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School of Surgical and Reproductive Sciences, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK

(Requests for offprints should be addressed to J Bailey; Email: jarrod.bailey{at}ncl.ac.uk)

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Activating transcription factor 2 (ATF2), a ubiquitously expressed member of the basic region leucine zipper (bZIP) family of transcription factors activated by mitogen activated protein kinase (MAPK) pathways, is important in the mediation of cellular stress responses, development and transformation. We have previously reported the differential expression of active ATF2 in the human myometrium throughout pregnancy and labour, and identified and partially characterized a novel splice variant ATF2-small (ATF2-sm). To further understand the role of these factors in the myometrium, we have used gene microarrays to define the target genes in cultured myometrial cells stably-transfected with ATF2 and ATF2-sm cDNAs. Many of the genes identified appear to have potential roles in regulating myometrial function and include proteins involved in G-protein receptor signalling, cytokine signalling, transcriptional regulation, cell-cycle control, formation of the extracellular matrix and cytoskeletal architecture. ATF2 was found to affect the expression of 204 genes; 113 being up-regulated and 91 down-regulated whereas the novel ATF2-sm factor altered the expression of 55 genes; expression was increased in 29 cases and decreased in 26. A further 25 genes affected by ATF2-sm were identified by suppression subtractive hybridisation (SSH). Notably, the genes affected by ATF2 and ATF2-sm appear to belong to discrete groups: only two genes were affected by both factors.

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
Activating transcription factor 2 (ATF2) is a ubiquitously expressed member of the basic region leucine zipper (bZIP) transcription factor family (Landschulz et al. 1988, Ziff EB 1990), which includes other factors such as cyclic-AMP response-element binding protein (CREB) (Hoeffler et al. 1988), the highly spliced cyclic-AMP response-element modulator protein (CREM) (Foulkes et al. 1991), and other members of the ATF family. ATF2 plays an important role in mediating cellular stress responses, development and transformation (Hai et al. 1989, Maekawa et al. 1989, Abdel-Hafiz et al. 1992, Karin & Hunter 1995, Reimold et al. 1996, Ronai et al. 1998, Maekawa et al. 1999, van Dam & Castellazzi 2001), and its trans-activation potential is enhanced via phosphorylation at amino acid residues Thr69 and Thr71 (Davis 2000, Fuchs et al. 2000) by the mitogen activated protein kinases (MAPK) JNK/SAPK, p38 (Derijard et al. 1994, Gupta et al. 1995, Livingstone et al. 1995, van Dam et al. 1995, Raingeaud et al. 1996, Read et al. 1997, Chang & Karin 2001, Kyriakis & Avruch 2001), and the Ras effector pathways Raf-MEK-ERK and Ral-RalGDS-Src-p38 (Ouwens et al. 2002). It modulates the expression of downstream-affected genes by binding either as a homo- or hetero-dimer with other members of the bZIP family (Hai & Curran 1991) to cAMP response elements (CREs) and Activator Protein-1 (AP-1) sites within their promoter regions (Montminy et al. 1986, Lee et al. 1987, Yamamoto et al. 1988). Transcriptional activation is promoted via the intrinsic histone acetyl transferase (HAT) activity of ATF2 (Kawasaki et al. 2000), and by recruitment of co-activators such as p300/CBP (Cho et al. 2001). Interaction of ATF2 with p300/CBP and the basal transcriptional machinery is dependent upon phosphorylation at Ser121 within its p300 interaction domain (Kawasaki et al. 1998), mediated by protein kinase C (PKC); it has been suggested that Ser121-unphosphorylated ATF2 is transcriptionally silent, necessitating the use of constitutively active ATF2 mutants in transcription studies (Steinmuller & Thiel 2003). However, we have reported highly significant trans-activation of CRE-containing reporter genes by transient transfection of an ATF2-expressing plasmid into both myometrial and COS-7 cell lines (Bailey et al. 2002). Furthermore, we present data here to indicate promiscuous transcriptional modification in myometrial cell lines stably transfected with ATF2 constructs.

Recently we have shown that various members of the bZIP transcription factor family including ATF2 are differentially expressed in the human myometrium during gestation and parturition (Bailey et al. 2002). However, in addition to the well characterized 60 000 mol wt ATF2 protein, we also reported the existence of a novel putative splice-variant designated ATF2-small (ATF2-sm) (Bailey et al. 2000), showing potent trans-activation properties. To our knowledge, this is the first alternatively-spliced variant of ATF2 to be identified in human tissue. Although comprising 432 bp and translating to a polypeptide of 144 amino acids with a calculated mol wt of 15 408, in vitro translation experiments and western blotting of human myometrial tissue homogenates consistently demonstrated an apparent mol wt of approximately 28 000; reasons for this are discussed in detail with supporting evidence in Bailey et al.(2002). Of particular interest was the fact that western blot profiling of the ATF2 and ATF2-sm species in pregnant non-labouring myometrium, sampled from the upper and lower uterine segments (as detailed in Fig. 1a), indicated that both isoforms were spatially expressed within the body of the uterus; ATF2 is expressed only in the lower segment, whereas there exists a gradient of expression of the ATF2-sm protein with the highest level in the upper segment. This may correlate with the known contractile and relaxatory properties of these uterine regions at term and at the onset of parturition. Although no definite exonic structure of the ATF2 gene has been reported, a predicted model has been proposed (Ensembl gene ID ENSG00000115966, Fig. 1b) comprising 12 exons and five functional domains, including a proline-rich domain (involved in protein–protein interaction), two glutamine-rich domains (involved in transcriptional trans-activation), a HAT domain and a bZIP domain involved in CRE-binding. Despite comprising only the first two and last two putative exons, and being devoid of most exons coding for functional domains, the novel ATF2-sm protein was found to possess CRE-binding activity and to trans-activate CRE-directed transcription of a reporter gene in myometrial and COS-7 cells to an equivalent degree to that of the full-length ATF2. To further characterize the roles of ATF2 and ATF2-sm in regulating myometrial gene expression during foetal maturation and parturition, we have stably transfected myometrial cell lines with plasmid constructs expressing the individual proteins, and made use of microarray, suppression subtractive hybridization (SSH) and semi-quantitative/real time RT-PCR experiments to identify downstream target genes that may be under their transcriptional control.

View larger version (42K): [in this window] [in a new window]   Figure 1 (a) Spatial expression of the ATF2 and ATF2-sm transcription factors in the human myometrium during pregnancy. Western blotting of myometrial tissue extracts with an anti-ATF2 antibody revealed a post-conception switch from uniform expression to spatially differential expression of these factors. The full-length ATF2 protein was found to be present in a gradient from the lower uterine segment to the fundus; the novel ATF2-sm splice-variant, in contrast, was present in a gradient from the fundus to the lower segment. (b) Predicted functional domains of the ATF2 and ATF2-sm proteins, indicated by shading. P, proline-rich domain; Q, glutamine-rich domains involved in trans-activation; HAT, histone acetyl-transferase domain; bZIP, basic-region leucine-zipper domain.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

Human myometrial tissue biopsies were collected from pregnant non-labouring (P) women at elective caesarean section and from non-pregnant (NP) pre-menopausal women (ages 32–46 yr) during hysterectomies performed for benign gynaecological disorders such as menorrhagia or dysmenorrhoea. NP tissue samples were taken from the middle of the uterine wall, about 5–10 mm away from the endometrial or serosal surfaces: upper segment samples were taken from the corpus and lower segment samples were taken from close to the cervix, although it is generally realised that the lower uterine segment is poorly defined in the NP state. P samples were taken using laparoscopic biopsy forceps from the side of the uterus opposite the placental bed to minimize the inclusion of deciduas, again from the corpus (upper segment) and close to the cervix (lower segment). These myometrial samples were then snap frozen in liquid nitrogen and stored at –70 °C. Written consent was obtained from all women and ethics approval for the study was granted by the Newcastle and North Tyneside Health Authority Ethics Committee.

Establishment of stable myometrial cell lines

Primary cultures of human myometrial cells were initiated by treating small pieces of P myometrium with collagenase solution (1 mg/ml collagenase, 0.02 mg/ml DNase, 0.2 mg/ml trypsin inhibitor, elastase and BSA fraction V, in Hank’s balanced salt solution free of Ca⁺ and Mg²⁺ ions). Samples were incubated with gentle shaking for 4 h, then transferred to a new tube to which 5 ml complete medium plus 10% FCS had been added. After centrifugation at 1000 g for 10 min, the supernatant was decanted and discarded, the cells were resuspended in 15 ml of fresh complete medium plus 10% FCS and cells were then seeded into flasks. After incubation at 37 °C for 1 h to allow fibroblast attachment, the medium, now containing primarily myometrial cells with few contaminating fibroblasts, was transferred to new flasks and incubated for 24 h at 37 °C. The growth medium was then replaced to inhibit fibroblast growth, with MEM D-Valine medium (Gibco) plus 10% FCS, containing 50 U/ml of penicillin and 50 µg/ml of streptomycin. Growth medium was replaced with fresh medium every 2–3 days, and after reaching confluence, cells were subcultured at a ratio of 1:3.

ATF2 and ATF2-sm full length plasmids were cloned as described by (Bailey et al. (2002). Transfections were performed upon low passage number (2 or 3) myometrial cells at 70% confluence using 4 µl Mirus TransIT-LT1 lipid-based transfection reagent (Cambridge Bioscience, Cambridge, UK) per 1 µg plasmid. Typically, 6 µg plasmid was used per transfection, mixed with 24 µl LT-1 reagent and 200 µl serum-free medium and incubated at room temperature (RT) for 5 min. This complex was added to the cells and incubated at 37 °C for 4 h, after which time it was removed and fresh medium (+ serum) added. Incubation at 37 °C was continued for 48 h, after which time the selective agent G418 was added to a final concentration of 80 µg/ml. The final concentration of G418 was increased to 250 µg/ml for subsequent selection and maintenance of isolated stably-transfected colonies. Transfection efficiencies were consistently above 80%.

Cell staining/immunocytochemistry

Cultured myometrial cells were stained to allow morphological examination using haematoxylin and eosin stains. Cells grown on cover-slips were rinsed with 1x PBS, fixed in cold methanol for 10 min, washed with water and then exposed to haematoxylin for 1 min. Following a quick water wash, cells were exposed to Scott’s water (0.04 M NaCO₃, 0.08 M MgSO₄) for 1 min. Another water wash was performed, then the cells were incubated in eosin stain for 2 min. After a final brief wash in water, the slips were allowed to dry and mounted in DPX resin on microscope slides for viewing.

To determine the level of fibroblast contamination in the smooth-muscle cell cultures, immunocytochemistry was performed using Fibroblast Antigen (Ab-1; Oncogene Research Products, CA, USA) (Saalbach et al. 1997) and the DAKO ChemMate APAAP detection kit with a Vector Blue Alkaline Phosphatase Substrate Kit III (Vector Laboratories, CA, USA). Cells were cultured on cover-slips and fixed as above, then incubated in normal rabbit serum for 10 min. After a brief rinse with TBS, cells were incubated in Ab-1 primary antibody for 1 h at a 50µl/ml dilution (10µg/ml final concentration) at room temperature. After three 5 min rinses in 1xTBS, cells were exposed to the secondary-link antibody for 45 min. Following three more 5 min rinses in 1xTBS, the cells were incubated in APAAP for 20 min, followed by another round of washing in 1xTBS. One drop of the Levamisole chromagen was then added to the cells, and colour development was monitored microscopically. Chromagenesis was halted by rinsing with water.

RNA isolation

Total RNA was isolated from approximately 1x10⁷ cells using TRI-Reagent (Sigma-Aldrich) according to the manufacturer’s protocol. The resulting pellet was resuspended in 100 µl RNase-free H₂O and incubated at 55 °C for 10 min to aid this process. This total RNA was then applied to an RNeasy mini-column (Qiagen), and subjected to RNase-free DNase treatment using the RNase-free DNase set (Qiagen). The remainder of the manufacturer’s protocol was then followed for RNA cleanup, eluting with 25 µl of RNase-free H₂O. Total RNA was quantified by spectrophotometry and used for microarray analysis or SSH.

Confirmation of ATF2/ATF2-sm expression levels in stably-transfected cell lines

Levels of ATF2/ATF2-sm expression in stably transfected cell lines were examined and compared with one another by sqRT-PCR using the Superscript one-step RT-PCR system (Invitrogen) in a total volume of 25 µl with 5% (v/v) RNaseOUT (Invitrogen) RNase inhibitor, and 100 ng total RNA template prepared as described above. Primers were as described previously by Bailey et al.(2002), and reaction conditions as follows: reverse transcription step=50 °C for 30 min, 94 °C for 2 min. Amplification step=30 cycles of (94 °C 15 s, 50 °C 30 s, 68 °C 1 min), followed by 68 °C for 9 min. 5 µl of each reaction was subjected to agarose gel electrophoresis and RNA samples from those colonies displaying the highest ATF2/ATF2-sm expression were selected for microarray analysis and SSH.

Affymetrix microarray analysis

Experimental design, execution, and subsequent data analysis were all carried out according to the guidelines of the Minimum Information About a Microarray Experiment (MIAME) document, developed by the Microarray Gene Expression Data Society (http://www.mged.org/miame) (Brazma et al. 2001).

Target preparation

A total of 10 µg RNA was used for each target. Each sample was used as a basis to synthesize double-stranded cDNA, which in turn was used to synthesize biotin-labelled cRNA for hybridization to the microarray chips. The Affymetrix protocol was followed, and the steps are summarized briefly here:

cDNA synthesis
First-strand cDNA synthesis was achieved using the SuperScript Choice system (Invitrogen) and the Gene-Chip T7-Oligo(dT) primer at 42 °C for 1 h. Second strand cDNA was synthesized from this using E.coli DNA ligase, polymerase I and RNase H at 16 °C for 2 h.

Synthesis of biotin-labelled cRNA
This was performed according to the protocol, using the Affymetrix Enzo BioArray High Yield RNA Transcript Labelling Kit (Affymetrix, High Wycombe, Herts, UK); 20 µg of this labelled and purified cRNA was then fragmented (according to the protocol) prior to the hybridization step.

Hybridization
The hybridization cocktail was prepared according to the Affymetrix instructions, and incubated with the array at 45 °C for 16 h in rotisserie oven at 60 r.p.m.

Washing, staining and scanning
Washing and staining was performed in the Affymetrix Fluidics Station 400 according to the standard format of the single stain protocol for eukaryotic targets.

Data analysis
Data from the chip hybridizations was processed using the Affymetrix microarray suite 4.0 software. Human 133 A microarray chips were hybridized with biotin-labelled cRNA from the following myometrial cell-lines: (i) Control non-transfected primary cultures (Ctrl); two replicates (ii) Control cells stably-transfected with empty vector with no insert downstream of a CMV promoter (CMV–); three replicates (iii) Cells stably-transfected with a plasmid to express ATF2 (ATF2); three replicates (iv) Cells stably-transfected with a plasmid to express ATF2-sm (ATF2-sm); three replicates. Raw data files direct from the Affymetrix chip scanner were imported into the GeneSpring program (Silicon Genetics)) for each chip. These data sets were transformed by converting all signal values below 0.01 to 0.01, then subjected to per chip normalization to the 50th percentile and per gene normalization to the median. To determine a baseline profile of gene expression with which to compare the ATF2 and ATF2-sm results, an initial comparison was made between cell-lines (i) and (ii), using data from the primary non-transfected cells as a baseline and data from the CMV– empty vector transfected cells as the experimental set. This established which genes were affected by the stable transfection of the vector, and which therefore should be excluded from the main analyses involving the ATF2 constructs. According to related literature (Mayanil et al. 2001, Gibellini et al. 2002), an appropriate cut-off point for this purpose is in the region of greater than or equal to a 1.5—2-fold change in expression; 1.5 was used in our analysis. The remaining data set following this exclusion was then used as a baseline for comparisons with ATF2 and ATF2-sm expression. This was achieved by performing a parametric one-way analysis of variance (ANOVA) on the normalized data assuming equal variances, incorporating the Benjamini and Hochberg False Discovery Rate multiple testing correction (Hochberg & Benjamini 1990) set at a rate of 0.05. The resultant list of genes differentially expressed between the two control classes was then filtered to include only those genes showing a fold change in expression of 1.5 or greater. These genes were then subtracted from the list of genes represented on the Affymetrix Human 133 A chip (22 283), resulting in the elimination of genes whose expression was altered by the process of stable transfection and selection. The remaining data set following this exclusion was then used as a final baseline for comparisons with ATF2/ATF2-sm expression. These comparisons involved much the same process; data was normalized as above, then compared with gene expression levels in the final baseline by ANOVA using the same criteria. The results were then filtered to include only those genes whose expression altered by a fold change of two or greater.

SSH and differential screening

SSH was performed according to the published protocols using the PCR-Select cDNA subtraction kit and PCR-Select differential screening kit (BD Biosciences Clontech, Oxford, UK). Briefly, mRNA was isolated from the stably-transfected ATF2-sm cell-line using the Poly(A)Pure mRNA Isolation Kit (Ambion Europe Ltd., Huntingdon, Cambs, UK) 2 µg was used to synthesize ds-cDNA. Following RsaI digestion and ligation of adaptors to tester cDNA, two rounds of hybridization were performed with excess driver cDNA to subtract non-differentially expressed molecules, then two rounds of adaptor-directed suppression PCR to selectively amplify the cDNAs representing differentially expressed genes. These products were then cloned into the pCR4-TOPO vector (Invitrogen) to form a subtracted cDNA library, and subtracted and unsubtracted probes were synthesized from these PCR products for use in the library screening procedure. Briefly, transformed bacterial colonies from the library construction step were picked, and grown overnight in 96-well plates in LB AP⁺ medium. 1 µl of each culture was then used as a template for adaptor-directed PCR amplification of the plasmid inserts, of which 5 µl was spotted onto a Hybond N membrane. Four replicate membranes were produced for probing with forward subtracted, reverse subtracted, unsubtracted tester and unsubtracted driver probes. These probes were synthesized by random primer labelling of 75 ng of the appropriate cDNA, and hybridized to the membranes in sealed trays with shaking at 72 °C after pre-hybridizing with PerfectHyb Plus buffer (Sigma-Aldrich) containing 0.2x SSC and 50 µl blocking solution (10 mg/ml sheared salmon sperm DNA, 0.3 mg/ml oligonucleotides corresponding to nested primers and complementary sequences). After washing and drying, the membranes were subjected to autoradiography. Clones representing differentially expressed genes were identified according to the combination of hybridization results for the four different probes summarized in the PCR-Select differential screening kit protocol, and identified by DNA sequencing.

Semi-Quantitative RT-PCR (SQ RT-PCR)

In order to support the results obtained from the microarray and SSH experiments, a selection of candidate genes were examined for differential expression as a result of the over-expression of ATF2 and/or ATF2-sm by sqRT-PCR. The primers used for this were as follows, where F=forward primer, R=reverse primer. GAPDH: F=CTGCCGTCTAGAAAAACC, R=CCA CCTTCGTTGTCATACC; MTATP6: F=TGTTCG CTTCATTCATTGCC, R=ATGTGTTGTCGTGCA GGTAGA; CYB: F=CCCAATACGCAAAATTAA CCC, R=CGGATGCTACTTGTCCAATGA; HTM: F=ATGGACGCCATCAAGAAGAA, R=GAAAATG TCCTACAATGTGCA; CDH2: F=GCCTCCATGT GCCGGATA, R=TCTCGGTCCAAAACAGCAAT; ZNF297b: F=AAGGCACAGGCTGAATCTGTT, R=TCTGTGCCCAGATTTGCATT; LIM: F=GAG TCACTTGTCAGCCCTTGT, R=ACATTGTTCC GAATGGGCTT; TES: F=ATTGATGGGCTTA GGTCACGA, R=TGAGGGGGAAAAAAGAAGTG; RPL15: F=TAAGCCAAGATGGGTGCATA, R= CAAATTGACCCTGGACAACA; GPR63: F=CGC TGCCCTTCGCTTGA, R=GCAGTATCAGGTCC CATTGATG; GNA15: F=CGGGCCTACTATGAG CGTC, R=ACGTAGCCCTCCTCGGTGAT; GAS1: F=CCGCCGCCTCATCTGCT, R=GCCTCGGCG TACTGGTTG; MAP7: F=CGGCTCTCCTCT TCATCTGC, R=GAACGAATGTGTGGGCGTC; HSP70B': F=TTCGACAACCGGCTCGTG, R=TTG TTCCCGCTCAGGTCCTT; KIF20: F=GACAGG AAGATCAGGGTTGTGTC, R=CAAAAGAGTCCT TGGGTGCTT; CALCRL: F=TGAGGACTCAATT CAGTTGGGAGT, R=CCATCCATCCCAGGTTC TGTTG and GAB2: F=ACAGCCGACTTCACCG AGCTTC, R=CAGACCGGCCTGCACTCTCT

sqRT-PCR was performed using the SuperScript OneStep RT-PCR system. 50 ng DNase-treated total RNA from stably-transfected cells, or 5 ng mRNA from pooled non-pregnant myometrial tissue or pregnant non-labouring myometrial tissue was used as template according to the manufacturer’s protocol, and a total of 28, 30 and 32 cycles of PCR were performed at an appropriate annealing temperature for each particular primer pair to ascertain the best quantitative signal for each target sequence following agarose gel electrophoresis (2% E-gels, Invitrogen) of the products. Image analysis and band intensities were analyzed with the Intelligent Quantifier program (Bio Image Systems Inc., Jackson, MI, USA).

Real-time RT-PCR analysis

Forward primers were designed using LUX primer design software (www.invitrogen.com) and were as follows. ATF2: GNA15=5'-GACGCCGGGCCTGC TATGACCGC-3' and ATF2-sm: HSP70B'=5'-CACGATTCGACAACCGGCTCGG-3'. Reverse non-LUX labelled primers were as above for SQ RT-PCR. Reactions were performed using the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen). Reactions were set up in 25 µl volumes according to the manufacturer’s protocol, and cycled as follows: 50 °C for 15 s: 95 °C for 2 s: then 45 cycles of 95 °C for 15 s and 60 °C for 30 s. Commercially available LUX primers (Invitrogen) were used to amplify GAPDH as a reference gene. Melting curve analysis was performed to ensure reaction specificity, and relative quantitation was determined by standard curve analysis.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Stable transfection and microarray data

Following the stable transfection and selection process, cell-lines were examined to verify that (a) they were predominantly composed of myometrial smooth-muscle cells and that fibroblast contamination had been successfully kept to a minimum, and (b) that the cells had not been in any way damaged, i.e. were in a healthy state with no gross morphological changes. Figure 2a and b shows the results of these examinations following the appropriate cell-staining procedures, indicating not only low-level or zero presence of fibroblasts in the cultures (less than 1%), but also a ‘normal’ morphology in the stably-transfected cell-lines when compared with the non-transfected control.

View larger version (57K): [in this window] [in a new window]   Figure 2 (a) Immunocytostaining of human myometrial and fibroblast cultured cells using anti-fibroblast specific antigen (Ab-1) mAb ASO2 (oncogene). (i) Note intense anti-ASO2 reactivity with fibroblast cells, in contrast to the light staining with myometrial cells in (ii). Negative controls, omitting the primary ASO2 mAb, are shown in (iii) and (iv) for fibroblast and myometrial cells, respectively. (b) Haematoxylin and eosin (H&E) staining of stably-transfected myometrial cells expressing (i) ATF2 and (ii) ATF2-sm. Morphology was compared with control non-transfected (iii) and empty-vector stably-transfected (iv) cells, and showed no difference. (c) RT-PCR analysis of isolated stably-transfected colonies to determine abundance of ATF2 and ATF2-sm mRNAs. Total RNA was isolated from these cell lines, and used as a template for RT-PCR of the ATF2 and ATF2-sm isoforms. Expression levels were compared with control non-transfected (–) and empty-vector stably-transfected (C) cells. Those with the highest degrees of ATF2 and ATF2-sm expression (*) were used for SSH analysis and as the three replicate RNA populations for the microarray analysis.

View larger version (107K): [in this window] [in a new window]   Figure 3 (a) Total RNA samples were isolated from stably-transfected cell-lines expressing the ATF2 gene, and hybridized to Affymetrix Human 133 A microarray chips as described in Materials and methods. Following scanning and data analysis, those genes found to be up- or down-regulated by 2-fold or greater in comparison to baseline expression were classified according to their function by GeneSpring software, and are listed here. (b) Total RNA samples were isolated from stably-transfected cell-lines expressing the ATF2-sm isoform, and hybridized to Affymetrix Human 133 A microarray chips as described in Materials and methods. Following scanning and data analysis, those genes found to be up- or down-regulated by 2-fold or greater in comparison to baseline expression were classified according to their function by GeneSpring software, and are listed here.

From the total of 259 genes found to be significantly altered as a result of the expression of either ATF2 factor, only two were affected by both: transient receptor potential cation channel, subfamily C, member 4 (TRPC4) which was up-regulated by both isoforms, and calcitonin receptor like (CALCRL) which was down-regulated by both. The genes in all lists were classified and grouped according to their functions as determined by the GeneSpring software; where genes belonged to more than one functional group, the results tables were manually trimmed to prevent multiple entries of these particular genes. Many of these genes are described in detail in the discussion section.

SSH of ATF2-sm stably transfected myometrial cells

Dot-blot screening of 100 transformed bacterial colonies resulting from this procedure gave rise to 25 putative differentially expressed clones, as determined from the blot patterns with probes derived from (i) forward subtracted, (ii) reverse subtracted, (iii) unsubtracted driver and (iv) unsubtracted tester cDNAs (Fig. 4). These genes were identified by sequencing of the clones represented by the colonies on the dot-blots, and are listed in Table 2.

View larger version (138K): [in this window] [in a new window]   Figure 4 SSH results revealed differentially expressed genes in ATF2-sm stably transfected cells. Briefly, libraries of putative differentially expressed genes were constructed following the subtraction procedure, and transformed into bacterial cells for amplification. The inserts in the library plasmids were then amplified from these bacterial cultures by PCR, and spotted onto membranes for screening with forward subtracted, reverse subtracted, unsubtracted driver and unsubtracted tester probes. Examples of the resultant blots are shown here. The likelihood of differential expression of any particular clone was indicated by the pattern and intensity of the signals from the four blots; three instances are shown here, where =probably differentially expressed; =probably differentially expressed if intensity difference between forward subtraction and others is high; =not differentially expressed. Boxed area=negative controls.

The validity of the results from the microarray and SSH experiments was examined by performing a panel of semi-quantitative RT-PCR reactions. Four randomly-selected genes were chosen from the results of the ATF2 and ATF2-sm microarray experiments for each factor and, by semi-quantitative RT-PCR, their mRNA levels assessed in control cultured myometrial cells containing empty vector, the stably-transfected myometrial cell-line used for the array hybridization, pooled non-pregnant (NP) tissue and pooled pregnant non-labouring (P) tissue (n=6; Fig. 5). In each case, the level of expression in stably-transfected cells compared with control cells reflected the results from the microarrays: those genes found to be up- or down-regulated by expression of ATF2 and ATF2-sm by microarray analysis also showed increased or decreased expression respectively in the stable cell-lines compared with control cells by RT-PCR. In addition, the expression of these genes was found to be significantly altered between the pooled NP and P tissue, reflecting the in vivo biological situation. Of the eight genes examined, four were expressed at a higher level in NP tissue and four at a higher level in P tissue. Real-time RT-PCR analysis of two of these genes, GNA15 and HSP70B', also indicated significant up-regulation of their expression in stable cell-lines in concordance with the microarray data, and also increased expression in NP tissue compared with P tissue reflecting the in vivo situation.

View larger version (20K): [in this window] [in a new window]   Figure 5 Semi-quantitative and quantitative RT-PCR was performed to verify differential expression of a random selection of genes revealed in the microarray analyses. Comparisons were made between (a) total RNA templates isolated from control empty vector stably-transfected cell lines and cell lines stably transfected with ATF2 and ATF2-sm constructs, and (b) mRNA templates isolated from pooled (n=6) NP and P myometrial tissue samples. 5 ng of mRNA or 50 ng of total RNA was used as template in RT-PCR reactions using the SuperScript OneStep RT-PCR system (Invitrogen) for 28, 30 and 32 PCR cycles, and the linear-range products were analyzed by 2% agarose gel electrophoresis, as shown above. In each case, the level of expression in stably-transfected cells compared with control cells reflected the results from the microarrays: those genes found to be up- or down-regulated by expression of ATF2 and ATF2-sm by microarray analysis also showed increased or decreased expression, respectively, in the stable cell-lines compared with control cells by RT-PCR. In addition, the expression of these genes was found to be significantly altered between the pooled NP and P tissue, reflecting the in vivo biological situation. Of the eight genes examined, four were expressed at a higher level in NP tissue and four at a higher level in P tissue. Real-time RT-PCR analysis of two of these genes, GNA15 and HSP70B', also indicated significant up-regulation of their expression in stable cell-lines in concordance with the microarray data, and also increased expression in NP tissue compared with P tissue reflecting the in vivo situation.

View larger version (52K): [in this window] [in a new window]   Figure 6 Semi-quantitative RT-PCRs of selected differentially expressed candidate genes from the SSH screen of ATF2-sm stably-transfected cells. One-tube RT-PCR was performed using total RNA isolated from NP and P lower segment myometrial tissue samples as template, using 28, 30 and 32 PCR cycles to obtain a representative band from the linear amplification range after agarose gel electrophoresis. Bands were visualized using a gel documentation system at equivalent integrations, and scans of these are shown above. In each case, candidate positive genes from the SSH screen, i.e. those up-regulated in ATF2-sm stably-transfected cells, were found to be more highly expressed in the NP myometrium.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

The results revealed a diverse range of gene categories in terms of biological processes, cellular components and molecular functions that are regulated by these bZIP factors. A small selection of these genes is detailed here, in terms of their patterns of expression in the presence of the ATF2 bZIP transcription factors and how these relate to myometrial function during gestation and labour.

TNFR9 was observed to be up-regulated by ATF2. Genes from the tumour necrosis factor receptor superfamily (TNFR) have significant roles in the myometrium due to the importance of TNF in gestation; this is expressed by myometrial stromal cells during pregnancy, although the major source in the myometrium is thought to be macrophages and other leukocytes (Yelavarthi et al. 1991, Tabibzadeh et al. 1994, Young et al. 2002). It stimulates the production of arachadonic acid and prostaglandins (in particular the stimulatory PGF1) (Pollard & Mitchell 1996a, b, Hertelendy et al. 2002); up-regulates matrix metalloproteinase-9 (MMP-9), which is involved in tissue remodelling of the myometrium during labour (Roh et al. 2000); increases adenylyl cyclase (AC) activity in myometrial cells (Gogarten et al. 2003); and may prepare the myometrium for labour by regulating the cyclic-ADP–ribose-signalling (cADPR) pathway which controls the development of intracellular Ca²⁺ transients (Barata et al. 2004).

ATF2-sm increased the expression of the heat shock 70 kDa protein (HSPA6/HSP70B'); heat shock proteins form associations with steroid receptors, including those for oestrogen and progesterone, and modulate their functions (Komatsu et al. 1997). HSPs 70 and 90 have been shown to increase dramatically during labour in the ovine myometrium, and possibly inhibit progester-one receptors and activate oestrogen receptors (Wu et al. 1996).

In the signal transduction/integrin receptor signalling class, ATF2 caused an increase in expression of the calcium channel, voltage dependent, P/Q type, alpha 1A subunit (CACNA1A), and a decrease in the expression of the regulator of G-protein signalling 14 (RGS14). With regard to CACNA1A, the importance of the regulation of calcium homeostasis in the myometrium and its relation to uterine contractility is self-evident. RGS proteins interact with the Gq and Gi proteins to accelerate GTPase activity and RGS2, for example, is known to increase in cultured human myometrial cells in response to oxytocin via an increase in intracellular Ca²⁺ concentration, and also increases during pregnancy and decreases just prior to labour in rat myometrium in response to progesterone levels (Park et al. 2002, Suarez et al. 2003).

The expression of various integrins and cadherins are known to alter in the human uterus during pregnancy (Brackin et al. 2002, Charpigny et al. 2003); for example, ICAM-1 has been found to be up-regulated in the myometrium 10.5-fold during labour (Ledingham et al. 2001), and E-selectin (SELE; endothelial adhesion molecule 1) has been shown to increase during labour (Thomson et al. 1999). In this respect ATF2-sm down-regulated expression of integrin 3 (ITGA3).

Among the other functional classes of genes affected by ATF2 and ATF2-sm over-expression were the following: gonadotropin inducible transcription repressor-3 (GIOT-3) was down-regulated by ATF2, which may lead to an increase in its expression in the transition from the non-pregnant to the pregnant state as ATF2 expression decreases. This could also be augmented by the increase in myometrial hCG levels during pregnancy. The gene expression of four collagen types was altered by ATF2; XIII1 and IV3 were increased, whereas VI1 and XVII1 were decreased. Collagen is important in the fibrillar network of the uterus, where it provides mechanical support for its structural components and a favourable milieu for their activities (Goranova et al. 1993). Fibrils have been shown to reorganize in response to the hormonal changes that take place during pregnancy, and also to be degraded by myofibroblastic interstitial cells in preparation for the labouring and postpartum stages to allow the development of elastic fibres and basement membranes (Nishinaka & Fukuda 1991). Three forms of collagen type VI (one of which was down-regulated by ATF2 here) have been shown to be present at high levels in murine myometrium during pregnancy (Dziadek et al. 1995). In addition, ATF2 caused a decrease in the expression of the MMP-20. The expression of MMPs in the myometrium is regulated by TGF-ß1 (Ma & Chegini 1999), and is absolutely dependent on the presence of serotonin, which is induced by IL-1 (Dumin et al. 1998).

Protein kinase C (PKC), involved in the mediation of endothelin-induced uterine contractions (Oriji & Keiser 1996) and the regulation of oxytocin-mediated myometrial contractions (Phillippe 1994), is of critical importance in the myometrium. It acts to promote a contractile state by reducing the activity of adenylyl cyclase and therefore cAMP accumulation (Grammatopoulos et al. 1996, Grammatopoulos & Hillhouse 1999), and by reducing guanylyl cyclase activity and therefore cGMP accumulation, to neutralize relaxatory pathways. ATF2, however, caused a decrease in expression of the (iota) isoform; this may be involved in cytokine-induced NF-kappa B activation (Anthonsen et al. 2001).

G protein coupled receptors (GPCRs) comprise the largest family of receptors in the uterus; they can be stimulatory or inhibitory, and their interaction with heterotrimeric GTP-binding proteins enables them to induce the hydrolyzation of GTP and to modulate the activity of a wide variety of enzymes and ion channels that affect uterine contractility. Some GPCRs are coupled to phospholipase C, which generates the second messengers inositol 1,4,5-trisphosphate (IP₃) and diacyl-glycerol. These act to stimulate contractions via the mobilization of calcium in the sarcoplasmic reticulum in the case of IP₃, and in the case of diacylglycerol to activate PKC and the MAPK cascades (Bernal 2003). Other GPCRs are coupled to adenylyl cyclase, which promotes the accumulation of cAMP which acts to promote uterine relaxation via the inactivation of myosin light-chain kinase (MLCK) and the action of the cAMP-dependent bZIP transcription factors. In addition, uterine contractility can also be enhanced via the activation of Rho GTPases, and the subsequent action of Rho kinases to potentiate the effect of MLCK (Moore et al. 2000, Moran et al. 2002). GPCR63 was up-regulated by ATF2.

S100 calcium-binding protein A13 (S100A13) has been shown to mediate the stress-induced release of IL-1 (Mandinova et al. 2003); this interleukin induces the expression of serotonin in the uterus, which in turn allows the expression of MMPs in myometrial cells (Dumin et al. 1998). The importance of collagen and MMPs in the uterus has already been discussed; S100A13 was up-regulated by ATF2.

The gene for caveolin 2 (CAV2) was up-regulated by ATF2; caveolin expression in the myometrium of the pregnant rat is known to be suppressed in the first half of pregnancy, after which it increases up to delivery, increasing the number of caveolae present in myometrial cells (Turi et al. 2001). This has been shown to be dependent upon levels of oestrogen and progesterone.

There is mounting evidence that cytokines are critically important in the modulation of uterine activity and that the regulation of parturition is, to a large degree, an inflammatory process. A rise in proinflammatory cytokines and the infiltration of leukocytes into the uterine tissues at labour are well documented. They are known to be largely derived from the placenta and extra-placental membranes (gestational tissues) (Bowen et al. 2002), although the cytokine-inducible expression of interleukins in uterine smooth muscle cells suggests that the myometrium itself may be a significant contributor of inflammatory mediators (Helmer et al. 2002). The chemokine (C-C motif) receptor 4 (CCR4) was up-regulated by ATF2.

Endothelin receptors were affected by ATF2; receptor type A, which is preferentially expressed in the upper segment of the uterus and thought to exert a stimulatory effect with regard to contractility, was found to be down-regulated.

Any variance in the expression of oestrogen receptors will, taking into account the importance of the hormone and its wide ranging effects upon other factors involved in the control of uterine gene expression and contractility, exert promiscuous and far reaching effects. It is therefore interesting to note the increased expression of oestrogen receptor 2 (ESR2/ESR-beta) with over-expression of ATF2, and to compare this to the detected increase in expression of the alternative oestrogen receptor 1 (ESR1/ESR-alpha) associated with over-expression of the ATF2-related bZIP transcription factor CREB in myometrial cells (Bailey et al. 2004), which has been found to be prevalent in the non-pregnant myometrium but almost absent in pregnant non-labouring and spontaneous labouring tissue (Bailey et al. 2000). Interestingly, a significant minority of genes found to be affected by the over-expression of ATF2 and ATF2-sm in myometrial cells in this study are also affected by CREB (Bailey et al. 2004): 25% of the genes affected by ATF2, and 38% of the genes affected by ATF2-sm.

An overlap also exists between the groups of genes affected by ATF2 and ATF2-sm. This is represented in the form of a Venn diagram in Fig. 7. The number of genes commonly affected by the two ATF2 proteins amounts to only two, or just under 1% and just over 3% of genes affected by ATF2 and ATF2-sm respectively. However, these two genes may well be of great importance with regard to myometrial contractility; one, TRPC4 encodes a component of store-operated calcium entry (SOCE) channels that play an important role in myometrial calcium homeostasis (Dalrymple et al. 2002), and was up-regulated by both ATF2 isoforms. The other, CALCRL is related to the CGRP, which induces dose-dependent relaxation in spontaneously contracting myometrium from pregnant women, an effect that is diminished in myometrium obtained from patients during labour and in the non-pregnant states (Dong et al. 1999). This gene was down-regulated by both ATF2 isoforms.

View larger version (8K): [in this window] [in a new window]   Figure 7 The ATF2 and ATF2-sm factors regulate highly discrete groups of genes; only two were regulated by both proteins. However, these two genes may well be of great importance with regard to myometrial contractility; one, TRPC4, encodes a component of store-operated calcium entry (SOCE) channels that play an important role in myometrial calcium homeostasis, and was up-regulated by both ATF2 isoforms. The other, CALCRL, is related to the CGRP, which induces dose-dependent relaxation in spontaneously contracting myometrium from pregnant women, an effect that is diminished in myometrium obtained from patients during labour and in the non-pregnant states. This gene was down-regulated by both ATF2 isoforms.

The spatio-temporal expression of ATF2 bZIP transcription factors raises the concern of their modes of action; not only are these factors responsive to different stimuli and activated by different pathways, but their dimerization codes bring into play several promoter elements in the downstream genes affected by them. Although the ATF2 and ATF2-sm proteins, in common with the related CREB/CREM factors, are substrates for PKA-mediated phosphorylation in vitro, the ATF2 proteins are insensitive to cAMP/PKA stimulation, and instead are a major target of MAPK cascades, upon which it depends for its activation. These MAPK cascades, which are instigated for example in response to the presence of inflammatory cytokines (well-characterized modulators of uterine gene expression), are classified into three subfamilies; the p38 cascade, the c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade, and the extra-cellular signal-regulated protein kinase (ERK) cascade. All three of these cascades act in response to a variety of stresses to cause the phosphorylation and activation of ATF2 and increase its transcriptional activity, and the type of cascade involved is dependent upon the type of stimulatory factor (Hayakawa et al. 2003). The latter acts through the binding of mitogens or growth factors at the cell membrane, recruiting the Ras GTPase which activates the Raf Ser/Thr kinase. This in turn activates MAPK/ERK kinase (MEK) which phosphorylates and activates ERK1 and ERK2. These factors translocate to the nucleus where they activate a range of transcription factors that control cellular processes such as growth and differentiation, including ATF2. With regard to dimerization, ATF2 for example forms dimers with NF-B and c-Jun; such dimers have greatly altered promoter motif binding properties (Wagner & Green 1994), and variable affinities to CRE contexts. ATF2:c-Jun heterodimers have a great affinity for AP1 sites compared with CREs, and the two constituent proteins may show either an antagonistic relationship to one another, or be present in certain tissues at inversely proportional levels. We have previously shown that the ATF2-sm protein also heterodimerizes with c-Jun in the myometrium, though the consequences of this are not presently known (Bailey et al. 2002).

In support of our hypothesis that ATF2/ATF2-sm proteins may play an important role involved in modulating myometrial gene expression and functionality, we have made an effort to link genes affected by these proteins to what is known of the in vivo physiology of the uterus during gestation and labour.

	Acknowledgements

 
This work was funded by a grant made available from the Wellcome Trust (grant 062928).

	References

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Received 4 August 2004
Accepted 13 September 2004
Made available online as an Accepted Preprint 4 October 2004

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