Mol. Hum. Reprod. Advance Access originally published online on January 23, 2008
Molecular Human Reproduction 2008 14(3):181-191; doi:10.1093/molehr/gan004
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Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells
1Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, Hyogo Prefecture 650-0017, Japan 2Center for Biomedical Research, The Population Council, New York, NY 10021, USA
3Correspondence address. Tel: +81-78-382-6000; Fax: +81-78-382-6019; E-mail: maruo{at}kobe-u.ac.jp
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Abstract |
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Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT–PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10–8 M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations
10–7 M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells. Key words: CDB-2914/collagen/extracellular matrix metalloproteinase inducer/leiomyoma/progesterone receptor modulator
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Introduction |
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Uterine leiomyomas are benign clonal tumors originating from smooth muscle cells of the uterus. A neoplastic transformation of myometrium to leiomyoma likely involves somatic mutations of myometrial cells and complex interactions of sex steroids and local growth factors (Walker and Sewart, 2005). Traditionally, estrogen has been thought to be the major promoter of leiomyoma growth. However, accumulating evidence indicates that progesterone also plays a vital role in the regulation of leiomyoma growth by modulating cell proliferation and apoptosis induction (Matsuo et al., 1997; Shimomura et al., 1998; Kurachi et al., 2001; Maruo et al., 2004).
CDB-2914(17-acetoxy-11-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a novel progesterone receptor (PR) modulator that binds to PR with high affinity and has little or no antiglucocorticoid activity (Attardi et al., 2002). CDB-2914 has been developed as a progesterone antagonist for the use in a variety of clinical applications, including the treatment of uterine leiomyomas, endometriosis, dysfunctional uterine bleeding and cancer (Blithe et al., 2003). The precise mechanism underlying action of CDB-2914 on uterine leiomyoma growth is unknown. Nevertheless, we have recently demonstrated that CDB-2914 not only inhibits the growth of cultured human uterine leiomyoma cells and induces apoptosis of these cells (Xu et al., 2005), but also inhibits the induction of angiogenic factors such as vascular endothelial growth factor and adrenomedullin and their receptors (Xu et al., 2006).
It is likely that leiomyomas growth results from an increase in cell proliferation, deposition of the extracellular matrix (ECM) and growth promoting action of local growth factors in paracrine and/or autocrine manner (Walker and Sewart, 2005). One of the distinctive features of uterine leiomyomas is the presence of abundant fibrous connective tissue elements in the ECM. The ECM of uterine leiomyomas consists primarily of collagen, fibronectin and proteoglycan (Stewart et al., 1994; Wola
ska et al., 1998; Berto et al., 2003; Catherino et al., 2004; Malik and Catherino, 2007). Fibrillar collagens such as type I, II, III, IV and V collagens are the most abundant collagens and function as structural proteins (Byers, 2000). Type III collagen is known to be distributed in type I collagen-containing tissues (Gelse et al., 2003). Types I and III collagen mRNAs were shown to be elevated in uterine leiomyomas relative to the adjacent myometrium in the proliferative phase of the menstrual cycle (Stewart et al., 1994). Furthermore, a microarray analysis demonstrated that pro-alpha1 (III) collagen and pro-alpha1 (I) collagen mRNAs were increased 2-fold in uterine leiomyomas compared with myometrium, suggesting the involvement of collagen deposition in characterizing the etiology of uterine leiomyoma enlargement (Wang et al., 2003). In this context, leiomyomas could be viewed as a fibrotic disease.
ECM metalloproteinase inducer (EMMPRIN/CD147) is composed of two immunoglobulin domains in the extracellular region, a single transmembrane domain and a short cytoplasmic domain (Gabison et al., 2005). EMMPRIN is widely expressed on human tumor cells, including fibroblasts (Sun and Hemler, 2001), endometrium (Noguchi et al., 2003), human placenta and fetal membranes (Li et al., 2004), and human ovary and ovarian endometriosis (Smedts et al., 2006). A recombinant EMMPRIN was shown to stimulate cultured fibroblasts to produce matrix metalloproteinase (MMP)-1, MMP-2, MMP-3 (Kanekura et al., 2002) and stimulate the production of MMP-9 in monocytes and MMP-2 in smooth muscle cells (Schmidt et al., 2006). Gene silencing of EMMPRIN by small-interfering RNA was demonstrated to hinder lipopolysaccharide-induced monocyte secretion of MMP-9, indicating a predominant role of EMMPRIN in MMP-9 induction (Schmidt et al., 2006).
Among the MMP family, three subclasses, namely collagenases, gelatinases and MT-MMPs, are involved in native fibrillar collagen degradation (Gioia et al., 2007). Soluble fibrillar collagen I is enzymatically processed by collagenases such as MMP-1, MMP-8 and MMP-13 (Chung et al., 2004; Inada et al., 2004), gelatinase A (MMP-2) (Aimes and Quigley, 1995), gelatinase B (MMP-9) (Allan et al., 1995) and MMP-14 (Ohuchi et al., 1997). MMP-3 is also an activator of other MMPs such as MMP-1 and MMP-9. Dou et al. (1997) reported that MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2 and types I and III collagens were maximally expressed in leiomyoma and myometrium during the progesterone-dominant secretory phase of the menstrual cycle.
The tissue inhibitors of MMPs (TIMPs) are endogenous inhibitors of MMPs and efficiently inhibit the enzymatic activity by binding to the catalytic domain of the MMPs (Olson et al., 2000). The C-terminal domain of TIMPs is important for the protein–protein interaction and binding to MMPs, thereby regulating the MMP activation process (Lambert et al., 2004). TIMP-1 and TIMP-2 can bind MMP-9 and MMP-2, respectively, through the C-terminal hemopexin-like domain (Olson et al., 2000).
The turnover of the ECM is regulated by the combined action of MMPs and TIMPs (Visse and Nagase, 2003). The dysregulation of the ECM remodeling has been thought to play a role in uterine leiomyoma growth. However, it remains unknown whether EMMPRIN is expressed in uterine leiomyoma cells and myometrial cells and acts to modulate the induction of MMPs and TIMPs.
The present study was designed to investigate the effects of PR modulator CDB-2914 on the expression of the ECM components, including EMMPRIN, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-13, TIMP-1, TIMP-2 and types I and III collagens in cultured human uterine leiomyoma cells in comparison with cultured myometrial cells. In addition, to explore the role of EMMPRIN in CDB-2914-induced modulation of the expression of ECM components in cultured leiomyoma cells, we examined the effects of RNA interference (RNAi) of EMMPRIN on the expression of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, TIMP-1, TIMP-2 and types 1 and III collagens at mRNA and/or protein levels in those cells.
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Tissue collection
Twenty-three samples of human uterine leiomyoma tissues and myometrium were obtained from Japanese women with regular menstrual cycles who underwent hysterectomy at Kobe University Hospital. Informed consent was obtained from each patient before surgery for the use of uterine leiomyoma tissues and myometrium for the present study. The Institutional Review Board approved the use of uterine leiomyoma tissues and myometrium for culture experiments. The patients ranged in age from 30–48 years, with a mean age of 40.6 years, and had received no hormonal therapy for at least 6 months before surgery. The histological diagnosis of each uterine specimen was examined. Samples were excluded from the study if accurate menstrual cycle dates could not be assigned or if unexpected pathology was found (e.g. adenomyosis). Each uterine specimen was examined by a pathologist for histological evaluation. Endometrial tissues were obtained from the extirpated uterus, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (1950). Twelve samples were collected from the proliferative phase of the menstrual cycle and eleven samples were from the secretory phase of the menstrual cycle.
Cell culture
Uterine leiomyoma tissues and myometrium were obtained in the proliferative phase or secretory phase of the menstrual cycle. The central parts of leiomyoma tissues were collected with a careful removal of pseudo-capsules and fibrous septa materials. Tissues obtained were dissected from endometrial layers, cut into small pieces and digested in 0.2% collagenase (wt/vol) at 37°C for 3–5 h (Matsuo et al., 1997). The collagenase treatment is shown to provide a pure population with smooth muscle cell characteristics without stromal or glandular epithelial cell contamination (Matsuo et al., 1997). The leiomyoma and myometrial cells were collected by centrifugation at 460 g for 5 min and washed three times with phosphate buffered saline containing 1% antibiotic solution. The cell viability was determined by trypan blue exclusion test. The isolated leiomyoma cells and myometrial cells were plated at densities of 
1 x 106 cells/dish in 10 cm2 culture dishes and 1 x 104 cells/well in 24-well tissue culture plates. The isolated leiomyoma and myometrial cells in culture dishes were subcultured at 37°C for 120 h in a humidified atmosphere of 5% CO2–95% air in phenol red-free DMEM supplemented with 10% fetal bovine serum (vol/vol; Invitrogen Life Technologies, Inc., Grand Island, NY, USA). The isolated cells were subcultured in phenol red-free DMEM supplemented with 10% fetal bovine serum for 120 h. The monolayer cultures reaching 70% confluence were treated with graded concentrations (10–8, 10–7 and 10–6 M) of CDB-2914 (HRA Pharma, Paris, France), in serum-free, phenol red-free DMEM. CDB-2914 was dissolved in absolute ethanol. The final concentration of ethanol in culture media was <0.01%, and the same concentration of ethanol was used as a vehicle in control cultures.
Western blot analysis
Proteins were extracted from cultured leiomyoma cells and myometrial cells as described previously (Shimomura et al., 1998). The cells were lysed at 4°C for 20 min using a lysis buffer consisting of 150 mM NaCl, 2 mM phenylmethylsulphonylfluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/l aprotinin, 0.1% sodium dodecyl sulfate and 50 mM Tris–HCl, pH 7.5. Whole cell lysates were subsequently centrifuged at 13 000 g for 30 min at 4°C, and the supernatants were collected. Protein contents in the supernatants were determined by the Bradford assay. Each 100 µg aliquot of the protein extracts was electrophoresed on a 10% SDS–PAGE under reducing conditions. The proteins were electrophoretically transferred from gels to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The blots were exposed overnight to primary antibodies, followed by incubation for 1 h with horse-radish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech, Arlington Heights, IL, USA). The antigen–antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Biosciences, UK). Membranes were visualized by exposure to X-OMAT film (Eastman Kodak Co., Rochester, NY, USA). The radioautograms were scanned and quantified with ChemiImager 4400 (Astec Co., Ltd., Osaka, Japan). The experiments were repeated with at least three different cultured specimens in triplicate with similar results, and the reported results are representative. The relative values of each protein were normalized with actin values form the same samples.
The following primary antibodies were used in this study: EMMPRIN (sc-9753), MMP-1 (sc-6837), MMP-8 (sc-8849), MMP-13 (sc-12363), TIMP-1 (sc-6832), TIMP-2 (sc-9905), type I collagen (sc-8784), type III collagen (sc-8781) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and actin (MS-1295-P0) (Neo Marker).
Real-time RT–PCR analysis
Total RNA was isolated using the RNeasy Protect kit (Qiagen, Inc., Chatsworth, CA, USA). First strand cDNA was synthesized from 2 µg total RNA using an Omniscript RT Kit (Qiagen, Inc.). The primer of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and GADPH equipped with Light Cycler-Primer Set (Search-LC, Heidelberg, Germany) were used. Real-time RT–PCR was performed in a LightCycler® platform (Roche Molecular Diagnostics) with 20 µl glass capillaries, using 10 µl of a reaction mix that contained 2 µl of LightCycler® FastStart DNA MASTER PLUS SYBR Green I (Roche), 6 µl of RNase-free water, 2 µl of each primer set and 10 µl of RNA extract (10 ng RNA/µl). Each sample was analyzed in duplicate in three independent real-time RT–PCR assays. Cycling conditions included incubation at 95°C for 10 min, and 35 cycles of 95°C for 10 s, 68°C for 10 s and 72°C for 16 s. Synthesis of a DNA product of the expected size was confirmed by melting curve analysis using the LightCycler software (Roche, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression served as an endogenous control.
MMP activity assay
Endogenous MMP-1, MMP-2, MMP-3 and MMP-9 activity was assayed by activity assay (Amersham Biosciences) according to the manufacturer's instructions. In brief, we applied 100 µl/well of each untreated and CDB-2914 treated-medium sample in duplicate into 96 microwells microtiter plates precoated with anti-MMP antibody. MMP-1, MMP-2, MMP-3 and MMP-9 were captured by MMP-specific antibodies. All standards were measured by incubating with 4-aminophenyl mercuric acetate (0.025 mM for MMP-1, 0.5 mM for MMP-2 and 1 mM for MMP-3 and MMP-9) and endogenous activity of samples was measured with assay buffer alone. The concentration of endogenous active MMP-1, MMP-2, MMP-3 and MMP-9 in each sample was measured using a microplate reader set at 405 nm. The amount of active MMP in all samples was determined by interpolation from the standard curve.
Small interfering RNA (siRNA) targeting of EMMPRIN genes
The siRNA for human EMMPRIN mRNA target sequences was synthesized by NIPPON EGT (NIPPON EGT'S OLIGO, Toyama, Japan). The EMMPRIN-specific siRNA sequence was AGUCGUCAGAACACAUCAAdTdT. Non-specific siRNA [(UUCUCCGAACGUGUCACGU)d(TT)], which does not match to any sequence of the human genome, was used as a negative control. After reaching 70% confluence, cultured leiomyoma cells were transfected with EMMPRIN siRNA for 48 h using siScreen Custum Plate (ALLIANCE Technology, Inc., Osaka, Japan) according to the manufacturer's instruction. At the end of transfection, the medium was replaced with serum-free, phenol red-free DMEM and cultured leiomyoma cells were treated with 10–7 M CDB-2914 for 24 h for Western blot analysis and/or real-time RT–PCR analysis.
Statistical analysis
The data were expressed as the mean ± SD from at least three independent experiments. Statistical significance was determined using Student's t-test and one-way ANOVA. A difference with a P < 0.05 was considered statistically significant.
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Effects of graded concentration of CDB-2914 on the protein contents of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells and myometrial cells
To examine whether CDB-2914 modulates the expression of the ECM components in cultured leiomyoma cells and myometrial cells, the effects of CDB-2914 on EMMPRIN, MMP-1, MMP-8, MMP-13, TIMP-1, TIMP-2, type I collagen and type III collagen protein contents in leiomyoma cells and myometrial cells cultured for 24 h were assessed by Western blot analysis (Figs 1
–4).
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One-way ANOVA of the indices for EMMPRIN, MMP-1, MMP-8, TIMP-1, TIMP-2 and types I and III collagen protein contents showed significant effects of CDB-2914 concentrations (P < 0.001) in cultured leiomyoma cells.
Compared with untreated control cultures, treatment with CDB-2914 at concentrations 10–8 M significantly (P < 0.05) increased EMMPRIN (Fig. 1), MMP-1 and MMP-8 protein contents (Fig. 2) in cultured leiomyoma cells. However, CDB-2914 treatment did not affect MMP-13 protein content in leiomyoma cells (Fig. 2). Compared with untreated control cultures, TIMP-1 and TIMP-2 protein contents were significantly (P < 0.05) decreased in cultured leiomyoma cells treated with CDB-2914 at concentrations 10–7 M (Fig. 3). Treatment with CDB-2914 at concentrations 10–8 M significantly (P < 0.05) decreased type I collagen and type III collagen protein contents in cultured leiomyoma cells compared with untreated control cultures (Fig. 4).
In normal myometrial cells, however, treatment with graded concentrations of CDB-2914 did not affect EMMPRIN, MMP-1, MMP-8, MMP-13, TIMP-1, TIMP-2, type I collagen and type III collagen protein contents (Figs 1–4).
Effects of graded concentrations of CDB-2914 on mRNA levels of MMPs and TIMPs in cultured leiomyoma cells and myometrial cells, assessed by real-time RT–PCR analysis
The effects of graded concentrations of CDB-2914 on MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA in leiomyoma cells and myometrial cells cultured for 24 h were assessed by real-time RT–PCR (Figs 5 and 6).
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One-way ANOVA of the indices for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA showed significant effects of CDB-2914 concentrations (P < 0.001) in cultured leiomyoma cells.
Compared with untreated control cultures, treatment with CDB-2914 at concentrations 10–8 M significantly (P < 0.05) increased MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels in cultured leiomyoma cells (Fig. 5). Treatment with CDB-2914 at concentrations 10–7 M significantly (P < 0.05) decreased TIMP-1 and TIMP-2 mRNA levels in cultured leiomyoma cells compared with untreated control cultures (Fig. 6). However, CDB-2914 treatment did not affect MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA levels in cultured myometrial cells (Figs 5 and 6).
Effects of graded concentrations of CDB-2914 on the activity of MMPs in the medium of cultured leiomyoma cells, assessed by MMP activity assay
The effects of graded concentrations of CDB-2914 on endogenous activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium of leiomyoma cells cultured for 24 h were assessed by MMP activity assay (Fig. 7). One-way ANOVA of the indices for active MMP-1, MMP-2, MMP-3 and MMP-9 showed significant effects of CDB-2914 concentrations (P < 0.001) in cultured leiomyoma cells.
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Compared with untreated control cultures, treatment with CDB-2914 at concentrations
10–8 M significantly (P < 0.05) increased active MMP-1, MMP-2, MMP-3 and MMP-9 levels in the medium of cultured leiomyoma cells (Fig. 7).
Effects of EMMPRIN RNA interference on CDB-2914-induced modulation of the ECM components in cultured leiomyoma cells
To explore the role of EMMPRIN in CDB-2914-induced modulation of the ECM components in cultured leiomyoma cells, we used the siRNA technique to knockdown EMMPRIN gene in these cells. Real-time RT–PCR analysis demonstrated that the transfection of cultured leiomyoma cells with siRNA EMMPRIN (siEMMPRIN) suppressed EMMPRIN mRNA amount compared with the cells transfected with non-specific siRNA control (siControl) cells with the inhibition rate being 80% (Fig. 8A). Western blot analysis also revealed that RNAi of EMMPRIN suppressed EMMPRIN protein content by 72% (Fig. 8B).
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RNAi of EMMPRIN in cultured leiomyoma cells suppressed 10–7 M CDB-2914-induced increase in EMMPRIN protein content compared with siControl cells treated with CDB-2914 (Fig. 8). In the absence of the addition of CDB-2914, MMP-1 and MMP-8 protein contents were significantly (P < 0.05) decreased in cultured leiomyoma cells transfected with siEMMPRIN compared with siControl cells (Fig. 9). However, RNAi of EMMPRIN resulted in a significant (P < 0.01) inhibition of CDB-2914-induced increase in MMP-1 and MMP-8 protein contents (Fig. 9). In contrast, TIMP-1 and TIMP-2 protein contents were significantly (P < 0.05) increased in siEMMPRIN cells treated with 10–7 M CDB-2914 compared with siControl cells treated with CDB-2914 (Fig. 9). RNAi of EMMPRIN resulted in the significant (P < 0.05) increase in types I collagen and type III collagen protein contents in cultured leiomyoma cells treated with CDB-2914 compared with siControl cells treated with CDB-2914 (Fig. 9).
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RNAi of EMMPRIN caused a significant (P < 0.01) down-regulation of CDB-2914-induced induction of MMP-1, MMP-2, MMP-3, MMP-9 mRNA levels (Fig. 10) and a significant (P < 0.01) up-regulation of CDB-2914-induced reduction of TIMP-1 and TIMP-2 mRNA levels in siEMMPRIN cells compared with siControl cells treated with CDB-2914 (Fig. 10).
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In the present study, we demonstrated that PR modulator CDB-2914 up-regulated the expression of EMMPRIN and MMPs in cultured leiomyoma cells and activity of MMPs in the medium and down-regulated the expression of TIMP-1, TIMP-2, type I collagen and type III collagen in these cells without affecting the expression of these ECM components in cultured myometrial cells. This is the first evidence showing that CDB-2914 can modulate the expression of the ECM components in cultured leiomyoma cells, but not in cultured myometrial cells. This reinforces the cell-type specific action of CDB-2914 on leiomyoma cells. Furthermore, a series of dose-dependency studies and RNAi experiments indicated an essential role of EMMPRIN in CDB-2914-induced modulation of the expression of MMPs, TIMPs and collagens in cultured leiomyoma cells.
EMMPRIN is a highly glycosylated cell surface transmembrane protein associated with the surface of tumor cells. EMMPRIN is expressed as core protein (27 kDa), less glycosylated form (–32 kDa) and highly glycosylated form (45–65 kDa) (Tang et al., 2004). A highly glycosylated form of EMMPRIN is known to be involved in the stimulation of MMP production (Sun and Hemler, 2001; Tang et al., 2004). Our study demonstrated that a highly glycosylated form of EMMPRIN was present in both cultured leiomyoma cells and myometrial cells. However, CDB-2914 treatment augmented the protein content of a highly glycosylated form of EMMPRIN with molecular weight of 58 KDa in cultured leiomyoma cells, but not in cultured myometrial cells. This suggests that CDB-2914-mediated increase in a highly glycosylated form of EMMPRIN may induce the expression of MMPs in cultured leiomyoma cells. Actually, EMMPRIN is shown to stimulate the production of interstitial collagenases (MMP-1), gelatinase A and B (MMP-2 and MMP-9) and stromelysin-1 (MMP-3) in tumor cells and fibroblasts (Taylor et al., 2002; Gabison et al., 2005).
MMPs are proteinases that participate in the promotion of the ECM degradation (Visse and Nagase, 2003). Under normal physiological conditions, the activity of MMPs is precisely regulated at the transcriptional and post-transcriptional levels and is controlled at the protein level via their activators, inhibitors and their cell surface localization (Sternlicht and Werb, 2001). Although a possible involvement of sex steroid hormones in regulating the expression of MMPs and TIMPs in uterine leiomyomas has been postulated (Dou et al., 1997), the direct effects of progesterone and PR modulator on the expression of MMPs and TIMPs in uterine leiomyoma cells have not been investigated so far.
The present study demonstrated that CDB-2914 treatment up-regulated MMP-1, MMP-2, MMP-3, MMP-8 and MMP-9 levels in cultured leiomyoma cells and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium of these cells and down-regulated TIMP-1, TIMP-2 and types I and III collagen levels without affecting the levels of MMPs, TIMPs and collagens in cultured myometrial cells.
CDB-2914-induced MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 expression in cultured leiomyoma cells were consistent with the inactive forms of zymogens, but CDB-2914 treatment activated MMP-1, MMP-2, MMP-3 and MMP-9 in the medium of these cells. Since most proMMPs are secreted from the cells and activated extracellularly (Visse and Nagase, 2003), the release of CDB-2914-induced proMMPs to the extracellular spaces surrounding uterine leiomyomas and the subsequent activation of MMPs may act to degrade types I and III collagens in the ECM. In this context, our results support the possibility that CDB-2914 treatment down-regulates types I and III collagens through a mechanism dependent of MMP-mediated degrading action of collagens. Additional study is necessary to clarify how CDB-2914 down-regulates collagen levels in cultured leiomyoma cells in an MMP-dependent manner.
Moreover, the decreased synthesis of TIMP-1 and TIMP-2 in cultured leiomyoma cells in response to CDB-2914 treatment may attenuate an ability of TIMPs to directly inhibit MMPs in the extracellular spaces and cause a shift toward proteolytic collagen degradation, leading to a reduced deposition of collagens in the ECM of uterine leiomyomas.
However, the precise mechanism by which CDB-2914 modulates the expression of the ECM components in cultured leiomyoma cells remains to be elucidated. Nonetheless, we have previously demonstrated that the concomitant treatment with CDB-2914 attenuated the progesterone-induced stimulatory effects on the expression of proliferating nuclear cell antigen (Xu et al., 2005), vascular endothelial growth factor and adrenomedullin proteins (Xu et al., 2006) in cultured leiomyoma cells. This fact suggests that CDB-2914 may exert its effects on the expression of the ECM components in cultured leiomyoma cells through antagonizing progesterone at the PR level.
Taken together, it seems reasonable to speculate that CDB-2914 treatment up-regulates EMMPRIN and MMPs levels and down-regulates TIMPs and collagen levels in cultured leiomyoma cells, thereby modulating the ECM turnover in favor of collagenolysis in cultured leiomyoma cells.
To explore the role of EMMPRIN in CDB-2914-induced modulation of the expression of the ECM components in cultured leiomyoma cells, we next examined the effects of EMMPRIN on the expression of MMPs, TIMPs and collagens by using a specific siRNA against EMMPRIN. In this study, RNAi of EMMPRIN abrogated CDB-2914-mediated induction of MMP-1, MMP-2, MMP-3, MMP-8 and MMP-9 at mRNA and protein levels and CDB-2914-mediated reduction in TIMP-1, TIMP-2 and types I and III collagen at mRNA and/or protein levels in cultured leiomyoma cells. These results suggest that EMMPRIN may play a vital role, at least in part, in the regulation of both CDB-2914-mediated up-regulation of MMPs and CDB-2914-mediated down-regulation of TIMPs and collagens in cultured leiomyoma cells.
Interstitial collagens such as types I and III collagens can be specifically cleaved by the collagenases. Three mammalian collagenases such as MMP-1, MMP-8 and MMP-13 can directly cleave the Gly975-Leu976 bond on the a1(II) chain, generating the characteristic three quarter and one quarter length fragments. After the cleavage, the collagen fragments become susceptible to the further degradation by other enzymes, including MMP-3, MMP-2 and MMP-9 (Gioia et al., 2007).
RNAi of EMMPRIN reversed CDB-2914-mediated reduction in types I and III collagen levels in cultured leiomyoma cells, suggesting a possible association of EMMPRIN with CDB-2914-mediated down-regulation of collagens in cultured leiomyoma cells. Since tumor cell-derived EMMPRIN was reported to stimulate fibroblasts to degrade type I collagen in an MMP-dependent fashion (Rosenthal et al., 2005), our results suggested that CDB-2914 treatment increased the expression of proMMPs intracellularly and the activity of secreted MMPs in the medium of cultured leiomyoma cells. It is postulated that EMMPRIN may be involved in the MMP-dependent pathway by which CDB-2914 down-regulates types I and III collagens.
The data presented here suggest the differential regulation of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells and myometrial cells in response to CDB-2914 treatment. The reason for the differential actions of CDB-2914 between two types of cells remains poorly understood. Nonetheless, the differences in PR isoform A (PR-A) and PR-B levels in cultured leiomyoma cells and myometrial cells and the difference in CDB-2914-induced modulation of PR-A and PR-B expression in these cells may be attributable to the differential actions of CDB-2914. The relative levels of PR-A and PR-B expression in the cells are believed to be critical for appropriate cellular response to progesterone (Vegeto et al., 1993). Two PR isoforms have different transcriptional activities (Tung et al., 1993; Vegeto et al., 1993; Wen et al., 1994); PR-B functions as a transcriptional activator of progesterone-responsive genes, whereas PR-A functions as a ligand-dependent repressor of PR-B transcriptional activity (Vegeto et al., 1993). Actually, we have previously demonstrated that PR-B protein content was elevated in cultured untreated leiomyoma cells compared with cultured untreated myometrial cells with no difference in PR-A protein content (Chen et al., 2006). Additionally, CDB-2914 treatment was shown to increase PR-A and decrease PR-B protein content in cultured leiomyoma cells compared with control cultures, whereas no significant changes in PR isoform contents were observed in cultured myometrial cells (Xu et al., 2006). Another explanation of the differential actions of CDB-2914 may be the difference in the recruitment of nuclear receptor coactivators and corepressors between two types of cells. Further study is warranted to elucidate the effects of CDB-2914 on nuclear coregulator expression in cultured leiomyoma cells and myometrial cells.
In conclusion, we provided the first evidence that CDB-2914 treatment induces the expression of EMMPRIN, MMP-1, MMP-2, MMP-3, MMP-8 and MMP-9 and inhibits the expression of TIMP-1, TIMP-2 and types I and III collagens in cultured leiomyoma cells without comparable effects on cultured myometrial cells. Furthermore, it became evident that EMMPRIN may play a vital role, at least partly, in up-regulating MMPs and down-regulating TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914-mediated induction of EMMPRIN, disruption of the MMPs/TIMPs balance and reduced collagen synthesis may result in a reduced deposition of collagens and an enhanced degradation of collagens in the extracellular spaces of leiomyomas, thereby impairing the tissue integrity and causing the reduction in the expansive activity of uterine leiomyomas. The elucidation of the differential regulatory mechanisms of CDB-2914 between leiomyoma cells and myometrial cells is essential for the clinical application of CDB-2914 to the treatment of uterine leiomyomata in the future.
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This work was supported in part by Grants-in-Aid for Scientific Research 18390449 from the Japanese Ministry of Education, Science and Culture (from July 2006 to March 2009) and by Laboratoire HRA Pharma to Maruo T (from April 2006 to March 2007).
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Submitted on November 1, 2007; resubmitted on January 7, 2008; accepted on January 11, 2008.
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