Autocrine Activation of the Local Insulin-Like Growth Factor I System Is Up-Regulated by Estrogen Receptor (ER)-Independent Estrogen Actions
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内分泌学杂志 2005年第2期
Vascular Biology Institute, Department of Medicine, University of Miami School of Medicine (M.K., M.P., I.H.S., A.R., A.F., S.J.E.), Miami, Florida 33136; and Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University (H.W.), Tel Aviv 69978, Israel
Address all correspondence and requests for reprints to: Dr. Sharon J. Elliot, Vascular Biology Institute, University of Miami School of Medicine, 1600 N.W. 10th Avenue, RMSB, Room 1043-R104, Miami, Florida 33136. E-mail: selliot@med.miami.edu.
Abstract
Autocrine activation of the IGF-I system in mesangial cells (MC) promotes glomerular scarring in a model of type 1 diabetes. Although estrogens protect against progressive nondiabetic glomerulosclerosis (GS), women with diabetes seem to loose the estrogen-mediated protection against cardiovascular disease. However, little is known about the local IGF-I system and its interactions with estrogens in the pathogenesis of type 2 diabetic GS. Therefore, we examined db/db B6 (db/db) mice, a model of type 2 diabetes and diabetic GS. The IGF-I system was activated in the glomeruli and MC of female diabetic db/db mice, but not in nondiabetic db/+ littermates. We found increased IGF-I receptor (IGFR) expression and activation, including activation of MAPK. Surprisingly, estrogens, via an estrogen receptor (ER)-independent mechanism(s), increased IGFR expression, IGFR and insulin receptor substrate phosphorylation, and extracellular signal-regulated kinase activation in db/db MC. In contrast, ER expression was decreased in MC and glomeruli of db/db mice. Treatment with a neutralizing antibody to IGF-I or the MAPK inhibitor PD98059 increased ER expression and transcriptional activity. This suggests that the local prosclerotic IGF-I system is activated in type 2 diabetes and diminishes ER-mediated protection against GS. Although estrogens may stimulate protective ER signaling, they also activate the IGF-I system via ER-independent mechanisms in db/db MC. The later estrogen effects appear to outweigh the antisclerotic effects of ER activation. This may in part account for loss of estrogen protection against the progression of diabetic GS in women with type 2 diabetes.
Introduction
DIABETES IS THE leading cause of end-stage renal disease (ESRD) in industrialized countries (1). Diabetic glomerulosclerosis (GS), the hallmark of advanced diabetic kidney disease, is a scarring process that includes extracellular matrix (ECM) accumulation in the mesangium (2, 3). Mesangial cells (MC), the vascular smooth muscle-like cells in the glomerulus, play a central role in the pathogenesis of diabetic GS, because they synthesize ECM as well as the ECM-degrading matrix metalloproteinases (3). An imbalance between MC ECM synthesis and degradation has been postulated to be responsible for the glomerular scarring process (4).
There is mounting evidence that autocrine and/or paracrine activation of the IGF-I signaling system in MC contributes to the development of diabetic GS (5, 6, 7). In a model of type 1 diabetes, autocrine activation of the IGF-I signaling cascade regulates ECM turnover in a manner that could lead to ECM accumulation in the glomerulus (6, 8, 9). Autocrine activation of the IGF-I system has also been proposed to initiate a prosclerotic response in type 2 diabetic nephropathy, but this has not been extensively studied (10).
The incidence of nondiabetic cardiovascular diseases, including chronic kidney disease, which progresses to ESRD, is lower in premenopausal women than in age-matched men (11, 12). In a recent meta-analysis, Neugarten and co-workers (13) found a gender-specific difference and a more favorable outcome in women with chronic kidney disease, IgA nephropathy, and membranous nephropathy. Based on epidemiological data, estrogens have been suggested to play a major role in protection against the development and progression of large and small vessel disease in women during their reproductive years (14). In line with these observations, we previously reported that the renal glomerulus is a target tissue for estrogens and that estrogens protect against the development and progression of GS in nondiabetic mice (15, 16). We also showed that the susceptibility to develop GS in response to injury is inversely correlated with the level of glomerular estrogen receptor (ER) expression (16).
However, women with diabetes seem to loose the potentially estrogen-mediated protection against developing cardiovascular disease (17). Although the incidence of coronary events in women is generally low before menopause, young women with diabetes between the ages of 20–34 yr have a 33-fold higher relative risk for acute myocardial infarction than nondiabetic women. In contrast, men, whose rate of coronary events is already higher in the nondiabetic state compared with age-matched women, have only a 9-fold increase in risk once diabetes develops (18). The time to progress to ESRD due to diabetic nephropathy tended to be shorter in African-American women than in men with advanced diabetic kidney disease in a Mississippi-based population (19). Recent data from the United States Renal Data System also suggest that estrogens do not protect women with diabetes from diabetic GS, because there is no difference in the incidence of ESRD due to diabetes among men and women between 20–64 yr of age (20). Interestingly, progression of diabetic nephropathy is accelerated during pregnancy in over 40% of women with preexisting diabetic kidney disease and moderate to severe renal insufficiency (21). This may suggest that elevated sex steroid levels play a deleterious role in the disease process. However, the molecular mechanisms responsible for the loss of estrogen protection in diabetes-induced cardiovascular disease, including diabetic GS, are not well understood.
Direct interactions and cross-talk between the estrogen and IGF-I signaling pathways have been described in various vascular and nonvascular tissues (22, 23, 24, 25, 26). The IGF-I system exerts tissue-specific stimulatory and inhibitory effects on the estrogen signaling pathways and vice versa (23, 24, 26). For example, estrogens down-regulate components of the IGF-I signaling cascade, whereas IGF-I activates the ER in aortic/vascular smooth muscle cells (27, 28). Because little is known about the interactions between the estrogen and IGF-I signaling pathways in the pathogenesis of type 2 diabetic GS, we examined db/db B6 mice, a model of type 2 diabetes mellitus, with diabetic nephropathy (29, 30).
In this study we examined whether there was autocrine activation of the IGF-I system and whether estrogens could modulate the activation of the local IGF-I signaling cascade in glomeruli and mesangial cells of female mice with diabetes. We also explored the effects of the autocrine-activated IGF-I system on the expression of mesangial ER, which mediate the protective effects of estrogens in the glomerulus and MC.
Materials and Methods
Reagents
The reagents used for real-time PCR were purchased from PerkinElmer Applied Biosystems (Foster City, CA). Culture media and supplements were obtained from Invitrogen Life Technologies (Grand Island, NY). Charcoal-stripped fetal bovine serum (FBS) was purchased from HyClone (Pittsburgh, PA). 17?-Estradiol (E2), 17-estradiol (17), anti -smooth muscle actin, ?-galactosidase substrate, HEPES, phenylmethylsulfonylfluoride, aprotinin, leupeptin, benzamidine, EDTA, sodium pyrophosphate, sodium fluoride, sodium orthovanadate, Triton X-100, Tween 20, BSA (fraction V), glycerol, and NaCl were purchased from Sigma-Aldrich Corp. (St. Louis, MO). ICI 182,780 (ICI) was obtained from Tocris (Ballwin, MO). PD98059 (PD) was purchased from Calbiochem (La Jolla, CA). Tissue culture plates were obtained from Nunclon (Nalge Nunc, Rochester, NY). Reagents for SDS-PAGE and immunoblotting were obtained from NOVEX (San Diego, CA). Nitrocellulose membranes were purchased from Amersham Biosciences (Piscataway, NJ). All antibodies [IGF-I receptor (IGFR), insulin receptor substrate 1 (IRS-1), phosphotyrosine (PY99), extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), ER, H-184, and ER? Y-19], and protein A/G-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Nitrocellulose membranes (Hybond ECL) and films (Hyperfilm ECL) for chemiluminescence detection were obtained from Amersham Biosciences. TransFast and lysis buffer were purchased from Promega Corp. (Madison, WI). The IGF-I neutralizing antibody was purchased from R&D Systems, Inc. (Minneapolis, MN). mouse/rat enzyme immunoassay was purchased from Diagnostics Systems Laboratories (Webster, TX).
Model
Female db/db (C57BL/6J-m Leprdb/2+) mice and their lean (Leprdb/+) nondiabetic littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). The diabetes mutation, Leprdb, is a point mutation in the leptin receptor gene, Lepr, that promotes abnormal splicing and thereby causes a shortening of the intracellular domain of the receptor (31). In the C57BL/6 inbred background, the phenotype of the homozygous Leprdb mutation is characterized by obesity, hyperglycemia, and sustained hyperinsulinemia, associated with marked hypertrophy and hyperplasia of ?-cells (32). These mice develop diabetic nephropathy with an increased albumin excretion rate by 12 wk of age and glomerular lesions by 16 wk of age (29, 33, 34) The lesions are similar to those observed in humans with diabetes.
Glomerular isolation
Glomeruli were microdissected from kidneys of 4- to 6-month-old female diabetic (db/db) and nondiabetic littermate mice (db/+). Diabetic mice were obese and had severe untreated hyperglycemia (>300 mg/dl) before death.
Real-time PCR
Amplification and measurement of target RNA were performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as previously described (16). TaqMan probes and primers for amplification of the specific transcripts were designed using Primer Express 1.5. Quantitative RT-PCR was performed in a one-step buffer system according to the manufacturer’s instructions. The sequences of the probes for ER and ER? have been previously described (16). The sequences of the IGFR were: forward primer, GAG GGT GGC CTT CTG GAC A; reverse primer, TAT ACT GCC AGC ACA TGC GC; and probe, VIC-CGG ACA ACT GCC CTG ATA TGC TGT TTG A. Each sample was normalized to the 18S content as previously described (16). Standard curves for each molecule were generated using serial dilutions (0.001–100 ng) of mRNA from mouse uterus. PCR assays were conducted in duplicate for each sample. Data are expressed as the percentage of db/+ mRNA isolated from 100 glomeruli and represent the mean ± SEM of four animals for each group.
Isolation, propagation, and characterization of mesangial cell lines
MC were isolated from db/db mice and control littermates, as previously described (6). Each line of cells was derived from an individual mouse. All experiments were performed on two individual cell lines of db/db and two individual cell lines of littermate controls between passages 6 and 18. Growth curves were performed to determine whether there was a difference in proliferation as described previously for diabetic MC. Briefly 5000 cells/well were plated in 24-well plates (Nalge Nunc) in DMEM/Ham’s F-12 medium with 20% FBS as previously described (6). Duplicate wells were trypsinized and counted every 24 h for 7 d.
Cell culture and experimental design
Four days before the collection of protein, cells were plated in T75 cm2 flasks in phenol red-free DMEM/Ham’s F-12 medium (B medium) containing 20% dextran-charcoal stripped FBS as previously described (15). Twenty-four hours before collection, the medium was replaced with B medium containing 0.1% BSA and vehicle control (0.001% ethanol), increasing concentrations of E2 (0.1–10 nM), ICI (1 μM), ICI followed 1 h later by E2 (1 nM), or 17 (1 nM) for 24 h. In time-course experiments, E2 was added for 10 min, 6 h, and 24 h, and protein was collected. In some experiments cells were exposed to 40 μM PD or 12.5 μg IGF-I neutralizing antibody for 24 h.
Immunoprecipitation and Western blotting
Cell lysates were processed, and samples were resolved by electrophoresis on 6% (IGFR and IRS-1) or 10% (ER, ER?, ERK, and Perk1/2) polyacrylamide gels as previously described (9). Electrotransfer of proteins from the gel to the nitrocellulose was performed by electroelution and immunoblotting as previously described. Immunoreactive bands were determined by exposing the nitrocellulose blots to a chemiluminescence solution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 min, followed by exposure to X-OMAT AR film (Eastman Kodak Co., Rochester, NY). The film was scanned and saved on computer disks for densitometric analysis using Image J software from NIH.
For immunoprecipitation (IP) experiments, 80–100 μg protein extract were incubated with an antibody against ER?, and 250 μg protein extract were incubated with antibodies against IGFR or IRS-1 for 1 h at 4 C, followed by the addition of protein A/G-agarose overnight. The resulting protein-antibody conjugate was centrifuged at 4 C and washed four times with PBS, pH 7.4. The final pellet was resuspended in PBS, sample buffer was added, and the mixture was boiled for 3 min before analysis as described above. Blots were exposed to IGFR, IRS-1, or PY99 antibodies. To control for nonspecific antibody binding, 250 μg BSA were IP.
Transfection studies
For transient transfection experiments, glomerular MC were transfected with a luciferase-based reporter construct containing four consecutive synthetic estrogen response elements (4x ERE; provided by David Shapiro). In addition, cells were transfected with luciferase reporter vectors under control of the full-length [p(–2350/+640)Luc] or proximal [p(–476/+640)Luc] IGFR promoter (35). The former construct was generated by subcloning 2350 bp of 5'-flanking sequences and 640 bp of the 5'-untranslated region of the rat IGFR gene into the p0LUC luciferase reporter vector (nucleotide +1 corresponds to the transcription start site). The later construct includes 476 bp of 5'-flanking sequences and 640 bp of 5'-untranslated sequences. The basal activities of both constructs were previously reported (35). Cells were transfected using Trans-Fast as previously described (16). The corresponding empty vectors were used as controls. Transfection efficacy was adjusted by cotransfection with phosphorylated Rous sarcoma virus-?-galactosidase (0.3–0.5 μg/well). After transfection, cells were incubated for 24 h in the presence of vehicle, 1 nM E2, ICI, ICI followed by E2, or 17. For luciferase and galactosidase assays, cells were lysed in 100 μl reporter lysis buffer at room temperature. Light emission was detected using a luminometer (AutoLumatPlus, PerkinElmer) after addition of luciferin to 40 μl cell lysate. Values were expressed as arbitrary light units normalized to the ?-galactosidase activity of each sample.
Measurement of baseline IGF-I, IGF-II peptide, and IGF-binding proteins (IGFBPs)
Supernatants from db/db and db/+ cells were collected and processed as previously described (6). After Sep-Pak (Waters, Milford, MA) extraction, IGF-I peptide was assayed using the mouse/rat enzyme immunoassay (Diagnostics Systems Laboratories).
IGF-II was measured using a mouse-specific, two-site ELISA (Drs. D. Hwang and P. Lee, David Geffen School of Medicine, University of California, Los Angeles, CA). The detection limit for this assay is 0.3 ng/ml, with inter- and intraassay coefficients of variation of less than 10%. Medium samples (9 ml) were extracted using Sep-Pak C18 cartridges, lyophilized, then reconstituted to 0.2 ml for assay. The recovery of added mIGF-II from reconstituted medium samples was >90%.
IGFBPs were assessed by ligand blot as described by Horney et al. (36). Briefly, supernatants from db/db and db/+ cells were subjected to trichloroacetic acid precipitation, loaded normalized to protein amount, and electrophoresed on 12% sodium dodecyl sulfate nonreducing polyacrylamide gels. After transfer of protein and washes, the blots were exposed to biotinylated IGF-I (a gift from S. Rosenzweig) and developed with chemiluminescence as described for Western blots.
Statistical analysis
Transfection studies and Western analysis experiments for IGFR, ERs, ERK, and pERK were performed three to five times on two individual cell lines of db/db and db/+ mice. All real-time PCR experiments were performed either on glomerular RNA extracted from four individual mice per group or on two independent MC lines. Comparison between groups was performed using one-way ANOVA and Tukey’s multiple comparison test (for PCR experiments and phosphorylation experiments) or t test (for all other analyses).
Results
IGFR, ER, and ER? mRNA expression in microdissected glomeruli
IGFR mRNA expression was approximately 2-fold higher in the glomeruli isolated from db/db mice than in those of littermate controls, as assessed by real-time RT-PCR (P < 0.05; Table 1). In contrast, ER expression in db/db glomeruli was only about 10% of the ER mRNA levels found in db/+ glomeruli (P < 0.05; Table 1). ER? mRNA expression was not significantly different between the two.
TABLE 1. Expression of IGFR and ER mRNA in 100 glomeruli microdissected from db/db and db/+ mice
MC characterization
To examine and compare the potential interactions between the estrogen and IGF-I signaling pathways in the glomeruli of diabetic and nondiabetic mice, MC were isolated from microdissected glomeruli from db/db (n = 2) or db/+ (n = 2) mice. MC were identified by the presence and the typical filamentous distribution pattern of -smooth muscle actin. Cells were used between passages 6 and 18. Growth curves were performed to determine the proliferation rate. MC lines isolated from diabetic db/db mice had an increased proliferation rate compared with control cell lines (data not shown). This was similar to our previous findings for MC isolated from nonobese diabetic mice, a model of type I diabetes (6).
Baseline IGFR mRNA and protein expression and ERK activation in db/db and db/+ MC
Baseline levels of IGFR mRNA expression were determined in vitro in MC isolated from female diabetic db/db mice and their db/+ littermate controls. The levels of IGFR mRNA were about 2-fold higher in db/db than in db/+ MC as assessed by real-time RT-PCR (Fig. 1A). As shown in the representative Western blot (Fig. 1B), this correlated with the 2.8-fold higher level of IGFR protein expression in db/db MC (P < 0.05). A parallel increase in ERK activation was also present in db/db MC (Fig. 1C). Treatment with a neutralizing antibody against IGF decreased ERK1 and ERK2 activation by about 50% and 40%, respectively (Fig. 1D). Baseline densitometry units of total ERK1 (1.023 ± 0.13 vs. 1.08 ± 0.23; n = 5) and ERK2 (0.875 ± 0.22 vs. 0.65 ± 0.20; n = 5) were not different between db/db and db/+ MC.
FIG. 1. Baseline IGFR mRNA (A) and protein (B) expression and ERK activity (C) are increased in db/db MC. A, IGFR mRNA expression was determined by real-time RT-PCR. The ratios of IGFR/18S were calculated from the data of three individual experiments and were expressed as a percentage of db/+. B, IGFR protein expression. Five micrograms of protein were separated by 6% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with an antibody to the IGFR. As shown in the representative Western blot, a band with an approximate molecular mass of 97 kDa was detected in db/+ (lane 1) and db/db (lane 2) MC. In the graph, the mean ± SEM from three individual experiments were expressed as a percentage of db/+ MC ;*, P < 0.05). C, ERK activity. Western blots were probed with antibodies to pERK and ERK. As shown in the representative Western blot, bands were detected at the approximate molecular masses of 44 kDa (ERK1) and 42 kDa (ERK2) in db/+ (lane 1) and db/db (lane 2) MC. The graph shows the ratio of pERK/total ERK calculated from the data from five individual experiments, expressed as a percentage of db/+ MC (;*, P < 0.05). D, IGFR activation with a neutralizing antibody (ab) decreases ERK activation in db/db MC. The db/db MC were treated with an IGF-I-neutralizing antibody, and Western analysis was performed as described in Materials and Methods. The inset shows a representative Western blot of untreated (lane 1) db/db MC and db/db MC treated with IGF-I neutralizing antibody (lane 2). The ratio of pERK/total ERK was calculated from the data from two individual experiments and is expressed as a percentage of the vehicle (V) control value (*, P < 0.05).
Baseline determination of IGF-I and IGF-II peptide and IGFBPs
There was no difference in baseline IGF-I peptide synthesis in db/db vs. db/+ MC (9.77 ± 1.88 vs. 9.59 ± 2.47 ng/106 cells). The db/db MC tended to secrete more IGF-II (db/db MC, 0.7024 ± 0.03; db/+ MC, 0.5648 ± 0.04 ng/106 cells; P = 0.075; n = 3), but this did not reach statistical significance. Ligand blotting revealed a band with an approximate molecular mass of 32 kDa and a doublet at 45 kDa in both db/db and db/+ MC supernatants (37). Based on the molecular masses, we postulated these to be IGFBP-2 and IGFBP-3, respectively. IGFBP-2 was increased in db/db (180 ± 40%) compared with db/+ MC cells.
Regulation of IGFR protein by estrogens
Estrogens regulate IGFR expression in various tissues, but this phenomenon has not been examined in MC (26, 28, 38, 39). Therefore, db/db and db/+ MC were treated with E2 for 24 h (Fig. 2, A and B). Treatment with physiological concentrations of E2 (0.1 and 1 nM) increased IGFR protein levels in db/db MC, but decreased IGFR protein levels in the MC of db/+ littermate controls (Fig. 2).
FIG. 2. IGFR protein expression is increased in db/db (A), but not in db/+ (B), MC after treatment with E2 for 24 h. db/db (A) or db/+ (B) MC were treated with either vehicle or E2 (0.1 and 1 nM) as described in Materials and Methods. Cell lysates of db/db (A) or db/+ (B) were electrophoresed and probed with an antibody to the IGFR. Shown are representative Western blots. The graphs show data from six individual collections, expressed as a percentage of the vehicle control value for each cell type. Each bar is the mean ± SEM of data from two individual cell lines each isolated from db/db (A) and db/+ (B) mice. *, P < 0.05; **, P < 0.005 (compared with vehicle-treated cells). C, The increase in IGFR protein is mediated by ER-independent effects of estrogens in db/db MC. The db/db MC were treated with 1 nM E2, 1 μM (ICI, a combination of 1 nM E2 and 1 μM/1 nM (ICI/E2), or 1 nM 17 as described in Materials and Methods. Cell lysates were analyzed as described above. A representative Western blot is shown. The graph shows data from three individual experiments. *, P < 0.05; **, P < 0.005 (compared with vehicle-treated cells). ##, P < 0.005 (E2- or ICI-treated cells compared with ICI/E2-treated cells).
To determine whether regulation of the IGFR protein was an ER-mediated effect, we incubated db/db and db/+ MC with E2, 17, ICI (1 μM), or ICI followed 1 h later by 1 nM E2-containing medium. We found that ICI, 17, and E2 increased IGFR protein levels by up to 2-fold in db/db MC. The combination of ICI and E2 had an additive effect and raised IGFR protein levels nearly 3-fold in db/db MC. Interestingly, equimolar concentrations of E2, the most potent natural estrogen, and its transcriptionally inactive stereoisomer, 17, were equally effective in increasing IGFR protein levels (Fig. 2C). In contrast, in db/+ MC, ICI prevented the estrogen-induced down-regulation of IGFR, suggesting that this was an ER-mediated effect (data not shown).
Transcriptional activity of the IGFR promoter
To determine whether the estrogen-mediated, but ER-independent, increase in IGFR levels was due to up-regulation of IGFR transcription, we transfected db/db MC with a luciferase reporter vector that was under the transcriptional control of either a shorter proximal version or the full-length IGFR promoter (35). There was no increase in luciferase activity after estrogen stimulation when db/db MC were transfected with the shorter p(–476/+640)luciferase (Luc) promoter construct (Fig. 3A). When cells were transfected with the full-length p(–2350/+476)Luc IGFR promoter construct, we found that treatment with E2 or 17 was equally effective and increased luciferase activity by approximately 50% (Fig. 3B; P < 0.05). These results suggest that the estrogen-responsive region of the IGFR promoter is located upstream of bp –476 in the 5'-flanking region.
FIG. 3. Transcriptional regulation of IGFR expression in db/db MC. db/db MC were transfected with Luc-based reporter plasmids under the transcriptional control of either the proximal construct (A) or the full-length IGFR promoter (B). One hour after transfection, the cells were treated as described in Materials and Methods. Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db and db/+ mice. Triplicate wells were collected for each treatment in each experiment. E2 and 17 dose, 1 nM concentration. *, P < 0.05. n = 5 individual collections.
There was a trend for higher IGFR promoter activity when the transfected cells were treated with ICI alone or in combination with E2, but this did not reach statistical significance. There was no additive effect of ICI/ E2 on IGFR promoter activity, in contrast to the observed increase in IGFR protein expression in db/db MC after ICI/ E2 treatment.
E2-induced activation of IGFR and IRS-1
Activation of the IGFR leads to autophosphorylation of the ?-subunit and subsequent tyrosine phosphorylation of the IRS-I (40). Because we found that E2 increased IGFR levels in db/db MC, we wanted to determine whether estrogens also modulated this signaling event. Western analysis revealed that after 24 h, E2 (0.1 and 1 nM) increased IGFR tyrosine phosphorylation approximately 1.3- and 1.6-fold, respectively (Fig. 4A; P < 0.05; n = 3). E2-induced phosphorylation of IRS-1 also increased up to 3.2-fold over that with vehicle (Fig. 4B; P < 0.05; n = 3). To control for nonspecific binding of antibodies, BSA was IP at the same time as cell lysates and run on the blots (data not shown).
FIG. 4. E2-induced tyrosine phosphorylation of IGFR and IRS-1. db/db MC were treated with vehicle (V) or 0.1 or 1 nM E2 as described in Materials and Methods. Cell lysates were immunoprecipitated (IP) with an IGFR antibody (A) or IRS-1(B) and analyzed for IGFR, IRS-1, and PY99, respectively. Graphs depict the mean ± SEM of data from three individual experiments for IGFR and four experiments for IRS-1. Data are expressed as a percentage of vehicle control phosphorylation. All data were statistically significant compared with vehicle (*, P < 0.05).
Time course of ERK phosphorylation
In many cell types, including MC, estrogens increase ERK activation in a rapid and transient fashion (41). To assess whether the estrogen-induced ERK was sustained in db/db MC over a longer period of time, we examined ERK activation after treatment with E2 for 10 min, 6 h, and 24 h. Total ERK levels did not change over time, as shown in the representative Western blot (Fig. 5A). There was a rapid increase in ERK activation after 10 min of treatment, as previously reported. After a return to baseline levels at 6 h, there was a second increase in ERK phosphorylation after 24 h of treatment. This suggests that E2 regulates ERK1/2 activation in a biphasic fashion, namely via a rapid nongenomic activation at 10 min and a second mechanism that may involve gene activation after 24 h of treatment (Fig. 5, B and C).
FIG. 5. Time course of ERK activation in db/db MC. A, db/db MC were treated with either vehicle or E2 (0.1–1 nM) for 10 min, 6 h, or 24 h. Shown is a representative Western blot of pERK and total ERK (A). The graphs represent the ratio of pERK/total ERK1 (B) or pERK/total ERK2 (C) at 10 min (), 6 h (), and 24 h () as calculated from five experiments. Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db mice.
ERK activation at 24 h is ER independent
Baseline ERK phosphorylation was higher in db/db than in db/+ MC, although total ERK was not different between the groups (see Fig. 1). Treatment with E2 increased ERK1/2 activation in db/db MC (Fig. 6, A and B). The transcriptionally inactive stereoisomer, 17, and the most potent natural estrogen, E2, were equally effective in increasing ERK1/2 phosphorylation (Fig. 6, C and D), whereas the complete ER antagonist ICI did not block the stimulatory actions of E2 in db/db MC. This suggests that estrogen-induced ERK activation is mediated via an ER-independent mechanism in db/db MC. There was no estrogen-induced activation of ERK1/2 in db/+ MC (Fig. 6, A and B, ).
FIG. 6. Effect of estrogen on ERK1/2 activation in db/db and db/+ MC. Estrogen activates ERK1 (A) and ERK2 (B) in db/db, but not db/+, MC. The activation in db/db cells is ER independent (C and D). Cell lysates (2.5 μg) treated with vehicle or E2 (0.1 and 1 nM) isolated from db/db () or db/+ () MC were separated by SDS-PAGE, and immunoblotting was performed using antibodies specifically directed against pERK and total ERK1 (A) or ERK2 (B). The ratios of pERK/total ERK were calculated from five experiments and expressed as a percentage of the vehicle control. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db and db/+ mice. ****, P < 0.0005; **, P < 0.005; *, P < 0.05 (compared with vehicle-treated cells). db/db MC ERK1 (C) and ERK2 (D) activations are ER independent. Representative Western blot of db/db MC treated as described in Materials and Methods. Data are expressed as a percentage of the vehicle control value of the ratio of pERK/total ERK. E2 and 17, 1 nM in graphs C and D. *, P < 0.05 (compared with vehicle-treated cells).
Western analysis of ER and ER?
We have previously shown that sclerosis-prone mice have decreased glomerular (in vivo) and mesangial (in vitro) ER expression (16). However, the effects of diabetes on glomerular and mesangial ER expression are unknown. To determine whether ER protein expression was decreased in MC isolated from mice with diabetic nephropathy, we studied ER protein expression in MC isolated from female diabetic db/db mice and their db/+ littermate controls. As shown in the representative Western blot, a band was evident at approximately 67 kDa using an antibody that recognizes the N terminus of the ER subtype. The graph shows that ER was approximately 50% lower in db/db than in db/+ MC (db/db, 44.03 ± 9.8%; db/+, 100 ± 0.07%; n = 5; P < 0.005; Fig. 7A). Eighty micrograms of protein were IP with an antibody against ER?, and a band was identified at 55 kDa. ER? protein expression was approximately 20% lower in db/db (Fig. 7B; 79.78 ± 4.22%; n = 3) than in db/+ MC (100 ± 1.47%; n = 3; P < 0.005).
FIG. 7. ER (A) and ER? (B) protein expression is lower in db/db than in db/+ MC. Twenty micrograms of db/+ (lane 1) and db/db (lane 2) cell lysates were analyzed by Western blot, and representative gels are shown. Data are expressed as the mean ± SEM of five individual experiments, expressed as a percentage of protein of db/+ MC (; **, P < 0.005). The transcriptional activity of ERs is lower in db/db (C) compared with db/+ (D) MC. Transfection experiments were performed as described in Materials and Methods using a Luc-based reporter construct plasmid under the transcriptional control of 4x ERE. One hour after transfection, cells were treated with vehicle (v) or E2 (0.1 and 1 nM). Data are expressed as the mean ± SEM percentage of the vehicle control value (V) for db/db (C) and db/+ (D) MC (*, P < 0.05 compared with V).
Transcriptional activity of ER
To assess the transcriptional activity of the MC ERs, we transfected db/db and db/+ MC with a Luc-based reporter gene construct that is under the transcriptional control of a promoter containing four consecutive synthetic ERE. We found that the transcriptional activity of the ERs was lower in db/db than in db/+ MC after stimulation with E2 (Fig. 7, C and D).
Inhibition of ERK activation increases ER expression and ER activity
Activation of the MAPK signaling pathway decreases ER expression in breast cancer cells (42). Thus, we postulated that the increased baseline ERK activation decreased ER expression in db/db MC. Treatment of db/db MC with the MAPK inhibitor PD for 24 h decreased ERK activation as expected and increased ER expression by approximately 50% (Fig. 8, A, lanes 1 and 2, and Fig. 8B; 148.5 ± 17.3% of control; n = 4). ER? expression did not increase (data not shown). The 1.5-fold increase in ER expression observed after PD treatment for 24 h was followed by a 2-fold increase in the baseline/ER-mediated transcriptional activity in db/db MC in transfection experiments using the ERE reporter construct (Fig. 8C). In contrast, ER expression did not change in db/+ MC after PD treatment (Fig. 8A, lanes 3 and 4). In addition, treatment with the IGF-I-neutralizing antibody increased ER protein expression activity 30% (Fig. 8D).
FIG. 8. Blocking ERK activation increases ER expression and activity in db/db MC. MC were treated with 40 μM PD for 24 h. Twenty micrograms of cell lysate were analyzed by Western blot as described in Materials and Methods. A, Representative Western blot of ER is shown. db/db (lanes 1 and 2) and db/+ littermate control (lanes 3 and 4) MC treated with PD (lanes 2 and 4). The arrow denotes the ER band. B, Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db mice. *, P < 0.05. n = 4 individual collections. C, Cells were treated with 40 μM PD98059 overnight before transfection with 4x ERE. Transcriptional activation increased 2-fold after ERK activation was blocked. *, P < 0.05. n = 2 individual collections. D, ER expression increased after cells were treated with a neutralizing antibody to IGF-I as described in Materials and Methods (n = 2 individual collections). The inset shows a representative Western blot.
Discussion
To determine whether autocrine activation of the glomerular/mesangial IGF-I signaling pathway(s) is involved in the development of type 2 diabetic GS, we studied female diabetic C57BL6 db/db (db/db) mice and their nondiabetic littermates. The db/db mice on a C57BL6 genetic background are a model of type 2 diabetes and, importantly, develop diabetic GS. A caveat of this db/db mouse model of diabetes is the fact that these mice remain hyperinsulinemic due to pancreatic islet/?-cell hyperplasia during their entire life span. In contrast, the development and progression of type 2 diabetes in humans are characterized by initial hyperinsulinemia and subsequent ?-cell failure, leading to relative and absolute insulin deficiency. However, despite the differences in endocrine pancreas function, the functional and morphological changes associated with the development of diabetic GS are very similar in humans and db/db mice. Similarly, the time it takes to develop diabetic GS, i.e. 16 wk in db/db mice vs. 10 yr in humans, respectively, appears to be roughly analogous when one considers the average life span of mice and humans (24 months vs. 70 yr). Thus, db/db mice appear to be an appropriate model to study the pathogenesis of diabetic GS.
IGFR mRNA expression was higher in glomeruli isolated from young female diabetic db/db mice than in those from nondiabetic female db/+ littermates. We had previously reported that increased IGFR expression was part of the autocrine-activated IGF-I system mediating a prosclerotic response in MC of NOD mice with type 1 diabetes (6). The present data represent in vivo evidence that intrinsic activation of the local glomerular IGF-I signaling pathway is also involved in the pathogenesis of type 2 diabetic GS.
To examine the intracellular components of the IGF-I system and the potential cross-talk between IGF-I and estrogens, we isolated and propagated MC from the glomeruli of female db/db mice and their nondiabetic db/+ littermate controls. IGFR mRNA and protein levels were elevated in the MC of db/db mice, confirming that the MC phenotype in culture was similar to that found in intact glomeruli in vivo. Interestingly, db/db and db/+ MC synthesized similar amounts of IGF-I. This suggests that increased mesangial IGFR expression in db/db MC is the principal cause of the autocrine activation of the glomerular/mesangial IGF-I system rather than higher IGF-I peptide secretion. In contrast, in the NOD model of type 1 diabetes, MC of diabetic mice expressed higher levels of both IGF-I and IGFR than those of nondiabetic animals (9). It remains to be seen whether increased glomerular/mesangial IGF-I synthesis represents a mechanism or feature that distinguishes type 1 from type 2 GS or whether this finding is solely restricted to NOD and db/db mice.
However, levels of an IGF-BP with an approximate molecular mass of 32 kDa, which most likely represents IGFBP-2, were increased in supernatants of db/db MC. We previously reported an increase in IGFBP-2 in glomeruli isolated from mice with streptozotocin-induced diabetes (43). IGFBP-2 has been shown to inhibit IGF action, predominantly that of IGF-II, in many cell types. Interestingly, there was no difference in IGF-II secretion between db/db and db/+ MC, suggesting that it was not responsible for activation of the IGF system in db/db mice. IGFBP-2 can also bind to ECM components and enhance the effects of the IGF system (37, 44). In fact, recent microarray analysis of prostate cancer and brain tumor tissues revealed increased IGFBP-2 expression (44). Furthermore, IGFBP-2 has been shown to enhance the growth of prostate tumor cells and to inhibit the growth of normal prostate cells (44). However, the relevance of increased mesangial IGFBP-2 expression for diabetic GS remains to be explored in future studies.
Because activation of the mesangial MAPK signaling cascade by the local IGF-I/IGFR system has been implicated in the development of renal hypertrophy and type 1 diabetic GS (7, 9, 30, 45), we studied the activation/phosphorylation of its downstream ERK1/2 components. The baseline levels of phosphorylated ERK1/2 were increased in db/db, but not in db/+, MC, which paralleled the level of IGFR expression. Importantly, treatment with an IGF-I-neutralizing antibody decreased ERK1/2 activation in db/db MC. This suggests that the MAPK signaling cascade is also used by the autocrine-activated glomerular/mesangial IGF-I system in type 2 diabetes (5, 10) and appears to contribute to the development of type 2 diabetic GS.
Direct interactions and cross-talk between the IGF-I and estrogen signaling pathways exist in several tissues and disease processes and involve genomic and nongenomic effects (23, 24, 27, 38, 46, 47). These interactions have not been explored in type 1 or type 2 diabetic GS, but may have important implications for its pathogenesis. Therefore, we studied whether estrogen treatment modulates the activity of the local IGF-I system in diabetic MC. Unexpectedly, we found an opposite response to E2 treatment in db/db and db/+ MC. E2 down-regulated IGFR expression in MC from nondiabetic db/+ mice via an ER-mediated mechanism similar to what was previously described in vascular smooth muscle cells (28). In contrast, E2 increased IGFR expression in db/db MC. To test whether this was an ER-mediated effect, we treated db/db MC with 17, an ER transcriptionally inactive stereoisomer; ICI, a complete ER antagonist; and a combination of ICI and E2. Both 17 as well as ICI increased IGFR expression similar to E2, suggesting that the estrogen-induced increase in IGFR was due to an ER-independent mechanism (46, 48). Interestingly, the combination of E2 and ICI had an additive effect on IGFR protein expression. Several recent studies have reported ER-independent effects of estrogens in neurons and breast cancer cells (47, 48, 49, 50). Although a potential third ER subtype and/or a surface ER or direct interaction of steroids with the DNA replication machinery have been suggested to explain these cell type-specific effects, there is little concrete evidence in vivo to confirm any of these mechanisms at present (51, 52, 53). Interestingly, those estrogen molecules that have a hydroxyl group in the C3 position of their phenolic A ring can serve as antioxidants by scavenging hydroxyl radicals (54, 55). This leads to the formation of nonphenolic quinols that have no affinity to ERs (55). Such antioxidant capabilities of estrogens could be responsible for the observed ER-independent effects of E2, 17, and ICI, because all three molecules have a hydroxyl group in the phenolic A ring (56). The additive effect of the ICI/E2 combination on IGFR protein expression could simply be explained by the increased availability of scavenging hydroxyl groups doubling the antioxidant potential. Interestingly, Frasor and colleagues (57) reported that on a small number of genes in breast cancer cells, raloxifene, tamoxifen, and ICI act as either full or partial agonists.
To determine whether estrogens regulated IGFR expression at the transcriptional level, we transfected luciferase-based reporter genes under the regulatory control of either a proximal or a full-length IGFR promoter into db/db MC. Estrogen treatment did not increase luciferase activity in db/db MC transfected with the short promoter fragment. In contrast, E2 and 17 similarly increased the transcriptional activity of the full-length IGFR promoter by approximately 50%. Thus, estrogens control IGFR transcription in diabetic MC via regulatory elements located between bp –476 and –2350 upstream of the transcription start site. The fact that the biologically inert stereoisomer 17 was as potent as E2 in activating the full-length IGFR promoter suggests that the estrogen-induced increase in IGFR translation is also mediated via ER-independent mechanisms. Although there was a trend for higher IGFR promoter activity, the effects of ICI alone or in combination with E2 on the IGFR promoter did not reach statistical significance. Along those lines, there was no additive effect of ICI/ E2 on IGFR promoter activity, in contrast to the observed increase in IGFR protein expression in db/db MC after ICI/ E2 treatment.
Taken together, we suggest that the estrogen-induced increase in IGFR mRNA and protein expression is mediated via different, but ER-independent, mechanisms in diabetic db/db MC. These ER-independent actions of estrogens are currently not well characterized, but appear to be of great importance in the diabetic milieu.
To determine whether this estrogen-mediated, but ER-independent, increase in IGFR levels translated into downstream signaling events, we examined phosphorylation of IGFR and IRS-I as well as ERK activity in db/db MC after estrogen treatment. Phosphorylation of IGFR and IRS-1 increased in the presence of E2, confirming the occurrence of downstream signaling as well as signal amplification after estrogen-mediated IGFR activation (39).
In addition, estrogen treatment increased ERK phosphorylation in cells isolated from db/db mice. Estrogen activation of ERK has been shown in many cell types, including MC (50, 58), although the effects appear to occur rapidly and disappear within 60 min (41). We performed a time course to determine whether there were rapid as well as sustained effects of estrogen treatment. Treatment over a 10-min period with physiological E2 concentrations increased ERK1/2 phosphorylation. The levels of ERK phosphorylation returned to baseline at 6 h and increased again at 24 h in the presence of E2 or 17. Because ICI did not block this E2-mediated effect, ERK appears to be activated via an ER-independent mechanism. A delayed activation of ERK by estrogens has been previously described in human endothelial cells via an ER-dependent autocrine stimulation of fibroblast growth factor (59). Herein, we found that estrogens stimulated ERK activation in diabetic db/db MC in a biphasic and ER-independent manner.
In previous studies, we have shown that MC express both ER subtypes, ER and ER?, that ER is the predominant subtype in the glomerulus, and that the susceptibility to GS is inversely correlated with the level of glomerular/mesangial ER expression (15, 16). In other words, estrogen protection against scarring is predominantly mediated by ER. However, estrogen-mediated protection of the cardiovascular system, including the glomerulus, appears to be lost in the presence of diabetes in both human and experimental disease (11, 14, 20). The lower levels of ER and ER? in the glomerulus and MC of db/db compared with those of db/+ mice are consistent with our previous observation that low glomerular/mesangial ER expression is associated with the propensity to develop GS. Similarly, the transcriptional activity of ER appeared to be diminished in db/db MC.
Because MAPK activation decreases ER expression in breast cancer cells (22, 42), we hypothesized that activation of the MAPK signaling cascade by the local IGF-I system could be responsible for the decrease in ER expression in db/db MC. Treatment of db/db MC with the MAPK inhibitor PD for 24 h increased ER expression by approximately 50%. In addition, the 1.5-fold higher ER expression was accompanied by an approximately 2-fold increase in baseline transcriptional activity in transfection experiments using an ERE-responsive reporter gene. Similar results were found when db/db MC were treated with an IGF-I-neutralizing antibody that blocks activation of the local IGF-I system. This suggests that in type 2 diabetes, autocrine activation of the IGF-I system down-regulates ER expression and estrogen/ER responsiveness via activation of the MAPK signaling cascade as part of a prosclerotic response in MC. Importantly, the decreased ER expression in db/db MC is reversible after blocking PD-sensitive IGF-I signaling pathways. Thus, estrogens display a paradoxical effect in diabetic MC, namely, stimulation of the local IGF-I system via an ER-independent mechanism, which, in turn, may further decrease the already insufficient ER-mediated protection against diabetic GS in susceptible individuals.
In summary, there is autocrine activation of the local prosclerotic IGF-I signaling system in glomeruli and MC of young female diabetic db/db mice with severe type 2 diabetic GS. Increased MAPK activity is an important downstream component of the prosclerotic IGFR-mediated signaling events, similar to what is seen in type 1 diabetic nephropathy. Importantly, in db/db MC isolated from female diabetic mice, estrogens exhibit a paradoxical prosclerotic effect(s) via an ER-independent mechanism(s). Estrogens increase IGFR expression and stimulate prosclerotic signaling events, including IGFR/IRS-I complex formation and ERK phosphorylation, in an ER-independent manner in diabetic MC.
Furthermore, ER expression, which inversely correlates with the susceptibility to GS, is decreased in the glomeruli and MC of db/db mice. Autocrine activation of the IGF-I signaling pathway accounts for the low ER expression in db/db MC, because treatment with either an IGF-I-neutralizing antibody or the MAPK inhibitor PD increases ER expression and ER-mediated transcriptional activity. Estrogen treatment could lead to an additional decrease in ER-mediated protection against GS, because estrogens stimulate the local IGF-I system in db/db MC via an ER-independent mechanism. These data may, at least in part, account for the fact that premenopausal women with type 2 diabetes are not protected by estrogens against progressive diabetic nephropathy.
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Address all correspondence and requests for reprints to: Dr. Sharon J. Elliot, Vascular Biology Institute, University of Miami School of Medicine, 1600 N.W. 10th Avenue, RMSB, Room 1043-R104, Miami, Florida 33136. E-mail: selliot@med.miami.edu.
Abstract
Autocrine activation of the IGF-I system in mesangial cells (MC) promotes glomerular scarring in a model of type 1 diabetes. Although estrogens protect against progressive nondiabetic glomerulosclerosis (GS), women with diabetes seem to loose the estrogen-mediated protection against cardiovascular disease. However, little is known about the local IGF-I system and its interactions with estrogens in the pathogenesis of type 2 diabetic GS. Therefore, we examined db/db B6 (db/db) mice, a model of type 2 diabetes and diabetic GS. The IGF-I system was activated in the glomeruli and MC of female diabetic db/db mice, but not in nondiabetic db/+ littermates. We found increased IGF-I receptor (IGFR) expression and activation, including activation of MAPK. Surprisingly, estrogens, via an estrogen receptor (ER)-independent mechanism(s), increased IGFR expression, IGFR and insulin receptor substrate phosphorylation, and extracellular signal-regulated kinase activation in db/db MC. In contrast, ER expression was decreased in MC and glomeruli of db/db mice. Treatment with a neutralizing antibody to IGF-I or the MAPK inhibitor PD98059 increased ER expression and transcriptional activity. This suggests that the local prosclerotic IGF-I system is activated in type 2 diabetes and diminishes ER-mediated protection against GS. Although estrogens may stimulate protective ER signaling, they also activate the IGF-I system via ER-independent mechanisms in db/db MC. The later estrogen effects appear to outweigh the antisclerotic effects of ER activation. This may in part account for loss of estrogen protection against the progression of diabetic GS in women with type 2 diabetes.
Introduction
DIABETES IS THE leading cause of end-stage renal disease (ESRD) in industrialized countries (1). Diabetic glomerulosclerosis (GS), the hallmark of advanced diabetic kidney disease, is a scarring process that includes extracellular matrix (ECM) accumulation in the mesangium (2, 3). Mesangial cells (MC), the vascular smooth muscle-like cells in the glomerulus, play a central role in the pathogenesis of diabetic GS, because they synthesize ECM as well as the ECM-degrading matrix metalloproteinases (3). An imbalance between MC ECM synthesis and degradation has been postulated to be responsible for the glomerular scarring process (4).
There is mounting evidence that autocrine and/or paracrine activation of the IGF-I signaling system in MC contributes to the development of diabetic GS (5, 6, 7). In a model of type 1 diabetes, autocrine activation of the IGF-I signaling cascade regulates ECM turnover in a manner that could lead to ECM accumulation in the glomerulus (6, 8, 9). Autocrine activation of the IGF-I system has also been proposed to initiate a prosclerotic response in type 2 diabetic nephropathy, but this has not been extensively studied (10).
The incidence of nondiabetic cardiovascular diseases, including chronic kidney disease, which progresses to ESRD, is lower in premenopausal women than in age-matched men (11, 12). In a recent meta-analysis, Neugarten and co-workers (13) found a gender-specific difference and a more favorable outcome in women with chronic kidney disease, IgA nephropathy, and membranous nephropathy. Based on epidemiological data, estrogens have been suggested to play a major role in protection against the development and progression of large and small vessel disease in women during their reproductive years (14). In line with these observations, we previously reported that the renal glomerulus is a target tissue for estrogens and that estrogens protect against the development and progression of GS in nondiabetic mice (15, 16). We also showed that the susceptibility to develop GS in response to injury is inversely correlated with the level of glomerular estrogen receptor (ER) expression (16).
However, women with diabetes seem to loose the potentially estrogen-mediated protection against developing cardiovascular disease (17). Although the incidence of coronary events in women is generally low before menopause, young women with diabetes between the ages of 20–34 yr have a 33-fold higher relative risk for acute myocardial infarction than nondiabetic women. In contrast, men, whose rate of coronary events is already higher in the nondiabetic state compared with age-matched women, have only a 9-fold increase in risk once diabetes develops (18). The time to progress to ESRD due to diabetic nephropathy tended to be shorter in African-American women than in men with advanced diabetic kidney disease in a Mississippi-based population (19). Recent data from the United States Renal Data System also suggest that estrogens do not protect women with diabetes from diabetic GS, because there is no difference in the incidence of ESRD due to diabetes among men and women between 20–64 yr of age (20). Interestingly, progression of diabetic nephropathy is accelerated during pregnancy in over 40% of women with preexisting diabetic kidney disease and moderate to severe renal insufficiency (21). This may suggest that elevated sex steroid levels play a deleterious role in the disease process. However, the molecular mechanisms responsible for the loss of estrogen protection in diabetes-induced cardiovascular disease, including diabetic GS, are not well understood.
Direct interactions and cross-talk between the estrogen and IGF-I signaling pathways have been described in various vascular and nonvascular tissues (22, 23, 24, 25, 26). The IGF-I system exerts tissue-specific stimulatory and inhibitory effects on the estrogen signaling pathways and vice versa (23, 24, 26). For example, estrogens down-regulate components of the IGF-I signaling cascade, whereas IGF-I activates the ER in aortic/vascular smooth muscle cells (27, 28). Because little is known about the interactions between the estrogen and IGF-I signaling pathways in the pathogenesis of type 2 diabetic GS, we examined db/db B6 mice, a model of type 2 diabetes mellitus, with diabetic nephropathy (29, 30).
In this study we examined whether there was autocrine activation of the IGF-I system and whether estrogens could modulate the activation of the local IGF-I signaling cascade in glomeruli and mesangial cells of female mice with diabetes. We also explored the effects of the autocrine-activated IGF-I system on the expression of mesangial ER, which mediate the protective effects of estrogens in the glomerulus and MC.
Materials and Methods
Reagents
The reagents used for real-time PCR were purchased from PerkinElmer Applied Biosystems (Foster City, CA). Culture media and supplements were obtained from Invitrogen Life Technologies (Grand Island, NY). Charcoal-stripped fetal bovine serum (FBS) was purchased from HyClone (Pittsburgh, PA). 17?-Estradiol (E2), 17-estradiol (17), anti -smooth muscle actin, ?-galactosidase substrate, HEPES, phenylmethylsulfonylfluoride, aprotinin, leupeptin, benzamidine, EDTA, sodium pyrophosphate, sodium fluoride, sodium orthovanadate, Triton X-100, Tween 20, BSA (fraction V), glycerol, and NaCl were purchased from Sigma-Aldrich Corp. (St. Louis, MO). ICI 182,780 (ICI) was obtained from Tocris (Ballwin, MO). PD98059 (PD) was purchased from Calbiochem (La Jolla, CA). Tissue culture plates were obtained from Nunclon (Nalge Nunc, Rochester, NY). Reagents for SDS-PAGE and immunoblotting were obtained from NOVEX (San Diego, CA). Nitrocellulose membranes were purchased from Amersham Biosciences (Piscataway, NJ). All antibodies [IGF-I receptor (IGFR), insulin receptor substrate 1 (IRS-1), phosphotyrosine (PY99), extracellular signal-regulated kinase (ERK), phosphorylated ERK (pERK), ER, H-184, and ER? Y-19], and protein A/G-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Nitrocellulose membranes (Hybond ECL) and films (Hyperfilm ECL) for chemiluminescence detection were obtained from Amersham Biosciences. TransFast and lysis buffer were purchased from Promega Corp. (Madison, WI). The IGF-I neutralizing antibody was purchased from R&D Systems, Inc. (Minneapolis, MN). mouse/rat enzyme immunoassay was purchased from Diagnostics Systems Laboratories (Webster, TX).
Model
Female db/db (C57BL/6J-m Leprdb/2+) mice and their lean (Leprdb/+) nondiabetic littermates were purchased from The Jackson Laboratory (Bar Harbor, ME). The diabetes mutation, Leprdb, is a point mutation in the leptin receptor gene, Lepr, that promotes abnormal splicing and thereby causes a shortening of the intracellular domain of the receptor (31). In the C57BL/6 inbred background, the phenotype of the homozygous Leprdb mutation is characterized by obesity, hyperglycemia, and sustained hyperinsulinemia, associated with marked hypertrophy and hyperplasia of ?-cells (32). These mice develop diabetic nephropathy with an increased albumin excretion rate by 12 wk of age and glomerular lesions by 16 wk of age (29, 33, 34) The lesions are similar to those observed in humans with diabetes.
Glomerular isolation
Glomeruli were microdissected from kidneys of 4- to 6-month-old female diabetic (db/db) and nondiabetic littermate mice (db/+). Diabetic mice were obese and had severe untreated hyperglycemia (>300 mg/dl) before death.
Real-time PCR
Amplification and measurement of target RNA were performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems) as previously described (16). TaqMan probes and primers for amplification of the specific transcripts were designed using Primer Express 1.5. Quantitative RT-PCR was performed in a one-step buffer system according to the manufacturer’s instructions. The sequences of the probes for ER and ER? have been previously described (16). The sequences of the IGFR were: forward primer, GAG GGT GGC CTT CTG GAC A; reverse primer, TAT ACT GCC AGC ACA TGC GC; and probe, VIC-CGG ACA ACT GCC CTG ATA TGC TGT TTG A. Each sample was normalized to the 18S content as previously described (16). Standard curves for each molecule were generated using serial dilutions (0.001–100 ng) of mRNA from mouse uterus. PCR assays were conducted in duplicate for each sample. Data are expressed as the percentage of db/+ mRNA isolated from 100 glomeruli and represent the mean ± SEM of four animals for each group.
Isolation, propagation, and characterization of mesangial cell lines
MC were isolated from db/db mice and control littermates, as previously described (6). Each line of cells was derived from an individual mouse. All experiments were performed on two individual cell lines of db/db and two individual cell lines of littermate controls between passages 6 and 18. Growth curves were performed to determine whether there was a difference in proliferation as described previously for diabetic MC. Briefly 5000 cells/well were plated in 24-well plates (Nalge Nunc) in DMEM/Ham’s F-12 medium with 20% FBS as previously described (6). Duplicate wells were trypsinized and counted every 24 h for 7 d.
Cell culture and experimental design
Four days before the collection of protein, cells were plated in T75 cm2 flasks in phenol red-free DMEM/Ham’s F-12 medium (B medium) containing 20% dextran-charcoal stripped FBS as previously described (15). Twenty-four hours before collection, the medium was replaced with B medium containing 0.1% BSA and vehicle control (0.001% ethanol), increasing concentrations of E2 (0.1–10 nM), ICI (1 μM), ICI followed 1 h later by E2 (1 nM), or 17 (1 nM) for 24 h. In time-course experiments, E2 was added for 10 min, 6 h, and 24 h, and protein was collected. In some experiments cells were exposed to 40 μM PD or 12.5 μg IGF-I neutralizing antibody for 24 h.
Immunoprecipitation and Western blotting
Cell lysates were processed, and samples were resolved by electrophoresis on 6% (IGFR and IRS-1) or 10% (ER, ER?, ERK, and Perk1/2) polyacrylamide gels as previously described (9). Electrotransfer of proteins from the gel to the nitrocellulose was performed by electroelution and immunoblotting as previously described. Immunoreactive bands were determined by exposing the nitrocellulose blots to a chemiluminescence solution (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 min, followed by exposure to X-OMAT AR film (Eastman Kodak Co., Rochester, NY). The film was scanned and saved on computer disks for densitometric analysis using Image J software from NIH.
For immunoprecipitation (IP) experiments, 80–100 μg protein extract were incubated with an antibody against ER?, and 250 μg protein extract were incubated with antibodies against IGFR or IRS-1 for 1 h at 4 C, followed by the addition of protein A/G-agarose overnight. The resulting protein-antibody conjugate was centrifuged at 4 C and washed four times with PBS, pH 7.4. The final pellet was resuspended in PBS, sample buffer was added, and the mixture was boiled for 3 min before analysis as described above. Blots were exposed to IGFR, IRS-1, or PY99 antibodies. To control for nonspecific antibody binding, 250 μg BSA were IP.
Transfection studies
For transient transfection experiments, glomerular MC were transfected with a luciferase-based reporter construct containing four consecutive synthetic estrogen response elements (4x ERE; provided by David Shapiro). In addition, cells were transfected with luciferase reporter vectors under control of the full-length [p(–2350/+640)Luc] or proximal [p(–476/+640)Luc] IGFR promoter (35). The former construct was generated by subcloning 2350 bp of 5'-flanking sequences and 640 bp of the 5'-untranslated region of the rat IGFR gene into the p0LUC luciferase reporter vector (nucleotide +1 corresponds to the transcription start site). The later construct includes 476 bp of 5'-flanking sequences and 640 bp of 5'-untranslated sequences. The basal activities of both constructs were previously reported (35). Cells were transfected using Trans-Fast as previously described (16). The corresponding empty vectors were used as controls. Transfection efficacy was adjusted by cotransfection with phosphorylated Rous sarcoma virus-?-galactosidase (0.3–0.5 μg/well). After transfection, cells were incubated for 24 h in the presence of vehicle, 1 nM E2, ICI, ICI followed by E2, or 17. For luciferase and galactosidase assays, cells were lysed in 100 μl reporter lysis buffer at room temperature. Light emission was detected using a luminometer (AutoLumatPlus, PerkinElmer) after addition of luciferin to 40 μl cell lysate. Values were expressed as arbitrary light units normalized to the ?-galactosidase activity of each sample.
Measurement of baseline IGF-I, IGF-II peptide, and IGF-binding proteins (IGFBPs)
Supernatants from db/db and db/+ cells were collected and processed as previously described (6). After Sep-Pak (Waters, Milford, MA) extraction, IGF-I peptide was assayed using the mouse/rat enzyme immunoassay (Diagnostics Systems Laboratories).
IGF-II was measured using a mouse-specific, two-site ELISA (Drs. D. Hwang and P. Lee, David Geffen School of Medicine, University of California, Los Angeles, CA). The detection limit for this assay is 0.3 ng/ml, with inter- and intraassay coefficients of variation of less than 10%. Medium samples (9 ml) were extracted using Sep-Pak C18 cartridges, lyophilized, then reconstituted to 0.2 ml for assay. The recovery of added mIGF-II from reconstituted medium samples was >90%.
IGFBPs were assessed by ligand blot as described by Horney et al. (36). Briefly, supernatants from db/db and db/+ cells were subjected to trichloroacetic acid precipitation, loaded normalized to protein amount, and electrophoresed on 12% sodium dodecyl sulfate nonreducing polyacrylamide gels. After transfer of protein and washes, the blots were exposed to biotinylated IGF-I (a gift from S. Rosenzweig) and developed with chemiluminescence as described for Western blots.
Statistical analysis
Transfection studies and Western analysis experiments for IGFR, ERs, ERK, and pERK were performed three to five times on two individual cell lines of db/db and db/+ mice. All real-time PCR experiments were performed either on glomerular RNA extracted from four individual mice per group or on two independent MC lines. Comparison between groups was performed using one-way ANOVA and Tukey’s multiple comparison test (for PCR experiments and phosphorylation experiments) or t test (for all other analyses).
Results
IGFR, ER, and ER? mRNA expression in microdissected glomeruli
IGFR mRNA expression was approximately 2-fold higher in the glomeruli isolated from db/db mice than in those of littermate controls, as assessed by real-time RT-PCR (P < 0.05; Table 1). In contrast, ER expression in db/db glomeruli was only about 10% of the ER mRNA levels found in db/+ glomeruli (P < 0.05; Table 1). ER? mRNA expression was not significantly different between the two.
TABLE 1. Expression of IGFR and ER mRNA in 100 glomeruli microdissected from db/db and db/+ mice
MC characterization
To examine and compare the potential interactions between the estrogen and IGF-I signaling pathways in the glomeruli of diabetic and nondiabetic mice, MC were isolated from microdissected glomeruli from db/db (n = 2) or db/+ (n = 2) mice. MC were identified by the presence and the typical filamentous distribution pattern of -smooth muscle actin. Cells were used between passages 6 and 18. Growth curves were performed to determine the proliferation rate. MC lines isolated from diabetic db/db mice had an increased proliferation rate compared with control cell lines (data not shown). This was similar to our previous findings for MC isolated from nonobese diabetic mice, a model of type I diabetes (6).
Baseline IGFR mRNA and protein expression and ERK activation in db/db and db/+ MC
Baseline levels of IGFR mRNA expression were determined in vitro in MC isolated from female diabetic db/db mice and their db/+ littermate controls. The levels of IGFR mRNA were about 2-fold higher in db/db than in db/+ MC as assessed by real-time RT-PCR (Fig. 1A). As shown in the representative Western blot (Fig. 1B), this correlated with the 2.8-fold higher level of IGFR protein expression in db/db MC (P < 0.05). A parallel increase in ERK activation was also present in db/db MC (Fig. 1C). Treatment with a neutralizing antibody against IGF decreased ERK1 and ERK2 activation by about 50% and 40%, respectively (Fig. 1D). Baseline densitometry units of total ERK1 (1.023 ± 0.13 vs. 1.08 ± 0.23; n = 5) and ERK2 (0.875 ± 0.22 vs. 0.65 ± 0.20; n = 5) were not different between db/db and db/+ MC.
FIG. 1. Baseline IGFR mRNA (A) and protein (B) expression and ERK activity (C) are increased in db/db MC. A, IGFR mRNA expression was determined by real-time RT-PCR. The ratios of IGFR/18S were calculated from the data of three individual experiments and were expressed as a percentage of db/+. B, IGFR protein expression. Five micrograms of protein were separated by 6% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with an antibody to the IGFR. As shown in the representative Western blot, a band with an approximate molecular mass of 97 kDa was detected in db/+ (lane 1) and db/db (lane 2) MC. In the graph, the mean ± SEM from three individual experiments were expressed as a percentage of db/+ MC ;*, P < 0.05). C, ERK activity. Western blots were probed with antibodies to pERK and ERK. As shown in the representative Western blot, bands were detected at the approximate molecular masses of 44 kDa (ERK1) and 42 kDa (ERK2) in db/+ (lane 1) and db/db (lane 2) MC. The graph shows the ratio of pERK/total ERK calculated from the data from five individual experiments, expressed as a percentage of db/+ MC (;*, P < 0.05). D, IGFR activation with a neutralizing antibody (ab) decreases ERK activation in db/db MC. The db/db MC were treated with an IGF-I-neutralizing antibody, and Western analysis was performed as described in Materials and Methods. The inset shows a representative Western blot of untreated (lane 1) db/db MC and db/db MC treated with IGF-I neutralizing antibody (lane 2). The ratio of pERK/total ERK was calculated from the data from two individual experiments and is expressed as a percentage of the vehicle (V) control value (*, P < 0.05).
Baseline determination of IGF-I and IGF-II peptide and IGFBPs
There was no difference in baseline IGF-I peptide synthesis in db/db vs. db/+ MC (9.77 ± 1.88 vs. 9.59 ± 2.47 ng/106 cells). The db/db MC tended to secrete more IGF-II (db/db MC, 0.7024 ± 0.03; db/+ MC, 0.5648 ± 0.04 ng/106 cells; P = 0.075; n = 3), but this did not reach statistical significance. Ligand blotting revealed a band with an approximate molecular mass of 32 kDa and a doublet at 45 kDa in both db/db and db/+ MC supernatants (37). Based on the molecular masses, we postulated these to be IGFBP-2 and IGFBP-3, respectively. IGFBP-2 was increased in db/db (180 ± 40%) compared with db/+ MC cells.
Regulation of IGFR protein by estrogens
Estrogens regulate IGFR expression in various tissues, but this phenomenon has not been examined in MC (26, 28, 38, 39). Therefore, db/db and db/+ MC were treated with E2 for 24 h (Fig. 2, A and B). Treatment with physiological concentrations of E2 (0.1 and 1 nM) increased IGFR protein levels in db/db MC, but decreased IGFR protein levels in the MC of db/+ littermate controls (Fig. 2).
FIG. 2. IGFR protein expression is increased in db/db (A), but not in db/+ (B), MC after treatment with E2 for 24 h. db/db (A) or db/+ (B) MC were treated with either vehicle or E2 (0.1 and 1 nM) as described in Materials and Methods. Cell lysates of db/db (A) or db/+ (B) were electrophoresed and probed with an antibody to the IGFR. Shown are representative Western blots. The graphs show data from six individual collections, expressed as a percentage of the vehicle control value for each cell type. Each bar is the mean ± SEM of data from two individual cell lines each isolated from db/db (A) and db/+ (B) mice. *, P < 0.05; **, P < 0.005 (compared with vehicle-treated cells). C, The increase in IGFR protein is mediated by ER-independent effects of estrogens in db/db MC. The db/db MC were treated with 1 nM E2, 1 μM (ICI, a combination of 1 nM E2 and 1 μM/1 nM (ICI/E2), or 1 nM 17 as described in Materials and Methods. Cell lysates were analyzed as described above. A representative Western blot is shown. The graph shows data from three individual experiments. *, P < 0.05; **, P < 0.005 (compared with vehicle-treated cells). ##, P < 0.005 (E2- or ICI-treated cells compared with ICI/E2-treated cells).
To determine whether regulation of the IGFR protein was an ER-mediated effect, we incubated db/db and db/+ MC with E2, 17, ICI (1 μM), or ICI followed 1 h later by 1 nM E2-containing medium. We found that ICI, 17, and E2 increased IGFR protein levels by up to 2-fold in db/db MC. The combination of ICI and E2 had an additive effect and raised IGFR protein levels nearly 3-fold in db/db MC. Interestingly, equimolar concentrations of E2, the most potent natural estrogen, and its transcriptionally inactive stereoisomer, 17, were equally effective in increasing IGFR protein levels (Fig. 2C). In contrast, in db/+ MC, ICI prevented the estrogen-induced down-regulation of IGFR, suggesting that this was an ER-mediated effect (data not shown).
Transcriptional activity of the IGFR promoter
To determine whether the estrogen-mediated, but ER-independent, increase in IGFR levels was due to up-regulation of IGFR transcription, we transfected db/db MC with a luciferase reporter vector that was under the transcriptional control of either a shorter proximal version or the full-length IGFR promoter (35). There was no increase in luciferase activity after estrogen stimulation when db/db MC were transfected with the shorter p(–476/+640)luciferase (Luc) promoter construct (Fig. 3A). When cells were transfected with the full-length p(–2350/+476)Luc IGFR promoter construct, we found that treatment with E2 or 17 was equally effective and increased luciferase activity by approximately 50% (Fig. 3B; P < 0.05). These results suggest that the estrogen-responsive region of the IGFR promoter is located upstream of bp –476 in the 5'-flanking region.
FIG. 3. Transcriptional regulation of IGFR expression in db/db MC. db/db MC were transfected with Luc-based reporter plasmids under the transcriptional control of either the proximal construct (A) or the full-length IGFR promoter (B). One hour after transfection, the cells were treated as described in Materials and Methods. Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db and db/+ mice. Triplicate wells were collected for each treatment in each experiment. E2 and 17 dose, 1 nM concentration. *, P < 0.05. n = 5 individual collections.
There was a trend for higher IGFR promoter activity when the transfected cells were treated with ICI alone or in combination with E2, but this did not reach statistical significance. There was no additive effect of ICI/ E2 on IGFR promoter activity, in contrast to the observed increase in IGFR protein expression in db/db MC after ICI/ E2 treatment.
E2-induced activation of IGFR and IRS-1
Activation of the IGFR leads to autophosphorylation of the ?-subunit and subsequent tyrosine phosphorylation of the IRS-I (40). Because we found that E2 increased IGFR levels in db/db MC, we wanted to determine whether estrogens also modulated this signaling event. Western analysis revealed that after 24 h, E2 (0.1 and 1 nM) increased IGFR tyrosine phosphorylation approximately 1.3- and 1.6-fold, respectively (Fig. 4A; P < 0.05; n = 3). E2-induced phosphorylation of IRS-1 also increased up to 3.2-fold over that with vehicle (Fig. 4B; P < 0.05; n = 3). To control for nonspecific binding of antibodies, BSA was IP at the same time as cell lysates and run on the blots (data not shown).
FIG. 4. E2-induced tyrosine phosphorylation of IGFR and IRS-1. db/db MC were treated with vehicle (V) or 0.1 or 1 nM E2 as described in Materials and Methods. Cell lysates were immunoprecipitated (IP) with an IGFR antibody (A) or IRS-1(B) and analyzed for IGFR, IRS-1, and PY99, respectively. Graphs depict the mean ± SEM of data from three individual experiments for IGFR and four experiments for IRS-1. Data are expressed as a percentage of vehicle control phosphorylation. All data were statistically significant compared with vehicle (*, P < 0.05).
Time course of ERK phosphorylation
In many cell types, including MC, estrogens increase ERK activation in a rapid and transient fashion (41). To assess whether the estrogen-induced ERK was sustained in db/db MC over a longer period of time, we examined ERK activation after treatment with E2 for 10 min, 6 h, and 24 h. Total ERK levels did not change over time, as shown in the representative Western blot (Fig. 5A). There was a rapid increase in ERK activation after 10 min of treatment, as previously reported. After a return to baseline levels at 6 h, there was a second increase in ERK phosphorylation after 24 h of treatment. This suggests that E2 regulates ERK1/2 activation in a biphasic fashion, namely via a rapid nongenomic activation at 10 min and a second mechanism that may involve gene activation after 24 h of treatment (Fig. 5, B and C).
FIG. 5. Time course of ERK activation in db/db MC. A, db/db MC were treated with either vehicle or E2 (0.1–1 nM) for 10 min, 6 h, or 24 h. Shown is a representative Western blot of pERK and total ERK (A). The graphs represent the ratio of pERK/total ERK1 (B) or pERK/total ERK2 (C) at 10 min (), 6 h (), and 24 h () as calculated from five experiments. Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db mice.
ERK activation at 24 h is ER independent
Baseline ERK phosphorylation was higher in db/db than in db/+ MC, although total ERK was not different between the groups (see Fig. 1). Treatment with E2 increased ERK1/2 activation in db/db MC (Fig. 6, A and B). The transcriptionally inactive stereoisomer, 17, and the most potent natural estrogen, E2, were equally effective in increasing ERK1/2 phosphorylation (Fig. 6, C and D), whereas the complete ER antagonist ICI did not block the stimulatory actions of E2 in db/db MC. This suggests that estrogen-induced ERK activation is mediated via an ER-independent mechanism in db/db MC. There was no estrogen-induced activation of ERK1/2 in db/+ MC (Fig. 6, A and B, ).
FIG. 6. Effect of estrogen on ERK1/2 activation in db/db and db/+ MC. Estrogen activates ERK1 (A) and ERK2 (B) in db/db, but not db/+, MC. The activation in db/db cells is ER independent (C and D). Cell lysates (2.5 μg) treated with vehicle or E2 (0.1 and 1 nM) isolated from db/db () or db/+ () MC were separated by SDS-PAGE, and immunoblotting was performed using antibodies specifically directed against pERK and total ERK1 (A) or ERK2 (B). The ratios of pERK/total ERK were calculated from five experiments and expressed as a percentage of the vehicle control. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db and db/+ mice. ****, P < 0.0005; **, P < 0.005; *, P < 0.05 (compared with vehicle-treated cells). db/db MC ERK1 (C) and ERK2 (D) activations are ER independent. Representative Western blot of db/db MC treated as described in Materials and Methods. Data are expressed as a percentage of the vehicle control value of the ratio of pERK/total ERK. E2 and 17, 1 nM in graphs C and D. *, P < 0.05 (compared with vehicle-treated cells).
Western analysis of ER and ER?
We have previously shown that sclerosis-prone mice have decreased glomerular (in vivo) and mesangial (in vitro) ER expression (16). However, the effects of diabetes on glomerular and mesangial ER expression are unknown. To determine whether ER protein expression was decreased in MC isolated from mice with diabetic nephropathy, we studied ER protein expression in MC isolated from female diabetic db/db mice and their db/+ littermate controls. As shown in the representative Western blot, a band was evident at approximately 67 kDa using an antibody that recognizes the N terminus of the ER subtype. The graph shows that ER was approximately 50% lower in db/db than in db/+ MC (db/db, 44.03 ± 9.8%; db/+, 100 ± 0.07%; n = 5; P < 0.005; Fig. 7A). Eighty micrograms of protein were IP with an antibody against ER?, and a band was identified at 55 kDa. ER? protein expression was approximately 20% lower in db/db (Fig. 7B; 79.78 ± 4.22%; n = 3) than in db/+ MC (100 ± 1.47%; n = 3; P < 0.005).
FIG. 7. ER (A) and ER? (B) protein expression is lower in db/db than in db/+ MC. Twenty micrograms of db/+ (lane 1) and db/db (lane 2) cell lysates were analyzed by Western blot, and representative gels are shown. Data are expressed as the mean ± SEM of five individual experiments, expressed as a percentage of protein of db/+ MC (; **, P < 0.005). The transcriptional activity of ERs is lower in db/db (C) compared with db/+ (D) MC. Transfection experiments were performed as described in Materials and Methods using a Luc-based reporter construct plasmid under the transcriptional control of 4x ERE. One hour after transfection, cells were treated with vehicle (v) or E2 (0.1 and 1 nM). Data are expressed as the mean ± SEM percentage of the vehicle control value (V) for db/db (C) and db/+ (D) MC (*, P < 0.05 compared with V).
Transcriptional activity of ER
To assess the transcriptional activity of the MC ERs, we transfected db/db and db/+ MC with a Luc-based reporter gene construct that is under the transcriptional control of a promoter containing four consecutive synthetic ERE. We found that the transcriptional activity of the ERs was lower in db/db than in db/+ MC after stimulation with E2 (Fig. 7, C and D).
Inhibition of ERK activation increases ER expression and ER activity
Activation of the MAPK signaling pathway decreases ER expression in breast cancer cells (42). Thus, we postulated that the increased baseline ERK activation decreased ER expression in db/db MC. Treatment of db/db MC with the MAPK inhibitor PD for 24 h decreased ERK activation as expected and increased ER expression by approximately 50% (Fig. 8, A, lanes 1 and 2, and Fig. 8B; 148.5 ± 17.3% of control; n = 4). ER? expression did not increase (data not shown). The 1.5-fold increase in ER expression observed after PD treatment for 24 h was followed by a 2-fold increase in the baseline/ER-mediated transcriptional activity in db/db MC in transfection experiments using the ERE reporter construct (Fig. 8C). In contrast, ER expression did not change in db/+ MC after PD treatment (Fig. 8A, lanes 3 and 4). In addition, treatment with the IGF-I-neutralizing antibody increased ER protein expression activity 30% (Fig. 8D).
FIG. 8. Blocking ERK activation increases ER expression and activity in db/db MC. MC were treated with 40 μM PD for 24 h. Twenty micrograms of cell lysate were analyzed by Western blot as described in Materials and Methods. A, Representative Western blot of ER is shown. db/db (lanes 1 and 2) and db/+ littermate control (lanes 3 and 4) MC treated with PD (lanes 2 and 4). The arrow denotes the ER band. B, Data are expressed as a percentage of the vehicle control value. Shown are the mean ± SEM of cell lysates collected from two individual cell lines isolated from db/db mice. *, P < 0.05. n = 4 individual collections. C, Cells were treated with 40 μM PD98059 overnight before transfection with 4x ERE. Transcriptional activation increased 2-fold after ERK activation was blocked. *, P < 0.05. n = 2 individual collections. D, ER expression increased after cells were treated with a neutralizing antibody to IGF-I as described in Materials and Methods (n = 2 individual collections). The inset shows a representative Western blot.
Discussion
To determine whether autocrine activation of the glomerular/mesangial IGF-I signaling pathway(s) is involved in the development of type 2 diabetic GS, we studied female diabetic C57BL6 db/db (db/db) mice and their nondiabetic littermates. The db/db mice on a C57BL6 genetic background are a model of type 2 diabetes and, importantly, develop diabetic GS. A caveat of this db/db mouse model of diabetes is the fact that these mice remain hyperinsulinemic due to pancreatic islet/?-cell hyperplasia during their entire life span. In contrast, the development and progression of type 2 diabetes in humans are characterized by initial hyperinsulinemia and subsequent ?-cell failure, leading to relative and absolute insulin deficiency. However, despite the differences in endocrine pancreas function, the functional and morphological changes associated with the development of diabetic GS are very similar in humans and db/db mice. Similarly, the time it takes to develop diabetic GS, i.e. 16 wk in db/db mice vs. 10 yr in humans, respectively, appears to be roughly analogous when one considers the average life span of mice and humans (24 months vs. 70 yr). Thus, db/db mice appear to be an appropriate model to study the pathogenesis of diabetic GS.
IGFR mRNA expression was higher in glomeruli isolated from young female diabetic db/db mice than in those from nondiabetic female db/+ littermates. We had previously reported that increased IGFR expression was part of the autocrine-activated IGF-I system mediating a prosclerotic response in MC of NOD mice with type 1 diabetes (6). The present data represent in vivo evidence that intrinsic activation of the local glomerular IGF-I signaling pathway is also involved in the pathogenesis of type 2 diabetic GS.
To examine the intracellular components of the IGF-I system and the potential cross-talk between IGF-I and estrogens, we isolated and propagated MC from the glomeruli of female db/db mice and their nondiabetic db/+ littermate controls. IGFR mRNA and protein levels were elevated in the MC of db/db mice, confirming that the MC phenotype in culture was similar to that found in intact glomeruli in vivo. Interestingly, db/db and db/+ MC synthesized similar amounts of IGF-I. This suggests that increased mesangial IGFR expression in db/db MC is the principal cause of the autocrine activation of the glomerular/mesangial IGF-I system rather than higher IGF-I peptide secretion. In contrast, in the NOD model of type 1 diabetes, MC of diabetic mice expressed higher levels of both IGF-I and IGFR than those of nondiabetic animals (9). It remains to be seen whether increased glomerular/mesangial IGF-I synthesis represents a mechanism or feature that distinguishes type 1 from type 2 GS or whether this finding is solely restricted to NOD and db/db mice.
However, levels of an IGF-BP with an approximate molecular mass of 32 kDa, which most likely represents IGFBP-2, were increased in supernatants of db/db MC. We previously reported an increase in IGFBP-2 in glomeruli isolated from mice with streptozotocin-induced diabetes (43). IGFBP-2 has been shown to inhibit IGF action, predominantly that of IGF-II, in many cell types. Interestingly, there was no difference in IGF-II secretion between db/db and db/+ MC, suggesting that it was not responsible for activation of the IGF system in db/db mice. IGFBP-2 can also bind to ECM components and enhance the effects of the IGF system (37, 44). In fact, recent microarray analysis of prostate cancer and brain tumor tissues revealed increased IGFBP-2 expression (44). Furthermore, IGFBP-2 has been shown to enhance the growth of prostate tumor cells and to inhibit the growth of normal prostate cells (44). However, the relevance of increased mesangial IGFBP-2 expression for diabetic GS remains to be explored in future studies.
Because activation of the mesangial MAPK signaling cascade by the local IGF-I/IGFR system has been implicated in the development of renal hypertrophy and type 1 diabetic GS (7, 9, 30, 45), we studied the activation/phosphorylation of its downstream ERK1/2 components. The baseline levels of phosphorylated ERK1/2 were increased in db/db, but not in db/+, MC, which paralleled the level of IGFR expression. Importantly, treatment with an IGF-I-neutralizing antibody decreased ERK1/2 activation in db/db MC. This suggests that the MAPK signaling cascade is also used by the autocrine-activated glomerular/mesangial IGF-I system in type 2 diabetes (5, 10) and appears to contribute to the development of type 2 diabetic GS.
Direct interactions and cross-talk between the IGF-I and estrogen signaling pathways exist in several tissues and disease processes and involve genomic and nongenomic effects (23, 24, 27, 38, 46, 47). These interactions have not been explored in type 1 or type 2 diabetic GS, but may have important implications for its pathogenesis. Therefore, we studied whether estrogen treatment modulates the activity of the local IGF-I system in diabetic MC. Unexpectedly, we found an opposite response to E2 treatment in db/db and db/+ MC. E2 down-regulated IGFR expression in MC from nondiabetic db/+ mice via an ER-mediated mechanism similar to what was previously described in vascular smooth muscle cells (28). In contrast, E2 increased IGFR expression in db/db MC. To test whether this was an ER-mediated effect, we treated db/db MC with 17, an ER transcriptionally inactive stereoisomer; ICI, a complete ER antagonist; and a combination of ICI and E2. Both 17 as well as ICI increased IGFR expression similar to E2, suggesting that the estrogen-induced increase in IGFR was due to an ER-independent mechanism (46, 48). Interestingly, the combination of E2 and ICI had an additive effect on IGFR protein expression. Several recent studies have reported ER-independent effects of estrogens in neurons and breast cancer cells (47, 48, 49, 50). Although a potential third ER subtype and/or a surface ER or direct interaction of steroids with the DNA replication machinery have been suggested to explain these cell type-specific effects, there is little concrete evidence in vivo to confirm any of these mechanisms at present (51, 52, 53). Interestingly, those estrogen molecules that have a hydroxyl group in the C3 position of their phenolic A ring can serve as antioxidants by scavenging hydroxyl radicals (54, 55). This leads to the formation of nonphenolic quinols that have no affinity to ERs (55). Such antioxidant capabilities of estrogens could be responsible for the observed ER-independent effects of E2, 17, and ICI, because all three molecules have a hydroxyl group in the phenolic A ring (56). The additive effect of the ICI/E2 combination on IGFR protein expression could simply be explained by the increased availability of scavenging hydroxyl groups doubling the antioxidant potential. Interestingly, Frasor and colleagues (57) reported that on a small number of genes in breast cancer cells, raloxifene, tamoxifen, and ICI act as either full or partial agonists.
To determine whether estrogens regulated IGFR expression at the transcriptional level, we transfected luciferase-based reporter genes under the regulatory control of either a proximal or a full-length IGFR promoter into db/db MC. Estrogen treatment did not increase luciferase activity in db/db MC transfected with the short promoter fragment. In contrast, E2 and 17 similarly increased the transcriptional activity of the full-length IGFR promoter by approximately 50%. Thus, estrogens control IGFR transcription in diabetic MC via regulatory elements located between bp –476 and –2350 upstream of the transcription start site. The fact that the biologically inert stereoisomer 17 was as potent as E2 in activating the full-length IGFR promoter suggests that the estrogen-induced increase in IGFR translation is also mediated via ER-independent mechanisms. Although there was a trend for higher IGFR promoter activity, the effects of ICI alone or in combination with E2 on the IGFR promoter did not reach statistical significance. Along those lines, there was no additive effect of ICI/ E2 on IGFR promoter activity, in contrast to the observed increase in IGFR protein expression in db/db MC after ICI/ E2 treatment.
Taken together, we suggest that the estrogen-induced increase in IGFR mRNA and protein expression is mediated via different, but ER-independent, mechanisms in diabetic db/db MC. These ER-independent actions of estrogens are currently not well characterized, but appear to be of great importance in the diabetic milieu.
To determine whether this estrogen-mediated, but ER-independent, increase in IGFR levels translated into downstream signaling events, we examined phosphorylation of IGFR and IRS-I as well as ERK activity in db/db MC after estrogen treatment. Phosphorylation of IGFR and IRS-1 increased in the presence of E2, confirming the occurrence of downstream signaling as well as signal amplification after estrogen-mediated IGFR activation (39).
In addition, estrogen treatment increased ERK phosphorylation in cells isolated from db/db mice. Estrogen activation of ERK has been shown in many cell types, including MC (50, 58), although the effects appear to occur rapidly and disappear within 60 min (41). We performed a time course to determine whether there were rapid as well as sustained effects of estrogen treatment. Treatment over a 10-min period with physiological E2 concentrations increased ERK1/2 phosphorylation. The levels of ERK phosphorylation returned to baseline at 6 h and increased again at 24 h in the presence of E2 or 17. Because ICI did not block this E2-mediated effect, ERK appears to be activated via an ER-independent mechanism. A delayed activation of ERK by estrogens has been previously described in human endothelial cells via an ER-dependent autocrine stimulation of fibroblast growth factor (59). Herein, we found that estrogens stimulated ERK activation in diabetic db/db MC in a biphasic and ER-independent manner.
In previous studies, we have shown that MC express both ER subtypes, ER and ER?, that ER is the predominant subtype in the glomerulus, and that the susceptibility to GS is inversely correlated with the level of glomerular/mesangial ER expression (15, 16). In other words, estrogen protection against scarring is predominantly mediated by ER. However, estrogen-mediated protection of the cardiovascular system, including the glomerulus, appears to be lost in the presence of diabetes in both human and experimental disease (11, 14, 20). The lower levels of ER and ER? in the glomerulus and MC of db/db compared with those of db/+ mice are consistent with our previous observation that low glomerular/mesangial ER expression is associated with the propensity to develop GS. Similarly, the transcriptional activity of ER appeared to be diminished in db/db MC.
Because MAPK activation decreases ER expression in breast cancer cells (22, 42), we hypothesized that activation of the MAPK signaling cascade by the local IGF-I system could be responsible for the decrease in ER expression in db/db MC. Treatment of db/db MC with the MAPK inhibitor PD for 24 h increased ER expression by approximately 50%. In addition, the 1.5-fold higher ER expression was accompanied by an approximately 2-fold increase in baseline transcriptional activity in transfection experiments using an ERE-responsive reporter gene. Similar results were found when db/db MC were treated with an IGF-I-neutralizing antibody that blocks activation of the local IGF-I system. This suggests that in type 2 diabetes, autocrine activation of the IGF-I system down-regulates ER expression and estrogen/ER responsiveness via activation of the MAPK signaling cascade as part of a prosclerotic response in MC. Importantly, the decreased ER expression in db/db MC is reversible after blocking PD-sensitive IGF-I signaling pathways. Thus, estrogens display a paradoxical effect in diabetic MC, namely, stimulation of the local IGF-I system via an ER-independent mechanism, which, in turn, may further decrease the already insufficient ER-mediated protection against diabetic GS in susceptible individuals.
In summary, there is autocrine activation of the local prosclerotic IGF-I signaling system in glomeruli and MC of young female diabetic db/db mice with severe type 2 diabetic GS. Increased MAPK activity is an important downstream component of the prosclerotic IGFR-mediated signaling events, similar to what is seen in type 1 diabetic nephropathy. Importantly, in db/db MC isolated from female diabetic mice, estrogens exhibit a paradoxical prosclerotic effect(s) via an ER-independent mechanism(s). Estrogens increase IGFR expression and stimulate prosclerotic signaling events, including IGFR/IRS-I complex formation and ERK phosphorylation, in an ER-independent manner in diabetic MC.
Furthermore, ER expression, which inversely correlates with the susceptibility to GS, is decreased in the glomeruli and MC of db/db mice. Autocrine activation of the IGF-I signaling pathway accounts for the low ER expression in db/db MC, because treatment with either an IGF-I-neutralizing antibody or the MAPK inhibitor PD increases ER expression and ER-mediated transcriptional activity. Estrogen treatment could lead to an additional decrease in ER-mediated protection against GS, because estrogens stimulate the local IGF-I system in db/db MC via an ER-independent mechanism. These data may, at least in part, account for the fact that premenopausal women with type 2 diabetes are not protected by estrogens against progressive diabetic nephropathy.
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