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Female Reproductive Tract |
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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endometrium, gene regulation, Jak/Stat, prolactin, rabbit, RUSH, secretoglobin, signal transducers, steroid hormones, uteroglobin, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the rabbit uterus, prolactin augments the progesterone-dependent increase in uteroglobin mRNA [4]. The search for responsible transcription factors [5] culminated in the cloning and characterization of RUSH [6]. The acronym identified two alternatively spliced rabbit uteroglobin promoter-binding proteins, RUSH-1
and ß, as SWI/SNF-related helicases/ATPases. Symbols that were later adopted for the human (SMARCA3), and mouse (Smarca3) orthologs, identified some of the same characteristics as the RUSH acronym: SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 3. When the human nomenclature is applied to the rabbit ortholog, the gene name is rabbit SMARCA3, and RUSH is retained as the trivial name.
RUSH is the only SMARCA3-related gene known to be regulated by steroids. Northern analysis showed that progesterone treatment increased the uterine content of RUSH message. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and ion-pair reversed-phase HPLC analysis confirmed a steroid-dependent alternative splicing mechanism [6] in which RUSH-1 is the progesterone-dependent splice variant and RUSH-1ß is the estrogen-dependent splice variant. Interrogation of the proximal promoter of the RUSH gene revealed a progesterone-responsive region that harbors an overlapping progesterone receptor half-site (PRE) and a nuclear factor Y (NF-Y) consensus site [7].
RUSH then is a progesterone-regulated, SWI/SNF-related transcription factor that binds to the proximal promoter of the uteroglobin gene. The target search was initially reduced from –170/–85 to –160/–110 [8]. Although this region was large and complex, the absence of Stat5a binding elements was compelling evidence that the prolactin receptor activated a different intracellular transducer. Cyclic amplification and selection of targets was used to identify the RUSH binding site (–126/–121), and chromatin immunoprecipitation confirmed site-specific binding of RUSH to the transcriptionally active promoter [9]. Transient transfection assays with mutant constructs showed that the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcriptional activation of the gene [9]. The demonstration that RUSH is a prolactin signal transducer and speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TriReagent was purchased from Molecular Research Center Inc. (Cincinnati, OH). Human chorionic gonadotrophin (hCG), agarose-conjugated alkaline phosphatase, and sodium orthovanadate were purchased from Sigma Chemical Company (St. Louis, MO). Protein tyrosine phosphatase (PTPase), the catalytic subunit of protein phosphatase 1 (PP1), and okadaic acid were purchased from Roche Applied Sciences (Indianapolis, IN). The Phosphoprotein Antibody Sampler Pack was purchased from Zymed Laboratories Inc. (South San Francisco, CA). The NucleoTrap Nucleic Acid Purification Kit was purchased from Clontech Laboratories Inc. (Palo Alto, CA). The ReactionReady First Strand cDNA Synthesis Kit C-01 and the Human MultiGene-12 RT-PCR Profiling Kit for the JAK/STAT Signaling Pathway were purchased from SuperArray Bioscience Co. (Frederick, MD). Human Discover Chips were purchased from TeleChem International Inc. (Sunnyvale, CA). The CyScribe Post-Labeling Kit was purchased from Amersham Biosciences (Piscataway, NJ). TranSignal TF-TF Interaction Arrays (MA5010 and MA5011) were purchased from Panomics (Redwood City, CA). Nitrocellulose transfer/immobilization membranes were purchased from Schleicher and Schuell (Keene, NH). Renaissance Western Blot Chemiluminescence Kit was purchased from NEN Life Science Products Inc. (Boston, MA).
Affinity-purified RUSH-1(ß) [6] and RUSH-1 [9] antipeptide antibodies have been extensively characterized. For immunoprecipitation, pooled aliquots of these antibodies (600 µl) were dialyzed against two changes x 1 L each of TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at 4°C. The final concentration of the antibody was 1 mg/ml.
Animal Treatments
All studies were conducted according to the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center. Adult female New Zealand white rabbits (6 mo of age) were housed for 3 wk before experimentation.
For RNA isolation, eight rabbits were divided into two groups. One group of animals (n = 3) was used as estrous controls. The second group of animals (n = 3) was made pseudopregnant (PSP) by intravenous injection of 15–20 IU hCG and cervical stimulation. These PSP rabbits were killed 5 days after treatment, and ovulation was confirmed by the presence of corpora lutea at necropsy. The third group of animals was treated with progesterone (3 mg/kg/24 h) for 4 days and killed 24 h after the last injection. Ethanol:corn oil (50:50) was used as the vehicle. For nuclear extract preparations, one estrous rabbit was treated with progesterone for 5 days and killed 24 h after the last injection.
RNA Isolation
Total RNA was isolated from the endometrium of the two progesterone-treated rabbits by the cold precipitation method of Han et al. [10]. Total RNA was isolated from the endometrium of the estrous and PSP rabbits with TriReagent according to the manufacturer's instructions. The integrity of each RNA sample was confirmed by electrophoretic fractionation through formaldehyde-containing agarose gels (1.2%) and ethidium bromide staining. Poly(A+)RNA was isolated with the NucleoTrap Acid Purification Kit. RNA concentrations were determined spectrophotometrically (A260). The A260/280 ratio for each sample was >1.8.
Kits and Previously Described Assays
To profile members of the JAK/STAT pathway, cDNA templates were prepared from poly(A+)mRNA with the ReactionReady First Strand cDNA Synthesis Kit according to the manufacturer's instruction. RNA samples from three animals were pooled for use in each synthesis reaction. Resultant cDNA templates were used with the Human MultiGene-12 RT-PCR profiling kit according to the manufacturer's instructions. TransSignal TF-TF Interaction arrays were used as previously described by Hewetson and Chilton [7]. Nuclear extract proteins were prepared from the endometrium as described by Kleis-SanFrancisco et al. [5]. UG200 (–194/+9) was 3'-end labeled on the coding strand at an XbaI site with [-32P]dCTP using the Klenow fragment of DNA polymerase I to a specific activity of 1–2 x 107 cpm/µg. Binding reactions were performed as previously described [5].
Dephosphorylation, Immunoprecipitation, and Western Analysis
In vitro dephosphorylation of nuclear extract proteins was achieved with three different enzymes and validated with specific inhibitors. Briefly, 100-µg aliquots of nuclear extract proteins were treated with 1) 4 U alkaline phosphatase at 37°C to remove all phosphate groups, 2) 16 mU PTPase ± sodium orthovanadate (250 µM) at 37°C to selectively remove phosphate groups bound to tyrosine residues, or 3) 2 mU PPI ± okadaic acid (2.5 picomoles) at 30°C to selectively remove phosphate groups bound to serine/threonine residues. Assay temperatures were predetermined by the manufacturer. Optimal amounts of enzyme and incubation times were determined empirically. Convenient incubations that resulted in complete dephosphorylation were conducted in a time range from 6 h to overnight.
Nuclear extracts (10 µg) were incubated overnight at 4°C with 10-µl aliquots of affinity-purified RUSH antibodies, followed by incubation for 2 h at 4°C with a 50% slurry of protein A-Sepharose. Immunoprecipitated proteins and molecular size standards were fractionated by SDS/PAGE on 10% minigels and transferred to nitrocellulose membranes. Membranes were processed for Western analysis. Briefly, membranes were blocked in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.6) with 0.1% Tween 20 (TBST) and 2% powdered milk. Membranes were incubated overnight at 4°C in the same buffer containing horseradish peroxidase-conjugated rabbit antiphosphotyrosine (2 µg/ml) antibodies and washed 3 x 30 min in TBST. RUSH-specific phosphotyrosine signals were detected by chemiluminescence.
Gene Expression Profiling
Individual RNA samples were processed for interspecies microarray hybridization with Human Discover Chips, which profile 380 major genes from 30 functional groups. The CyScribe Post-Labeling Kit was used to generate CyDye-bound cDNA probes from amino allyl-labeled cDNAs synthesized from poly(A+)RNA samples. To compare hormone effects, two fluorescent dyes, Cy3 and Cy5, were used to generate relative expression measures in directly competitive hybridization assays. For these assays, probes from an estrous animal were labeled with Cy3 (excitation wave length 532 nm; green fluorescence) and probes from a progesterone-treated animal were labeled with Cy5 (excitation wave length 633 nm; red fluorescence). To control for dye bias, probes from a prolactin + progesterone-treated animal were labeled with Cy3. For dual-color microarray hybridizations (42°C), two probes were combined in the hybridization solution. This permitted simultaneous detection of hybridization signals and comparative analysis of gene expression levels. Microarrays were scanned and quantified with the ScanArray Express Laser confocal scanner and data were analyzed with the GeneTraffic Duo microarray analysis tools.
Transcription Factor (TF) Interaction Arrays
TransSignal TF-TF Interaction Array I was processed according to the manufacturer's instructions. Briefly, 50 µg of nuclear extract protein from the progesterone-treated rabbit was incubated with biotin-labeled double-stranded oligonucleotide probes, i.e., a library of cis-elements. RUSH plus affiliated transcription factors were immunoprecipitated with a cocktail (1 µg) of affinity-purified RUSH antibodies and magnetic protein G beads. For the negative control, the antibodies were replaced with normal IgG. Free cis-elements and nonspecific binding proteins were washed away. RUSH-affiliated biotin-labeled probes were eluted from the magnetic beads and hybridized to TranSignal Protein/DNA Array membranes. The genes on the array were spotted in duplicate at a specific concentration and again at a 1:10 dilution. This approach produced an interaction profile for RUSH and 54 different transcription factors, including Stat 1, 3, 4, 5, and 6.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Stat expression was evaluated with Human Discover Chips. These microarrays were made with 70-mer sense oligonucleotides for 380 genes printed in duplicate. Hybridization of full-length DNA probes across the entire length of long single-stranded targets for Stats 1, 2, 3, 4, and 5a enhanced gene specificity. Dual-probe hybridization allowed ratio analysis of experimental/control signals for each gene on each array. To compare hormone effects, two fluorescent dyes, Cy3 and Cy5, were used to generate replicate expression measures in two individual hybridization assays. Probes from an estrous animal were labeled with Cy3 (excitation wave length 532 nm; green fluorescence) and probes from a progesterone-treated animal were labeled with Cy5 (excitation wave length 633 nm; red fluorescence). Red and green channels were displayed in a single image or two-color overlay, in which changes in gene expression appeared as red or green spots against a background of yellow spots that correspond to transcripts that are present at equal concentrations in the two samples. As shown in Figure 2, Stat5a as well as such genes as cyclin-dependent kinase 5 (CDK5) are constitutively expressed in the rabbit endometrium. Stats 1–4 were also constitutively expressed (data not shown). The hybridization of two additional microarrays with Cy3-labeled probes from a prolactin + progesterone-treated animal and Cy5-lableled probes from a progesterone-treated animal increased the reliability of the microarray results and confirmed that Stat5a was constitutively expressed (data not shown) regardless of the hormonal milieu.
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Potential physical associations between RUSH and non-RUSH proteins, especially Stat5, were evaluated with TransSignal TF-TF Interaction Arrays. Nuclear extract, the source of RUSH and putative binding partners, was incubated with a library of cis-elements. RUSH-affiliated transcription factors with their corresponding cis-elements were coimmunoprecipitated with RUSH antibodies. Cis-elements were eluted and hybridized to arrays. As shown in Figure 3, there is no physical interaction between RUSH and Stat5. In fact, there was no physical affiliation between RUSH and any of the other Stat family members (Stats 1, 3, 4, 5, and 6) tested (data not shown). In contrast, a strong physical interaction occurred between RUSH and the nuclear phosphoprotein, early growth response protein 1 (Egr-1).
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The potential requirement for RUSH proteins to be phosphorylated to bind to DNA was evaluated in gel-shift assays. Optimum conditions for the dephosphorylation of native RUSH in nuclear extracts with alkaline phosphatase, serine-threonine-specific phosphoprotein phosphatase 1 (PP1) ± okadaic acid, and protein tyrosine phosphatase (PTPase) ± sodium orthovanadate were determined empirically. Test and control nuclear extracts were immunoprecipitated with RUSH antibodies and immunoblotted with antiphosphotyrosine antibodies to verify treatment effects. As shown in Figure 4, no phosphotyrosine-containing proteins were detectable after nuclear extracts are treated with either alkaline phosphatase or PTPase. Coincubation of nuclear extracts with PTPase and its inhibitor sodium orthovanadate protected against tyrosine-specific dephosphorylation. By comparison, serine-threonine-specific protein dephosphorylation with PP1 had no effect on phosphotyrosine detection. Gel-shift assays confirmed that in vitro dephosphorylation of RUSH in nuclear extracts with either alkaline phosphatase or PTPase selectively inhibited RUSH-DNA complex formation (Fig. 5). Coincubation with the PTPase and sodium orthovanadate preserved RUSH-DNA complex formation. By comparison, serine-threonine-specific protein dephosphorylation with PP1 had little or no effect on RUSH-DNA binding.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR profiling for Jak/Stat pathway-specific genes confirmed the potential for growth factor and cytokine signaling in the endometrium. Amplicons for Jak2 and Stat family members 3, 4, 5a, and 6 were expressed regardless of the hormonal milieu. Subsequent to this survey, interspecies microarrays that were used for differential expression profiling confirmed that Stat5a is constitutively expressed. This finding is not surprising because Stat5a activation in the mammary gland requires neither transcription nor translation. In contrast, combinatorial regulation of RUSH includes robust transcriptional activation by progesterone [7] and steroid-dependent posttranscriptional processing [6].
Although phosphorylation of a single tyrosine residue is required for Stat5a dimerization, serine phosphorylation [13] and threonine glycosylation [14] are also important for Stat5a function. Interactions between Stats and other nuclear factors are central to the integration of several signal-transduction pathways. The synergy between prolactin and glucocorticoid hormones requires the physical interaction of the glucocorticoid receptor with the N-terminus of Stat5a [15]. The p300/CBP, a putative integrator of the cross talk between nuclear receptors and AP-1, interacts with the C-terminus of Stat5a [16]. RUSH also participates in physical interactions with other proteins, including an atypical P-type ATPase in the inner nuclear envelope [17] and the transcription factor GATA-4 [9]. However, the notion of a protein-binding interaction between Stat5a and RUSH was eliminated by the TF-TF assays. This finding would negate the necessity to determine, by some means such as in situ analysis, whether or not RUSH and Stat5a are coexpressed in the same cell type, except the possibility that Stat5a stimulates another factor, which then associates with RUSH, has not been eliminated. Clearly, the identification of a strong physical interaction between RUSH and Egr-1 prompted a reexamination of the region of the uteroglobin promoter (–175/–80) that contains the authentic RUSH site (–126/ –121). Analysis with MatInspector V2.2 [18] failed to identify any Egr-1 consensus sites. Interest in a RUSH-Egr-1 interaction was piqued by the fact that Egr-1 plays an important role in activating the LHß promoter [19] and, in that regard, is critical for female fertility [20]. Egr-1 has been identified in the human [21] and rodent [22] uterus, and it is known to interact physically with the tumor suppressor p53 [23].
Seven primary mechanisms control transcription: Hedgehog, Wnt, transforming growth factor-ß, receptor tyrosine kinase, NOTCH, Jak/Stat, and nuclear receptors [24]. Although these mechanisms involve diverse pathways, they all require ligand-receptor binding and signal-related transcription factors to activate target genes. In the endometrium where Stat5a is present, prolactin activates the uteroglobin gene in the absence of Stat-binding elements. We have proffered Jak/RUSH as an alternative to the Jak/Stat pathway. The final phase of this study showed that tyrosine phosphorylation was required for RUSH-DNA binding, thus adding a further level of complexity to the molecular mechanism of RUSH action. A physical affiliation between Jak2 and RUSH was previously established by coimmunoprecipitation [9]. However, an authentic Jak/RUSH pathway is predicated on the ability of Jak2 to phosphorylate RUSH proteins. The goal of future experiments will be to directly test the hypothesis that Jak2 phosphorylates specific tyrosine residues in RUSH.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Beverly S. Chilton, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th Street—MS 6540, Lubbock, TX 79430. FAX: 806 743 2990; beverly.chilton{at}ttuhsc.edu
Received: 30 April 2004.
First decision: 2 June 2004.
Accepted: 22 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß, two uteroglobin promoter-binding proteins. Mol Endocrinol 1996 10:1335-1349[Abstract]
-mediated signaling. Cell 1999 96:121-130[CrossRef][Medline]This article has been cited by other articles:
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  A. Hewetson and B. S. Chilton Progesterone-Dependent Deoxyribonucleic Acid Looping between RUSH/SMARCA3 and Egr-1 Mediates Repression by c-Rel Mol. Endocrinol., April 1, 2008; 22(4): 813 - 822. [Abstract] [Full Text] [PDF]
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