mechanisms for enhancing or counterregulating hormone action. In the case of insulin, the cross-talk with LH-mediated pathways resulted in direct interactions between insulin and LH signaling systems at the levels of JAK2 and STAT5b. Simultaneous stimulation with both hormones led to increased tyrosine phosphorylation of JAK2 and STAT5b. In contrast, no effect on STAT1 phosphorylation was observed compared with acute insulin or LH administration. These results suggest that the positive cross-talk between insulin and LH signaling was due in part to additive effects on JAK2 activation and divergence of association between JAK2 and STAT1 and -5b. Another possible reason for this difference is differential insulin/LH signal amplification. STAT5b can be activated either by insulin through the insulin receptor in a JAK-independent fashion (36, 37) or with LH receptor by LH.
In conclusion, we have provided evidence for rapid direct effects of LH administration in vivo on intracellular signaling pathways demonstrating the existence of an additional signaling pathway stimulated by LH in the rat ovary in vivo. We also observed a convergence of LH and insulin signaling at the level of MAPK without synergism. Moreover, our results indicate direct and positive cross-talk between LH and insulin at the levels of JAK2, STAT-5b, and IRS-1 tyrosine phosphorylation and AKT serine phosphorylation. This mechanism may serve to potentiate the activities of both LH and insulin pathways and to increase stimulation in physiological processes such as the regulation of steroidogenesis that are under the combined control of both hormones.u@-4!z, 百拇医药
Acknowledgmentsu@-4!z, 百拇医药
We thank Mr. L. Janeri, Mr. J. L. Santos, and Mrs. L. Silva for technical assistance, and the National Institute of Diabetes and Digestive Kidney Diseases National Hormone and Pituitary Program and A. F. Parlow for the LH and hCG.
Received July 11, 2002.z4#da\, 百拇医药
Accepted for publication October 29, 2002.z4#da\, 百拇医药
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