Molecular Human Reproduction, Vol. 9, No. 10, 611-623,
October 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Promyelocytic leukaemia zinc finger protein (PLZF) is a glucocorticoid- and progesterone-induced transcription factor in human endometrial stromal cells and myometrial smooth muscle cells
Submitted on May 9, 2003; accepted on June 20, 2003
1 IHF Institute for Hormone and Fertility Research, University of Hamburg, 20251 Hamburg, 2 Institute of Pathology, Department of Gynaecopathology, University Hospital of Hamburg, 20246 Hamburg, Germany 3 Present address: ALLERGOPHARMA, Joachim Ganzer KG, 21465 Reinbek, Germany 4 Present address: Endokrinologikum Hamburg, 20251 Hamburg, Germany
5 To whom correspondence should be addressed at: Endokrinologikum Hamburg, Falkenried 88, 20251 Hamburg, Germany. e-mail: gellersen{at}endokrinologikum.com
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Abstract |
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The promyelocytic leukaemia zinc finger (PLZF) protein belongs to the family of Krüppel-like zinc finger proteins. It is a transcriptional repressor involved in cell cycle control and has been implicated in limb development, differentiation of myeloid cells, and spermatogenesis. Little is known about the regulation of PLZF expression. In search for mediators of progesterone signalling in the female reproductive tract, we discovered induction of PLZF mRNA in primary cultures of human endometrial stromal cells and myometrial smooth muscle cells (SMC) in response to progesterone. Surprisingly, dexamethasone was a more potent inducer of PLZF expression than progesterone and elicited a sustained up-regulation of PLZF mRNA levels within 2 h. Immunofluorescence showed localization of PLZF to the nuclei of dexamethasone-treated SMC. In uterine biopsies, nuclear staining for PLZF was found in myometrial cells and endometrial stromal cells of the secretory phase. The transcriptional start site of the PLZF gene was located to position –5801 in SMC. Transfected promoter constructs containing up to 4.1 kb of 5'-flanking DNA were not induced by activated glucocorticoid or progesterone receptor. In contrast, co-transfection of c-jun and c-fos expression vectors resulted in stimulation of reporter gene activity, indicating an involvement of AP-1 transcription factors in PLZF expression.
Key words: endometrium/glucocorticoid/myometrium/PLZF/progesterone
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Introduction |
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The promyelocytic leukaemia zinc finger protein (PLZF) gene (Zfp145) was first identified in acute promyelocytic leukaemia (APL) where a reciprocal chromosomal translocation t(11;17)(q23;q21) results in a fusion with the RARA gene encoding retinoic acid receptor
(RAR) (Chen et al., 1993). The wild-type PLZF gene encodes a transcription factor with nine zinc fingers of the C2-H2 type (Chen et al., 1993) which makes it a member of the Krüppel-like zinc finger protein family (Chowdhury et al., 1987). The PLZF protein contains an N-terminal BTB/POZ (bric-a-brac/tramtrack/broad complex, poxvirus and zinc finger) domain and a second repressor domain (RD2) (Li et al., 1997) which is able to recruit the ETO (for Eight-Twenty One) protein (Melnick et al., 2000). Both PLZF and ETO can bind proteins such as SMRT (silencing mediator for retinoid and thyroid-hormone receptors), N-CoR (nuclear receptor co-repressor), Sin3 and HDACI (histone deacetylase I) to form a multiprotein complex mediating transcriptional repressor function of PLZF (Breems-de Ridder et al., 2000; Melnick et al., 2000). PLZF–/– mice display musculoskeletal-limb defects and impaired spermatogenesis (Melnick and Licht, 1999; Barna et al., 2000). PLZF has been implicated in forebrain organization and hindbrain segmentation (Avantaggiato et al., 1995; Cook et al., 1995) and in haematopoiesis. High PLZF protein levels are present in the nuclei of human CD34+ bone marrow progenitors, but not in mature peripheral blood mononuclear cells (Reid et al., 1995). In addition, PLZF mRNA is readily detectable in human promyelocytic cell lines (KG-1, HL-60, NB-4) and disappears upon all-trans-retinoic acid (RA)-induced differentiation in HL-60 and NB-4 cells, suggesting down-regulation of PLZF expression as a prerequisite for granulopoiesis (Chen et al., 1993). In contrast, the PLZF expression level rises during megakaryocytic development, where it plays a stimulatory role by activating thrombopoietin receptor (TpoR) expression (Labbaye et al., 2002).
The genomic sequence and structure of the PLZF gene have been elucidated (Zhang et al., 1999). The gene contains six exons and five introns and its mRNA, with a length of 
8 kb, includes an unusually long 5'-untranslated region (UTR) of 6 kb entirely located in exon 1 which also encodes the POZ domain, the RD2, a proline-rich region and part of the first zinc finger. Several alternative splicing products lacking different portions of exon 1 have been described, resulting in the absence of the POZ domain, various portions of the proline-rich region, or a large proportion of the 5'-UTR (Zhang et al., 1999).
A PLZF binding motif with similarity to the lex operator sequence was found in the promoter of the cyclin A2 gene which has therefore been proposed as a PLZF target gene (Li et al., 1997; Sitterlin et al., 1997). PLZF binds to and represses the cyclin A2 promoter and is involved in the regulation of cell cycle progression and growth suppression (Yeyati et al., 1999). Furthermore, a PLZF binding site was identified in the promoter of the interleukin-3 receptor (IL-3R) subunit. Overexpression of PLZF in a murine haematopoietic cell line leads to reduced expression of IL-3R (Ball et al., 1999). PLZF may also be involved in establishing Hox gene expression boundaries in the developing limb bud by transcriptional repression of HoxD gene expression (Barna et al., 2002).
In addition to our limited knowledge of PLZF target genes, little is known about the mechanisms regulating PLZF expression. Recently, the binding of the oncoprotein Evi-1 to an Evi-1-like site in the PLZF promoter was shown to be essential for full promoter activity in myeloid cell lines (Takahashi and Licht, 2002). Knowledge of PLZF target genes and regulation of PLZF expression itself are of profound clinical relevance because both fusion products of the t(11;17)(q23;q21) translocation in APL, PLZF-RAR and RAR-PLZF, are critical in developing full-blown leukaemia (He et al., 2000), and this form of APL responds poorly to RA treatment (Guidez et al., 1998). While the DNA-binding specificity of the RAR-PLZF fusion protein is determined by the PLZF zinc fingers, placing PLZF target genes under the control of the RAR transactivation domain, the reciprocal fusion protein PLZF-RAR, produced under the control of the PLZF promoter, presumably causes aberrant regulation of RAR target genes.
In a screening approach to identify Krüppel-like transcription factors from bovine female reproductive tissues, we isolated the bovine homologue of PLZF from an ovarian cDNA library. RNase protection assays indicated that in bovine endometrium, PLZF is expressed in a cycle-dependent manner, increased transcript levels coinciding with elevated progesterone levels (N.Walther, unpublished observation). This prompted us to investigate whether PLZF was expressed in the human endometrium in a progesterone-dependent fashion. Under the influence of luteal progesterone, the human endometrium undergoes a profound process of differentiation which serves to prepare the uterine lining for blastocyst implantation (Lessey, 2000). Other important roles of progesterone in reproduction are the maintenance of quiescence in the myometrium of pregnancy and mammary gland development (Graham and Clarke, 1997; Conneely and Lydon, 2000). Generally, the effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear hormone receptor superfamily. Many responses to progesterone in vivo as well as in cultured primary uterine cells occur in a delayed fashion, pointing against a direct transcriptional effect of liganded PR on a considerable number of presumed progesterone target genes. In addition to up-regulating a panel of genes, progesterone also suppresses the expression of a different set of genes including estrogen receptor (ER) and matrix metalloproteinases (Graham and Clarke, 1997). The molecular mechanisms by which the activated PR controls gene expression are still the subject of investigation (Beato and Klug, 2000; Boonyaratanakornkit et al., 2001; Christian et al., 2002). Notably, the transactivation function of PR can be modulated by interaction with a member of the Krüppel-like family of transcription factors (KLF9) in endometrial epithelial cells (Zhang et al., 2003).
Recently, expression of another member of the steroid hormone receptor family, the glucocorticoid receptor (GR), has been shown in the human endometrium and demonstrated to be restricted to the stromal compartment (Bamberger et al., 2001). Glucocorticoids (GC) act on cell proliferation and differentiation as evidenced by the generation of GR-deficient mice. GR–/– animals die shortly after birth from respiratory failure due to impaired development and maturation of the lung (Cole et al., 1995). In female reproduction, GC play a pivotal role in controlling mammary gland development and milk protein synthesis (Reichardt et al., 2001). In the rat myometrium, GR and 11ß-hydroxysteroid dehydrogenase type 1 are co-localized and might enhance local GC actions to facilitate parturition (Burton et al., 1996). However, the role of GC in regulation of endometrial function remains open to speculation.
The aim of this study was to characterize steroid-dependent regulation of PLZF expression in human uterine cells to elucidate a putative role in the steroid signalling pathway.
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Cell culture
Primary cultures of uterine cells were prepared from tissues obtained from cycling women at the time of hysterectomy for uterine prolapse or leiomyomata. Informed consent was obtained, and the study was approved by the local ethics committee. Purified human endometrial stromal cells (ESC) were prepared from five individuals as previously described (Gellersen et al., 1994). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 g/l glucose)/Ham’s F-12 (Bio Whittaker/Sigma) at a ratio of 1:1, supplemented with 10% dextran-coated, charcoal-treated, steroid-depleted fetal calf serum (DCC-FCS), 100 IU/ml penicillin, 100 mg/ml streptomycin (Life Technologies), 1 mg/ml insulin (Sigma), and 10–9 mol/l 17ß-estradiol (Sigma). Cells of the first passage were used for RNA extraction and transient transfections. Primary cultures of human myometrial smooth muscle cells (SMC) were prepared from nine individuals as reported elsewhere (Bonhoff and Gellersen, 1996). The cells were maintained in growth medium consisting of DMEM/Ham’s F-12, 10% FCS, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 10–9 mol/l 17ß-estradiol.
The KG-1 cell line (human Caucasian bone marrow myelogenous leukaemia, ECACC No. 86111306; European Collection of Cell Cultures, UK) was cultured in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies), 20% FCS, and antibiotics as above. The cell lines HL-60 (human Caucasian promyelocytic leukaemia; ECACCNo. 85011431) and Jurkat (human leukaemic T cell lymphoblast; ECACC No. 88042803) were cultured in IMDM with 10% FCS and antibiotics. The cell line T47D (human breast ductal carcinoma; ECACC No. 85102201) was cultured in DMEM/Ham’s F-12 supplemented with 10% FCS, antibiotics, and 7 µg/ml insulin. For stimulation experiments, FCS in the respective maintenance media was replaced by DCC-FCS, and cells received 2 µmol/l RA, or 250 nmol/l of dexamethasone (DEX), hydrocortisone, medroxyprogesterone acetate (MPA) (Sigma), progesterone (ICN author please give address on first mention) and the antagonists RU486 or ZK98299 (kindly provided by Schering AG, Germany), unless indicated otherwise. Control cultures received vehicle.
RNA isolation, RT–PCR, Southern blot analysis
Total RNA was isolated from cultured cells using the guanidinium isothiocyanate phenol procedure (peqGOLD RNAPureTM; Peqlab) following the manufacturer’s recommendations. First strand cDNA was synthesized from 5 µg total RNA using oligo(dT)-primed extension with SuperscriptTM RT II (Life Technologies). After completion, the 20 µl reaction mix was made up to 100 µl.
The following oligonucleotides were used as PCR primers (positions relative to the translational start site and GenBank accessions or references are given): sense and antisense primers for amplification of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (288/308 and 554/575; HUMGAPDH), for human PLZF cDNA (–75/–54 and 1133/1158) (Chen et al., 1993), for human cyclin A2 cDNA (814/837 and 1190/1213; HSCYCLINA), for human GR cDNA (1827/1847 and 2398/2417) (Hurley et al., 1991). Two different primer pairs were used for human PR cDNA: located in the 5' region specific to PR-B (–117/–94 and 274/294), or in the ligand binding domain (LBD) shared by PR-B and PR-A (2191/2211 and 2608/2631) (Misrahi et al., 1987). One microlitre of cDNA served as a template for PCR amplification. Amplification mode for GAPDH cDNA: 94°C for 4 min, followed by 25 cycles of 95°C for 30 s, 50°C for 30 s, 72°C for 30 s and a final extension at 72°C for 10 min. Amplification mode for PLZF cDNA: 95°C for 5 min, followed by touchdown, three cycles each with annealing temperatures of 72, 69, 66, 63°C and 30 cycles with 60°C for 40 s, denaturation at 94°C for 15 s and extension at 72°C for 1 min, and a final extension at 72°C for 5 min. Amplification mode for cyclin A2 cDNA: 95°C for 4 min, followed by 30 cycles of 94°C for 15 s, 56°C for 30 s, 72°C for 90 s and a final extension at 72°C for 10 min. Amplification mode for PR-LBD: 95°C for 4 min, followed by 30 cycles of 95°C for 45 s, 50°C for 45 s, 72°C for 1 min and a final extension at 72°C for 10 min. Amplification modes for PR-B and GR cDNA: as for PR-LBD but with annealing temperatures of 64°C and 52°C respectively. Amplified products were examined by electrophoresis in 1% agarose gels and were transferred to positively charged nylon membrane (Roche Applied Science, Germany) unless indicated otherwise. Southern hybridization was performed with internal oligonucleotides labelled with terminal deoxynucleotidyl transferase and digoxygenin-11-dUTP and detected with the DIG Luminescent Detection Kit (Roche Applied Science). The following oligonucleotides were used as probes (positions are given relative to the start ATG): hGAPDH sense primer 392/412; hPLZF antisense primer –21/–42; human cyclin A2 antisense primer 1000/1023; hPR-LBD antisense primer 2371/2401; hPR-B sense primer 130/156; hGR antisense primer 1906/1935 (Hollenberg et al., 1985). Direct visualization of PCR products in agarose gels was performed by staining with SYBR Gold (Molecular Probes) and detection in a Typhoon 8600 imager (Molecular Dynamics).
Relative quantitative RT–PCR with 18S internal standard
Total RNA was isolated as described above. Random-primed cDNA was synthesized from 1 µg total RNA with ImProm-IITM Reverse Transcription System (Promega) following the manufacturer’s instructions. The following oligonucleotides were used as PCR primers for human PLZF cDNA: 1400/1423 and 1568/1588 (positions relative to the translational start site) (Chen et al., 1993). Primers for amplification of human 18S cDNA were part of the QuantumRNATM 18S Internal Standards Kit (Ambion). To allow non-radioactive detection, dNTP included dCTP and Cy5-dCTP (Amersham Pharmacia) at a 10:1 ratio. Amplification mode for simultaneous amplification of 18S and PLZF cDNA: 95°C for 3 min, followed by touchdown, three cycles each with annealing temperatures of 69, 66, 63, 60°C and 21 cycles with 60°C for 30 s, denaturation at 95°C for 30 s and extension at 72°C for 30 s, and a final extension at 72°C for 10 min. For controlled reduction of the amplification efficiency of the 18S PCR template, the CompetimerTM technology (Ambion) was employed, mixing the 18S cDNA-specific PCR primers and competimer at a 2:8 ratio. Amplified products were resolved by electrophoresis in 5% TBE gels (Criterion; BioRad), detected in a Storm phosphoimager (Molecular Dynamics) and quantified by densitometry using the NIH Image 1.62 software.
Real time quantitative PCR
One microlitre cDNA prepared by oligo(dT) priming was used for real time quantitative PCR using the LightCycler Instrument and the LightCycler-FastStart DNA Master SYBR Green I Kit (Roche Applied Science). The following oligonucleotides were used as PCR primers: sense and antisense primers for amplification of human GAPDH cDNA as described above, and for human PLZF cDNA 1321/1344 and 1575/1594 (positions relative to the translational start site) located in the 3' region (Chen et al., 1993). Standard curves for both GAPDH and PLZF were generated from dilution series. Real time PCR was performed on both cDNA and standard in order to extrapolate the obtained relative values. For normalization, extrapolated relative abundance values of PLZF were divided by the extrapolated values derived from GAPDH in each corresponding cDNA. To obtain the relative differences in mRNA abundance between the samples, the control sample was designated as 1 and the relative expression levels of the other samples were calculated relative to this sample. The experimental protocol followed the manufacturer’s recommendations with minor modifications of the amplification of the target cDNA: target temperature (°C)/ incubation time (s)/ acquisition mode; 95/10/none, 56/10/none, 72/25/none, 85/5/single.
Northern blot analysis
For Northern blot analysis including gel electrophoresis, transfer and hybridization, the NorthernMaxTM Kit, a formaldehyde-based system, was used. Synthesis of the cRNA probe and non-isotopic labelling was performed by Strip-EZTM RNA and BrightStarTM Psoralen-Biotin Kits, and the non-isotopic detection by BrightStarTM BioDetectTM following the manufacturer’s instructions (all from Ambion). For generation of a template for cRNA synthesis, region –65 to +2056, containing the entire protein coding region of the PLZF cDNA, was amplified from ESC RNA by RT–PCR using primers which introduced a 5'-HindIII and a 3'-XbaI site. The PCR product was restricted with these enzymes and cloned into the respective sites of pGEM-3Zf(+) (Promega) to yield plasmid pGEM-PLZF(2.1). After linearization with ApaI, cRNA was synthesized with T7 RNA polymerase to produce a 1.5 kb probe complementary to the 3'-portion of PLZF mRNA including the proline-rich domain and the zinc finger region. A plasmid containing 400 bp of rat cyclophilin cDNA in pGEM-4 (kindly provided by Dr G.E.DiMattia, London Regional Cancer Center, London, Ontario) was linearized with EcoRI at the 5'-end of the insert and used as a template for cRNA synthesis with T7 RNA polymerase. Both probes and RNA molecular weight markers (0.24–9.49 kb; Life Technologies) were labelled with psoralen-biotin as mentioned above. Total RNA (50 µg/lane) from cultured ESC, SMC and KG-1 cells was electrophoresed along with RNA molecular weight markers in a 1% agarose gel at 23 V overnight. Membranes were hybridized simultaneously with cRNA probes for PLZF and cyclophilin.
5'-RACE (rapid amplification of cDNA ends) and promoter analysis
In order to predict the promoter region of the PLZF gene, computerized analysis of genomic sequences supported by the TSSW and TSSG prediction programs was performed (Wingender, 1994; Solovyev et al., 1995) (available at: service{at}bchs.uh.edu). Total RNA extracted from SMC, stimulated with DEX for 4 h, and from KG-1 cells were used as templates to determine the 5' end of PLZF transcripts using a 5'/3' RACE Kit (Roche Applied Science). First strand cDNA synthesis and nested/anchored PCR were performed following the manufacturer’s instructions with antisense PLZF-specific primers (priming: –5595/–5615; nested 1: –5695/–5716; nested 2: –5755/–5776) according to the most recently published mRNA sequence (Zhang et al., 1999). The predicted 5'-flanking DNA was scanned for transcription factor binding sites using the TFSEARCH program (http://molsun1.cbrc.aist.go.jp/research/db/TFSEARCH.html) or the AliBaba 2.1 software (http://wwwiti.cs.uni-magdeburg.de/grabe/alibaba2) and the TRANSFAC system (http://transfac. gbf.de) (Wingender et al., 2001).
Reporter constructs and expression vectors
All luciferase gene reporter constructs are based on the pGL3-Basic vector (Promega). The human PLZF promoter sequences containing the transcriptional start sites determined by 5'-RACE were generated by PCR. Genomic DNA was used as a template to produce four PLZF promoter fragments with different lengths of 5'-flanking region. The upstream oligonucleotides correspond to positions –9802/–9825, –7913/–7935, –6865/–6888 and –6346/–6368 relative to the start ATG codon; the downstream oligonucleotide was complementary to –5750/–5772. The PCR products of 4.1, 2.2, 1.1 and 600 bp were purified using the crystal violet gel system from the TOPO-XL-PCR Cloning Kit and were cloned into the pCR-XL-TOPO vector (Invitrogen). Inserts in 3'–5' orientation were excised with EcoRV/SpeI and subcloned into pGL3-Basic digested with Ecl136II/NheI. Inserts were verified by sequencing; all promoter constructs contain a nucleotide exchange from G to T at position –6054 relative to the start ATG, indicating a polymorphism in the genomic DNA template. The progesterone/glucocorticoid-responsive reporter gene construct PRE/–32/luc3 has been described previously (Greenland et al., 2000). The reporter plasmid 3xAP1/luc3, containing three AP-1 consensus elements in front of the minimal Ig
promoter in pGL3-Basic, was kindly provided by Dr R.Schmid (University of Ulm, Germany).
For generation of the expression vector pcDNA/c-jun, carrying the human c-jun cDNA, human genomic DNA was amplified with Expand polymerase (Roche Applied Science) using primers 5'-TCAGGCAGACAGAC AGACACAGC-3' (sense) and 5'-GTTAACGAAAGCAGGCCAGAAAGA-3' (antisense). The PCR product was digested with Acc65I and NotI, and the resulting fragment spanning the sequence –704/+1213 relative to the start ATG of c-jun mRNA was cloned into pcDNA3.1(+) (Invitrogen). Human c-fos cDNA was amplified by RT–PCR with Pfx polymerase (Life Technologies) from Jurkat cells which had been stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (5 x 10–8 mol/l) and Ionomycin (1 µg/ml) (Sigma) for 12 h. The primers were: 5'-GAGCAACTGAGAAGCC AAGACTGAG-3' (sense) and 5'-TAAGGAGAAAGAGAAAAGAGA CACA-3' (antisense). The PCR product spanning the sequence –140/+1457 relative to the start codon was digested with NotI at position –110 and ligated into the NotI and EcoRV sites of pcDNA3.1(–). Human GR cDNA was excised from pRS-hGR (kindly provided by Dr R.M.Evans, Howard Hughes Medical Institute, USA) with Acc65I/XhoI and inserted into the respective sites of pcDNA3.1(+) to obtain the expression vector pcDNA/GR. The human PR-B and PR-A cDNA were excised from hPR1 and hPR2 (kindly provided by Dr P.Chambon, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Strasbourg, France) (Kastner et al., 1990) with EcoRI and subcloned into the BstXI site of pRc/CMV (Invitrogen) with the aid of BstXI adapters. From these constructs (N-hPR1 and N-hPR2), inserts were excised with Acc65I/XbaI and inserted into the respective sites of pcDNA3.1(+) (Invitrogen) to obtain the expression vectors pcDNA/hPR-B and pcDNA/hPR-A.
Transient transfections
Transient transfections of ESC and SMC were performed by the calcium phosphate co-precipitation method as described previously (Gellersen et al., 1997) in triplicates using 24-well dishes. A maximum of 0.75 µg of the reporter construct with the largest insert, or equimolar amounts of constructs with shorter inserts, and 0.05 µg of each expression vector were used unless indicated otherwise. Controls received equimolar amounts of empty expression vector, and total DNA was kept constant by addition of promoterless plasmid DNA. Medium was replaced 20 h later and 250 nmol/l DEX or progesterone was added for stimulation experiments. Cell harvest was performed 24 h after medium replacement, and luciferase activity measured with the Luciferase Reagent Kit (Promega).
Immunofluorescence, immunohistochemistry
For immunofluorescence studies, SMC in DCC-FCS medium at 80% confluence were treated with 250 nmol/l DEX or left untreated for 24 h. They were washed twice with phosphate-buffered saline (PBS) and then fixed with methanol for 5 min at room temperature. Cells were washed again with PBS and preincubated in normal goat serum (NGS) for 10 min. The cells were then incubated for 1 h at room temperature with anti-PLZF monoclonal antibody (Ab-1; Oncogene) diluted 1:50 in PBS or with NGS as a control. After washing with 0.001 mol/l PBS, cells were incubated for 1 h at room temperature with Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) diluted 1:100 with PBS/2% NGS, washed again and stored in PBS. Stained cells were visualized with a fluorescence microscope (Nikon Epiphot, Germany).
For immunohistochemistry, surgical specimens which had been routinely fixed in 4% buffered formalin and embedded in paraffin were used. Tissue samples were obtained from three different women of reproductive age (35, 40 and 44 years) in the proliferative, mid- and late secretory phase of normal menstrual cycles. The patients underwent hysterectomy for uterine leiomyoma in two cases and for cervical carcinoma in one case. Serial sections of 4–6 µm were cut from paraffin blocks containing normal endometrial tissue and mounted on aminopropyltriethoxysilane-coated slides, deparaffinized in xylene and rehydrated in graded alcohols. For antigen retrieval, the slides were microwaved for 20 min in 20 mmol/l Tris, 10 mmol/l citrate, 13 mmol/l EDTA, pH 7.8. Immunohistochemistry was then performed in an automated system (Dako Autostainer; Dako, Denmark) with the ChemMateTM Peroxidase/DAB Detection Kit (Dako). PLZF monoclonal antibody (Ab-1; Oncogene) was used at a dilution of 1:50. Sections were counterstained with haematoxylin. For negative controls the primary antibody was omitted.
All investigations described in the paper were conducted in accordance with the guidelines proposed in The Declaration of Helsinki (http://www.wma.net).
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Results |
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Regulated expression of PLZF in human endometrial stromal cells and myometrial smooth muscle cells
In order to examine PLZF gene expression in the human uterus and potential regulation by nuclear receptors, we prepared cultures of primary ESC and SMC and extracted total RNA after treatment of the cells with progesterone, DEX or RA. Northern blot analysis with a cRNA probe to the 3' part of PLZF mRNA (Figure 1) detected a PLZF transcript of
8 kb in KG-1 myelogenous leukaemia cells, which served as a positive control because of their high basal PLZF expression (Figure 2A). In uterine cells, PLZF expression was detectable after DEX treatment for 48 h in ESC and 24 h in SMC (Figure 2A). To gain higher sensitivity and be able to distinguish possible splice variants, we used RT–PCR analysis for PLZF followed by Southern hybridization with an internal oligonucleotide. The primers were chosen such that the amplified region encompasses 1236 bp of exon 1 including a few bases of 5'-UTR and the sequences coding for the POZ and proline-rich domains. Within this region, three alternative splice variants of the PLZF gene are known (AS II–IV) in addition to transcripts containing the complete exon 1 (Zhang et al., 1999) (Figure 1). RT–PCR analysis revealed that DEX and progesterone stimulated PLZF expression in human ESC and SMC and that there was a stronger increase after DEX than after progesterone treatment (Figure 2B). Treatment with RA only resulted in a very weak signal whereas no PLZF mRNA was detectable in untreated cells. The amplified product was 1236 bp in size and therefore represents the variant which contains the entire exon 1, whereas products corresponding to AS II–IV were not detected. Identical results were obtained using hydrocortisone instead of the synthetic glucocorticoid DEX, and the synthetic progestin MPA instead of progesterone, confirming the much higher potency of GC as compared to progestins in up-regulating PLZF expression (data not shown).
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The presence of PLZF protein in cultured SMC was then assessed by indirect immunofluorescence. After 24 h of DEX treatment, speckled PLZF immunoreactivity was detected in the nuclei in a pattern typical of the punctate nuclear localization of PLZF and PLZF-RAR
chimeras previously described in other cell systems (Reid et al., 1995; Dong et al., 1996) (Figure 2C). In order to investigate PLZF expression in the human uterus in vivo, immunohistochemistry was performed (Figure 3). Strong nuclear staining for PLZF was found in the stromal compartment of the endometrium (Figure 3d, g) and in the myometrium (Figure 3f, i) during the mid- and late secretory phase of the menstrual cycle. In contrast, no PLZF was detected in uterine tissue obtained in the proliferative phase (Figure 3a, c). Faint staining for PLZF was detected in the glandular epithelium of the late secretory phase (Figure 3g).
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To determine whether the effects of DEX and progesterone on PLZF expression in vitro were mediated by the GR or PR respectively, we treated SMC with DEX, progesterone and the antagonists RU486 and ZK98299 alone or in combination. RT–PCR and Southern blot analysis showed that the strong induction of PLZF expression by DEX was fully antagonized by RU486, a GR/PR antagonist, but not by ZK98299, which has a reduced ability to antagonize the GR compared to RU486 and is a strong PR antagonist (Chwalisz et al., 2000). The low level of PLZF expression obtained after progesterone stimulation appeared slightly reduced by both RU486 and ZK98299 (Figure 4A). Employing a different experimental approach, the same oligo(dT)-primed cDNA as used in Figure 4A were subjected to real time PCR, and PLZF cDNA levels were normalized to GAPDH cDNA levels (Figure 4B). Finally, cDNA was synthesized by random-priming and analysed by relative quantitative PCR with internal 18S standards (data not shown). All three techniques yielded comparable results.
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It has been reported that RA-induced differentiation of HL-60 and NB-4 promyelocytic cells is accompanied by down-regulation of PLZF (Chen et al., 1993). We therefore tested whether RA would repress DEX- or progesterone-induced PLZF expression in SMC. RT–PCR/Southern blot analysis revealed that RA only had a minor, if any, repressive effect on the basal or steroid-induced levels of PLZF transcripts in SMC (Figure 4C). These results were confirmed by real time PCR (Figure 4D) and by relative quantitative PCR with internal 18S standards (data not shown).
After having demonstrated that PLZF mRNA is up-regulated by DEX and progesterone, we investigated the time- and dose-dependency of these responses. To determine the onset of PLZF expression after adding DEX to ESC and SMC, a time-course between 15 min and 4 h was performed and showed the rapid induction of PLZF expression within 2 h in ESC and 3 h in SMC (Figure 5A). In SMC treated with 1 pmol/l, 1 nmol/l or 1 µmol/l DEX for 4 h or progesterone for 24 h, PLZF expression was highest with the highest dose of steroid employed (Figure 5B). In an extended time-course, using an intermediate dose of 250 nmol/l DEX or progesterone, PLZF expression in SMC reached a plateau within 4 h of treatment with either steroid and remained high for 
72 h (Figure 5B). A more detailed dose–response study revealed that maximal induction of PLZF expression was achieved by 50 nmol/l DEX or progesterone within 4 h (Figure 5C).
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Recently, cyclin A2 has been put forward as a candidate target gene of PLZF. A growth inhibitory effect of PLZF has been demonstrated in a murine myeloid cell line, in NIH3T3 murine fibroblasts, and the human leukaemic cell line NB-4 (Yeyati et al., 1999). To examine this in our cell system, we amplified cyclin A2 cDNA from those SMC cultures which had been treated with 250 nmol/l DEX for 4–72 h, or with different doses of DEX for 4 h (Figure 5B). Although there was a marked increase in PLZF mRNA levels after 4 h of treatment with 1 µmol/l DEX, no change in cyclin A2 expression was observed. With prolonged treatment, in the presence of persistently high levels of PLZF mRNA, there seemed to be a slight decrease in cyclin A2 mRNA levels between 48 and 72 h.
We then wanted to assess whether PLZF expression was also steroid-dependent in other cell types. We used KG-1 myelogenous leukaemia cells for which a high basal PLZF expression had been reported (Chen et al., 1993), and T47D breast cancer cells which have high endogenous levels of PR and are a widely used model to study progestin responses in mammary cells (Nordeen et al., 1989). KG-1 cells were treated with RA, DEX, progesterone or vehicle for 4 and 24 h (Figure 6A); T47D cells were treated with DEX, progesterone or vehicle for 2, 4 and 24 h (Figure 6B). RT–PCR showed no regulation of PLZF expression in KG-1 cells. In T47D cells, PLZF expression was readily detectable in control cultures, markedly induced by DEX and weakly by progesterone within 2 h of treatment. PLZF mRNA levels remained elevated for 24 h.
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T47D cells in our hands showed DEX responsiveness, although a lack of GR has previously been reported for this cell line (Nordeen et al., 1989). In order to confirm expression of GR and PR in the cell types employed in this study, we performed RT–PCR using primers specific for the ligand-binding domain of GR (GR-LBD), for PR-B, and for the ligand-binding domain common to PR-A and PR-B (PR-LBD) (Figure 7). Amplification of PR-LBD was included to detect PR-A mRNA in cells which might not express PR-B. As expected, PR was found highly expressed in T47D cells. PR mRNA was not or barely detectable in the haematopoietic cell lines KG-1, HL-60 and Jurkat, while ESC and SMC displayed an intermediate level of expression. In contrast, GR was strongly expressed in all cell types, including T47D cells, but not in HL-60 which also displayed a significantly lower level of GAPDH mRNA. Therefore, the lack of PLZF up-regulation in response to DEX, seen in KG-1 cells, was not due to absence of GR expression.
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Analysis of the PLZF promoter
The PLZF transcript detected by Northern blot analysis in human ESC, SMC and KG-1 cells was
8 kb in size (see Figure 2A). This is believed to result from an unusually long 5'-UTR of 6 kb in addition to 2 kb of translated region (Zhang et al., 1999). To date, the true 5'-end of the PLZF transcript has not been determined experimentally. Computerized searches for transcription start sites in the PLZF gene predicted a putative start site with a TATA box at position –5979 relative to the translational start site (Zhang et al., 1999; and our own data). To verify this prediction experimentally, 5'-RACE was performed using cDNA from KG-1 cells and SMC (Figure 8A). The RACE was initiated by reverse priming 400 bases downstream of the putative transcription start site, followed by two rounds of nested PCR. We obtained two independent clones with the KG-1 cDNA and five clones with the SMC cDNA. Sequencing located the transcription start site of KG-1 cells at –5858 and of SMC at –5801 relative to the ATG start codon. Alignment of the human 5'-flanking DNA with the corresponding mouse genomic sequence revealed a striking degree of homology within the first 185 bp adjacent to the start site (Figure 8B). This highly conserved region contains the putative TATA box and a GC-rich element conferring tissue-specificity of expression (Takahashi and Licht, 2002). The sequence further upstream, including the recently characterized Evi-1-like site (Takahashi and Licht, 2002), displays a significantly lower degree of homology.
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Within 4.1 kb of 5'-flanking DNA, search for transcription factor binding sites predicted potential binding sites for GR and activating protein-1 (AP-1) (Figure 8C). For transfection analysis, we cloned various portions of the putative PLZF promoter region, ranging from 4.1 to 0.6 kb in length and including 50–100 bp of predicted 5'-UTR, in front of the luciferase reporter gene (Figure 8C). SMC were transfected with three PLZF promoter constructs (PLZF-6368/luc3, PLZF-6888/luc3 and PLZF-9825/luc3) in the absence or presence of co-expressed GR
, PR-B or PR-A, and were treated with DEX or progesterone, respectively, or left untreated (Figure 9). The three promoter constructs did not respond to the steroid stimulus even in the presence of the GR or PR-B. Interestingly, all promoters responded slightly to PR-A, commonly considered a repressor of transcription, upon progesterone treatment. In contrast, a control construct driven by a palindromic PRE/GRE (PRE/-32/luc3) was strongly induced through co-transfected GR in response to DEX, and through co-transfected PR-B in response to progesterone. Co-transfection of PR-A, on the other hand, repressed the progesterone-dependent induction of reporter gene activity mediated through endogenous PR, in the absence of co-transfected steroid receptor.
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In order to assess whether AP-1 is involved in PLZF promoter control, ESC were transfected with all four PLZF promoter constructs and the AP-1 proteins c-jun/c-fos, in the absence or presence of co-expressed GR
, and were treated with DEX or left untreated (Figure 10). All promoter constructs failed to respond to GR but were induced by AP-1 even in the presence of GR and even though only the three largest promoter constructs contain an AP-1 consensus site. The glucocorticoid-responsive control reporter construct PRE/–32/luc was massively induced by GR upon addition of DEX, and a control construct driven by AP-1 consensus elements was also highly inducible by c-jun/c-fos.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Studies on the expression and function of PLZF have been focused on fetal development and haematopoiesis (Chen et al., 1993; Avantaggiato et al., 1995; Cook et al., 1995; Reid et al., 1995; Barna et al., 2000; Rego and Pandolfi, 2001). PLZF is a negative regulator of cell cycle progression and appears to be involved in differentiation (Yeyati et al., 1999). Upon RA-induced granulocytic differentiation of promyelocytic cells, PLZF mRNA levels are down-regulated (Chen et al., 1993). Here we demonstrate for the first time expression of PLZF in uterine cells, and a steroid-dependent up-regulation of PLZF mRNA. Notably, PLZF was up-regulated in a more pronounced fashion by GC than by progesterone in human ESC and SMC. This phenomenon was observed in all 14 individual tissue preparations examined. While our study was under way, a microarray-based search for genes differentially regulated by DEX and the progestin R5020 in T47D human breast cancer cells was reported which had revealed PLZF as one of the genes displaying this unusual pattern of regulation (Wan and Nordeen, 2002). Thus, our findings in human uterine cells have been shown in a different cell system.
By immunofluorescence, we demonstrated the nuclear accumulation of PLZF in distinct speckles in DEX-treated SMC. Up-regulation by GC was mediated by the classical GR because the GR antagonist RU486 abrogated the DEX-induced PLZF expression. In addition, both the GR and the PR were found to be expressed in ESC, SMC and T47D cells. While PLZF was up-regulated in ESC, SMC and T47D cells by steroids, no alteration of the PLZF expression level could be detected in steroid-treated KG-1 cells although these cells have endogenous GR. This indicates a cell type-specific control of PLZF expression possibly resulting from the interaction of steroid receptors with cell-specific co-factors (McKenna et al., 1999; Takahashi and Licht, 2002).
We not only demonstrated the accumulation of PLZF protein in steroid-stimulated primary cultures of SMC, but also for the first time showed the expression of PLZF in the human uterus in vivo. The protein showed exclusive nuclear localization in the stromal compartment of the endometrium and in myometrial cells in biopsies taken in the secretory phase of the menstrual cycle. It is conceivable that different relative levels of GR and PR in the intact uterus compared to isolated cells in culture might impart a discordancy of steroid sensitivity in vivo versus in vitro. Our immunohistochemical analysis of a limited number of biopsies suggests a cycle-dependent up-regulation of PLZF which would be compatible with progesterone-mediated control. These observations still need to be confirmed on a larger number of dated specimens. In addition, RT–PCR analysis detected high levels of PLZF mRNA in human first trimester decidua and term placenta (data not shown), pointing to sustained expression of PLZF throughout pregnancy. The presence of GR and PR mRNA and protein in endometrial stromal cells throughout the cycle, and in decidua, has been demonstrated (Henderson et al., 2002).
Although in primary cell cultures the induction of PLZF mRNA in response to DEX or progesterone occurred within 2–3 h, our studies argue against a direct transcriptional effect of activated PR or GR on the PLZF promoter. Transfected promoter constructs carrying up to 4.1 kb of 5'-flanking sequence were not induced in response to DEX or progesterone, even when GR or PR-B respectively were co-transfected, and the putative GR/PR binding sites in this region are only half-palindromic glucocorticoid/progesterone response elements (GRE/PRE). It is conceivable that GRE are located outside the promoter region tested by us, or that GR binds to the promoter only when tethered by other transcription factors which are limiting in the transfected cells and were not provided by co-transfection. A cooperative interaction has been reported for GR and jun/jun dimers, or for GR and STAT5 (Cella et al., 1998; Webster and Cidlowski, 1999; Reichardt et al., 2001). However, in co-transfections of GR and c-jun (see Figure 10), or GR and constitutively active STAT5 (data not shown), we did not obtain ligand-dependent activation of PLZF promoter/reporter gene constructs. Alternatively, the action of GR or PR may be non-genomic, particularly in response to high steroid concentrations (Borski, 2000). Significant evidence has accumulated over the past few years regarding the ability of steroid hormone receptors to engage in non-genomic actions (Koukouritaki et al., 1996; Falkenstein et al., 2000; Hodel, 2001). Liganded GR or PR can modulate the MAPK pathway by interaction with cytosolic signalling molecules (Migliaccio et al., 1998; Croxtall et al., 2000; Boonyaratanakornkit et al., 2001). Downstream events may include induction of an intermediate transcription factor(s) which then binds to and activates the PLZF promoter. However, others have recently demonstrated that the DEX- or R5020-induced elevation of PLZF transcript levels in T47D cells cannot be blocked by cycloheximide or actinomycin D, arguing for the involvement of a pre-existing factor in the up-regulation of PLZF mRNA (Wan and Nordeen, 2002). In addition, GC have been shown to stabilize mRNA (Rosewicz et al., 1994; Uchijima et al., 1999). The intriguingly long 5'-UTR of the PLZF mRNA has not been functionally characterized as yet but may be implicated in regulated expression of PLZF (Pesole et al., 2001). The recent identification of an entirely different class of steroid receptors, namely the membrane progestin receptors mPR-, -ß and -
, offers a new mechanistic basis for previously unexplained non-classical steroid actions. The structure of these mPR incidates that they belong to the family of G protein-coupled receptors and may thus link steroid signalling to second messenger pathways (Zhu et al., 2003).
Interestingly, transient transfections in ESC revealed an induction of PLZF expression by AP-1 transcription factors. Surprisingly, the AP-1-mediated transcriptional up-regulation could not be repressed by GR, a mechanism previously reported (Jonat et al., 1990; Schüle et al., 1990). The predicted AP-1 binding site in the PLZF promoter does not appear to be required for AP-1-mediated induction, because it does not lie within the shortest promoter construct PLZF-6368/luc3 which was clearly responsive to c-jun/c-fos. Alignment of the immediate 5'-flanking DNA of the human PLZF gene with the corresponding mouse genomic sequence, located on chromosome 9, revealed an intriguing extent of homology between coordinates –5858 (the most 5' transcriptional start site determined by us) and –6043, followed by less well conserved sequence. This may point to the presence of conserved regulatory elements in close vicinity to the transcription start site.
Candidate target genes of PLZF include the gene encoding cyclin A2, an important component of the cell cycle machinery, and the gene encoding the subunit of the IL-3R that mediates the IL-3 cytokine effect by stimulating the proliferation and differentiation of haematopoietic progenitor cells (Kosugi et al., 1995; Ball et al., 1999; Yeyati et al., 1999). However, we did not observe a repression of cyclin A2 (see Figure 5B) or IL-3R mRNA levels (data not shown) in DEX-treated SMC within 24 h in the presence of sharply increased PLZF mRNA levels. Numerous genes are down-regulated by GC and/or progesterone including the IL-8 gene which is repressed by both steroids in endometrial explants and the HOXA-10 gene which is down-regulated in SMC by progesterone (Read et al., 1989; Kelly et al., 1994; Lee et al., 1997; Cermik et al., 2001; Wan and Nordeen, 2002). Interestingly, HOXA-10 has been identified as a target gene of vitamin D3 receptor (VDR) up-regulation in myeloid differentiation (Rots et al., 1998), and PLZF suppresses VDR transcriptional activity by direct physical interaction (Ward et al., 2001). Since PLZF is up-regulated by GC and progesterone and is a transcriptional repressor, we propose that it may serve as a mediator of repressive effects of these steroids.
GC and progesterone on the one hand, and the transcription factor PLZF on the other hand are involved in processes of differentiation, apoptosis and cell cycle control. Thus, the GC- and progesterone-mediated up-regulation of PLZF expression shown by us in ESC and SMC may provide a missing link in steroid signalling pathways. Further, the identification of direct targets of PLZF will be important to elucidate its role in female reproduction.
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Acknowledgements |
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We thank Rita Kempf, Gabriele Rieck and Bianca Kelp for excellent technical assistance and Drs P.Chambon, R.Evans, R.Schmid and G.E.DiMattia for plasmids. We are indebted to Dr H.K.Pauli and Prof. C.Lindner, Elim Hospital Hamburg, for providing hysterectomized tissue and thank Olaf Nagel for advice with real time PCR. This work was supported by the Deutsche Krebshilfe (10-1301-Ge1).
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