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Department of Obstetrics, Gynaecology and Neonatology (S.M., P.V., D.L., A.A.), The "L. Mangiagalli" Hospital; Department of Obstetrics and Gynaecology, Clinica "Macedonio-Melloni" (M.V.) and Istituto Auxologico Italiano (A.M.D.B.), Cusano Milanino 20095, Italy
Address all correspondence and requests for reprints to: Dr. A. M. Di Blasio, Molecular Biology Laboratory, Istituto Auxologico Italiano, Via Zucchi 18, Cusano Milanino 20095, Italy. E-mail: a.diblasio{at}auxologico.it.
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Abstract |
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Objective: Using an oligonucleotide microarray-based hybridization analysis, we observed that a Wnt family member transcript, Wnt5b, was overexpressed in smooth muscle cells (SMC) derived from leiomyomas when compared with matched myometrial cells. Based on this finding and on previous observations, we have hypothesized that altered expression of specific Wnt family members might be involved in leiomyoma formation and/or growth.
Main Outcome Measures: The expression patterns of two members of the Wnt pathway, Wnt5b and secreted frizzled related protein (sFRP)1, were evaluated in myometrial SMC (n = 22) and in leiomyoma cells (n = 27) by real-time quantitative PCR. In addition, regulation of expression of the two molecules was examined.
Results: Compared with myometrial SMC, cells derived from leiomyomas had significantly higher levels of both Wnt5b and sFRP1 transcripts. When the data were analyzed as a function of the phase of the menstrual cycle, no significant difference in sFRP1 mRNA levels could be detected, whereas levels of Wnt5b transcript were significantly higher in the secretory phase in myometrial cells. Treatment with 9-cis retinoic acid significantly inhibited Wnt5b expression in myometrial SMC but not in their leiomyoma counterparts.
Conclusions: Specific Wnt signaling genes are overexpressed in leiomyoma cells. Moreover, in these cells, the regulation of Wnt5b expression by retinoids appears to be attenuated.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Recently, a modulator of Wnt signaling, secreted frizzled related protein (sFRP)1, was suggested to contribute to the development of uterine leiomyomas through an antiapoptotic effect (4). The Wnt family of secreted glycoproteins regulates growth and survival of several cell types. Aberrant activation of the Wnt signaling pathway has major oncogenic effects (5). Binding of Wnt to its receptor complex, consisting of a member of the frizzled family, leads to stabilization of ß-catenin by inhibiting the phosphorylating activity of the glycogen synthase kinase 3ß (6). Unphosphorylated ß-catenin translocates into the nucleus, where it activates target gene expression through interacting with T cell- and lymphoid-enhancing transcription factors in a process termed classical or canonical Wnt signaling cascade, as opposed to the recently described alternative effects on calcium uptake and protein kinase C activation (5).
In the present study, we have first applied the oligonucleotide microarray analysis to compare gene expression profiles between leiomyoma and myometrial smooth muscle cells (SMC). Among the various genes found to be differentially expressed, a Wnt family member transcript, Wnt5b, was found to be increased in leiomyoma. Hence, we have hypothesized that leiomyoma formation and/or growth might be associated with an enhanced expression of specific Wnt and frizzled family members. To investigate this issue, myometrial SMC and their leiomyoma counterparts were specifically evaluated for expression pattern and regulation of the sFRP1 gene, as a further confirmation of previous findings, and the Wnt5b gene to substantiate the microarray analysis.
The data presented herein indicate that both transcripts analyzed were increased in cells derived from leiomyomas. A negative regulation of the Wnt5b gene expression by 9-cis retinoic acid (RA) was also observed, but its extent was different in myometrial and pathologic samples. These findings, taken together with results derived from previous microarray surveys (7, 8, 9, 10, 11, 12, 13), lead us to postulate that the Wnt signaling pathway is essentially altered in cells from leiomyomas.
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Patients and Methods |
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The study included 22 premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. At the time of surgery, patients were not receiving any type of hormonal or drug therapy except for nonsteroidal antiinflammatory drugs. Histological evaluation of the endometrium and the patient’s last menstrual period were used to determine the phase of the menstrual cycle. The study protocol was approved by the Institutional Review Board of the Department of Obstetrics, Gynaecology and Neonatology, University of Milano. Patients were informed in detail regarding the aims and procedures of the study and gave their written consent to sample collection.
Tissue collection
Tissue was collected from leiomyomas and matched myometrium. In case of multiple leiomyomas, multiple biopsies were taken from the largest ones. The myometrium was removed at a distance of 2 cm from both the endometrium and the leiomyomas. Four patients had more than one leiomyoma; thus samples were collected from 27 leiomyomas. Specimens were assigned to the proliferative phase in 10 patients and to the secretory phase in 12 patients.
Cell preparation and culture
Samples of leiomyoma and myometrium were minced and incubated at 37 C for 2–3 h with 0.1% type A collagenase (Roche, Mi, Italy). Cell suspensions were then centrifuged at 600 x g for 5 min and washed several times. The cell pellet was resuspended in DMEM (Sigma, St. Louis, MO) containing 10% fetal bovine serum (Sigma). Cells were plated in plastic dishes and maintained in DMEM with 10% fetal bovine serum and 1% antibiotic-antimycotic solution (Sigma) at 37 C in 95% air-5% CO2. The medium was changed after 24 h to remove unattached cells and subsequently twice a week. Cells were cultured until confluency before harvesting for RNA preparation. Treatment with 9-cis RA (10–6 M) and 17ß-estradiol (E2) (10–7 to 10–6 M) was carried out with cells at confluence. Culture medium was removed and replaced by fresh medium, supplemented or not with the compounds, and the incubation was continued for 24 h before harvesting. For each condition, about 5 x 105 cells were used. Cells from myometrium and matched leiomyomas were always harvested simultaneously.
RNA extraction and preparation for hybridization on oligonucleotide microarrays
For microarray analysis, primary cultures of myometrial and leiomyoma cells were derived from tissues collected from four patients, two of them with two leiomyomas, for a total of six sample pairs. This was done to estimate the presence of differences in gene expression among leiomyomas of different patients and also among different leiomyomas of the same patient. At the time of surgery, two patients were in the proliferative phase of the menstrual cycle (A and B), and two were in the secretory phase (C and D). Total RNA (20 µg) was extracted from cultures of myometrium or leiomyoma using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). Preparation of in vitro transcription products, oligonucleotide array hybridization, and scanning were performed according to Affymetrix (Santa Clara, CA) protocols. For first-strand cDNA synthesis, 20 µg total RNA and 0.1 nM T7-tagged oligo-dT primer [GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(T24)] were denatured at 70 C for 10 min, cooled on ice, then reverse transcribed with 400 U Superscript II at 42 C for 1 h in first-strand buffer, 10 mM dithiothreitol, and 0.5 mM deoxynucleotide triphosphate mix in 20 µl total vol (all reagents from Invitrogen). Second-strand cDNA was completed by adding 40 U DNA polymerase I, 10 U Escherichia coli DNA ligase, 2 U RNase H, second-strand buffer, 200 µM each deoxynucleotide triphosphate, and nuclease-free water to 150 µl final vol and incubating 120 min at 16 C. Thereafter, 10 U T4 DNA polymerase was added and the incubation extended for 5 min further at 16 C. The reaction was then stopped by adding 0.5 M EDTA and the resulting cDNA extracted once with phenol/chloroform. One microgram of cDNA was used for in vitro transcription with a T7 RNA polymerase and labeled with biotinylated ribonucleotides according to the manufacturer’s instructions (Enzo Biochem, Inc., Diagnostic, Farmingdale, NY) to generate biotinylated cRNA targets. Labeled RNA was purified with RNeasy spin columns (QIAGEN, Hilden, Germany) and chemically fragmented for 35 min at 95 C.
Microarray profiling and data analysis
Analysis of gene expression was performed with Affymetrix high-density oligonucleotide array human HG-U133A chip, which contains 22,283 probe sets representing more than 14,500 well-substantiated human genes, whereas the remaining are expressed sequence tags. Hybridization and analysis of gene expression were performed at Biopolo, Bicocca University, Milano. Ten micrograms of fragmented biotinylated cRNA were hybridized for 16 h to HG-U133A microarrays; the arrays were then washed and stained with streptavidin-phycoerythrin (final concentration of 10 µg/ml). Signal amplification was performed using a biotinylated antistreptavidin antibody. Within the arrays, each individual transcript is represented by perfect match probes in conjunction with a corresponding set of mismatch probes. Data were collected by laser scanning, and pixel levels were analyzed with Microarray Suite version 5.0 software (Affymetrix). The analysis software calculates a set of absolute metrics for each transcript: a measure of the expression level (signal), a detection call for each transcript (present, absent, or marginal), and the level of confidence with which the sequence was detected. Parametric testing was applied to detect genes showing 1.5-fold or greater difference in expression between myometrial and leiomyoma cells, with P < 0.05 for statistical significance.
Evaluation of gene expression by real-time quantitative PCR analysis
Primary cultures of myometrial and leiomyoma cells were obtained from 22 patients, four of which had more than one leiomyoma. One microgram of total RNA was reverse transcribed for 2 h at 37 C using the High-Capacity cDNA archive kit (Applied Biosystems, Foster City, CA). The ABI Prism 7900 sequence detection system (Applied Biosystems) was used for real-time quantitative PCR analysis using hypoxanthine phosphoribosyltransferase (HPRT)-1 as an endogenous control. Real-time PCR was performed using specific primers and probes for sFRP1, Wnt5b, and progesterone receptor (PR) target genes (Assays-on-Demand Gene Expression Products, Applied Biosystems). Target and reference genes were amplified in separate wells in duplicate. Reaction conditions included 10 µl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 1 µl of primers and probes mixture, 50 ng template cDNA, and nuclease-free water to a 96-well reaction plate. The total reaction vol was 20 µl. The cycling conditions were as follows: 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 sec at 95 C followed by 1 min at 60 C. The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The 
cycle threshold (Ct) values from each sample were obtained by subtracting the values for the reference gene from the sample Ct. For each experimental sample, the 2–Ct has been calculated, and data have been graphically indicated as relative expression.
Detection of Wnt5b mRNA by qualitative PCR and sequence analysis
Two hundred nanograms of cDNA were amplified by a standard procedure. Oligonucleotide primers had the following sequence: human Wnt5b, forward 5'-AGATCGTGGACCAGTACATCTG-3'; reverse 5'-TTACGGAACCCATCTACATTCTG-3' (14). Wnt5b primers amplified a 507-bp fragment. After an initial denaturation step at 95 C for 5 min, PCR conditions for amplification were 38 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec, with a final extension at 72 C for 5 min. Integrity of RNA and absence of genomic contamination were assessed by amplification of the HPRT-1 gene with intron-spanning primers (forward 5'-GCTTGCTGGTGAAAAGGACC-3', reverse 5'-GTCAAGGGCATATCCTACAAC-3') according to the following PCR protocol: 94 C for 20 sec, 52 C for 20 sec, and 72 C for 20 sec for 34 cycles. The nucleotide sequence of the PCR products was confirmed by automatic sequence analysis on the ABI Prism 310 Genetic Analyzer (Applied Biosystems).
Western blot for estrogen receptor (ER)
and retinoid X receptor (RXR)
Samples of myometrial and leiomyoma cell cultures were treated with a lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates were subsequently centrifuged at 13,000 x g for 30 min and the supernatant collected. The protein concentration of each sample was determined using a commercial protein assay kit (BCA Protein Assay Kit; Pierce Biotechnology, Inc., Rockford, IL). Proteins resolved by SDS-PAGE were transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences Europe GMBH, Cologno Monzese, Italy). After brief washing in TBST [25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1% Tween 20], the membrane was blocked with 5% skim milk plus 5% BSA/TBST for 2 h at room temperature. The membrane was then incubated overnight at 4 C with an antibody raised against human ER (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:100 in 5% skim milk plus 5% BSA/TBST or with an anti-RXR antibody (Santa Cruz Biotechnology, Inc.) diluted 1:500. All subsequent steps were performed at room temperature. After 1 h washing with TBST, membranes were incubated for 1 h with peroxidase-conjugated antirabbit IgG (Amersham Biosciences Europe GMBH). For protein load control, anti-ß-actin mouse monoclonal antibodies were used. Secondary antimouse IgG antibody was used at 1:5000 dilution. Bound antibodies were visualized by chemiluminescence.
Statistical methods
Nonparametric Wilcoxon signed-rank test and Friedman’s test were used to compare quantitative variables that were not normally distributed. For normally distributed variables, the one-way ANOVA and the Fisher least-significant-difference test as posttest were used. P <0.05 was considered as statistically significant. Data were reported as median and interquartile range or mean ± SEM.
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Results |
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Oligonucleotide microarray analysis was used to compare gene expression profiles in leiomyoma vs. myometrial cells. To assess the purity of cultures analyzed and to exclude a substantial contamination of microvascular endothelial cells, we performed a flow cytometric analysis for CD31, which is a marker of this cell type (15). CD31-positive cells were less than 2% in both myometrial and leiomyoma cultures (data not shown). In line with these data, microarray analysis demonstrated that desmin and -smooth muscle actin genes, but not the gene coding for CD31 (PECAM-1), were expressed in the cultures examined.
Among the genes with altered expression pattern, the Wnt5b gene (NCBI accession NM030775) was selected for validation and regulation analysis on the basis of the following observations: 1) in our microarray analysis, the gene was called absent in all samples of myometrial SMC and present in five of six leiomyoma cell samples (Fig. 1
). Determination of relative expression in such cases is not correct because it implies calculations including data that primarily represent noise or near background intensities; 2) the soluble form of one component of the Wnt receptor family, sFRP1 (NCBI accession AF001900, Unigene accession Hs.7306), has been previously shown to be altered in leiomyoma (4); and 3) the gene appeared of particular biologic significance because different members of the Wnt family were shown to be differentially expressed between myometrium and leiomyoma tissue by other groups using microarray analysis (7, 8, 9, 10, 11, 12, 13) (Table 1).
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and in contrast to the data from microarray analysis, Wnt5b mRNA was detected in all samples of myometrial and leiomyoma cells. Microarrays analysis confirmed the detection of sFRP1 mRNA in all the samples of myometrial and leiomyoma cells evaluated, but relative expression was similar between the two cell types (data not shown). Therefore, to better clarify gene expression patterns of both members of the Wnt family, we proceeded with the quantification of their mRNA levels by real-time quantitative PCR. Secreted FRP1 and Wnt5b mRNA levels in leiomyoma cells and in matched myometrial controls as detected by real-time quantitative PCR
Expression levels of Wnt5b and sFRP1 were measured in myometrial and leiomyoma cells derived from 22 patients. In general, sFRP1 was more abundant than Wnt5b, with Ct values very close to those of the reference gene, whereas Ct values of Wnt5b were 10 or higher. Compared with myometrial SMC, cells derived from leiomyomas had significantly higher levels of both Wnt5b and sFRP1 (Figs. 2A and 3A), thus confirming that the degree of differential expression detected by real-time quantitative PCR or microarrays analysis varied, probably due to the lower sensitivity of the latter one. No significant difference in sFRP1 mRNA levels could be detected as a function of the different phases of the menstrual cycle in both myometrial and leiomyoma cells (Fig. 2B). Conversely, levels of Wnt5b mRNA were significantly higher in the secretory phase of the cycle in myometrial cells, whereas no differences could be found for leiomyoma cells (Fig. 3B).
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Previous data demonstrated a dose-dependent induction of sFRP1 mRNA in cells cultured from leiomyomas in response to treatment with E2 (4). Therefore, to evaluate whether Wnt5b overexpression in leiomyoma cells might be likewise associated with an high estrogenic condition, we tested the effect of E2 on the expression of Wnt5b in SMC derived from myometrium and leiomyoma. Myometrial and leiomyoma cells were stimulated with different concentrations of E2 for 24 h, and mRNA levels were measured by real-time PCR. In both cell types, levels of Wnt5b mRNA were nor affected by treatment with E2 (Fig. 4A). The positive control of these experimental conditions was the observed induction of PR gene expression, which is considered a classical marker of estrogen responsiveness (Fig. 4B). Furthermore, expression of ER in myometrial and leiomyoma SMC was demonstrated by Western blot analysis (Fig. 5).
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(Fig. 5). The rationale to use 9-cis RA is based on previous observations suggesting a specific role for the retinoid pathway in leiomyoma formation (1). Cells were treated for 24 h with 10–6 M 9-cis RA to activate retinoid signaling (1, 16). 9-cis RA did not affect sFRP1 gene expression in either cell type (Fig. 6B). However, treatment with 9-cis RA caused significant down-regulation of Wnt5b expression in myometrial, but not in leiomyoma, SMC (Fig. 6A).
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Discussion |
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Because microarray analysis identified the Wnt5b gene as overabundant in leiomyoma cells, this study was more specifically aimed to ascertain the potential involvement of the Wnt signaling cascade in the development of leiomyomas. Strong support for this hypothesis came from the observation that several genes that were reported to be differentially regulated in leiomyoma tissue by other authors did actually belong to the Wnt pathway (7, 8, 9, 10, 11, 12, 13). Table 1 shows a list of these genes detected in different studies using the microarray technology. Finally, an increased expression of sFRP1, a soluble modulator of the Wnt pathway, has been recently associated with leiomyomas, particularly under high estrogenic conditions (4).
Thus, we have investigated the expression pattern of Wnt5b and sFRP1 in cells derived from leiomyomas and from their myometrial counterparts applying the real-time quantitative PCR procedure. A significant increased expression of both Wnt5b and sFRP1 mRNAs in leiomyoma cells was confirmed by this approach. It has to be noted, however, that there is about 25% of samples in which this increase is minimal or absent, and this may account for the variation of the results observed. It is plausible and already suggested that leiomyoma growth and survival depend upon differences of local factors and the receptor pattern of each tumor (18). This also explains the heterogeneity of the leiomyoma growth, even within the same uterus, and the different response in tumor size under the same hormonal treatment.
It is also evident that some discrepancies have emerged between results from microarray analysis and those derived from real-time quantitative PCR. The limited sensitivity of the microarray technique is likely to explain most of these problems. In this context, it is interesting to note that whereas, in general, the different studies performed using this procedure have found an aberrant expression of several genes involved in the Wnt pathway in leiomyomas, among the studies there is very poor consistency for each of these specific genes (7, 8, 9, 10, 11, 12, 13). Similar discrepancies have also been reported in other situations (19). In view of these considerations, a cautious evaluation of results from microarray analysis is further recommended.
The possibility that the Wnt pathway may be altered in a consistent proportion of leiomyomas yields an important clue to the pathophysiology of this tumor, mainly because Wnt5b is a potent cancer-associated gene (14). The gene is present at a higher level in chronic lymphocytic leukemia (19) and is expressed in breast cancer and in tumors derived from upper gastrointestinal tract (14). The binding of a particular secreted Wnt ligand to its seven-transmembrane-type receptor of the Frizzled family on the cell surface activates the ß-catenin-T cell transcription factor pathway. Downstream, Wnt target genes include c-Myc, cyclin D1, and Tcf1. This cascade determines many important cell-fate decisions throughout development by controlling gene expression, cell behavior, cell adhesion, and cell polarity (5, 20). The exact mechanisms underlying alteration of Wnt signaling cascade in leiomyomas are unclear. One possibility is the recent demonstration that the Wnt/ß-catenin signaling positively regulates the function of peroxisome-activated receptor-
(21) that has been shown to be significantly overexpressed in leiomyoma relative to myometrium (1). Second, ß-catenin expression in leiomyomas is controversial, but a consistent up-regulation has been recently detected in a preliminary report using a specific array for cadherin family member genes (22, 23). The pathway is very complex; and, based on microarray procedure (7, 8, 9, 10, 11, 12), many proteins seem to be aberrantly expressed. Further studies are needed to elucidate this aspect.
Modulation of expression of the Wnt5b gene as a function of the menstrual cycle was not a totally unexpected finding. Previous studies have shown that expression of several members of the Wnt pathway can be differently regulated by steroid hormones in various cell types (14, 24, 25, 26). However, it is worth noting that whereas sFRP1 expression seems to be associated with leiomyoma in the context of high estrogenic conditions (4), Wnt5b mRNA levels were not regulated by E2 and, especially in myometrial cells, increased in the secretory phase of the cycle. Leiomyomas show proliferative activity in the luteal phase (2), and it might be possible that an altered regulation of Wnt5b expression between the two phases relates to their abnormal proliferation.
More interestingly, an aberrant regulation of Wnt5b expression in leiomyoma cells has been observed in relation to 9-cis RA treatment. Although in myometrial cells Wnt5b gene expression could be negatively modulated by incubation with the compound, cells from leiomyoma were more resistant to such treatment. The retinoid pathway seems to have a major role in leiomyomata development and growth (1, 13). In a model of leiomyoma in guinea pigs, high levels of all trans RA, together with E2 stimulation and a high expression of specific heterodimeric nuclear receptors, can induce the formation of the benign tumor. In the same conditions, 9-cis RA seems to prevent growth (1). The Wnt signaling is known to cooperate with RA receptor signaling to induce tumoral pathological states. The intersection between the two pathways occurs at the level of ß-catenin, which potentiates the activation of RA receptor-responsive elements by RA (27). The protective role of 9-cis RA in this context might be related to its ability to modulate RXR expression as a critical factor for the formation of specific nuclear receptor heterodimers on one hand (1) and to regulate the Wnt pathway on the other. Therefore, a defective response to 9-cis RA could be one of the factors that favor leiomyoma growth, through the activation of the Wnt signaling.
In conclusion, the results presented herein support the following observations: 1) specific members of the Wnt signaling pathway, which represent a mechanism that contributes to the progression of a high percentage of human cancers, are aberrantly expressed in leiomyoma cells; 2) in uterine SMC, one of the members of the Wnt signaling pathway, Wnt5b, is up-regulated in the secretory phase of the menstrual cycle; and 3) in uterine SMC, a negative regulation of the Wnt5b gene expression by 9-cis RA can be observed, but this effect is significant only in myometrial cells and not in pathologic samples.
Because increasing numbers of women are seeking less invasive therapies for symptomatic fibroid uteri, these findings might have, in the future, some relevance for the development of new therapeutic strategies.
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Acknowledgments |
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Footnotes |
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1 S.M. and P.V. contributed equally and are listed in alphabetical order. 
Abbreviations: E2, 17ß-Estradiol; ER, estrogen receptor; HPRT, hypoxanthine phosphoribosyltransferase; PR, progesterone receptor; RA, retinoic acid; RXR, retinoid X receptor; sFRP, secreted frizzled related protein; SMC, smooth muscle cells.
Received February 9, 2005.
Accepted June 10, 2005.
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, retinoid X receptor , and all-trans retinoic acid than myometrium. Cancer Res 59:5737–5744 in MKN45 cells and up-regulation of WNT5B by ß-estradiol in MCF-7 cells. Int J Mol Med 10:345–349[Medline]
and -ß expression in microvascular endothelial cells and smooth muscle cells of myometrium and leiomyoma. Mol Hum Reprod 8:770–775 activity in colon cancer cells. Proc Natl Acad Sci USA 102:1460–1465 in human uterine leiomyoma. J Clin Endocrinol Metab 86:454–457This article has been cited by other articles:
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Y. Zhao, Y. Wen, M. L. Polan, J. Qiao, and B. H. Chen Increased expression of latent TGF-{beta} binding protein-1 and fibrillin-1 in human uterine leiomyomata Mol. Hum. Reprod., May 1, 2007; 13(5): 343 - 349. [Abstract] [Full Text] [PDF]
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