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Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
(Requests for offprints should be addressed to F J DeMayo; Email: fdemayo{at}bcm.tmc.edu)
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Abstract |
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Introduction |
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The ovarian steroid hormone progesterone (P4) is an essential regulator of reproductive events associated with all aspects of the establishment and maintenance of pregnancy (Clarke & Sutherland 1990, Lydon et al. 1995). The physiologic affects of P4 are mediated through progesterone receptors (PGRs) that are expressed as two isoforms, PGR-A and PGR-B, that arise from the same gene (Mulac-Jericevic et al. 2000, Conneely et al. 2002). PGR is a transcription factor belonging to the nuclear receptor superfamily (Evans 1988, O’Malley & Conneely 1992, Tsai & O’Malley 1994). The fertility defects exhibited by the PRKO mice unequivocally demonstrated the critical importance of P4 and its receptor in establishment and maintenance of pregnancy (Lydon et al. 1995, 1996).
Although the impact of the P4-PGR axis on murine uterine function has been extensively investigated, only a few P4-PGR-regulated genes have been identified. These include genes encoding amphiregulin (Areg) (Das et al. 1995), histidine decarboxylase (Hdc) (Paria et al. 1998), homeo box A10 (Hoxa10) and homeo box A11 (Hoxa11) (Lim et al. 1999), calcitonin (Calca) (Kumar et al. 1998, Zhu et al. 1998), calbindin-D9K (Nie et al. 2000), Indian hedgehog (Ihh) (Takamoto et al. 2002), hypoxia-inducible factor 1 (Hif1a; Daikoku et al. 2003) and immune-responsive gene 1 (Irg1) (Cheon et al. 2003). These target genes have been identified by testing candidate genes (Das et al. 1995), by differential library screening (Zhu et al. 1998) and by DNA microarray approaches (Cheon et al. 2002, Takamoto et al. 2002). The advent of the last named, high-density DNA microarray technology, has immensely improved the ability to identify PGR-regulated genes in the uterus.
We recently used oligonucleotide microarrays to identify the genes with expression regulated by the P4-PGR axis in the mouse uterus (Jeong et al. 2005). PRKO and wild-type mice were ovariectomized and then treated with 1 mg P4 or vehicle (sesame oil) every 12 h. Groups of mice were killed 4 h after the first P4 injection (acute treatment) or 4 h after the fourth injection of P4 (chronic treatment). Using this methodology, we identified several genes regulated by PGR in the uterus in response to P4 (Jeong et al. 2005). Interestingly, we found that the expression of Clca3 (chloride channel calcium-activated 3) mRNA was suppressed significantly by P4 and PGR.
The calcium-activated chloride channels (CLCA) family appears to mediate a calcium-activated chloride conductance in a variety of tissues, including epithelium (Evans & Marty 1986, Huang et al. 1993, Arreola et al. 1996), smooth muscle (Amedee et al. 1990, Clapp et al. 1996), skeletal muscle (Hume & Thomas 1989) and neurons (Barnes & Hille 1989, Hallani et al. 1998). Six members of this family have been identified, cloned and partially characterized in the mouse: Clca1 (Gandhi et al. 1998, Romio et al. 1999), Clca2 (Lee et al. 1999), Clca3 (Komiya et al. 1999), Clca4 (Elble et al. 2002), Clca5 and Clca6 (Abdel-Ghany et al. 2003, Beckley et al. 2004, Evans et al. 2004). Structural analysis indicates significant similarities among different CLCA family members, including protein sizes of 902–943 amino acids and four or five transmembrane regions (Pauli et al. 2000, Jentsch et al. 2002). However, the tissue and cellular distribution patterns appear to be unique to each protein.
Clca3 (also termed gob-5) has been identified in goblet cells throughout the intestinal tract by in situ hybridization (Komiya et al. 1999). The Clca3 transcript was also detected by Northern blot in the murine trachea and uterus without identification of the respective cell types. However, the function and regulation of Clca3 have not been reported to date, and the biologic processes in which it is involved are unclear. In this study, we analyzed the spatiotemporal expression and regulation of Clca3 in the response to P4 and E2 in early pregnant uterus.
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Materials and Methods |
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Mice were maintained in the designated animal care facility at Baylor College of Medicine according to the institutional guidelines for the care and use of laboratory animals. Thirty-six wild-type and 12 PRKO mice at 6 weeks of age were ovariectomized. Two weeks later, ovariectomized wild-type mice were injected with one of the following: vehicle (sesame oil), P4 (1 mg/mouse), E2 (0.1 µg/mouse) or P4 plus E2. The injections were at 0 h and at 12-h intervals thereafter, the animals being killed at 4, 16, and 40 h (wild-type) or 4 and 40 h (PRKO) (n=3 animals per genotype per treatment). Hormone injection was repeated every 12 h for the 16- and 40-h samples to prevent the effect of hormone degradation by metabolism. The mice were anesthetized with Avertin (2,2,-tibromoethyl alcohol; Sigma-Aldrich) and killed by cervical dislocation under anesthetic at 4, 16 or 40 h (4 h after fourth injection) to collect the uteri. Multiple-timed pseudopregnant female mice were achieved by treating female mice with a superovulatory regimen of gonadotropin and mating with vasectomized male mice. Hormonal induction of ovulation was used to synchronize the cycle of mice for multiple-timed pregnant females. Briefly, wild-type females were administered 5 IU pregnant mare’s serum gonadotropin (VWR Scientific Products, West Chester, PA, USA), followed 48 h later by 5 IU human chorionic gonadotropin (Organon, West Orange, NJ, USA), and placed with a vasectomized male mouse. The morning of vaginal plug was designated as day 0.5. Uterine tissues were flash frozen at the time of dissection and stored at –80 °C for RNA or fixed with 10% (v/v) formalin for in situ hybridization.
Quantitative real-time PCR
RNA was extracted from uterine tissues with the RNeasy total RNA isolation kit (Qiagen). Expression levels of Clca3 mRNA were measured by real-time RT–PCR TaqMan analysis with the ABI Prism 7700 Sequence Detector System according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Prevalidated probes and primers for Clca3 and 18S RNA were purchased from Applied Biosystems. RT–PCR was performed with One-Step RT–PCR Universal Master Mix reagent and TaqMan Gene Expression Assays (Applied Biosystems) according to the manufacturer’s instructions. Standard curves were generated by serial dilution of a preparation of total RNA isolated from whole mouse uterus. All real-time PCR was done with the three independent RNA sets. mRNA quantities were normalized against 18S RNA with ABI rRNA control reagents. Statistical analyses used one-way ANOVA followed by Tukey’s post hoc multiple range test with the Instat package from GraphPad (San Diego, CA, USA). P values of < 0.05 were considered statistically significant, and values of < 0.01 highly significant. In the figures, S.E.M. is indicated in all bar graphs.
In situ hybridization
The protocol for in situ hybridization was essentially as described previously by Simmons et al.(1989). Uterine tissues were fixed in 10% (v/v) formalin. After overnight fixation at room temperature, tissues were dehydrated through a series of ethanol and then processed for paraffin embedding. Paraffin sections were mounted onto poly-L-lysine-coated slides (VWR Scientific Products, West Chester, PA, USA), and used for in situ hybridization. The riboprobes were generated by in vitro transcription of amplified DNA products containing the T7 polymerase promoter sequence flanking the desired nucleotide primer sequence, using 35S-UTP (Promega). Slides were incubated for 7 min at room temperature in Proteinase K (20 µg/ml) in a buffer containing 50 mM Tris and 5 mM EDTA (pH 8). Slides were then acetylated with acetic anhydride, dehydrated and exposed to either denatured antisense or sense probes in hybridization buffer (50% (v/v) formamide, 10% (w/v) dextran sulfate, 5 Denhardt’s solution, 300 mM NaCl, 5 mM EDTA (pH 8), 20 mM Tris (pH 8) and 0.05 mg/ml yeast tRNA). Hybridization was performed at 55 °C overnight in a humidity chamber containing 5 SSC and 50% (v/v) formamide. Hybridized slides were exposed to 20 µg/ml RNase A for 30 min at 37 °C. Slides were washed in 50% (v/v) formamide, 2 SSC and 100 mM 2-mercaptoethanol, followed by 2 SSC at 55 °C for 30 min, dehydrated in a graded series of ethanol in 0.3 M ammonium acetate, and exposed to Biomax MR film overnight (Kodak). The following morning, slides were dipped in autoradiography emulsion (Amersham) and placed at 4 °C in a light-proof box for several days. After development, slides were counterstained with hematoxylin.
Construction of Clca3-luciferase-expression vectors
The 891 bp (–907 to –17) of 5'-flanking region of the Clca3 gene were PCR-amplified from mouse genomic DNA as a template, using the 5'-PCR primer, 5'-GAA GACCAAAAGGATGAAAATGAC-3', and the 3'-PCR primer, 5'- GGGAAGCTTTGGAAAGGGCTGGGTG TAGAAG -3'. The resulting PCR product was subcloned into the pCR II TA TOPO cloning vector (Invitrogen). The 891 bp Clca3 fragment was liberated from the pCR II TA TOPO vector and subcloned into the luciferase gene in the pGL3 Basic vector (Promega).
Two deletion constructs, 528 bp (–544 to –17) and 319 bp (–335 to –17) promoter, were generated via PCR with the same 3' PCR primer and two different 5' PCR primers: –544 (5'-GATGCTGAGGAGAAATGTGGA GTT-3'), and –335 (5'-TGAGGAACCAGATTAG GAT-3'). The resulting PCR product was subcloned into the luciferase gene in the pGL3 basic vector (Promega).
Transient transfection assay
HEC-1A cells were seeded in 24-well plates and cultured according to ATCC recommendations until they were approximately 60–70% confluent. Cell cultures were maintained in media containing charcoal-stripped fetal calf serum (FCS). The cells were transfected with Superfect (Qiagen) with 1 µg reporter gene, and 10 or 50 ng estrogen receptor alpha (Esr1) or estrogen receptor beta (Esr2). Cells were treated with either E2 (10–8 M), or vehicle (ethanol) for 24 h. Cells were harvested for luciferase assay 24 h after addition of the E2 or ethanol. Cell extracts were prepared after 24-h transfection and assayed by a luciferase reporter system (Promega). Protein concentration was used to correct for differences in transfection efficiencies. The data represent two independent experiments, each performed in triplicate. Statistical analyses were by one-way ANOVA followed by Tukey’s post hoc multiple-range test with the Instat package from GraphPad. P values of < 0.05 were considered statistically significant and values of < 0.01 highly significant. The S.E.M. is indicated in all bar graphs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously identified Clca3 as a potential chronic P4-and PGR-regulated gene in the murine uterus by DNA microarray analysis (Jeong et al. 2005). In this study, we analyzed the P4 and PGR regulation of Clca3 in the murine uterus. For further determination of the reliance of Clca3 expression on PGR function, ovariectomized wild-type and PRKO mice were injected with P4 or vehicle (sesame oil). Uteri were collected from the mice after 4 h (acute treatment) or 40 h (chronic treatment) of hormone treatment, and the expression of Clca3 was investigated by real-time RT–PCR. As shown in Fig. 1
, the mRNA transcript of Clca3 was detected in the uteri of wild-type ovariectomized mice. However, the Clca3 transcript was significantly decreased after chronic treatment with P4 in the wild-type mice (10%). To establish whether the P4 regulation of Clca3 is PGR dependent, we analyzed the expression of the Clca3 in the uteri of wild-type and PRKO mice. After chronic P4 treatment, downregulation of the Clca3 transcription was not detected in the PRKO mice (Fig. 1). We also did not detect any change of Clca3 expression in the wild-type mice after the acute P4 treatment, although a significant effect was seen at 16 h in the wild-type mice. These results were consistent with our microarray data identification of Clca3 gene as a late responsive gene. These results show that Clca3 mRNA expression is downregulated by both P4 and PGR in chronic treatment, but not in acute treatment.
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, using the antisense Clca3 probe, we observed a hybridization signal in the luminal and glandular epithelium cells of the uterine section obtained from vehicle-treated wild-type uterus. The observed signal was not uniform but indicated distinct regions of signal intensity in the luminal and glandular uterine epithelium. These signals were not detected in the chronic P4-treated, wild-type samples. To establish whether the P4 downregulation of the Clca3 gene is mediated through PGR, ovariectomized PRKO and wild-type female mice were treated with P4 or vehicle. After chronic P4 treatment, the uteri were collected, and in situ hybridization was performed. As expected, hybridizing signals for Clca3 transcripts were detected in the P4-treated PRKO uterine sections (Fig. 3). These results indicate that PGR is essential for Clca3 gene downregulation in luminal and glandular epithelium cells.
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P4 antagonizes E2 actions, such as the stimulation of proliferation of the epithelial cells in the mouse uterus (Martin et al. 1973b, Huet-Hudson et al. 1989). To determine whether P4 antagonizes the effect of E2 in Clca3 expression, we treated ovariectomized female mice with E2 or E2 plus P4 for 4, 16, or 40 h. Real-time RT–PCR analysis was performed with uterine RNA after E2 or E2 plus P4 treatment. As shown in Fig. 4, P4 repressed Clca3 mRNA levels 16 and 40 h after treatment. The expression of Clca3 mRNA was increased 3.65-, 5.52- and 87.02-fold 4 and 16 h after E2 treatment and after 40 h of chronic E2 treatment respectively. The results suggest that E2 induces the expression of Clca3 in the uterus. Figure 4 also shows that treatment of mice with E2 plus P4 significantly inhibited the induction of Clca3 by E2. These results indicate that the expression of Clca3 is induced by E2, but the induction of mRNA expression by E2 is inhibited by P4 in the uterus.
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). The signal of Clca3 was increased at 16 h, and then a significant induction of Clca3 mRNA was observed in the luminal epithelium and glandular epithelium cells at 40 h. To determine the spatial regulation between E2 and P4 in the Clca3 expression, we performed in situ hybridization on the E2 plus P4-treated uteri (Fig. 5B). The weak epithelial signals were detected at 4 h, and then the signals were increased at 16 h. However, we could not detect any signal at 40 h in uterine sections. These results suggest that P4 is a downregulator of Clca3 expression and E2 is a positive regulator in the luminal and glandular epithelium.
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The above analysis identified the pharmacologic regulation of Clca3 by the ovarian steroids E2 and P4. We next established the expression profile of Clca3 mRNA in mouse uteri during early pregnancy with real-time RT–PCR to measure levels of Clca3 and compare the expression to two other uterine P4-responsive genes, amphiregulin (Areg) (Das et al. 1995) and patched homolog 1 (Ptch1) (Takamoto et al. 2002) from pseudo-pregnant samples. As shown in Fig. 6A, the strong expression of Clca3 detected on day 0.5 declined sharply on days 1.5 and 2.5 (5.59% and 1.38% respectively) and was undetectable after day 3.5. The levels of P4 were elevated on day 2.5 during early pregnancy, as shown by the induction of P4-responsive genes Areg and Ptch1 (Fig. 6B and C respectively). These results suggest that strong downregulation of Clca3 expression occurs in the uterus on day 2.5 of gestation, presumably in response to P4.
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). Consistent with the real-time RT–PCR results, the Clca3 mRNA transcripts were strongly expressed in luminal and glandular epithelial cells at day 0.5. However, the specific hybridization signal was not observed in the epithelial cells of uterine sections obtained from day 2.5. These results further confirm that Clca3 transcription is repressed in the luminal and glandular epithelial cells of pregnant uteri. Moreover, these spatial patterns of Clca3 expression are similar to the ones observed in the uteri of ovariectomized mice in response to P4 treatment. These results suggest that the repression of Clca3 may be important during early pregnancy.
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To investigate the molecular mechanisms underlying Clca3 gene expression regulation by P4 and E2, we examined the Clca3 promoter and 5'-flanking region for potential sequences that are recognized by PGR and estrogen receptor (ESR). Although no palindromic PRE (progesterone response element) could be identified in the 2 kb 5'-flanking region, this region contains one palindromic ERE (estrogen response element) and one half-ERE, the most proximal of which is present within the –420 (Fig. 8A). This palindromic ERE of Clca3 was shown to be conserved in mouse, rat, man, cow and dog (Fig. 8B).
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). Under these conditions, it was shown that Esr1, and not Esr2, activated, in a dose-dependent manner, the 891 bp promoter of Clca3 in HEC-1A cells (Fig. 8C). We could not detect regulation of the Clca3-luciferase reporter when cotransfected in HEC-1A cells with expression vectors for Pgr-A or Pgr-B (data not shown). For further identification of the region of Esr1 regulation of the Clca3 promoter, cotransfection experiments in HEC-1A cells were conducted with deletion fragments of the Clca3 promoter fused to the luciferase reporter. These reporters contained 528 and 319 bp of the Clca3 promoter. The fragment containing 891 and 528 bp of the promoter region showed enhanced luciferase activity by E2. However, deletions to –335 bp, which eliminated the only full ERE site, resulted in 58% loss of E2 induction of luciferase activity. These results suggest that the conserved ERE region of Clca3 is important for Esr1 regulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The analysis of Clca3 in mouse uterus shows that the gene is expressed in the luminal and glandular compartments of the endometrium. The expression of this gene is not uniform throughout the epithelial compartments, but shows focal regions of intensity. This may reflect a cellular specificity to the expression of this gene. Although there is little information on the expression of cell specific markers in uterine epithelial cell types, Clca3 may represent a means of detecting subpopulations of uterine epithelial cell types.
Our results indicate that the expression of Clca3 is stimulated by E2 and repressed by P4. The expression studies during pregnancy show that Clca3 is highest at day 0.5, and its expression decreases as pregnancy progresses, when P4 levels are rising. This decrease in Clca3 is opposite to the expression of genes stimulated by P4, as well as the expression of Pgr (Tan et al. 1999, Takamoto et al. 2002), validating in vivo physiologic repression of this gene by P4. In silico analysis of the 10 kb region flanking 5' to the proximal promoter region of Clca3 could not detect any conserved PRE. However, one conserved ERE was detected at -418 ~ -407, and one half site was detected at –176 ~ –171 of the 5' region of the mouse gene (detection by TESS (Transcription Element Search System; www.cbil.upenn.edu/tess)). The consensus ERE motif is a palindromic repeat of the sequence GGTCA separated by three nucleotides (Klinge 2001). These palindromic repeats are separated by one nucleotide, but conserved in five different species, including mouse, rat, man, cow and dog (Fig. 8B). We show significant E2 induction of the 891 and 528 bp promoter reporter construct, while induction was significantly reduced in the 319 bp promoter-reporter construction. Even though the 319 bp promoter fragment did not contain a full ERE consensus site, E2 induction was still observed, albeit significantly reduced. This residual regulation by E2 may be through activation of the ERE half-site at –176 ~ –171, or it may be through a mechanism independent of DNA binding (Aronica et al. 1994, Tesarik & Mendoza 1995, Le Mellay et al. 1997, Simoncini et al. 2004). Interestingly, the 0.5 kb region displays higher luciferase activity than the 1.0 kb full-length promoter region. This region may contain potential repressive elements between –543 and –1006, relative to ATG. The above results demonstrate that E2 regulates, through its receptor, Clca3 transcription.
These results suggest that this estrogen response in Clca3 may serve as a direct docking site for the estrogen receptor and provide direct regulation by estrogen. Since no conserved PRE was found, repression of PGR may be through an indirect mechanism. Since P4 inhibits the expression of Esr in the uterine epithelium, the expression of Clca3 may be by P4 inhibiting Esr expression. However, this does not exclude other transcription factors being inhibited by P4.
Clca3 has been identified in goblet cells of the intestinal tract by in situ hybridization (Komiya et al. 1999). Increasing evidence suggests that members of the CLCA family play a role in diseases with epithelial secretory dysfunction (Hashimoto et al. 2004). By use of the murine model of bronchial asthma, Clca3 was found to be a key regulator of the induction of mucus overproduction. In this model, antisense Clca3 therapy effectively suppressed the asthma phenotype, whereas Clca3 overexpression exacerbated the condition (Nakanishi et al. 2001). Changes in MUC1 glycoform expression have been related to a phase of the menstrual cycle and endometrial receptivity in a number of studies (Meseguer et al. 1998, Lagow et al. 1999, Aplin et al. 2001). Failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin and retention of PGR, particularly in epithelial cells (Horne et al. 2005). Downregulation of Clca3 by P4 during early pregnancy may be a means of initiating the decrease in mucus production by the endometrial epithelial cells in the uterus. The decrease in mucus production may initiate the window of receptivity for embryo attachment and implantation.
In summary, this study demonstrated Clca3 mRNA to be expressed in murine uterine luminal and glandular epithelial cells, but not in stroma cells in ovariectomized mice. The levels of this mRNA in the uterine luminal and glandular epithelial cells were significantly downregulated by P4 and upregulated by E2. E2-mediated induction was inhibited by P4 when the ovariectomized mice were treated with E2 and P4. The expression of Clca3 was turned off on day 1.5 of pregnancy. These results suggest that steroid hormonal regulation of Clca3 may be important in the uterus of mouse during early pregnancy.
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Acknowledgements |
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Funding |
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References |
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Received in final form 21 February 2006
Accepted 2 March 2006
Made available online as an Accepted Preprint 13 March 2006
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