Molecular Human Reproduction, Vol. 9, No. 11, 709-717,
November 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
PEP-19 overexpression in human uterine leiomyoma
Submitted on May 23, 2003; resubmitted on July 13, 2003. accepted on July 28, 2003
1 Department of Gynecology and Obstetrics and 2 Department of Clinical Molecular Biology, Faculty of Medicine, Kyoto University, Kyoto, 606-8507, Japan
3 To whom correspondence should be addressed at: Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, 54, Kawaharacho, Shogoin, Sakyoku, Kyoto, 606-8507, Japan. e-mail: sfu{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Although uterine leiomyomas represent one of the most common neoplasms in adult women, their pathogenesis remains poorly understood. A cDNA microarray analysis was performed to search for candidate genes expressed to a greater degree in leiomyoma compared with matched myometrium. A total of 15 candidate genes was obtained; neuron-specific protein PEP-19 (Purkinje cell protein 4; PCP 4) exhibited a striking difference in expression between leiomyoma and myometrium. Although PEP-19 expression has been reported exclusively in the central nervous system, the present study demonstrated that PEP-19 is also expressed in other human organs, including prostate, kidney and uterus. To clarify the role of PEP-19 in the pathogenesis of leiomyomas, PEP-19 expression was investigated for a series of human leiomyoma, as well as normal myometrium and leiomyosarcoma. PEP-19 mRNA and protein expression were much stronger in leiomyomas compared with normal myometrium, suggesting that PEP-19 might be involved in leiomyoma pathogenesis.
Key words: microarray/northern blot analysis/PEP-19/uterine leiomyoma
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Introduction |
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Uterine leiomyomas represent the most common neoplasm present in the female genital tract. Approximately 20–25% of women of reproductive years are afflicted with this disease (Buttram et al., 1981). Although leiomyomas are benign and rarely result in a lethal outcome, they frequently lead to infertility and menorrhagia (Bajekal et al., 2000). In addition, leiomyomas also represent the most common indication for hysterectomy in Japan, as well as in the USA (Farquhar et al., 2002). Despite the prevalence and tremendous influence on reproductive women, the pathogenesis of uterine leiomyomas has yet to be elucidated. It has been reported that leiomyomas grow under the influence of ovarian steroids, expressing receptors for such hormones (Brandon et al., 1993, 1995). Growth factors, such as epidermal growth factor, insulin-like growth factor (IGF) and their binding proteins, TGF-ß, and the receptors for such hormones, have also been reported to affect leiomyoma growth (Giudice et al., 1993; Vollenhoven et al., 1993; Harrison-Woolrych et al., 1994; Arici et al., 2000). Nonetheless, the factors that promote both initial leiomyoma development and regulate in-vivo growth remain poorly understood. Recently we reported that secreted frizzled related protein 1 (sFRP1), a Wnt signal-related gene, was found to be overexpressed in uterine leiomyoma, and we postulated that strong expression of sFRP1 under high estrogenic conditions may contribute to uterine leiomyoma development (Fukuhara et al., 2002). In the present work, a microarray analysis for leiomyoma and matched normal myometrium obtained from the same patient was performed to search for genes with elevated expression in leiomyomas. Among the candidate genes detected, PEP-19, a calmodulin regulatory protein found within neurons, exhibited the most striking difference in expression between leiomyoma and normal myometrium. Therefore, in an attempt to explore the relevance of PEP-19 to the pathogenesis of uterine smooth muscle tumours, we analysed the expression profile of PEP-19 in a series of leiomyoma, normal myometrium and leiomyosarcoma samples.
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Materials and methods |
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Tissue collection
Leiomyoma and matched myometrial tissue samples were obtained from patients who underwent hysterectomies for uterine leiomyoma in the Kyoto University Hospital. Patients (n = 37) ranged in age from 37 to 55 years (mean = 44.9 ± 4.3 years). Informed consent was obtained from each patient with use of consent forms; all protocols were approved by the local Human Investigation Committee. Myometrial and leiomyoma tissue samples were simultaneously obtained from the identical uterus. After completion of each hysterectomy, tissue specimens were immediately frozen in liquid nitrogen for subsequent RNA isolation. Endometrial dating was carried out according to the methods previously reported (Noyes et al., 1950).
Array hybridization
Poly A+ RNA was extracted from leiomyoma tissue and normal myometrium in one patient, aged 38 years and in the follicular phase, using a commercially available mRNA purification kit (Amersham Biosciences Corp., USA). The RNA aliquots were labelled with Cy3-dCTP and Cy5-dCTP. Labelled probes were mixed with microarray hybridization solution version 2 (Amersham Biosciences Corp.) and formamide to yield a final concentration of 50%. Each sample was hybridized onto the UniGEM V cDNA microarray (Incyte Genomics Inc., formerly Genomesystems Inc., USA; http://www.incyte.com). UniGEM V array contained 7800 unique and sequence-verified cDNA or expressed sequence tag (EST) elements. The Cy3:Cy5 signal ratio (leiomyoma/myometrium signal intensity) was calculated and analysed by GEM tools 2.4 (Genomesystems Inc.). We selected 12 genes that showed high signal value (>300) in leiomyoma and high expression signal ratio (
2.0) as the candidate genes. Three EST which showed high expression signal ratio (2.0) were also selected.
Northern blot analysis
Total RNA was prepared from surgical specimens by homogenization in Trizol reagent (Life Technologies, USA). Separation of total RNA (20 µg per lane) was performed with 1% agarose gel electrophoresis in the presence of formaldehyde. The separated RNA was then transferred to a nylon membrane (Hybond-N+; Amersham Biosciences Corp.) and cross-linked by UV light. The EcoRI-XhoI-restricted fragments of the selected genes were radiolabelled with [
32P]dCTP using the random priming technique. S26 ribosomal protein was also radiolabelled with [32P]dCTP for normalization. The membranes were then hybridized, washed and autoradiography was performed using intensifying screens at –70°C.
Anti-PEP-19 antibody
PEP-19 antiserum was received from Dr James Morgan, St Jude Children’s Research Hospital, Memphis, TN, USA. It was raised in rabbits immunized with the peptide as an immunogen. The peptide corresponding to amino acid residues 43–55 of PEP-19 (V-A-I-Q-S-Q-F-R-K-F-Q-K-K) was synthesized and used for the present work.
Immunostaining
Tissue specimens were dissected and immersed in 5% formaldehyde in 0.1 mol/l phosphate buffer pH 7.4 for 2 h at room temperature. Samples were then transferred to a phosphate-buffered saline solution containing 20% sucrose and kept overnight at room temperature. After fixation, 12 µm frozen sections were prepared and mounted on gelatin-coated glass slides. The ABC (avidin–biotin–horseradish peroxidase complex) method was performed to demonstrate PEP-19. Sections were incubated with an anti-PEP-19 rabbit antiserum at a dilution of 1/5000 for 24 h at room temperature, followed by incubation with biotinylated horse anti-mouse IgG and ABC complex (Vector Laboratories Inc., USA). After performing a nickel ammonium sulphate-intensified diaminobenzidine reaction, the sections were dehydrated in a graded series of alcohols, cleared in xylene, and protected with cover slips (Fukuhara et al., 2002).
Immunofluorescence labelling
First, 12 µm frozen sections of the tissue specimens were obtained and thaw-mounted on gelatin-coated glass slides. The slides were incubated at 4°C overnight with PEP-19 antibody at a dilution of 1/5000 for mouse brain tissue and 1/2000 dilution for human uterine tissues, which was followed by incubation for 1 h at room temperature with the secondary antibody, an anti-rabbit IgG antibody conjugated to fluorescein isothiocyanate (FITC; Dako Cytomation Denmark A/S, Denmark, used at a 1/100 dilution). Slides were then counterstained with 2 mg/ml propidium iodide (PI), and further digestion was performed with 1 mg/ml RNAase at 37°C for 30 min. The prepared slides were examined with a confocal microscope (Axiovert 200M; Carl Zeiss Co. Ltd, Germany).
Multiple tissue northern blot analysis
Multiple Tissue Northern (MTNTM) Blots were purchased from Clontech Co. (BD Biosciences Clontech, USA). Analysis was performed with premade northern blot membranes featuring Premium Poly A+ RNA from a variety of different human tissues.
Statistical methods
Statistical significance was assessed by the non-parametric Mann–Whitney U-test; P < 0.05 was considered significant.
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Results |
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Microarray analysis and confirmation with northern blot analysis
Microarray analysis demonstrated 15 candidate genes with a stronger expression in leiomyoma compared with matched control myometrium (Table I). To investigate whether such results are typical for leiomyoma, northern blot analysis of these genes was performed using five matched pairs of leiomyoma and myometrial tissue (Figure 1). Except for two EST that did not show clear bands, the average signal in five cases showed a tendency of the leiomyoma signal to be greater than the myometrium signal by northern blot analysis (Table I).
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All five cases exhibited higher expression of PEP-19 and sFRP1 in the leiomyoma compared with the myometrium. In three or four out of five cases examined, regulator of G protein signalling 5, endometrial bleeding associated factor, tenascin C, stromelycin 3, versican, collagen type VI
3, IGF2, TGFß3, IGF binding protein 5 (IGFBP 5), and expressed sequence tag EST (A) (GenBank accession number: AA527426) were expressed to a greater extent in the leiomyoma compared with the myometrium; no clear EST (B) (GenBank: AA528009) expression difference was noted; no EST (C) (GenBank: AA631903) band was observed. Such data supported the microarray analysis, especially with respect to PEP-19. Accordingly, attention was directed towards PEP-19 expression in uterine smooth muscle tumours.
PEP-19 mRNA expression in multiple human organs
PEP-19 mRNA expression was examined in multiple human organs. Among 16 organs examined, PEP-19 expression level was highest in brain, prostate and kidney, followed by uterine tissue (Figure 2).
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PEP-19 mRNA levels in myometrium and leiomyoma
PEP-19 mRNA levels in myometrium and leiomyoma were evaluated in 37 cases, of which 33 exhibited higher PEP-19 mRNA expression in the leiomyoma compared with the matched normal myometrium. Two cases in the luteal phase, one case in the menstruation phase, and one case who had pre-surgical estrogen and progestogen administration did not exhibit such a tendency (Figure 3). The most significant difference of PEP-19 expression was observed for myometrial and leiomyoma tissue in the follicular phase. Although PEP-19 expression for leiomyoma tended to be higher than that of myometrium in the luteal phase, the difference was not significant (Figure 4).
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Immunohistochemistry
As a positive control, immunohistochemistry for PEP-19 protein in the mouse brain was performed. PEP-19 was highly expressed in Purkinje fibres in the cerebellum and neurons in the hippocampus (Figure 5). Immunohistochemistry of human uterus revealed that leiomyoma cells stained more strongly in comparison with normal myometrium. PEP-19 was weakly expressed in the cytoplasm of myometrial smooth muscle cells. Vascular smooth muscle cells showed particularly strong staining (Figure 6).
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Confocal microscopy
In mouse brain, PEP-19 positive cells (cells with ‘green’ fluorescence in the FITC channel) can be observed. Nuclei emitted red fluorescence with PI. PEP-19 positive cells possessed large neural bodies and resembled Purkinje cells (Figure 7).
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PEP-19 positive cells are readily apparent in leiomyoma tissue. Double staining with PEP-19 and
smooth muscle actin (SMA) demonstrated that PEP-19 positive cells were also positive for SMA. The staining pattern in PEP-19 positive cells indicates that the protein was predominantly expressed in the cytoplasm, as opposed to the nucleus (Figure 8).
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PEP-19 mRNA levels in uterine leiomyosarcoma and cell lines
Northern blot analysis of PEP-19 expression was performed in three uterine leiomyosarcoma tissue samples, as well as the sarcoma cell lines, SKN, SKLMS1 and MESSA. No expression was detected, suggesting that PEP-19 might be down-regulated in leimyosarcoma and such sarcoma cell lines (Figure 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In order to elucidate genetic events involved in uterine leiomyoma pathogenesis, cDNA microarray analysis was performed to search for genes exclusively expressed in leiomyomas. A total of 15 candidate genes with stronger expression in leiomyoma tissue samples compared with matched normal myometrium samples was obtained. Among such genes, stromelycin 3, IGF 2, TGFß3, and IGFBP 5 have been previously reported to be overexpressed in leiomyoma compared with myometrium (Giudice et al., 1993; Vollenhoven et al., 1993; Palmer et al., 1998; Arici et al., 2000). The expression of such genes in leiomyomas was further evaluated with northern blot analysis, which revealed that stromelycin 3, IGF 2, TGFß3 and IGFBP 5 were expressed to a greater degree in leiomyoma compared with matched myometrium in three or four out of five samples. Among the 15 candidate genes detected by microarray analysis, PEP-19 exhibited the most striking expression difference between leiomyoma and myometrium. Furthermore, northern blot analysis confirmed that PEP-19 expression was markedly stronger for leiomyoma tissue compared with myometrial tissue in all five cases initially examined. Recently, Tsibris et al. (2002) demonstrated that expression of PEP-19, along with stromelycin3, IGF 2, IGFBP 5, versican and TGFß3, was up-regulated in leiomyoma relative to matched myometrium, although such results were not confirmed by other methods. The current study focused on PEP-19 expression in uterine smooth muscle tumours as detected by northern blotting, as well as immunohistochemistry.
The issue of interest was whether PEP-19 expression was restricted to the brain, where abundant expression of this protein has previously been reported, most notably in the cerebellum and olfactory bulbs. Ziai et al. (1988) reported that PEP-19 expression was present in the cerebellum of several vertebrates, including rats, mice, monkeys and humans, but remained undetectable in all non-neural tissues examined. Nonetheless, in an analysis of multiple human organs carried out for the present work, PEP-19 mRNA was also found to be highly expressed in the prostate, kidney and uterus. It is also of note that recently myelin proteolipid protein (PLP), one of the most abundant proteins in the central nervous system (CNS), was reported to be up-regulated in leiomyoma (Tsibris et al., 2002). Accordingly, in leiomyoma, genes previously thought to be exclusively expressed in the CNS have indeed been found to be expressed, suggesting a potential role in leiomyoma pathophysiology.
PEP-19 expression was more thoroughly investigated in an increased number of samples. Northern blot analysis demonstrated that PEP-19 expression was elevated in 33/37 (89.2%) cases. Furthermore, leiomyoma immunohistochemical staining revealed clear staining of PEP-19. To date, altered PEP-19 expression has been reported in brains with Huntington’s and Alzheimer’s disease (Slemmon et al., 1994; Utal et al., 1998), suggesting a potential role for this protein in the pathophysiology of such diseases. Nonetheless, the cellular functions of PEP-19 remain to be determined. PEP-19 belongs to a family of calmodulin regulatory proteins, including RC3/neurogranin and GAP-43/neuromodulin (Slemmon et al., 1996). All such proteins interact with the calcium-free form of calmodulin, sequestering calmodulin from calcium and preventing activation until certain threshold levels of calcium are attained (Gerendasy et al., 1997). Expression of PEP-19 has recently been shown to modulate calcium-dependent calmodulin kinase activity within cells (Johanson et al., 2000). Consequently, PEP-19 might affect a variety of cellular functions involved in the control of various neuronal processes including apoptosis (Yano et al., 1998). Indeed, it was recently reported that PEP-19 expression in PC12 pheochromocytoma cells inhibited cell death following apoptotic stimuli (Erhardt et al., 2000). Accordingly, PEP-19 might be involved in the control of apoptosis of leiomyoma cells, although such a postulation remains untested.
One of our hypotheses regarding leiomyoma pathogenesis is that leiomyomas may result from the proliferation of smooth muscle cells in the myometrial tissue that survive repeated ischaemic reperfusion stress experienced during the menstrual cycle (Fujii et al., 1999). In each luteal phase of the menstrual cycle, myometrial smooth muscle undergoes proliferative activity in preparation for pregnancy. If pregnancy does not occur, the proliferative activity of myometrial smooth muscle cells is interrupted at the time of menstruation. Myometrial contraction during menstruation, which results in the cessation of menstrual bleeding, induces a hypoxic state in myometrial smooth muscle cells, resulting in ischaemic injury. The injured and inappropriately repaired cells might represent progenitor leiomyoma cells, as is the case for various other neoplasms. If such a hypothesis were true, interference with appropriate apoptosis in damaged cells, caused by factors such as PEP-19, may contribute to leiomyoma pathogenesis. Recently, we reported that strong sFRP1 expression under high estrogenic conditions appeared to contribute to the development of uterine leiomyomas (Fukuhara et al., 2002). Although PEP-19 is also highly expressed in leiomyomas, expression does not appear to be related to estrogenic stimuli; i.e. expression is similar for all stages of the menstrual cycle, estrogen/progestogen administration, GnRH agonist treatment, and the post-menopausal state. Consequently, PEP-19 may act independently from factors that coordinate the menstrual cycle. Investigation of the possible functions of PEP-19 in uterine leiomyomas are also currently underway by examining uterine smooth muscle cells transfected with the gene.
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Acknowledgements |
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We are grateful to both Dr James Morgan for his gift of the PEP-19 antiserum and Ms Michiko Muraoka for her helpful technical support. This work was supported in part by Grants-in-Aid for Scientific Research (Nos. 13307047 and 13877272) from the Ministry of Education and Sports, Japan.
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