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Endocrinology Vol. 142, No. 9 3842-3849
Copyright © 2001 by The Endocrine Society

ARTICLES

E2-Induced Degradation of Uterine Insulin Receptor Substrate-2: Requirement for an IGF-I-Stimulated, Proteasome-Dependent Pathway

R. Gregg Richards, Diane M. Klotz, Mark R. Bush1, David K. Walmer and Richard P. DiAugustine

Hormones and Cancer Group, Laboratory of Molecular Carcinogenesis (R.G.R., D.M.K., M.R.B., R.P.D.) and Laboratory of Reproductive and Developmental Toxicology (D.K.W.), National Institute of Environmental and Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility (M.R.B., D.K.W.), Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Dr. Richard P. DiAugustine National Institute of Environmental and Health Sciences, National Institutes of Health, 111 TW Alexander Drive, MD D4–04, Research Triangle Park, North Carolina 27709.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The insulin receptor substrates are docking proteins that bind various receptor tyrosine kinases and signaling proteins. Previous studies have shown that E2 or progesterone can regulate the relative abundance of insulin receptor substrate-1 and -2 in cells and tissues. For instance, uterine insulin receptor substrate-2 was decreased markedly at 24 h after E2 treatment of mice. In the present study we used various in vivo experimental approaches to examine the mechanism by which E2 influences uterine insulin receptor substrate-2 expression. Uterine insulin receptor substrate-2 mRNA levels were diminished after E2 treatment, but this diminution did not account for the total reduction in insulin receptor substrate-2 protein, suggesting that the E2-induced decrease in insulin receptor substrate-2 is not regulated solely at the mRNA level. Cotreatment with progesterone prevented the E2-stimulated reduction in insulin receptor substrate-2 protein at 24 h after hormone exposure. In addition, MG-132 and epoxomicin, inhibitors of proteasomal protease activity, inhibited the E2-induced decrease in uterine insulin receptor substrate-2 protein levels, and this correlated to an increase in uterine protein ubiquitination. Insulin receptor substrate-2 protein was diminished in uteri of E2-treated insulin receptor substrate-1-null mutant mice, but not in E2-treated IGF-I-null mutant mice. Furthermore, E2-induced diminution of uterine insulin receptor substrate-2 protein was only partially inhibited in the presence of wortmannin, a PI3K inhibitor. Collectively, these data suggest that the E2-induced decrease in uterine insulin receptor substrate-2 requires IGF-I signaling, is not dependent solely on insulin receptor substrate-1 and PI3K, and is blocked by progesterone as well as by pharmacological inhibition of proteasomal protease activity. We speculate that the IGF-I-activated IGF-I receptor, in response to E2, directly or indirectly modifies insulin receptor substrate-2, probably through phosphorylation, leading to ubiquitination and subsequent degradation of this docking protein by the proteasome. This degradation could be a regulatory step to inhibit insulin receptor substrate-2-dependent signaling in the uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN RECEPTOR substrate-2 (IRS-2) belongs to a family of modular docking proteins that also includes IRS-1 (1), IRS-3 (2, 3, 4), and IRS-4 (5). These intracellular signaling proteins interact with the receptors for insulin, IGF-I, and certain cytokines (1). Also, IRS-1 has been shown to interact with {alpha}vß3 integrin (6) and 14-3-3 (7, 8). IRS-1 and IRS-2 are primary substrates and effectors of the insulin and IGF-I receptor (IGF-IR) tyrosine kinases (1). The carboxyl-terminal regions of IRS-1 and IRS-2 contain numerous tyrosine phosphorylation sites that bind distinct effector proteins, including enzymes (PI3K, SH2 domain-containing protein tyrosine phosphatase, and Fyn) and adaptor proteins (Grb2, Nck, and Crk) (1). In addition, the IRS proteins have proline-rich regions that can mediate binding with SH3 domain-containing proteins, such as the SH3-mediated interaction of Nck-1 and Nck-2 with IRS-1 (9). Given the diverse signaling capacity of an IRS protein, it is not surprising that members of this docking protein family have been shown to play a role in various physiological processes, including carbohydrate metabolism and pancreatic ß-cell function (10), somatic cell growth (11), and female reproduction (12).

Although the IRS proteins are important signaling intermediates in a wide array of biological responses, the regulation of these intracellular proteins has only recently begun to be addressed. Ovarian steroid hormones differentially affect IRS-1 and IRS-2 in various cells and tissues. For instance, E2, in an IGF-I-dependent manner, stimulates uterine IRS-1 tyrosine phosphorylation and the formation of an IGF-IR/IRS-1/PI3K complex, yet has no apparent effect on IRS-1 steady state levels in this organ (13, 14). In contrast, uterine IRS-2 protein levels markedly and transiently decrease after E2 exposure (14). By comparison, E2 increases IRS-1 and IRS-2 mRNA and protein levels in MCF-7 cells (15), whereas progesterone (P) stimulates IRS-2, but not IRS-1, production by HeLa cells (16). Collectively, these data demonstrate that E2 and P can alter the relative abundance of the IRS proteins and support a mechanism through which these ovarian steroid hormones can influence the biological effects of various growth factors and cytokines.

In the present study we use various in vivo experimental approaches, including pharmacological inhibitors and mutant mice, to investigate the hormonal regulation of uterine IRS-2 expression. Overall, our data show that the E2-induced decrease in uterine IRS-2 is inhibited by P and does not occur in the absence of IGF-I or in the presence of proteasomal protease inhibitors. We propose that IGF-I, in response to E2, stimulates uterine IRS-2 phosphorylation, creating a specific signal for ubiquitination and subsequent degradation of this docking protein. The IGF-I-dependent degradation of uterine IRS-2 may serve to negatively regulate signaling that is dependent on this docking protein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, hormone treatments, and preparation of uterine tissue extracts
Ovary-intact or ovariectomized (at 8 or 10 wk of age) CD-1 mice were provided by Charles River Laboratories, Inc. (Raleigh, NC). IGF-I wild-type (IGF-I+/+) and IGF-I-null mutant (IGF-I-/-) mice were generated in our laboratory from heterozygous IGF-I breeding pairs (MF1 x 129/Sv hybrids) provided by Dr. Argiris Efstradiatis (Columbia University, New York, NY). Tail genomic DNA was screened by PCR to determine the genotype. The postnatal survival rate and phenotype of IGF-I-/- females were similar to those previously reported (17). IRS-1 wild-type (IRS-1+/+) and IRS-1 null mutant (IRS-1-/-) mice were obtained from Taconic Farms, Inc. (Germantown, NY), with the permission of Dr. Ronald Kahn (Joslin Diabetes Center, Boston, MA). All of the wild-type, IGF-I-/-, and IRS-1-/- mice were ovariectomized at 8–13 wk of age.

At 11 and 12 wk of age, uteri and vaginal tissue were collected from CD-1 mice at various stages of the estrous cycle. For each mouse, a 5-mm long section from the middle region of one uterine horn and a piece of vaginal tissue were placed in 10% neutral buffered formalin. After fixation, the tissues were embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin for histology. The stage of estrous cycle was based on uterine and vaginal histology. The remaining uterine tissue was placed in 1 ml ice-cold solubilization buffer A [1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 50 mM Na2MoO4, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 4 µg/ml 4-amido-phenylmethylsulfonylfluoride (4-amido-PMSF) in 50 mM Tris-HCl, pH 7.4] and disrupted with a 30-sec burst of a tissue homogenizer at the highest setting. Each homogenate was centrifuged at 14,000 x g for 5 min at 4 C, and supernatants were stored at -80 C.

To elevate uterine stromal PR levels (18), one group of castrated CD-1 mice was treated once daily for 2 consecutive d with sc injections of 100 ng E2 (Sigma, St. Louis, MO). At 48 h after the second injection, the mice were treated once with vehicle (sesame oil), 1 µg E2, 1 mg P (Sigma), or E2 and P. A second group of castrated CD-1 mice was treated with a single sc injection of E2 (1 µg) or vehicle (PBS/1% ethanol). At 6 h after treatment with E2 or vehicle, a subgroup of the mice received an ip injection of MG-132 (10 mg/kg; Calbiochem, San Diego, CA), epoxomicin (3 mg/kg; Affiniti Research Products Ltd., Mamhead, UK), or vehicle (50 µl dimethylsulfoxide) every 2 h for a total of 10 h. A third group of ovariectomized CD-1 animals received an sc injection of wortmannin (0.2 mg/kg; Alexis Biochemicals, San Diego, CA) or vehicle (50 µl sesame oil) at 1.5 and 0.5 h before treatment with 1 µg E2 or vehicle (PBS/1% ethanol). These mice were then treated with wortmannin or vehicle every hour for 6 h after E2/vehicle treatment and then every 3 h for the remainder of the treatment period. In addition, each IGF-I+/+, IGF-I-/-, IRS-1+/+, and IRS-1-/- mouse received a single sc injection of either E2 (20 µg/kg) or vehicle (PBS/1% ethanol). All mice were treated no earlier than 14 d after castration. Each uterus was collected at various times after the last treatment and homogenized in ice-cold solubilization buffer A as indicated above, and supernatants of homogenates were stored at -80 C. E2-induced effects on uterine IRS-2 protein levels were comparable between E2-primed and nonprimed mice as well as between mice treated with E2 at either 18 or 24 h before tissue collection. All surgical and treatment procedures were conducted in compliance with the guidelines of the NIEHS, NIH animal care and use committee.

Patient population and preparation of human endometrial extracts
The inclusion and exclusion criteria used to establish the patient population in this study have been described previously (19). In accordance with the guidelines of the internal review board committee at Duke University Medical Center, human uterine tissues were obtained at hysterectomy from women of reproductive age with spontaneous menstrual cycles occurring every 26–35 d. A full-thickness endometrial specimen was first obtained and fixed in formaldehyde for determination of the stage of the menstrual cycle by a board-certified pathologist. The remaining endometrium from the fundus was gently scraped using a scalpel and immediately placed into solubilization buffer B (1% Triton X-100, 2 mM EDTA, 2 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 50 mM Na2MoO4, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 4 µg/ml 4-amido-PMSF in 20 mM HEPES, pH 7.4) on ice. The tissue was then disrupted with a tissue homogenizer. After centrifugation of the homogenate at 21,000 x g for 1 min, the supernatant was stored at -80 C.

Immunoprecipitations and Western analyses
Aliquots of supernatants from human endometrial extracts (1.5 mg protein) or mouse uterine extracts (0.3–1.2 mg protein) were incubated with anti-IRS-1 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-IRS-2 (Upstate Biotechnology, Inc.), anti-IGF-IR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or anti-Grb10 (Santa Cruz Biotechnology, Inc.) antibodies for 2 h at 4 C. After incubation with the antibody, supernatants were incubated with protein A-Sepharose (CL-4B, Amersham Pharmacia Biotech, Piscataway, NJ) for an additional 2 h at 4 C. Antibody/protein A-Sepharose pellets were washed three times with solubilization buffer A without sodium deoxycholate, then boiled for 5 min in Laemmli sample buffer. After boiling, precipitates were stored at -20 C. In those experiments in which IRS-2 precipitates were analyzed for ubiquitin immunoreactivity, as detailed below, 5 mM N-ethylmaleimide was included in the solubilization and immunoprecipitation buffers. Protein concentrations were determined by the Pierce Chemical Co. (Rockford, IL) bicinchoninic acid protein assay.

After washing with solubilization buffer, a subset of the IRS-2 precipitates was washed an additional three times with 50 mM Tris-HCl, pH 8, containing 150 mM NaCl, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 4 µg/ml 4-amido-PMSF, then resuspended in 30 µl alkaline phosphatase buffer (50 µM CaCl2, 0.1 mM MgCl2, 50 mM NaCl, 20 µg/ml aprotinin, 20 µg/ml leupeptin, and 4 µg/ml 4-amido-PMSF in 25 mM Tris-HCl, pH 7.9). Precipitates were incubated at 37 C for 1 h with or without 5 U calf intestinal alkaline phosphatase (New England Biolabs, Inc., Beverly, MA). Immediately after incubation, IRS-2 precipitates were boiled for 5 min in Laemmli sample buffer and placed at -20 C.

Immunoprecipitates or 20-µl aliquots of mouse uterine extracts were boiled for an additional 2 min, subjected to SDS-7.5% PAGE, and then transferred to polyvinylidene fluoride (Immobilon-P) membrane (Millipore Corp., Bedford, MA). The membrane was blocked with either 5% bovine albumin/Tris-buffered saline and 0.1% Tween 20 or 3% nonfat dry milk/PBS and probed with horseradish peroxidase-conjugated antiphosphotyrosine monoclonal antibody (PY20, ICN Biomedicals, Inc., Costa Mesa, CA) or with rabbit polyclonal antibodies to IRS-1, IRS-2, IGF-IR, ubiquitin (Sigma), ubiquitin-protein conjugates (Affiniti Research Products Ltd.), Akt (New England Biolabs, Inc.), phospho-Akt (Ser473, New England Biolabs, Inc.), or Grb10. Blots probed initially with a rabbit polyclonal antibody were washed and incubated with a horseradish peroxidase-conjugated donkey antirabbit IgG secondary antibody (Amersham Pharmacia Biotech). Immunoreactive proteins were detected using enhanced chemiluminescence (Amersham Pharmacia Biotech). Where indicated, the ChemiImager 4000 Low Light Imaging System (Alpha Innotech Corp., San Leandro, CA) was used for quantitation of band density. Also, where indicated, relative density values were statistically compared using t test and were considered significantly different at P < 0.05.

RNA isolation and ribonuclease (RNase) protection assays
In a subset of the castrated CD-1 mice that were treated with E2 only, uteri were removed at 0 (untreated mice), 6, 12, 18, and 24 h after treatment. Tissues were then homogenized in TRIzol (Life Technologies, Inc., Grand Island, NY), and total RNA was isolated according to the manufacturer’s instructions. RNA was assessed for integrity by agarose gel electrophoresis and quantified by measuring the absorbance at 260 nm. Five micrograms of RNA from each uterus were hybridized to 32P-labeled RNA probes complementary to mouse IRS-2 mRNA and mouse ß-actin mRNA, and RNase protection assays were performed using the RPA III kit (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. Briefly, total RNA (5 µg) was coprecipitated with 1 x 105 cpm of each high specific activity probe, resuspended in hybridization buffer, and incubated at 42 C overnight. Samples were then digested with RNases A and T1 for 30 min at 37 C. After digestion, samples were precipitated and resuspended in gel loading buffer, then subjected to electrophoresis on 6% polyacrylamide/7 M urea gels. After electrophoresis the gels were vacuum dried, and film was exposed to the gels at -80 C. In addition, dried gels were exposed to a PhosphorImaging screen (Molecular Dynamics, Inc., Sunnyvale, CA) for quantitation of radioactivity in protected bands. The IRS-2 probe corresponded to nucleotides 3044–3438 of the reported IRS-2 cDNA sequence (20), which is complementary to the region of the IRS-2 mRNA that encodes a portion of the carboxyl-terminus of the IRS-2 protein. The primer sequences used to create the probe were 5'-ACTTCCCCTTCCTCCTTAC-3' (forward) and 5'-GTGGTGGTAGAGGAAAAGG-3' (reverse).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in uterine IRS-2 protein levels during the human and mouse ovarian cycles correspond to inhibition of the E2-induced decrease in uterine IRS-2 by P
Based on previously reported findings that uterine IRS-1 and IRS-2 protein levels are differentially affected after E2 treatment of the castrated mouse (14), this study examined the relationship between menstrual/estrous cycle-associated fluctuations in ovarian production of steroid hormones and changes in the levels of these docking proteins in human and mouse uteri. At the preovulatory stage, when serum E2 levels peak and circulating P reaches a nadir (21, 22), IRS-2 was barely detectable in human or mouse uterine extracts (Fig. 1AGo). In contrast, abundant IRS-2 was observed at the postovulatory stage (Fig. 1AGo) when P levels in human uterine tissue (21) and mouse serum (22) were increasing. Despite elevated levels of this docking protein, tyrosine-phosphorylated IRS-2 was detected in only one of four human uterine samples examined from the postovulatory stage, and analysis of four corresponding uteri from the mouse did not reveal tyrosine-phosphorylated IRS-2 (data not shown).



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  		Figure 1. Variations in uterine IRS-2 levels during the human and mouse ovarian cycles coincide with P inhibition of the E2-induced decrease in uterine IRS-2. A and C, Human and mouse uterine extracts were obtained from the preovulatory (Pre) and postovulatory (Post) stages of the ovarian cycle. B, Uterine extracts were obtained from ovariectomized, E2 (E)-primed CD-1 mice at 24 h after treatment with vehicle (V), 1 µg E2, 1 mg P, or E2 plus P. Immunoprecipitates (IP) of IRS-2 and IRS-1 were separated by SDS-7.5% PAGE and immunoblotted with antibody to phosphotyrosine (PY), IRS-2, or IRS-1. Molecular mass markers are expressed in kilodaltons. Each lane in A and B corresponds to a different human subject or animal, and results are representative of four separate observations per ovulatory stage or treatment group. Each lane in C relates to an equal aliquot of the samples shown in A.

 
Based on the above results that showed that IRS-2 levels are most abundant during the postovulatory stage when P levels are elevated, the effect of this hormone on uterine IRS-2 protein levels in the absence or presence of E2 was investigated in the ovariectomized mouse. In agreement with previously reported data (14), uterine IRS-2 protein levels were significantly reduced (P < 0.05) at 24 h after E2 treatment compared with IRS-2 levels in uteri from vehicle-treated animals (Fig. 1BGo). However, when mice were treated with both P and E2, IRS-2 levels were similar (P > 0.05) to those observed for controls (Fig. 1BGo). Also, uterine IRS-2 protein levels in mice treated with P alone were comparable (P > 0.05) to IRS-2 levels in uteri from vehicle-treated animals (Fig. 1BGo). In contrast to that observed in the uterus, E2 exposure did not alter IRS-2 levels in the liver (data not shown), demonstrating tissue-specific modulation of IRS-2 protein levels by this hormone.

Uterine IRS-1 levels were relatively constant between the preovulatory and postovulatory stages of the human and mouse ovarian cycles (Fig. 1CGo). However, uterine IRS-1 was strongly tyrosine phosphorylated at the preovulatory stage, but contained a weak phosphotyrosine signal at the postovulatory stage (Fig. 1CGo). In addition, IGF-IR-bound, tyrosine-phosphorylated IRS-1 was detected in mouse uterine extracts primarily during the proliferative stage of the estrous cycle (data not shown). These data support the idea that the IGF-IR/IRS-1 signaling complex, probably in conjunction with PI3K (14), is an important mediator of E2 action in the uterus.

Estradiol effects on uterine IRS-2 mRNA levels
To determine whether the decrease in uterine IRS-2 protein in response to E2 corresponded to lower IRS-2 mRNA levels, RNase protection analyses were performed to measure uterine IRS-2 transcript levels at various times after exposure to E2. Hybridization of the IRS-2 RNA probe to uterine mRNA generated a distinct protected band at each time point examined, and hybridization of the ß-actin RNA probe to uterine mRNA was used as an internal control (Fig. 2AGo). At 6, 12, and 18 h after E2 treatment of mice, uterine IRS-2 mRNA levels were reduced 40–50% compared with those in control mice (0 h); in addition, IRS-2 mRNA levels were decreased slightly (15%) 24 h after estrogen exposure (Fig. 2B).



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  		Figure 2. Uterine IRS-2 mRNA levels are decreased after E2 treatment. Castrated CD-1 mice were treated with 1 µg E2 (E), and uteri were collected at various times after treatment as indicated above the lanes. Total RNA was isolated as described in Materials and Methods. A, RNase digestion of total RNA (5 µg each) after hybridization of RNA with the IRS-2 and ß-actin probes generated the protected bands at approximately 400 and 240 nucleotides (nt), respectively. The sizes of nucleotide standards (std) are indicated on the left. B, At each time point examined after treatment with E2, the uterine IRS-2 to ß-actin mRNA ratio is represented as a percentage of this ratio at 0 h (control, 100%). Similar results were obtained in two independent experiments.

 
E2-stimulated ubiquitination of uterine proteins coincides with proteasome-dependent degradation of IRS-2
Previous in vitro studies have shown that the insulin- induced (23, 24, 25, 26) and IGF-I-induced (27, 28) decreases in the cellular content of IRS-1 were dependent on proteasomal activity, and IRS-1 degradation was associated with increased ubiquitination of this docking protein (27, 28). In the present study we examined whether E2 stimulates ubiquitination of uterine proteins in the presence of the proteasome inhibitor MG-132 or in the presence of epoxomicin, a highly specific inhibitor of proteasomal protease activity (29). In addition, we determined whether these inhibitors prevent the decrease in uterine IRS-2 observed after treatment with E2. Immunoblots of whole uterine extracts from mice after treatment with vehicle, E2, or MG-132 showed weak ubiquitin immunoreactivity; however, an intense ubiquitin signal was observed in the 120–220 kDa range in animals treated with either MG-132 and E2 or epoxomicin and E2 (Fig. 3AGo, upper panel). No signal was detected in any of the treatment groups when nonspecific rabbit IgG was used as the primary antibody (data not shown). At 18 h after E2 exposure, uterine IRS-2 protein levels were significantly less (P < 0.05) than those observed for vehicle-treated mice (Fig. 3AGo, lower panel). In contrast, IRS-2 levels in castrated mice treated with both MG-132 and E2 were not changed (P > 0.05) at 18 h after hormone exposure (Fig. 3AGo, lower panel). Furthermore, treatment with epoxomicin inhibited the E2-induced diminution of this docking protein (Fig. 3AGo, lower panel). Uterine IRS-2 levels in mice treated with MG-132 alone were similar (P > 0.05) to IRS-2 levels in control mice (Fig. 3AGo, lower panel). Specific ubiquitin immunoreactivity, as assessed with two separate ubiquitin antibodies, was not detected in immunoblots of IRS-2 precipitates from uterine extracts obtained from mice treated with E2 and MG-132 (data not shown), even when extraction and immunoprecipitation of this docking protein were carried out in buffers containing N-ethylmaleimide. This compound inhibits isopeptidase activities that otherwise may be detrimental to the detection of ubiquitinated proteins (30). As these findings do not show direct ubiquitination of uterine IRS-2, the inhibitory effects of the proteasomal inhibitors on the E2-induced IRS-2 decrease may be through an indirect mechanism. On the other hand, it is possible that the IRS-2 antibody used in the present study has no or a very low binding affinity for the IRS-2/ubiquitin moiety and is not very effective for immunoprecipitation of this complex.



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  		Figure 3. Ubiquitination of uterine proteins is stimulated by E2 and is associated with proteasome-dependent degradation of IRS-2. A, At 6 h after treatment with 1 µg E2 (E) or vehicle (V), ovariectomized mice were treated with MG-132 (10 mg/kg), epoxomicin (EPOX; 3 mg/kg), or dimethylsulfoxide every 2 h for a total of 10 h. Mice were killed at 2 h after the last treatment (18 h after E2 or vehicle exposure). Equal aliquots of each uterine extract were separated by SDS-7.5% PAGE and immunoblotted with antibody to ubiquitin (Ub; upper panel) or IRS-2 (lower panel). B, Castrated mice were treated with either V or E, and uterine extracts were prepared 4 h after treatment. IRS-2 immunoprecipitates from each uterine extract were first incubated at 37 C for 1 h either in the absence (-) or presence (+) of alkaline phosphatase (Ptase), followed by separation by SDS-7.5% PAGE, and immunoblotted with antibody to IRS-2. As previously reported (14 ), uterine IRS-2 at 4 h after E2 exposure (B, lanes 3 and 4) displayed decreased electrophoretic mobility. By 18 h after E2 treatment (A, lower panel, lanes 3 and 4), the migration of IRS-2 was similar to that observed in control extracts. Differences in IRS-2 electrophoretic mobility at various times after E2 exposure are probably due to changes in IRS-2 phosphorylation status. Molecular mass markers are expressed in kilodaltons. Each lane in A and B corresponds to a different animal, and the results, except for the one animal treated with epoxomicin, are representative of at least four separate observations per treatment group.

 
Various studies have shown that IRS-1 and IRS-2 decrease in mobility when analyzed by SDS-PAGE after treatment of cells or whole animals with E2 (14), IGF-I (27), insulin (31), TNF{alpha} (32), or okadaic acid (33). This change in gel mobility has been attributed to increased phosphorylation of the docking protein. In the present study incubation of IRS-2 precipitates from uterine extracts of E2-treated (4 h) mice with calf intestinal phosphatase caused IRS-2 to migrate similarly to that of IRS-2 from vehicle-treated mice (Fig. 3BGo). Uterine IRS-2 not exposed to calf intestinal phosphatase after precipitation from uterine extracts obtained from E2-treated mice had decreased gel mobility compared with that of control mice (Fig. 3BGo). These findings demonstrate that the decrease in gel mobility of uterine IRS-2 after E2 exposure is due to phosphorylation of this docking protein.

Differential effects of E2 on uterine IRS-2 levels in wild-type, IGF-I-/-, IRS-1-/-, and wortmannin- treated mice
We have shown previously that activation of uterine IRS-1 signaling in response to E2 is due primarily to the IGF-I-stimulated uterine IGF-IR (14). To determine the importance of IGF-I and IRS-1 to the E2-induced decrease in IRS-2, we examined uterine IRS-2 in both IGF-I and IRS-1 null mutant mice after treatment with vehicle or E2. Uterine IRS-2 levels in IGF-I+/+ and IRS-1+/+ mice after E2 treatment were 70–90% less than those in vehicle-treated wild-type mice (Fig. 4Go, A and B). By comparison, there was no apparent reduction in uterine IRS-2 levels in E2-treated IGF-I-/- mice compared with levels of this docking protein in uteri of vehicle-treated IGF-I-/- mice (Fig. 4AGo). In contrast, IRS-2 levels were diminished in IRS-1-/- mice at 24 h after treatment with E2, but the percent reduction (58–62%) was less than that observed in IRS-1+/+ mice after E2 treatment (Fig. 4BGo).



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  		Figure 4. The extent of E2-induced degradation of uterine IRS-2 varies among wild-type, IGF-I-/-, and IRS-1-/- mice. Uterine extracts were obtained from castrated, wild-type (+/+) and null mutant (-/-) IGF-I (A) or IRS-1 (B) mice at 18 h (A) or 24 h (B) after treatment with vehicle (V) or 20 µg/kg E2 (E). IRS-2 immunoprecipitates were separated by SDS-7.5% PAGE and immunoblotted with antibody to IRS-2. Molecular mass markers are expressed in kilodaltons. Each lane corresponds to an individual animal, and the results in A represent two separate observations. Data from these experiments were not analyzed statistically due to the small number of observations (n = 2) for each treatment group.

 
Previous studies have shown that Grb10 interacts with the IGF-IR (34, 35) and insulin receptor (35, 36). In addition, this adaptor protein binds the ubiquitin-protein ligase Nedd4 (37), making Grb10 an attractive candidate as an IRS-1- independent signaling intermediate between the IGF-I-activated IGF-IR and subsequent association of IRS-2 with an ubiquitin protein ligase. However, in the present study data from coimmunoprecipitation experiments demonstrated no detectable specific interactions between Grb10 and either the uterine IGF-IR or IRS-2 in the absence or presence of E2 (data not shown), suggesting that this adaptor protein is not involved with the E2/IGF-I-induced degradation of uterine IRS-2.

As the increase in PI3K association with the uterine IGF-IR after E2 exposure is dependent on IRS-1 (14), the effect of the PI3K inhibitor, wortmannin, on the E2-induced degradation of uterine IRS-2 was investigated. Protein kinase B, or Akt, is a downstream target of PI3K (38); therefore, Akt phosphorylation was used as an indirect measure of the in vivo activity of PI3K. Phosphorylated (Ser473) Akt was significantly increased (P < 0.05) in uteri of E2-treated mice compared with that in control animals (Fig. 5Go, upper panel). Although Akt phosphorylation was not completely inhibited in mice treated with E2 in the presence of wortmannin, the reduction (63%) in phosphorylated Akt in animals treated with both E2 and wortmannin was significant (P < 0.05) compared with that observed in mice treated with E2 alone (Fig. 5Go, upper panel). Despite the observed inhibition of PI3K, as measured by Akt phosphorylation, uterine IRS-2 levels were significantly reduced (P < 0.05) in mice treated with both wortmannin and E2 compared with those in vehicle-treated mice (Fig. 5Go, lower panel). However, the E2-induced reduction (56%) of IRS-2 in wortmannin-treated mice was less (P < 0.05) than that in mice treated with E2 only (80% reduction; Fig. 5Go, lower panel). Uterine IRS-2 levels were not changed (P > 0.05) in animals treated with wortmannin only. Finally, wortmannin treatment did not inhibit the E2-induced increase in IGF-IR-bound, tyrosine-phosphorylated IRS-1 (data not shown), indicating that the PI3K inhibitor did not interfere with E2-stimulated, IGF-I-dependent activation of the IGF-IR.



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  		Figure 5. The PI3K inhibitor, wortmannin, inhibits uterine Akt phosphorylation after E2 treatment, but only partially inhibits IRS-2 degradation. Ovariectomized CD-1 mice were treated with sesame oil or 0.2 mg/kg wortmannin (W) before and after treatment with vehicle (V) or 1 µg E2 (E). Aliquots of uterine extracts obtained at 18 h after V or E2 treatment were separated by SDS-7.5% PAGE and immunoblotted with antibody to phospho (P)-Akt (upper panel), Akt (middle panel), or IRS-2 (lower panel). Molecular mass markers are expressed in kilodaltons. Each lane corresponds to an individual mouse, and the data are representative of four separate observations.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that E2 treatment of the ovariectomized mouse causes a transient, but marked, decrease in uterine IRS-2 (14). Levels of this docking protein were not diminished in lysates from cultures of Ishikawa endometrial cancer cells after treatment with E2, IGF-I, or both (Richards, R. G., and R. P. DiAugustine, unpublished data). Therefore, this study used a whole animal model to investigate E2 and P regulation of uterine IRS-2. We extend our previous finding (14) by demonstrating that the E2-induced reduction in uterine IRS-2 is regulated potentially at both the mRNA and posttranslational levels, with the latter involving proteasomal degradation. The specific effects of E2 on IRS-2 mRNA transcription, stability, and translation were not examined in the present study; thus, regulation of uterine IRS-2 transcript levels by E2 at any of these steps cannot be excluded.

We show that the E2-stimulated decrease in uterine IRS-2 does not occur in animals treated with both E2 and P, suggesting that P prevents the E2-induced decrease in IRS-2 expression, stimulates IRS-2 synthesis, or possibly both. The idea that P stimulates the synthesis of uterine IRS-2 is supported by the observation that this hormone increases IRS-2 mRNA and protein levels in HeLa cells (16). However, we observed no increase in uterine IRS-2 protein at 24 h after treatment with P, suggesting that this hormone preserves uterine IRS-2 expression through a mechanism(s) that inhibits the E2-induced decrease in IRS-2. Investigators have shown that P stimulates the uterine expression of mRNA and/or protein for the epidermal growth factor receptor (EGFR) ligands, amphiregulin (39) and heparin binding EGF-like growth factor (HB-EGF) (40), suggesting that the uterine EGFR is a potential mediator of P action in this organ. A recent study demonstrated that EGF inhibited the IGF-I-induced degradation of IRS-1 in prostate epithelial cells and blunted the IGF-I-dependent ubiquitination of IRS-1 (28). In future studies it will be interesting to examine whether P inhibition of E2-induced uterine IRS-2 degradation is mediated by an EGFR-dependent signal.

Modulation of uterine IRS-2 by the ovarian steroid hormones in the castrated mouse corresponded to low IRS-2 expression in uterine extracts from the preovulatory stage of both the human and mouse ovarian cycles and abundant IRS-2 at the postovulatory stage. By comparison, there was no appreciable, cycle-dependent change in the amount of uterine IRS-1 protein; however, tyrosine-phosphorylated IRS-1 was most evident at the preovulatory stage. These data corroborate our previous findings that uterine IRS-1 protein levels are not altered in the E2-treated castrated mouse and that E2, but not P, can enhance tyrosine phosphorylation of uterine IRS-1 (13, 14). Collectively, the present findings demonstrate that hormone-induced changes in uterine IRS-1 and IRS-2 in the castrated mouse reflect, both quantitatively and qualitatively, cycle-dependent regulation of these proteins in the human and mouse uterus and underscore the physiological relevance of the in vivo hormone ablation/replacement model used in this study. An emerging theme from several recent studies is that IRS-1 and IRS-2 have specialized, rather than redundant, roles in IGF-I/insulin signaling in various tissues and cells (10, 11, 41). In the present study the data further support the idea that uterine IRS-1 and IRS-2 have different functional roles in hormone-dependent signaling in this organ.

The decrease in uterine IRS-2 after E2 treatment appears to result predominantly from a posttranslational event that requires the protease activity of the 26S proteasome, as specific inhibition of this activity blocks the estrogen-induced decrease in this docking protein. Proteasome-mediated degradation of many proteins requires conjugation to the polypeptide ubiquitin, which leads to rapid destruction of the protein by the proteasome (42). Specific degradation signals, which can be sequence or structural features of the substrate, seem to be important for the degradation of proteins by the ubiquitin system (42). In the present study uterine IRS-2 appears to be selectively degraded in response to E2. Both IRS-1 and IRS-2 are present in the uterine epithelium (14), and both exhibit enhanced tyrosine phosphorylation and a gel mobility shift after E2 exposure (Ref. 14 and the present study), yet only IRS-2 decreases in response to the hormone. The shift in IRS-2 mobility after E2 treatment is due primarily to increased phosphorylation of the docking protein. Proteolysis of various proteins, such as I{kappa}B{alpha} (43), ß-catenin (44), and the platelet-derived growth factor receptor (45) is phosphorylation dependent, such that the ubiquitin enzyme complex only recognizes the phosphorylated substrate. The component of this ubiquitin complex that is responsible for substrate specificity is the ubiquitin-protein ligase (E3), which contains a specific protein-protein interaction domain such as the SCFß-TrCP F box (42), the Nedd4 WW domain (46), and c-Cbl SH2 domain (45). Analogous to the phosphorylation-dependent E3 recognition motifs in the signaling proteins described above, E2-enhanced phosphorylation of uterine IRS-2 at a specific site may signal the selective degradation of this docking protein. IRS-2 contains numerous tyrosine and serine/threonine phosphorylation sites as well as proline-rich regions (1), of which one or more of these motifs may be important for the specific interaction of the docking protein with an E3-dependent ubiquitination pathway.

Our findings from experiments with IGF-I-null mutant mice suggest that IGF-I is required for the E2-induced degradation of uterine IRS-2, assuming that modulation of other factors that may occur as a result of the Igf1 gene disruption does not significantly influence the effects of E2 on this docking protein. E2 increases IGF-I mRNA levels in the rodent uterus (47, 48), suggesting that stimulation of local IGF-I synthesis is a key intermediate step in the E2-induced pathway leading to IRS-2 degradation. Whether IGF-I can down-regulate uterine IRS-2 expression without additional E2- dependent signaling has not been determined. However, the recent findings that IGF-I alone induced proteasome-dependent degradation of IRS-1 in MCF-7 (27) and prostate epithelial cells (28) suggest that the interaction of this growth factor with IGF-IR in the uterine epithelial cell (49) is sufficient to initiate the specific degradation of IRS-2.

Several in vitro studies with various cell types have shown that the IGF-I- or insulin-induced degradation of IRS-1 depends on PI3K (25, 26, 27, 28). We have shown previously that the increase in PI3K association with the uterine IGF-IR in response to E2 was contributed mainly by IRS-1 (14). Our present data show that a significant amount (~60%) of the uterine IRS-2 was degraded after E2 exposure in the absence of IRS-1 and in the presence of diminished PI3K activity. However, this E2-induced decrease in IRS-2 was slightly moderated (~20%) compared with that observed in E2-treated controls. These findings suggest that IRS-1/PI3K-dependent and -independent pathways emanating from the activated uterine IGF-IR play a concerted role in the E2/IGF-I-induced down-regulation of uterine IRS-2 expression. In addition to IRS-1 and IRS-2, various studies have shown that the IGF-IR interacts with SH2 domain-containing adaptor proteins, including Grb10 (34) and Shc (50). However, we found no Grb10 (Ref. 14 and present study) bound to uterine IGF-IR/IRS-2 either in the absence or presence of E2, and previously reported findings showed no increase in Shc tyrosine phosphorylation or Shc-bound Grb2 in uteri from E2-treated mice (14). These findings suggest that Grb10- and Shc-associated signaling pathways are not needed for E2-induced, IGF-I-dependent degradation of IRS-2 in the uterus. In addition to Grb10 and Shc, SH2-B/APS (51) and c-Crk (52) have been shown to interact with the IGF-IR, and these adaptor proteins also can associate with c-Cbl (53, 54), which was recently shown to have ubiquitin-protein ligase activity (45). The interaction of the adaptor protein with the E3 seems to be important for the ubiquitin enzyme complex to target certain substrates for ubiquitination (55). Based on these findings, it is possible that an E2-induced, IGF-I-dependent signal specifically promotes the interaction of uterine IRS-2 with an E3 ligase, and an ancillary protein(s), such as SH2-B/APS or c-Crk, could facilitate this interaction.

In summary, we show that the E2-induced decrease in uterine IRS-2 expression: 1) is regulated primarily at the posttranslational level, 2) is dependent on proteasomal protease activity and IGF-I signaling, and 3) is not solely dependent on an IRS-1/PI3K pathway. Furthermore, we establish that E2/IGF-I stimulates uterine IRS-2 phosphorylation that is associated with hormone-dependent degradation of this docking protein. We speculate that phosphorylation of uterine IRS-2 at a specific site(s) mediates association with a putative uterine IRS-2 E3 enzyme. Due in part to the relative paucity of known E3s (55), the actual ubiquitin-protein ligase responsible for ubiquitination of uterine IRS-2 remains to be identified. Other in vitro experimental approaches, such as coculture of uterine epithelial cells with stromal cells or with extracellular matrix, may help identify specific signaling events and molecular interactions critical for IRS-2 degradation, such as the relevance of adaptor proteins, such as Crk and SH2-B, to the IGF-I-dependent ubiquitination of IRS-2. In addition, although the nature of the signal(s) through which P inhibits the E2-induced degradation of uterine IRS-2 is not known, the ability of this hormone to preserve uterine epithelial IRS-2 expression may be an important mechanism by which P inhibits estrogen action in the epithelium. Cellular proliferation is a major effect of E2 in the uterus, and a recent report showed that E2-stimulated uterine epithelial cell mitosis, but not DNA synthesis, was markedly reduced in the IGF-I-null mutant mouse (56). An important query is whether the E2-induced, IGF-I-dependent loss of uterine IRS-2 is an obligate, regulatory event for estrogen-stimulated, IGF-I-dependent mitosis in the epithelium of this organ. Identification of an IRS-2-dependent pathway(s) that plays a role in suppressing E2-stimulated uterine epithelial cell proliferation may provide a novel target for therapeutic intervention of proliferative diseases in this organ.


   Acknowledgments
 
We thank Dr. Andrew Wallace and Sylvia C. Hewitt for reviewing this manuscript. We also thank Toni Ward, James Clark, and Page Myers for excellent technical assistance.


   Footnotes
 
1 Present address: Division of Reproductive Endocrinology and Infertility, MCHG-OG, Madigan Army Medical Center, Tacoma, Washington 98431. Back

Abbreviations: EGFR, Epidermal growth factor receptor; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; P, progesterone; PMSF, phenylmethylsulfonylfluoride; RNase, ribonuclease.

Received December 18, 2000.

Accepted for publication May 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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