Mol. Hum. Reprod. Advance Access originally published online on March 14, 2007
Molecular Human Reproduction 2007 13(5):343-349; doi:10.1093/molehr/gam007
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Increased expression of latent TGF-ß binding protein-1 and fibrillin-1 in human uterine leiomyomata
1 Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA, USA 2 Department of Gynecology and Obstetrics, Peking University, Third Hospital, Beijing, China
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Stanford University, 300 Pasteur Drive, Room HH333, MC: 5317, Stanford, CA, USA. E-mail: bchen{at}stanford.edu
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Abstract |
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We compared latent TGF-ß binding protein-1 (LTBP-1) and fibrillin-1 (FBN-1) expression in leiomyomata and myometrium, correlated with leiomyomata size. We studied in vivo and in vitro effects of ovarian steroids using matched leiomyomata and myometrium samples from both phases of the menstrual cycle. Leiomyomata were divided into small (
2 cm), medium (3–5 cm) and large (
6 cm) groups. We validated LTBP-1 and FBN-1 expression using QPCR, western blot and immunohistochemistry. LTBP-1 and FBN-1 mRNA and protein expressions were higher in the medium-sized group compared with myometrium in the proliferative phase (P = 0.01; P = 0.01). FBN-1 mRNA expression was higher in the secretory phase (P = 0.01). LTBP-1 mRNA and protein expression was higher in the medium group compared with the small and large groups in the proliferative phase (P = 0.04; P = 0.04). No differences between groups were seen in FBN-1 expression in either phase. 17ß-eestradiol (E2) increased mRNA and protein expression of LTBP-1 and FBN-1 in cultured leiomyoma smooth muscle cells (LSMC) (P < 0.05). No change in FBN-1 and LTBP-1 expression was observed when cells were treated with E2 plus progesterone. Estrogen may be involved in LTBP-1 and FBN-1 expression in leiomyomata. Extracellular matrix metabolism may be different in medium-sized leiomyoma. Key words: E2/FBN-1/leiomyomata/LTGF-ß-1/myometrium/17ß-estradiol
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Introduction |
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Uterine leiomyomata are the most common benign tumours in women, occurring in 70% of reproductive-aged women (Walker, 2002). Although benign, leiomyomata can cause menorrhagia, anemia, reduced fertility and increased spontaneous abortion. They are the most frequent indication for hysterectomy in women aged 30–54 in the USA (Wilcox et al., 1994). Because leiomyomas occur only after menarche, grow during the reproductive years and markedly shrink in hypoestrogenic conditions, it is presumed that growth of these tumours is estrogen-dependent. However, the mechanisms of how estrogen regulates leiomyoma growth remain undefined.
One distinctive feature of leiomyomata is the presence of abundant extracellular matrix (ECM). In recent years, transforming growth factor ß (TGF-ß) has been widely accepted as a key factor in the pathological growth of fibrotic tissue, including leiomyomata (Dou et al., 1996; Branton and Kopp, 1999; Lee Byung-Seok and Nowak, 2001). TGF-ß stimulates the expression of ECM proteins and inhibits the proteolytic degradation of matrix components of the ECM in leiomyomata (Sozen and Arici, 2002; Luo et al., 2005). TGF-ß1, ß2 and ß3 isoforms, as well as their receptors, have all been detected in leiomyoma and myometrium (Dou et al., 1996; Tang et al., 1997). Estrogen has been shown to modulate the expression of TGF-ß1 in cultured leiomyoma cells (Takahashi et al., 1994; Chegini et al., 2002), while the effects of estrogen are also mediated by TGF-ß1 activity (Chegini et al., 2002). Therefore, interaction between TGF-ß1 and estrogen may play a critical role in leiomyomata pathophysiology.
TGF-ß1 is secreted from cells as large inactive complexes consisting of the latency-associated peptide (LAP), the latent TGF-ß binding protein (LTBP) and mature dimeric growth factor. TGF-ß1 binds to its receptor and exerts its biological functions only when activated. LTBP-1 is involved in the assembly, secretion and targeting of TGF-ß1 to the ECM space where it is stored or activated (Oklu and Hesketh, 2000; Mazzieri et al., 2005). In the absence of a specific LTBP, TGF-ß may be inefficiently secreted and unable to localize in the ECM. Fibrillin-1 (FBN-1) has also been implicated in TGF-ß activation (Kaartinen and Warburton, 2003).
LTBPs and fibrillins belong to the fibrillin/LTBP gene family. Both of them share similar eight-cysteine domains found only in this gene family (Hyytiainen et al., 2004). Previous studies indicate that LTBP-1 and FBN-1 interact with each other. (Isogai et al., 2003). Although currently there are no reports on LTBP-1 or FBN-1 expression in leiomyomata, researchers have demonstrated an association between LTBP-1 and tissue fibrosis (Maeda et al., 1993; Waltenberger et al., 1993a, b; Corchero et al., 2004). FBN-1 has also been associated with fibrosis of the heart and liver (Lorena et al., 2004; Bouzeghrane et al., 2005). Therefore, we hypothesize that LTBP-1 and FBN-1 may be involved in the pathogenesis of leiomyomata through alterations in ECM metabolism. We investigated LTBP-1 and FBN-1 mRNA protein expression in human leiomyomata compared to myometrium, and correlated these with leiomyomata size. To investigate the effect of ovarian steroids, we studied LTBP-1 and FBN-1 expression in leiomyoma and matched myometrium from both phases of the menstrual cycle, and in cultured leiomyoma smooth muscle cells (LSMC) stimulated with E2 and progesterone.
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Materials and methods |
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Tissue collection
The Institutional Review Board of Stanford University School of Medicine approved this study. After informed consents were obtained, biopsies of intramural leiomyomata and matched unaffected myometrium were collected from premenopausal women at the time of hysterectomy. Myometrial samples were taken from the uterine fundus, and 1 cm in depth from the endometrium. Leiomyomata were divided by size into three groups: small (
2 cm), medium (3–5 cm) and large (6 cm). Leiomyomata samples were taken midway between the centre and periphery of each tumour. We excluded women with endometriosis, malignancy, pelvic inflammatory disease, inflammatory bowel disease, and connective tissue disorders. None had received hormonal therapy for at least three cycles before surgery. Phase of menstrual cycle was verified by endometrial histology. Tissues were frozen in liquid nitrogen immediately after excision and stored at –80°C before RNA and protein extraction. Parts of the samples were immediately put into Dulbecco's modified Eagle's medium (DMEM) for subsequent tissue cultures.
Cell culture
Uterine leiomyomata tissues (leiomyoma size: 3–5 cm) and matched myometrial tissues were washed in DMEM, cut into small pieces, and digested in 0.2% collagenase (wt vol–1) at 37°C for 8 h (Shimomura et al., 1998). LSMC and myometrial smooth muscle cells (MSMC) were collected by centrifugation at 3000 rpm x 10 min and washed several times with DMEM. The isolated LSMC and MSMC were, respectively, plated in six well plates and cultured in DMEM supplemented with 10% FBS (vol vol–1) and 1% antibiotic solution (penicillin, streptomycin and fungizone) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Characterization of the cultured cells was done through immunostaining with monoclonal antibodies to muscle-specific protein desmin, to a class of intermediate filament protein present in SMC, vimentin, and to a cytoskeletal protein for epithelial cells, cytokeratin 19. Cells were stepped down to serum-free DMEM with 0.2% lactalbumin hydrolate for 48 h. Treatment with 17ß-estradiol (E2) (0, 0.1, 1 and 10 ng/ml–1) or E2 (10 ng ml–1), plus different progesterone concentrations (1, 10 and 100 ng ml–1) was started when the cultured cells approached 70% confluence. The monolayer cultures were maintained in serum-free DMEM for an additional 48 h after treatment. Supernatant from each culture was collected and concentrated by 100-fold with Centricon (Millipore Corp., Bedford, MA, USA).
Immunohistochemical staining
We performed immunohistochemical staining using an avidin-biotin alkaline phosphatase method to locate LTBP-1 and FBN-1 in leiomyomata and myometrium tissues as described previously (Chen et al., 2005). The leiomyomata and myometrium tissues were fixed with 10% formalin and embedded in paraffin. Sections of 5 µm thick tissue were deparaffinized with xylene, rehydrated through graded alcohols, and washed with Tris-buffered saline Tween-20 (TBS-T) (pH 7.4, 0.02% Tween-20). To reduce non-specific binding, slides were blocked with 5% normal horse serum (Sigma Chemical Company, St Louis, MO, USA) and 1% bovine serum albumin (BSA) in TBS-T and left in humidity chambers for 30 min at room temperature, then washed three times in TBS-T for 5 min each. They were then incubated overnight at 4°C with mouse anti-human LTBP-1 primary antibody (R&D Systems, Inc., Minneapolis, MN, USA) and mouse anti-human FBN-1 primary antibody (LAB Vision Corporation, Fremont, CA, USA) at dilutions of 1/10 and 1/20.
After being washed in TBS-T three times, sections were incubated with a secondary antibody, biotinylated horse anti-mouse IgG (dilution 1/50, Sigma Chemical Company) in humidity chambers for 60 min at room temperature. Negative controls were incubated with TBS-T that contained 5% horse serum and 1% BSA without primary antibody. To amplify the signal, sections were washed with TBS-T and then stained using the avidin-biotin alkaline phosphatase-staining method (Vector Laboratories, Inc., Burlingame, CA, USA). Endogenous alkaline phosphatase activity was inhibited by adding levamisole to the buffer used to prepare the substrate solution. Finally, the slides were counterstained with 25% hematoxylin. A red precipitate indicated positive staining by the primary antibody.
Immunofluorescence staining
Immunofluorescence staining of LSMC and matched unaffected MSMC was performed, as previously described, to verify expression of LTBP and FBN-1 (Chen et al., 2005). Briefly, LSMC and MSMC from uterine leiomyomata were cultured in four well chamber slides. The cells were fixed with 4% paraformaldehyde and treated with 5% Triton. After washing with TBS-T and blocking with 5% normal secondary host serum, the slides were incubated with mouse anti-human LTBP-1 primary antibody (1/20) (R&D Systems, Inc.) and mouse anti-human FBN-1 primary antibody (1/10) (LAB Vision Corporation) at 4°C overnight. Deletion of the primary antibody was used as a negative control. After washing, the slides were incubated with goat anti-mouse IgG tetramethylrhodamine isothiocyanate 1/50. To observe nuclei, we used 6-diamidino-2-pheylindole staining. The slides were washed three times and mounted with Vectashield (Vector, Foster City, CA, USA).
Total RNA extraction and cDNA synthesis
We extracted total RNA from the frozen tissues and cultured cells using the STAT-60TM reagent following the manufacturer's suggested protocol (TEL-TEST, Inc., Friendswood, TX, USA). RNA concentrations were measured spectrophotometrically in a GenQuant RNA/DNA calculator (Pharmacia Biotech, Cambridge, UK). For reverse transcription (RT), we used the Gen Amp RNA PCR kit (Perkin-Elmer, Foster City, CA, USA) (Chen et al., 2005). We prepared 19 µl of RT-Master Mix for each sample containing 5 mmol l–1 MgCl2, 1 x PCR buffer II, 1 mmol l–1 each of deoxy-NTP, 2.5 µl l–1 oligo-deoxythimidine, 20 IU ribonuclease inhibitor (all from Perkin-Elmer) and 100 IU Moloney murine leukaemia virus reverse transcriptase (Gibco BRL). RT of 1 µg RNA for cDNA synthesis was performed in total volume of 20 µl. RT-Master Mix in PCR tubes was covered with 50 µl of light white mineral oil (Sigma, St Louis, MO) and kept on ice until RT. RT reaction was carried out in the DNA Thermal Cycler 480 (Perkin-Elmer GeneAmp, PCR Instrument System, Branchburg, NJ, USA) using a programme with one 15-min RT cycle at 42°C, followed by 5 min at 99°C, then quenched at 4°C. After the reaction was completed, samples were stored at –20°C until real-time PCR.
Relative quantitative real-time PCR
Specific sequences of oligonucleotide primers for LTBP-1, FBN-1, hypoxanthine phosphoribosyltransferase-1 (HPRT-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used, as in Wen et al. (2006). These primers were synthesized by the Protein, Amino Acid and Nucleic Acid (PAN) Facility (Stanford University, Stanford, CA, USA).
We performed relative quantitative real-time PCR following the manufacturer's instructions (Mx3005PTM Real Time PCR System, Stratagene, La Jolla, CA, USA), and using Brilliant® SYBR® Green QPCR Master Mix, Optical 96-Well Reaction Plate (Stratagene). Two housekeeping genes, HPRT-1 and GAPDH mRNA, were used as endogenous controls at the same time. Each well contained 7.625 µl water, 12.5 µl of 2xMaster Mix, 1 µl of upstream primer (150 nM), 1 µl of downstream primer (150 nM), 0.375 µl of diluted reference dye (Stratagene) and 2.5 µl cDNA in a total volume of 25 µl. Corresponding real-time PCR efficiencies were calculated according to the equation E = 10[–1/slope]. The efficiency of these gene reactions, which were between 90% and 110%, are within the range accepted by the Mx3005PTM Real Time PCR System protocol (Stratagene). Target and reference genes were amplified in separate wells in duplicate. Controls without the RT step were included for each primer pair to check for any contaminants. Melting (dissociation) curves of PCR reactions were monitored to ensure that there was one single PCR product and no primer dimmers. The thermal cycling conditions for real-time PCR were: denaturation programme (95°C for 10 min), amplification and quantification programme repeated 40 times (95°C for 30 s, 55°C for 60 s, 72°C for 60 s with a single fluorescence measurement) and melting curve programme (55–95°C). Further testing by agarose gel electrophoresis confirmed that there was only one PCR product of the expected size. Relative quantification was performed using HPRT-1 (Mangioni et al., 2005) as a housekeeping gene (the same results were also seen using GAPDH as a housekeeping gene) and following target genes. The relative expression ratio (R) of a target gene is calculated based on efficiency and the CT deviation of an unknown sample versus a control (using myometrium cDNA, or untreated LSMC or untreated MSMC cDNA as a calibrator), and expressed in comparison to a reference gene (Pfaffl, 2001). The mean CT ratios are expressed as fold differences in gene expression compared to myometrium (gene expression = 1).
Total protein extraction and western blot analysis
Frozen tissues were cut into small pieces and homogenized in 0.5 ml of RIPA buffer (150 mmol l–1 NaCl, 1% N-40, 0.5% deoxycholate, 0.1% sodium dodecylsulfate, 50 mmol l–1 TRIS-hydrochloric acid, 2 mmol l–1 phenylmethylsulfonyl fluoride, pH 7.4), then transferred into small tubes and rotated at 4°C overnight. Solubilized protein was collected after centrifugation at 10 000g for 30 min. The supernatant from each culture was collected and concentrated by 100-fold with Centricon (Millipore Corp). Protein concentrations were determined by Protein Bradford Assay (Bio-Rad Laboratories, Hercules, CA, USA) (Chen et al., 2003).
LTBP-1 and FBN-1 protein expressions were semi-quantitated by western blot. Thirty micrograms of total protein extracted from frozen uterine tissues and 10 µg of total protein from the supernatant were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis or 4–15% Tris–HCL polyacrylamide gel electrophoresis under non-reducing conditions, and were blotted onto nitrocellulose membranes (Pierce, Rockford, IL, USA) in an electrophoretic transfer cell (Bio-Rad Laboratories). Blots were blocked with 5% non-fat milk in Tris–HCl with Tween (TBS-T) at RT for 1 h. Mouse anti-human LTBP-1 primary antibody (2 µg ml–1; R&D Systems) or mouse anti-human FBN-1 primary antibody (2 µg ml–1, LAB Vision Corporation) was incubated at 4°C overnight, and sheep anti-mouse immunoglobulin (IgG) horseradish peroxidase-conjugated antibody (1/5000; Amersham International PLC, Buckinghamshire, UK) were diluted in TBS-T, and incubated for 1 h at room temperature. Blots were developed by chemiluminescence. Densitometry of immunoreactive bands on the film was performed with Molecular Analyst software (Bio-Rad).
Statistical analysis
The data were analysed using one-way analysis of variance (ANOVA) followed by Tukey Kramer HSD post hoc analysis and Student's t-test (significance level 
< 0.05).
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Results |
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We evaluated a total of 60 leiomyomata and myometrium samples from 20 women with leiomyomata, of which 11 were in the proliferative (estrogen-only) and 9 in the secretory phase of the menstrual cycle (Table I).
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Our immunohistochemistry stains of leiomyoma and myometrial tissues show that LTBP-1 and FBN-1 were detected in the ECM and vascular endothelial cells in leiomyomata and myometrium. The distribution of LTBP-1 and FBN-1 are also very similar (Figure 1).
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LTBP-1 mRNA expression was higher in leiomyomata compared with matched myometrium in the proliferative phase, especially in the medium-sized group (P = 0.01). LTBP-1 mRNA expression was higher in the medium-sized group compared with the small or large groups in the proliferative phase (P = 0.04; P = 0.04) (Figure 2A and B). No difference was observed in LTBP-1 mRNA expression in the secretory phase between leiomyomata and myometrium or among the three leiomyomata groups. FBN-1mRNA expression was higher in leiomyomata compared with matched normal myometrium in both proliferative (Figure 3A and B) and secretory phases (data not shown). This difference was statistically significant in the medium-sized group (P = 0.01; P = 0.01).
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Consistent with the mRNA data, western blot analysis showed that LTBP-1 protein expression was higher in the medium-sized leiomyomata compared with myometrium in the proliferative phase (P = 0.01) (Figure 2C). LTBP-1 protein expression was also higher in the medium-sized group than the small or large groups in the proliferative phase (P = 0.02; P = 0.01) (Figure 2C). No difference in LTBP-1 expression was observed between leiomyomata and myometrium or among the three leiomyomata groups during the secretory phase. FBN-1 protein expression was higher in the medium-sized leiomyomata compared with matched myometrium in both the proliferative (P = 0.01) (Figure 3C) and secretory phases (P = 0.02).
Immunofluorescence stains confirmed that cultured LSMC and MSMC expressed LTBP-1 and FBN-1 proteins (Figure 4). To study the in vitro effect of ovarian steroids on LTBP-1 and FBN-1 expression, cultured LSMC and MSMC were stimulated with varying concentrations of E2 and progesterone. Treatment with E2 (0, 0.1, 1.0 and 10 ng ml–1) increased LTBP-1 mRNA and protein expressions in LSMC in a dose-dependent manner (P = 0.01; P = 0.01 at 10 ng ml–1 concentration) (data not shown). The same trend was observed for FBN-1 mRNA and protein expression (P = 0.02; P = 0.01) (data not shown). No changes in LTBP-1 and FBN-1 mRNA and protein expression were observed when cells were treated with E2 (10 ng ml–1) plus different progesterone concentrations (0, 1, 10 and 100 ng ml–1) (Figures 5 and 6). MSMC showed no dose response to estrogen or estrogen plus progesterone stimulation.
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Discussion |
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Leiomyomata contain abundant quantities of ECM, which provides the architectural framework for these tumours (Perkett et al., 1990). TGF-ß is believed to be important for leiomyoma growth since it stimulates synthesis and inhibits degradation of ECM (Sozen and Arici, 2002). Treatment with TGF-ß1 resulted in inhibition of DNA synthesis in MSMCs, whereas LSMCs showed no such inhibition (Lee Byung-Seok and Nowak, 2001). LTBP-1 is a large protein that binds to latent TGF-ß complexes and facilitates TGF-ß1 secretion and sequestration into the ECM (Oklu and Hesketh 2000; Mazzieri et al., 2005). We hypothesize that the differential expression of LTBP-1 in leiomyoma compared to matched myometrium may result in increased binding and targeting of TGF-ß1 into the ECM, thus allowing TGF-ß1 to exert its paracrine effects.
Increased LTBP-1 expression was observed solely during the proliferative (estrogen-only) phase. Our in vitro experiments demonstrated that 17ß-estradiol increased LTBP-1 mRNA and protein expression in LSMC, whereas treatment with E2 plus different concentrations of progesterone showed no change in LTBP-1 mRNA and protein expression. Current reports conflict as to estrogen's effect on LTBP expression. Rainbow trout LTBP (rtLTBP-3) is up-regulated 5-fold in response to 17ß-estradiol (Andersson and Eggen, 2006), whereas other studies showed that estradiol had no effect on LTBP-1 expression in transformed human lung fibroblasts (Weikkolainen et al., 2003). Little is known regarding the effect of ovarian steroids on LTBP-1 mRNA and protein expression in leiomyoma cells. So far, only LTBP-2 mRNA expression has been demonstrated to be differentially up-regulated in leiomyoma compared with myometrium (Wu et al., 2002). However, these authors did not document differences in protein expression. Our data show that estrogen differentially up-regulated LTBP-1 expression in LSMCs compared with MSMCs.
Fibrillins are major structural components of the microfibrils found in connective tissues (Sinha et al., 2002). They display tissue-specific architecture and provide a template on which TGF-ß-related growth factors can attach (Kaartinen and Warburton, 2003). LTBPs and fibrillins are members of a family of homologous molecules and LTBP-1 has been found to bind to FBN-1 in the ECM (Isogai et al., 2003). Previous studies have demonstrated that LTBPs are associated with FBN-1-containing microfibrils in various tissues, including the heart (Nakajima et al., 1997), arterial walls (Sinha et al., 2002), capsular opacification (Saika et al., 2001) and bone (Taipale et al., 1996). We found that FBN-1 expression in leiomyoma compared to myometrium, while increased, was not menstrual cycle-dependent; thus, indicating a structural role in leiomyoma ECM. Our immunohistochemistry data demonstrated that LTBP-1 and FBN-1 co-localized and stained strongly in leiomyoma ECM. Taken together, these data suggest that LTBP-1 may interact with FBN-1 in the ECM of leiomyoma.
FBN-1 has also been associated with fibrosis of the heart and liver (Lorena et al., 2004; Bouzeghrane et al., 2005). Consistent with these findings, studies of homozygous mice show that FBN-1 abnormalities result in abnormal ECM turnover and phenotypic modulation of SMCs (Bunton et al., 2001). Proteoglycans are differentially expressed in leiomyomata compared with myometrium (Sozen and Arici, 2002; Berto et al., 2003; Mitropoulou and Konstantinos, 2004). It is interesting to note that versican, a large chondroitin sulfate proteoglycan, binds to FBN-1 between calcium-binding epidermal growth factor-like domains 11 and 21 (Isogai et al., 2002). The authors suggest that versican may connect microfibrils, such as FBN-1, to water-retaining hyaluronan-rich matrix. This may account for some of the structural properties of the tumour. Topical application of 17ß-estradiol to young skin increased expression of ECM proteins, including FBN-1 (Son et al., 2005). Similarly, our data showed that FBN-1 expression is significantly higher in leiomyomata tissues and is up-regulated by 17ß-estradiol.
We investigated LTBP-1 and FBN-1 expression in three groups of leiomyoma based on size. To minimize differences due to location of the leiomyomata in the uterus, we selected only fundal myomas. Interestingly, the medium-sized group was found to have the highest expression of both proteins compared with the small- and large-sized groups. This may be a reflection of the differences in bioactivity in these tumours with respect to size. Estrogen receptor expression has been found to be higher in leiomyoma under 5 cm in diameter compared to those larger than 5 cm (Wei et al., 2006). Since LTBP-1 appears to be estrogen-sensitive, it is possible that the differential expressions seen between the different-sized groups are due to a lower level of estrogen receptor in the large-sized leiomyoma. The mechanism for growth may vary depending on the size of the tumours.
There are also spatial differences in the biologic activity within large (>10 cm) leiomyomata. Regions next to the periphery of the tumour have been found to be more biologically active in large (>10 cm) leiomyoma compared with central tissue (Wei et al., 2006). These large leiomyoma also have higher levels of gene products compared with small (<2 cm) leiomyoma, with the exception of estrogen and progesterone receptors. Our biopsies of the leiomyoma were taken halfway between the outer edge and centre of the tumour. This was done to keep the spatial relationship consistent between all the tumours and to minimize the potential for large variations in gene expression found in the periphery. Additionally, we wanted to reduce variations in LTBP-1 and FBN-1 expression as a result of vessel proliferation, which is found primarily in the periphery of the tumours.
In the present study, we verified that both LTBP-1 and FBN-1 were expressed in leiomyoma and myometrium ECM. Although LTBP-1 mRNA and protein expressions were found to be higher in leiomyoma compared with myometrium during the proliferative phase of the menstrual cycle, FBN-1 expressions remained higher in leiomyoma throughout the menstrual cycle. These findings are most prominent in the medium-sized tumours. In addition, LTBP-1 and FBN-1 expressions were both up-regulated in cultured leiomyoma SMCs by 17ß-estradiol compared to MSMC, whereas no effect was observed with 17ß-estradiol plus progesterone stimulation. These results suggest that estrogen may modulate leiomyoma ECM metabolism through differential expressions of LTBP-1 and FBN-1. Future studies are necessary to explore how these differential expressions affect the TGF-ß paracrine effects in the ECM of leiomyomata.
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Acknowledgement |
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The authors wish to thank Lorna Groundwater of the Stanford University School of Medicine for her aid in editing. This study was supported by the National Institutes of Health Grant #R01 AGO1790 to Mary Lake Polan and Bertha Chen.
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Submitted on November 17, 2006; resubmitted on January 12, 2007; accepted on January 22, 2007.
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