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Relaxin increases elastase activity and protease inhibitors in smooth muscle cells from the myometrium compared with cells from leiomyomas

2008; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2008.03.043

1556-5653

Bertha Chen, Yan Wen, Xiao Yu, Mary Lake Polan,

Endometriosis Research and Treatment

We investigated the effect of relaxin on extracellular matrix remodeling in cultured myometrial compared with leiomyoma smooth muscle cells. Relaxin increases elastase activity and protease inhibitor expressions in myometrial smooth muscle cells, but no effect was observed in leiomyoma cells. We investigated the effect of relaxin on extracellular matrix remodeling in cultured myometrial compared with leiomyoma smooth muscle cells. Relaxin increases elastase activity and protease inhibitor expressions in myometrial smooth muscle cells, but no effect was observed in leiomyoma cells. Leiomyomas are common uterine tumors that affect up to 77% of reproductive-aged women (1Cramer S. Patel A. The frequency of uterine leiomyomas.Am J Clin Pathol. 1990; 94: 435-438Crossref PubMed Scopus (768) Google Scholar). Compared with normal myometrium, leiomyomas contain large amounts of collagen, elastin, hyaluronic acid, and gycosaminoglycans. Tumor growth is associated with extracellular matrix (ECM) remodeling, which occurs via increased biosynthesis, decreased degradation, or both. Collagen is degraded by a family of enzymes called the matrix metalloproteinases (MMPs), which are regulated by locally produced tissue inhibitors of metalloproteinases (TIMPs). Expression of MMP-2, a protease capable of degrading collagen and elastin fibers, was found to be increased in leiomyoma compared with normal myometrium (2Wolanska M. Sobolewski K. Bankowski E. Matrix Metalloproteinases of human leiomyoma in various stages of tumor growth.Gynecol Obstet Invest. 2004; 58: 14-18Crossref PubMed Scopus (31) Google Scholar), suggesting active turnover. Leiomyoma growth also depends on ovarian hormones. Although estrogen tends to stimulate growth, progesterone appears to both stimulate and inhibit growth (3Maruo T. Ohara N. Wang J. Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis.Hum Repro Update. 2004; 10: 207-220Crossref PubMed Scopus (226) Google Scholar). Myoma growth occurs during the secretory phase of the menstrual cycle when circulating estrogen, progesterone, and relaxin are present (4Bourlev V. Pavlovitch S. Stygar D. Volkov N. Lindblom B. Olovsson M. Different proliferative and apoptotic activity in peripheral versus central parts of human uterine leiomyomas.Gynecol Obstet Invest. 2003; 55: 199-204Crossref PubMed Scopus (18) Google Scholar). Our study investigated the effect of relaxin on ECM remodeling using an in vitro cell model. Specifically, we examined elastase activity and protease inhibitor expressions (α1-antitrypsin [ATT], TIMP-1, and TIMP-2) in relaxin-stimulated smooth muscle cells cultured from myometrium and leiomyoma. The institutional review board of the Stanford University School of Medicine approved this study. We selected women with leiomyoma (size: 8 to 10 cm) in the secretory phase of the menstrual cycle. The phase of cycle was confirmed by endometrial histology. The participants had not received hormone therapy for at least three cycles before surgery. Matched uterine myometrial and leiomyoma tissues were obtained from patients undergoing hysterectomy for leiomyoma. Uterine myometrial tissues from a fresh specimen were excised distal to the tumor and 1 cm in depth from the endometrium. A representative cross-section was fixed in 10% buffered formalin for 16 hours, processed with paraffin embedding, and used for immunohistochemistry. Myoma tissues were taken midway between the center and periphery of the tumor. After excision, the tissues were put into tubes containing Dulbecco's modified Eagle's medium (DMEM) for the isolation of smooth muscle cells for immunofluorescence staining and tissue culture, as described previously elsewhere (5Zhao Y. Wen Y. Polan M.L. Qiao J. Chen B.H. Increased expression of latent TGF-β binding protein-1 and fibrillin-1 in human uterine leiomyomata.Mol Hum Reprod. 2007; 13: 343-349Crossref PubMed Scopus (20) Google Scholar). Smooth muscle cells from the myoma or myometrium were cultured in a four-well chamber slide. The cells were fixed with 4% paraformaldehyde and treated with 0.5% Triton. After washing with 0.02% Triton in tris-buffered saline (TBS-T) and blocking with 1% bovine-serum albumin in TBS-T, the slides were then incubated with rabbit anti-LGR7 or anti-LGR8 (1/100; Phoenix Pharmaceuticals, Belmont, CA) and mouse anti-desmin (1/20; Sigma Chemical, St. Louis, MO) primary antibody 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-TRITC (1/50) and goat anti-rabbit-IgG-FITC (1/320, Sigma) at room temperature for 1 hour. We used 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI) staining to observe nuclei. They were visualized and photographed with AxioCam (Zeiss, Oberkochen, Germany). The ATT staining was performed on paraffin-embedded myoma and myometrium using the avidin-biotin-peroxidase (ABC) method, as described previously (6Chen B. Wen Y. Yu X. Polan M.L. Elastin metabolism in pelvic tissues: is it modulated by reproductive hormones?.Am J Obstet Gynecol. 2005; 192: 1605-1613Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The slides were stained with rabbit anti-α1-antitrypsin (1/20; Sigma) primary antibody overnight at 4°C. Deletion of the primary antibody was used as a negative control. After rinsing with TBS-T, slides were incubated with a secondary antibody, goat anti-rabbit biotin conjugate (1/50; Vector Laboratories, Burlingame, CA). The slides were incubated with Vectastain ABC Kit (Vector Laboratories) reagent for 30 minutes at room temperature. Slides were counterstained with 25% of hematoxylin. They were visualized and photographed with AxioCam. Weigert's solution and Van Gieson's mixture of picric acid and acid fuchsin were used to stain elastin and collagen respectively on paraffin-embedded myometrium and myoma tissues, as described previously (6Chen B. Wen Y. Yu X. Polan M.L. Elastin metabolism in pelvic tissues: is it modulated by reproductive hormones?.Am J Obstet Gynecol. 2005; 192: 1605-1613Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Smooth muscle cells were isolated from leiomyoma and myometrial tissues, as described previously (5Zhao Y. Wen Y. Polan M.L. Qiao J. Chen B.H. Increased expression of latent TGF-β binding protein-1 and fibrillin-1 in human uterine leiomyomata.Mol Hum Reprod. 2007; 13: 343-349Crossref PubMed Scopus (20) Google Scholar). Briefly, the tissues were minced into 1 to 2 mm explants and digested with 200 units/mL collagenase (Invitrogen, Carlsbad, CA). Dispersed cells were then centrifuged at 1000 rpm × g for 10 minutes and washed twice in serum-free media. The cells were plated on a six-well plate in DMEM with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μ/mL of streptomycin in an atmosphere of 5% CO2/95% air. The purity of the cells was assessed by immunofluorescence using mouse anti-desmin monoclonal antibody (Sigma) (7Andersen J. Grine E. Eng C.L. Zhao K. Barbieri R.L. Chumas J.C. et al.Expression of connexin-43 in human myometrium and leiomyoma.Am J Obstet Gynecol. 1993; 169: 1266-1276Abstract Full Text PDF PubMed Scopus (69) Google Scholar). When cells were confluent, they were primed with 1 μM 17β-estradiol in 0.2% lactalbumin hydrolysate (Sigma) (8Palejwala S. Stein D.E. Weiss G. Monia B.P. Tortoriello D. Goldsmith L.T. Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signaling pathway.Endocrinology. 2001; 142: 3405-3413Crossref PubMed Scopus (0) Google Scholar) in DMEM for 72 hours to induce relaxin receptor expression. The medium was then removed and replaced with 0.2% lactalbumin hydrolysate in DMEM without or with various amounts of relaxin (0–100 ng/mL, a gift of Dr. Aaron Hsueh, Stanford University) for 48 hours. The conditioned media were concentrated with Centricon (Millipore Corp., Bedford, MA) and stored at −80°C until experiments were performed. Elastase activity in the conditional media was determined by the generation of free amino groups from succinylated elastin (6Chen B. Wen Y. Yu X. Polan M.L. Elastin metabolism in pelvic tissues: is it modulated by reproductive hormones?.Am J Obstet Gynecol. 2005; 192: 1605-1613Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Briefly, 50 μL of conditional media was added to 100 μg of succinylated elastin in 50 μM sodium borate buffer (pH 8.0, Sigma) and incubated at 37°C for 1 hour. Then 50 μL of a 0.03% solution of TNBSA (Sigma) were added to each reaction and left for 20 minutes at room temperature. The optical density of each reaction was determined using Molecular Devices microplate reader at 450 nm (Molecular Devices, Sunnyvale, CA). The elastase activity was standardized by protein concentration. Ten micrograms of total protein from conditional media were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under a reducing condition (TIMP-1 and TIMP-2) or nonreducing condition (ATT) and blotted onto nitrocellulose membranes (Pierce, Rockford, IL) in an electrophoretic transfer cell (Bio-Rad Laboratories, Hercules, CA) (9Chen B. Wen Y. Wang H. Polan M.L. Differences in estrogen modulation of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-1 expression in cultured fibroblasts from continent and incontinent women.Am J Obstet Gynecol. 2003; 189: 59-65Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Chen B. Wen Y. Yu X. Polan M.L. The role of neutrophil elastase in elastin metabolism of pelvic tissues from women with stress urinary incontinence.Neurourol Urodyn. 2007; 26: 274-279Crossref PubMed Scopus (27) Google Scholar). Blots were blocked with 5% nonfat milk at 4°C overnight. After blocking, the membrane was washed three times in PBS-T (phosphate-buffered saline, pH 7.4, and 0.1% Tween), then incubated with monoclonal anti-TIMP-1, TIMP-2 (1 μg/mL; Oncogene Research Products, Cambridge, MA), or 1:5000 rabbit anti-human α1-antitrypsin (Sigma) for 1 hour at room temperature, followed by three washes in PBS-T. The membrane was incubated in 1:5000 dilutions of sheep anti-mouse IgG or donkey anti-rabbit IgG (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) conjugated to horseradish peroxidase for 1 hour at room temperature, followed by three washes in PBS-T. Blots were developed by chemiluminescence. The optical density was determined by GEL-DOC 2000 (Bio-Rad). Statistical analysis was performed using JMP software (SAS Institute, Inc., Cary, NC). For each dose-response curve, analysis of variance and post hoc analysis (Tukey-Kramer honestly significant difference) were applied to determine whether there was a statistically significant difference between the means of cell groups treated and untreated with relaxin. P<.05 was considered statistically significant. Four premenopausal women with large myomas (greater than 8 cm) were recruited. Given that leiomyomas grow during the secretory phase, we only obtained tissues from women in the secretory phase of the menstrual cycle (confirmed by endometrial histology). We confirmed expression of relaxin receptors, LGR7 and LGR8, and ATT in our cultured smooth muscle cells and tissues. We also verified the presence of substrate (collagen and elastin) for the ECM proteases in both myometrium and myoma. Total elastase activity was significantly higher in smooth muscle cells cultured from the myometrium compared with leiomyoma (P<.05 at relaxin concentration of 10 ng/mL Fig. 1A). It is interesting that total elastase activity remained consistently low in smooth muscle cells cultured from leiomyomas, in spite of increasing relaxin concentration. With respect to ATT, an elastase inhibitor, smooth muscle cells cultured from myometrium showed a statistically significant positive dose-response curve with increasing concentrations of relaxin. The highest expression occurred at the 10 ng/mL relaxin concentration dose (P<.05, see Fig. 1B), which corresponds to the dose of highest total elastase activity. This pattern suggests increased ECM remodeling, with active degradation and inhibition occurring with relaxin stimulation. Again, smooth muscle cells cultured from leiomyomas did not show any dose response to relaxin. Both TIMP-1 and TIMP-2, inhibitors of MMP-2, showed increased expression with increasing relaxin concentrations in myometrial cells compared with myoma cells. Myoma smooth muscle cells remained consistently unchanged. The ECM remodeling depends on the constant interplay between proteases and their inhibitors. This complex system is further modulated by reproductive hormones, growth factors, and cytokines. Estrogen and/or progesterone have both been associated with leiomyoma growth (3Maruo T. Ohara N. Wang J. Matsuo H. Sex steroidal regulation of uterine leiomyoma growth and apoptosis.Hum Repro Update. 2004; 10: 207-220Crossref PubMed Scopus (226) Google Scholar, 11Blake R. Leiomyomata uteri: hormonal and molecular determinants of growth.J Natl Med Assoc. 2007; 99: 1170-1184PubMed Google Scholar). However, myomas have been observed to both increase and decrease in size with the use of a levonorgestrel-releasing intrauterine device, which creates a progestational environment in the uterus (12Maruo T. Ohara N. Matsuo H. Xu Q. Chen W. RSitruk-Ware R. et al.Effects of levonorgestrel-releasing IUS and progesterone receptor modulator PRM CDB-2914 on uterine leiomyomas.Contraception. 2007; 75: S99-S103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Likewise, some leiomyomas grow during pregnancy while many shrink or remain stable in size (13Aharoni A. Reiter A. Golan D. Paltiely Y. Sharf M. Patterns of growth of uterine leiomyomas during pregnancy. A prospective longitudinal study.Br J Obstet Gynaecol. 1988; 95: 510-513Crossref PubMed Scopus (79) Google Scholar, 14Strobelt N. Ghidini A. Cavallone M. Pensabene I. Ceruti P. Vergani P. Natural history of uterine leiomyomas in pregnancy.J Ultrasound Med. 1994; 13: 399-401Crossref PubMed Scopus (68) Google Scholar, 15Hammoud A. Asaad R. Berman J. Treadwell M. Blackwell S. Diamond M. Volume change of uterine myomas during pregnancy: do myomas really grow?.J Minim Invasive Gynecol. 2006; 13: 386-390Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Maruo et al. (12Maruo T. Ohara N. Matsuo H. Xu Q. Chen W. RSitruk-Ware R. et al.Effects of levonorgestrel-releasing IUS and progesterone receptor modulator PRM CDB-2914 on uterine leiomyomas.Contraception. 2007; 75: S99-S103Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) suggested that in addition to its stimulatory effects, progesterone may also exert inhibitory effects on leiomyoma cell growth and survival. This dual role appears to be modulated by the local growth factor environment. There is currently scant data on the effects of relaxin—a peptide hormone present during the secretory phase of the menstrual cycle and in pregnancy—on leiomyoma growth. Relaxin increases MMPs (MMP-2 and MMP-9) and their inhibitors (TIMP-1 and TIMP-2) in the pig uterus and cervix (16Lenhart J. Ryan P. Ohleth K. Palmer S. Bagnell C. Relaxin increases secretion of matrix metalloproteinase-2 and matrix metalloproteinase-9 during uterine and cervical growth and remodeling in the pig.Endocrinology. 2001; 142: 3941-3949Crossref PubMed Scopus (52) Google Scholar, 17Lenhart J. Ryan P. Ohleth K. Palmer S. Bagnell C. Relaxin increases secretion of tissue inhibitor of matrix metalloproteinase-1 and -2 during uterine and cervical growth and remodeling in the pig.Endocrinology. 2002; 143: 91-98Crossref PubMed Scopus (28) Google Scholar). Serine protease activity in these tissues is also increased during pregnancy (18Wang-Lee J.L. Lenhart J. Ohleth K. Ryan P. Bagnell C. Regulation of urokinase- and tissue-type plasminogen activator by relaxin in the uterus and cervix of the prepubertal gilt.J Reprod Fertil. 1998; 114: 119-125Crossref PubMed Scopus (13) Google Scholar). These changes in the ECM are thought to contribute to uterine growth. To further understand the role of reproductive hormones on the pathophysiology of leiomyomas, we investigated the effect of relaxin on ECM remodeling in myomas compared with normal myometrium. Given that MMP-2 activity has been reported to be higher in myoma compared with myometrium (2Wolanska M. Sobolewski K. Bankowski E. Matrix Metalloproteinases of human leiomyoma in various stages of tumor growth.Gynecol Obstet Invest. 2004; 58: 14-18Crossref PubMed Scopus (31) Google Scholar, 19Bogusiewicz M. Stryjecka-Zimmer J. Postawski K. Jakimiuk A. Techberger T. Activity of matrix metalloproteinase-2 and -9 and contents of their tissue inhibitors in uterine leiomyoma and corresponding myometrium.Gynecol Endocrinol. 2007; 23: 541-546Crossref PubMed Scopus (40) Google Scholar), we investigated the expression of inhibitors of MMP-2 (TIMP-1 and TIMP-2). Similarly, due to the observed increase in serine protease activity with relaxin, we examined total elastase activity and the associated inhibitor, ATT. There are two potential areas of weakness in this preliminary study. Bourlev et al. (4Bourlev V. Pavlovitch S. Stygar D. Volkov N. Lindblom B. Olovsson M. Different proliferative and apoptotic activity in peripheral versus central parts of human uterine leiomyomas.Gynecol Obstet Invest. 2003; 55: 199-204Crossref PubMed Scopus (18) Google Scholar) documented that leiomyoma growth tends to occur in the peripheral area of the tumor. The peripheral area of the leiomyoma is also where angiogenesis is most active (20Wei J. Zhang X. Chiriboga L. Yee H. Perle M. Mittal K. Spatial differences in biologic activity of large uterine leiomyomata.Fertil Steril. 2006; 85: 179-187Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), and it may exhibit higher levels of degradation due to this. Because of this concern, we obtained biopsy samples in the middle one-third of the myoma, away from the area of increased angiogenesis, rather than the outer one-third or central portions. This may contribute to the lack of response seen in the cultured myoma cells. Furthermore, estrogen and progesterone receptor levels have been shown to be reduced in cell cultures compared with whole tissues (21Zatiseva M. Vollenhoven B.J. Rogers A.W. In vitro culture significantly alters gene expression profiles and reduces differences between myometrial and fibroid smooth muscle cells.Mol Hum Reprod. 2006; 12: 187-297Crossref PubMed Scopus (74) Google Scholar). Cells were taken during the secretory phase and used at first pass. Estrogen stimulation was used to induce relaxin receptor expression. It is possible that our culture condition may have artificially altered relaxin receptor expression in the myoma cells and, thus, cell response. Future tissue studies are necessary for verification. In this in vitro study, we observed an increase in total elastolytic activity and a concurrent increase in protease inhibitor (ATT, TIMP-1, TIMP-2) expression in relaxin-stimulated myometrial smooth muscle cells compared with leiomyoma smooth muscle cells. This is consistent with increased ECM remodeling activity with active degradation and inhibition in the myometrium. It is interesting that the smooth muscle cells cultured from the leiomyomas did not show any response to relaxin, suggesting a stable and quiescent ECM. The interactions among estrogen, progesterone, and relaxin require further examination. The authors thank Lorna Groundwater for her invaluable editorial assistance with this manuscript.