Departments of Pediatrics (T.D., S.T., S.K.Da., S.K.De.), Cell and Developmental Biology, Pharmacology, Biochemistry (D.B.F.) and Cancer Biology (T.D., S.T., S.K.Da., S.K.De.), Division of Reproductive and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232; and Department of Biochemistry and Molecular Biology (D.F.S.), Mayo Clinic, Scottsdale, Arizona 85259
Address all correspondence and requests for reprints to: S. K. Dey, Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee 37232. E-mail: sk.dey{at}vanderbilt.edu.
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ABSTRACT |
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INTRODUCTION |
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Hoxa10 is one abdominalB-like homeobox gene that is located in the Hoxa cluster and expressed in the developing genitourinary tract during mouse embryogenesis (3). Its distinct role in development has been defined by gene targeting experiments (6, 7). Hoxa10-deficient mice exhibit male and female infertility. The proximal region of the uterus in Hoxa10/ mice shows partial homeosis into an oviduct-like structure. However, this is not the cause of infertility in these females. Hoxa10 is strongly expressed in the mouse uterine stroma during the receptive phase, and also during implantation and the ensuing decidualization (7). Impaired stromal cell proliferation and decidualization are severely compromised in Hoxa10/ mice, leading to pregnancy failure (8). Furthermore, embryo transfer experiments have conclusively shown the maternal requirement for Hoxa10 in both implantation and decidualization (7, 9).
Coordination between the establishment of uterine receptivity and implantation competency of the blastocyst is essential to the process of implantation. Ovarian progesterone (P4) and estrogen play key roles in these processes. Whereas preovulatory estrogen secretion induces epithelial cell proliferation on d 1 of pregnancy, rising P4 levels from the newly formed corpora lutea superimposed with ovarian estrogen secretion on d 4 directs stromal cell proliferation and epithelial cell differentiation, leading to uterine receptivity for implantation (10). We have previously shown that stromal cell proliferation in Hoxa10/ mice in response to ovarian P4 and estrogen is severely compromised, whereas epithelial cell proliferation remains normal in response to estrogen (9). These results provide evidence that stromal cell responsiveness to P4 with respect to cell proliferation is impaired in Hoxa10/ mice, and that Hoxa10 is intimately involved in mediating stromal cell proliferation. P4 is a strong inducer of Hoxa10 in the mouse uterine stroma, and its induction occurs in this uterine compartment within 4 h of P4 injection in a protein synthesis-independent manner. The up-regulation of Hoxa10 by P4 is attenuated by the progesterone receptor (PR) antagonist RU-486, suggesting a requirement for PR in this induction (11). Collectively, these results suggest that Hoxa10 induces genes that are vital for stromal cell proliferation and differentiation in a P4-dominant environment and that Hoxa10 functions as a mediator of P4 effects in implantation. However, several P4-responsive genes, such as PR itself, Hoxa11, and c-myc, respond normally to P4 in Hoxa10 null mice (9). Therefore the mechanism by which Hoxa10 regulates uterine stromal cell proliferation and differentiation during uterine receptivity, implantation, and decidualization still remains elusive.
In the present study, we first employed a differential-display proteomics analysis using two-dimensional (2D) difference gel electrophoresis (DIGE) on purified uterine stromal cells obtained from wild-type and Hoxa10/ mice on d 4 of pseudopregnancy to compare differential expression profiles of proteins for identifying potential signaling targets of Hoxa10. DIGE provides a powerful quantitative component to conventional 2D gel electrophoresis and is capable of monitoring subtle abundance changes with statistical confidence owing to the ability to compare replicate measurements (12, 13). Sensitive and accurate matrix-assisted laser desorption/ionization (MALDI), time-of-flight (TOF), and tandem TOF/TOF mass spectrometry (MS), coupled with database interrogation, provided identification of 29 proteins with significant differences in expression levels. We focused on the immunophilin FKBP52 (FK506 binding protein 4) for further analysis because of its known assembly with multiple steroid hormone receptors (14) and its role regulating steroid hormone receptor function (15). Consistent with our proteomics data, we observed that the expression of FKBP52 is down-regulated in the stroma of Hoxa10 mutant mice on d 4 of pseudopregnancy with unaltered expression in the luminal and glandular epithelia when compared with that of wild-type mice. We also observed that FKBP52 is uniquely expressed in the pregnant mouse uterus during the periimplantation period in a cell-specific manner. More importantly, FKBP52 expression in the stroma, but not in the epithelium, is regulated by P4. These results suggest that the immunophilin FKBP52 is an important player in the P4-Hoxa10 signaling axis in the periimplantation mouse uterus.
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RESULTS |
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Uterine FKBP52 Expression Is Cell Specific and Is Down-Regulated in Hoxa10/ Mice
Our first objective was to confirm our proteomic data generated on isolated stromal cells with respect to in vivo expression of FKBP52 in uteri of wild-type and Hoxa10/ mice. Immunostaining performed on sections of d 4 pseudopregnant mouse uteri showed that FKBP52 protein is present in the stroma as well as in the luminal and glandular epithelia in wild-type mouse uteri. However, its levels are low in the Hoxa10/ uteri, especially in the stroma, when compared with wild-type uteri (Fig. 4A). Western blotting also detected the presence of FKBP52 protein in whole uterine extracts with higher levels in wild-type compared with Hoxa10/ mice (Fig. 4B
). However, when compared with actin levels, these changes are not especially dramatic. It should be noted that actin expression fluctuates in the rat and mouse uterus with changing experimental conditions (25, 26). Our present results also show fluctuation of several actin-binding proteins between Hoxa10 mutant and wild-type stromal cells (Table 1
). We next asked whether the localization of FKBP52 mRNA follows the same pattern as the protein. Indeed, in situ hybridization showed that FKBP52 mRNA expression follows the pattern of immunolocalization, and its expression is down-regulated in Hoxa10/ uterine stroma (Fig. 4C
). Collectively, these results confirm the proteomic data and provide further evidence that stromal cell FKBP52 expression is compromised in uteri lacking Hoxa10.
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Progesterone and Estrogen Differentially Regulate FKBP52 Expression in the Uterus
Our results showing differential expression of FKBP52 on d 1 and d 4 of pregnancy suggested that this gene is regulated in the uterus by ovarian steroids in a cell-specific manner. Therefore, we further examined the expression of this gene in a more defined system, i.e. in ovariectomized mice after steroid hormone treatment. In situ hybridization was performed to determine uterine cell-specific expression of FKBP52 in response to the ovarian steroid hormones P4 and/or estrogen (Fig. 6A). We observed that FKBP52 expression is low in uteri of ovariectomized mice treated with oil (vehicle control) and that this expression is primarily restricted to the luminal and glandular epithelia with low to undetectable expression in the stroma. An injection of 17ß-estradiol (E2, 100 ng/mouse) up-regulated the expression level by 12 h with the pattern remaining unaltered. In contrast, an injection of P4 (2 mg/mouse) showed a prominent shift in the expression pattern from the luminal epithelium to the stromal cells at 12 h (Fig. 6A
); the expression was down-regulated by 24 h (data not shown). A combined injection of E2 and P4 showed an expression pattern similar to that of P4 alone but at somewhat lower levels (Fig. 6A
). Interestingly, P4 treatment markedly down-regulated the E2 induced epithelial expression. Comparative RT-PCR analysis of total RNA isolated from uteri harvested from similarly treated mice confirmed the in situ hybridization results, showing an increase of FKBP52 expression in E2, P4, and E2 plus P4 treatment at 12 h, with a return to basal levels at 24 h (Fig. 6B
). To see whether steroid hormones influence FKBP52 expression pattern in Hoxa10 mutant mice, ovariectomized mutant mice were similarly treated with P4 and/or E2. The results show that the pattern of FKBP52 expression mostly remains similar to those in wild-type mice at 12 h. However, FKBP52 expression levels in Hoxa10 mutant uteri are lower with the exception of its higher levels in the epithelium after E2 treatment similar to that observed in wild-type mice (Supplemental Fig. 1 published as supplemental data on The Endocrine Societys Journals Online web site at http://mend.endojournals.org). These results suggest that Hoxa10 influences P4 regulation of FKBP52 in the stroma.
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DISCUSSION |
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Thus, our objective in this study was to identify signaling pathways related to Hoxa10 in the uterine stroma using 2D DIGE as a global proteomics approach because it is a valuable tool with which to generate a landscape of protein expression profiles. Our results show at least 36 proteins within the resolving power of the gels that are differentially expressed between Hoxa10/ and wild-type uterine stromal cells with statistical confidence within the 99th percentile confidence interval (Students t test, n = 3). This expression profile led us to focus on the relationship of FKBP52 with Hoxa10 and its regulation in the uterus with respect to uterine biology and implantation. In adult mice, P4 priming is an absolute requirement for stromal cell proliferation in response to estrogen. Because this proliferation also requires the expression of Hoxa10, the down-regulation of FKBP52 in Hoxa10/ mice reveals a potential downstream and integral target of Hoxa10 in this process. Steroid receptors are dependent on interactions with chaperone and cochaperone proteins to maintain their functions (29). Hormone-free steroid receptors assemble into heterooligomeric complexes with several molecular chaperone components, including the major heat shock protein Hsp90 and Hsp90-associated cochaperones such as FKBP52 or related immunophilins. The Hsp90-binding immunophilins are characterized by a tetratricopeptide repeat domain that targets the immunophilin to Hsp90-receptor complexes, plus a peptidylprolyl isomerase domain to which FK506 or other immunosuppressant drugs bind (30). FKBP52 and FKBP51 compete for a common binding site on Hsp90 and thus compete for assembly with receptor complexes. FKBP52 can elevate the hormone-binding affinity of glucocorticoid receptor in an Hsp90- and peptidylprolyl isomerase-dependent manner; conversely, FKBP51 can reverse the action of FKBP52 and revert glucocorticoid receptor to a lower affinity (23). FKBP51 expression is directly inducible by glucocorticoids (31) or P4 (32, 33), and hormone-stimulated expression of this inhibitor can antagonize the actions of FKBP52, thus reducing secondary responses to hormones. Studies on New World primates, which display multihormone resistance and constitutively overexpress FKBP51, lend physiological support to the impact of FKBPs on steroid hormone receptors (34, 35). In a cDNA microarray approach to examine P4-induced genes in the uterus of ovariectomized mice, FKBP51 message was rapidly elevated followed by a subsequent elevation in FKBP52 expression (36). The lagging up-regulation of FKBP52, presumably resulting from P4-mediated induction of Hoxa10, might serve in part to compensate for elevated FKBP51 expression and maintain PR sensitivity in uterine stromal cells. Indeed, our observation of the localization of FKBP52 in the uterus correlates well with that of PR (37). Estrogen has been shown previously to regulate FKBP52 expression in MCF-7 breast cancer cells (38).
The unique temporal and cell-specific localization of FKBP52 in the mouse uterus during the periimplantation period suggests that this immunophilin is intimately involved with the dynamic changes in the uterus that occur during this period under the influence of ovarian steroids and locally produced paracrine, juxtacrine, and/or autocrine factors. Because P4 effects are correlated with epithelial cell differentiation, but stromal cell proliferation on d 4, it is speculated that FKBP52 expression in the epithelium and stroma directs these events in the context of cell types. This is consistent with similar expression pattern of nuclear PR in the uterus on this day (37). This speculation is consistent with reduced FKBP52 expression in Hoxa10/ mice with reduced stromal cell proliferation. The expression of PR, Hoxa10, and FKBP52 at similar sites of the stroma surrounding the implanting blastocyst on d 5 morning also suggest that Hoxa10, in association with FKBP52 under the influence of P4, primarily promotes stromal cell proliferation, which is necessary for subsequent decidual growth. This is supported by the findings that implantation and decidualization are defective in the uterus lacking Hoxa10 (7, 9). Expression of Hoxa10 and FKBP52 in peripheral cells of the SDZ, especially at the mesometrial pole, again suggests that FKBP52-Hoxa10 signaling influences proliferation of decidualizing stromal cells. However, the potential function of FKBP52 and Hoxa10 that are expressed in the undifferentiated stroma on d 8 is not clearly understood. It is unknown how the decidual cell growth is restricted, leaving a layer of undifferentiated stromal cells underneath the myometrium. Our recent results suggest that Wnt signaling plays a role in creating such a boundary in the uterus to prevent undifferentiated stromal cells from decidualization and restoring these undifferentiated precursor stromal cells for providing normal stromal tissue after the pregnancy is terminated (39). Whether FKBP52 in collaboration with Hoxa10 also plays a role in this event warrants further investigation.
Because P4 priming of the uterus is a requirement for estrogen to initiate implantation in mice and because P4 responsiveness is compromised in Hoxa10/ mice (9), the down-regulation of FKBP52 in stromal cells lacking Hoxa10 suggests a functional role for a Hoxa10-FKBP52 signaling axis in uterine receptivity and implantation. This is consistent with a recent global gene expression profiling study that has shown initial down-regulation followed by up-regulation of FKBP52 expression in the mouse uterus after P4 treatment (36). In this respect, the ovariectomized mouse model provides a valuable experimental paradigm to study the effects of estrogen and/or progesterone on gene expression. Differential steroid hormonal regulation of FKBP52 expression in a cell-specific pattern elucidates a possible signaling pathway between the epithelial and stromal compartments. Our findings of P4 regulation of FKBP52 expression in the stroma and that of estrogen in the epithelium suggest that different regulatory mechanisms are involved for differential cell-specific regulation. P4 effects in the uterus are mediated by both PR-A and PR-B. Whether P4 effects on uterine FKBP52 expression are mediated by PR-A, PR-B, or both will require further investigation. Nonetheless, the observation of differential regulation of FKBP52 by estrogen and P4 points toward a role for FKBP52 in epithelial-mesenchymal cross-talk to mediate a full complement of uterine functions in response to ovarian steroid hormones. Our findings of lower levels of FKBP52 expression in Hoxa10 mutant uteri after P4 treatment also suggest that optimal regulation of FKBP52 in the stroma by this steroid requires the presence of Hoxa10.
To elucidate a definitive role for FKBP52 in the uterus, however, requires genetic ablation of the gene. Indeed, FKBP52 mutant mice have recently been generated, and preliminary characterization shows that homozygous deletion of this gene results in both male and female sterility. Female FKBP52/ mice appear physically normal, but are infertile. Further analysis has shown that ovaries of FKBP52 null mice appear morphologically normal with full oogenesis and follicular development. Furthermore, female mutant mice manifest normal ovulation and fertilization, and homozygous null embryos develop to the blastocyst stage. However, no sign of implantation or decidual swellings are apparent on d 8 of pregnancy, suggesting either failure of implantation or failure of decidualization immediately after implantation (D. Smith, manuscript in preparation). Therefore, infertility associated with these mice probably does not result from defects in oogenesis, ovulation, or fertilization, but could potentially occur at the implantation stage. This provides further evidence that the Hoxa10-FKBP52 axis is critical to implantation and decidualization.
This study recognizes proteomics analysis as a powerful tool with which to determine relevant players, which then can be further expounded by genetic and molecular approaches. The availability of FKBP52 knockout mice will shed light regarding multifaceted roles of this protein in uterine receptivity, implantation, and decidualization. In this study, we also found other differentially regulated proteins between wild-type and Hoxa10/ stromal cells. Future studies will clarify the role of these differentially expressed proteins in the mouse stroma and perhaps provide deeper insights into our understanding of uterine biology during preimplantation and decidualization.
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MATERIALS AND METHODS |
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Primary Culture of Uterine Stromal Cells
Procedures for the isolation and culture of mouse uterine stromal cells, collected on d 4 of pseudopregnancy, followed the previously described methods with minor modifications (17, 18). In brief, uteri were cleaned of fat tissue, slit longitudinally, and cut into small pieces (23 mm). Uterine pieces were then washed several times with phenol red-free Hanks balanced salt solution (HBSS, Life Technologies, Gaithersburg, MD) with 100 µg/ml streptomycin, 100 U/ml penicillin, and 2.5 µg/ml amphotericin B (Sigma Chemical Co., St. Louis, MO). Tissues were digested with 6 mg/ml dispase (Life Technologies) and 25 mg/ml pancreatin (Sigma) for 1 h at 4 C, 1 h at room temperature, and 10 min at 37 C. After these digestion steps, tissues were immediately diluted in HBSS containing 10% fetal bovine serum (FBS) and mixed thoroughly to dislodge the sheet of luminal epithelial cells. The remaining tissues were washed twice in fresh medium (HBSS + antibiotics) and incubated again for subsequent digestion in fresh medium containing 0.5 mg/ml collagenase at 37 C for 30 min. At the end of digestion, tissues were immediately diluted in HBSS with 10% FBS and mixed thoroughly using a 25-ml pipette. The digested cells (primarily containing stromal cells) were passed through a 70-µm nylon filter to eliminate any remaining clumps of epithelial cells and centrifuged. The pellet was washed twice with HBSS before the initiation of primary culture. Cells were plated and cultured in phenol red-free DMEM and Hams F-12 nutrient mixture (1:1) with 10% charcoal-stripped serum and antibiotics. After an initial incubation for 1 h, the medium was removed along with free-floating cells, and the cells that remained adhered to the culture dishes were transferred to fresh medium (DMEM-F-12, 1:1) containing 10% charcoal-stripped FBS and antibiotics and cultured for 24 h in the absence of any steroid hormone.
2D DIGE
Cultured cells were washed with prechilled 1x PBS three times. RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.5% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8)] was then added to make cell lysates. Cell lysates were centrifuged at 13,800 x g for 10 min at 4 C. Protein concentrations were measured using Bio-Rad Dc protein assay reagents (Bio-Rad Laboratories, Richmond, CA) in a photospectrometer. The mixed internal standard methodology (13, 42) was used in these studies with the following modifications. Six independent protein isolations were prepared from both wild-type and Hoxa10/ mouse uterine stromal cells and cultured for 24 h as described above. Due to limited availability, all six Hoxa10/ sample extracts were pooled (342 µg total) before precipitation with methanol-chloroform and resuspended in 171 µl buffer [7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPS), 30 mM Tris, 5 mM magnesium acetate] for a final concentration of 2 mg/ml. To be consistent, all six wild-type samples were similarly pooled (763.5 µg total) before precipitation and resuspension in 381.75 µl buffer. For each pooled sample, three independent 50 µl (100 µg) aliquots were labeled with either Cy3 or Cy5 (N-hydroxysuccinimidyl ester derivatives, GE Healthcare, Milwaukee, WI) as described below. The remaining Hoxa10/ sample (21 µl, 42 µg) was then combined with 129 µl of wild-type sample (258 µg) to produce a 14:86 mixed-sample internal standard (300 µg total). Each 50 µl (100 µg) Hoxa10/ and wild-type sample was separately labeled with 200 pmol of either Cy3 or Cy5 DIGE fluors (2 µl of a 100 pmol working solution in dimethyl formamide), and the 14:86 mixed-sample internal standard (300 µg total) was labeled with 600 pmol of Cy2. The stoichiometry of Cy-dye labeling was purposefully low to ensure equal labeling of the same protein from each extract, and the dyes exhibit a sensitivity that is at least that of conventional silver staining (1 ng) under these conditions (43). Labeling was performed for 30 min on ice in the dark, after which the reactions were quenched with 2 µl of 10 mM lysine for every 200 pmol Cy-dye for 10 min on ice in the dark, followed by the addition of an equal volume of 2x rehydration buffer [7 M urea, 2 M thiourea, 4% CHAPS, 4 mg/ml dithiothreitol (DTT)] supplemented with 0.5% immobilized pH gradient (IPG) buffer 47. The samples were combined into tripartite Cy2/Cy3/Cy5-labeled mixtures such that each mixture had a wild-type 100 µg sample, a Hoxa10/ 100 µg sample, and 100 µg of the Cy2-labeled internal standard (Fig. 2
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The three sets of tripartite-labeled samples were each brought to 450 µl final volume with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2 mg/ml DTT, 0.5% IPG buffer 47, trace bromophenyl blue, BPB) and were passively rehydrated into 24 cm 47 IPG strips (Amersham Biosciences, Arlington Heights, IL) for 28 h at room temperature in the dark. First-dimension isoelectric focusing was carried out for a total of 91 kVh, followed by second-dimension 12% SDS-PAGE using IPGphor and Ettan DALT 12 units, respectively (GE Healthcare). The isoelectrically focused samples were reduced and alkylated with DTT and iodoacetamide and equilibrated into the second dimension-loading buffer (glycerol, SDS, urea, trace BPB) according to the manufacturers protocol. Second-dimension SDS-PAGE gels were precast with low-fluorescence glass plates, with one glass plate presilanized (bind-silane, GE Healthcare) to affix the polymerized gel to only one of the glass plates.
The Cy2, Cy3, and Cy5 components of each gel were individually imaged using mutually exclusive excitation/emission wavelengths of 488/520 nm for Cy2, 532/580 nm for Cy3, and 633/670 nm for Cy5 using a Typhoon 9410 Variable Mode Imager (GE Healthcare). After imaging for Cy-dye components, the nonsilanized glass plate was removed, and the gels were fixed in 50% methanol, 7% acetic acid for 2 h, and then incubated in Sypro Ruby in the dark overnight. This poststain visualizes approximately 97% of the unlabeled protein and ensures accurate protein excision, as the molecular weight and hydrophobicity of the Cy-dyes can influence protein migration during SDS-PAGE. Sypro Ruby images were acquired on the same imager using 457/610 nm wavelengths, as well as reimaged after excision to ensure accurate protein excision.
DIGE Analysis
DIGE technology provides relative quantification and statistical confidence in abundance changes of thousands of proteins resolved by 2D-gel separations (12, 13, 42, 43, 44, 45). Using a pooled-sample internal standard present on each DIGE gel for normalization, many samples can be quantitatively intercompared across multiple DIGE gels that are run coordinately (13, 42), allowing for experimental replicates to be run simultaneously for statistical confidence.
DeCyder 5.02 software (Amersham Biosciences) was used for simultaneous comparison of abundance changes across all three sample pairs with statistical confidence. The DeCyder Differential In-gel Analysis (DIA) module was used for background normalization and triple codetection of the Cy2/Cy3/Cy5 signals for each resolved protein spot feature, after which Cy3:Cy2 and Cy5:Cy2 volume ratios were calculated. These ratios were used by the DeCyder Biological Variation Analysis module, which simultaneously matched all nine protein-spot maps from the three gels, and calculated average abundance changes and Students t test P values for the variance of these ratios for each protein across all three DIGE gels. With this experimental design, all quantitative information is measured relative to the same internal standard present on all three gels, thereby allowing for the quantitative intercomparison of samples between the DIGE gels. Fold-abundance changes are reported, whereby a fold increase is calculated directly from the Hoxa10/:Hoxa+/+ volume ratio, and a fold decrease = 1/volume ratio.
Protein Identification by MS and Database Interrogation
Proteins of interest were robotically excised and processed for MALDI-TOF MS and TOF/TOF tandem MS using an Ettan Spot Handling Workstation (GE Healthcare). Briefly, gel plugs were excised and equilibrated with 50 mM ammonium bicarbonate and dehydrated with acetonitrile in a 96-well plate format. The proteins in the dehydrated gel plugs were digested in gel with 10 µl porcine modified trypsin protease (Promega Corp., Madison, WI) in 20 mM ammonium bicarbonate for 3 h at 37 C, and the resulting proteolytic peptides were extracted in two cycles of 60% acetonitrile-0.1% trifluoroacetic acid and dried. Peptides were reconstituted in 5 µl 60% acetonitrile-0.1% trifluoroacetic acid, and 0.5 µl of this mixture was applied to a MALDI target and mixed on target with 0.5 µl of -cyano 4-hydroxycinnamic acid matrix (5 mg/ml in 60% acetonitrile-0.1% trifluoroacetic acid, supplemented with 1 mg/ml ammonium citrate). MALDI-TOF MS and TOF/TOF tandem MS were performed on a Voyager 4700 (Applied Biosystems, Foster City, CA) using data-dependent tandem MS acquisition on the 10 most abundant ions present in each MALDI-TOF peptide mass map. MALDI-TOF peptide mass maps and accompanying tandem mass spectra were then collectively searched against the SWISS-PROT and NCBInr databases using GPS Explorer software (Applied Biosystems) running the MASCOT database search engine (Matrix-Science). MALDI-TOF peptide mass maps were internally calibrated to within 20 ppm mass accuracy using trypsin autolytic peptides (m/z = 842.51 and 2211.10). Searches were performed without constraining protein molecular weight or isoelectric point, and allowed for carbamidomethylation of cysteine, partial oxidation of methionine residues, and one missed trypsin cleavage. Identifications were accepted based on a tripartite evaluation that takes into account significant molecular weight search (MOWSE) scores, spectrum annotation, and observed vs. expected migration on the 2D gel (42).
Uterine Expression and Hormonal Regulation of FKBP52
In situ hybridization and immunohistochemistry were employed to examine the cell-specific expression of FKBP52 mRNA and protein, respectively, in sections of d 4 pseudopregnant uteri from wild-type or Hoxa10/ mice. To analyze Hoxa10 and FKBP52 expression in the uterus during the periimplantation period, wild-type uteri were collected in the morning (09001000 h) of d 1, d 4, d 5, and d 8 of pregnancy for in situ hybridization. To determine whether FKBP52 was regulated by E2 and P4, wild-type, PR/, ER/, or Hoxa10/ mice were ovariectomized and rested for 2 wk. They were treated with an injection of sesame oil (vehicle control; 0.1 ml/mouse), E2 (100 ng/mouse), P4 (2 mg/mouse), or a combination of E2 and P4. Mice were killed at indicated times, and uteri were collected for in situ hybridization and comparative RT-PCR.
Hybridization Probes
The cDNA clones for FKBP52 were generated by RT-PCR cloning with specific primers. The cDNA clone for Hoxa10 has been described previously (11). For in situ hybridization, sense and antisense 35S-labeled cRNA probes were generated using Sp6 and T7 polymerases, respectively. Probes had specific activities of approximately 2 x 109 dpm/µg.
In Situ Hybridization
In situ hybridization was performed as previously described by us (27). In brief, frozen sections (12 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% paraformaldehyde in PBS. After prehybridization, the sections were hybridized at 45 C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense or sense cRNA probes. Ribonuclease A-resistant hybrids were detected by autoradiography. Sections were poststained with eosin and hematoxylin. Sections hybridized with the sense probes did not exhibit any positive signals and served as negative controls.
Immunohistochemical Staining
Cultured mouse uterine stromal cells were stained for desmin- and vimentin-specific stromal cell markers in culture (20, 21). Cells were seeded in the chamber slides and cultured for 24 h and fixed in 10% formalin buffer. After washing in PBS, cells were immunostained by using an antibody against desmin (1:200) or vimentin (1:200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The localization of FKBP52 in formalin-fixed paraffin-embedded uterine sections was achieved by following the protocol as previously described (19, 27). Immunostaining was performed using a polyclonal (rabbit) antibody to FKBP52. A Histostain-SP kit (Zymed Laboratories, Inc., South San Francisco, CA) was used for staining. Red deposits indicated the sites of positive immunostaining.
Western Blotting
Proteins were extracted from whole uterus on d 4 of pseudopregnancy and on d 1, d 4, d 5, and d 8 of pregnancy by homogenization in RIPA buffer containing proteinase and phosphatase inhibitor. The homogenates were centrifuged at 9800 x g for 10 min at 4 C. The supernatants were separated and their protein concentrations measured. The supernatants (50 µg protein) were then boiled for 5 min in SDS sample buffer. The samples were run on 10% SDS-PAGE gels under reducing conditions and transferred onto nitrocellulose membranes. The membranes were blocked with 10% milk in Tris-buffered saline-Tween 20 (TBST) for 1 h at room temperature and then incubated in 1% milk containing anti-FKBP52 antibodies (1:5000) overnight at 4 C. After incubation, membranes were washed three times (15 min each) with TBST, incubated with goat antirabbit IgG conjugated with horseradish peroxidase (1:20,000) in 1% milk at room temperature, and washed three times (15 min each) with TBST. The bands were detected using an enhanced luminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL).
RT-PCR
Total RNA was extracted from mouse uteri using Trizol according to the manufacturers instruction. Reverse transcription with oligo(dT) priming was performed to generate cDNAs from 4 µg total RNA using Superscript II following the instruction provided by the manufacturer. DNA amplification was carried out with Taq DNA polymerase (Invitrogen, San Diego, CA) using the following primers: FKBP52 (437 bp), 5'-AGT GTG GGG AAG GAG AGG TT-3' and 5'-GCT CTT GCC AGG TCA AAG TC-3'; rPL7 (246 bp), 5'-TCA ATG GAG TAA GCC CAA AG-3' and 5'-CAA GAG ACC GAG CAA TCA AG-3'. PCR conditions were 95 C for 5 min and then 23 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 45 sec, followed by incubation at 72 C for 10 min for FKBP52 amplification. For rPL7, amplification was performed at 95 C for 5 min and then 23 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 45 sec, followed by incubation at 72 C for 10 min. Amplified fragments were separated by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. The intensity of each band was measured by Scion Image (Scion Corp., Frederick, MD), and intensity of FKBP52 was corrected by the intensity of rPL7.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 4, 2004
Abbreviations: CHAPS, 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; 2D, two-dimensional; DIGE, difference gel electrophoresis; DTT, dithiothreitol; E2, 17ß-estradiol; FBS, fetal bovine serum; FKBP52, FK506 binding protein 4; HBSS, Hanks balanced salt solution; Hsp, heat shock protein; IPG, immobilized pH gradient; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; P4, progesterone; PDZ, primary decidual zone; PR, progesterone receptor; SDS, sodium dodecyl sulfate; SDZ, secondary decidual zone; TBST, Tris-buffered saline-Tween 20; TOF, time of flight.
Received for publication August 24, 2004. Accepted for publication October 27, 2004.
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REFERENCES |
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