BIOLOGY OF REPRODUCTION (2012) 87(1):2, 1–6 Published online before print 4 April 2012. DOI 10.1095/biolreprod.111.098806

Simvastatin Decreases Invasiveness of Human Endometrial Stromal Cells1 Anna Sokalska,3,4 Amanda Cress,3 Kaylon L. Bruner-Tran,5 Kevin G. Osteen,5 Hugh S. Taylor,6 Israel Ortega,3,7 and Antoni J. Duleba2,3 3

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of California Davis, Sacramento, California 4 Division of Infertility and Reproductive Endocrinology, Department of Gynecology, Obstetrics and Gynecological Oncology, Karol Marcinkowski University of Medical Sciences, Poznan, Poland 5 Women’s Reproductive Health Research Center, Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 6 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 7 IVI-Madrid, Madrid, Spain INTRODUCTION

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation—geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)—the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.

Endometriosis is one of the most debilitating and poorly understood gynecologic disorders, associated with a broad range of symptoms including dysmenorrhea, chronic intermenstrual pelvic pain, dyspareunia, and infertility. While the etiology of endometriosis remains debatable, the dominant concept invokes retrograde menstruation (Sampson’s theory) [1]. The series of events leading to ectopic implantation of endometrial glands and stroma and development of symptomatic endometriosis involves multiple processes, including adhesion of endometrial tissues to intraperitoneal structures, invasiveness, angiogenesis, growth of ectopic lesions, stimulation by estrogens, as well as inflammation, oxidative stress, and immune dysfunction. The establishment of endometriotic implants requires a complex interaction between endometriotic tissue and host peritoneum. One of the key processes is the adhesion of endometriotic cells to the mesothelium. Endometriotic stromal cells demonstrate stronger adhesive properties to extracellular matrix proteins than normal eutopic endometrial cells [2]. CD44 is a transmembrane protein adhesion molecule that plays a role in the attachment of endometrial cells to the peritoneum [3] and may be crucial for the initiation of endometriosis [4]. As presented by Griffith et al. [5], menstrual endometrial stromal cells derived from women with endometriosis exhibit an increased rate of adherence to peritoneal mesothelium and elevated expression of several isoforms of CD44. Elevated levels of soluble forms of CD44 were also detected in the peritoneal fluid of patients with endometriosis [6]. Formation of endometriotic implants also requires increased invasive potential of the endometriotic cells. Invasion may be enhanced by excessive expression of matrix metalloproteinases (MMPs) leading to local destruction of the extracellular matrix and hence establishment of the disease [7]. Several MMPs are inappropriately expressed in the endometrium of women with endometriosis and are up-regulated by tumor necrosis factor alpha and interleukin 1 [7, 8]. The endometrium of women with endometriosis compared to healthy controls is characterized by increased mRNA levels of MMP2 and MMP3 and decreased mRNA expression of tissue inhibitor of metalloproteinase 2 (TIMP2) [8, 9]. These features of endometrial cells favor implantation of endometriotic tissue in the peritoneal cavity. Additionally, the continuous expression of several MMPs, especially MMP3, MMP7, and MMP2, and decreased expression of TIMP2 in endometriotic lesions plays

CD44, endometriosis, human endometrial stromal cells, invasion assay, metalloproteinases, simvastatin, tissue inhibitor of metalloproteinases

1 Supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development grant U54 HD052668. 2 Correspondence: Antoni J. Duleba, Department of Obstetrics and Gynecology, University of California Davis, 4869 Y Street, Sacramento, CA 95817. E-mail: [email protected]

Received: 3 January 2012. First decision: 3 January 2012. Accepted: 2 April 2012. Ó 2012 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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ABSTRACT

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a role in the establishment of endometriosis [3, 8, 10]. While the role of autoantibodies in endometriosis is still not well understood, it has been shown that a hemopexin domain expressed by MMPs, except MMP7, can be recognized and bound by T-like autoantibodies in women with endometriosis leading to dysregulation of MMPs and TIMPs in ectopic lesions [11]. Reduced sensitivity of MMPs to progesterone in the endometrium of women with endometriosis, combined with all the mechanisms listed above, is likely to contribute to the invasive potential of refluxed endometrial tissues [7]. Recently, we demonstrated that statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), reduce proliferation of human endometrial stromal (HES) cells [12]. We have also found that simvastatin protects against development of experimental endometriosis by human endometrial tissues using a nude mouse model [13]. Comparable beneficial effects of statins were also reported in other in vivo and in vitro systems [14–18]. The current study was designed to investigate whether simvastatin may affect adhesiveness and invasiveness of HES cells as well as expression of selected genes relevant to adhesiveness and invasiveness: MMP2, MMP3, TIMP2, and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates of isoprenylation—geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)—the effects of GGPP and FPP were also evaluated.

FIG. 1. HES cells invasion assay.

MATERIALS AND METHODS Human Endometrial Tissues Endometrial tissues were obtained from endometrial biopsies collected from 13 subjects (age 25–41 years: nine Caucasian, two Asian, and two African American); during the proliferative phase of the menstrual cycle (between 9th and 13th day of the menstrual cycle with endometrial thickness of at least 7 mm as determined by transvaginal ultrasound). The specimens were collected from women undergoing surgeries for benign conditions and healthy volunteers. The use of human tissues was approved by the University of California Davis Institutional Review Board, and informed consent was obtained from all the subjects. HES cells were isolated following enzymatic digestion of endometrial fragments and subsequently passing the cells through a 70-lm sieve (BD Falcon) [19]. Cells were then cultured at 378C in humidified atmosphere of 95% air and 5% carbon dioxide in phenol red-free Dulbecco modified Eagle medium (DMEM; Gibco) with 1% antibiotic, 10% charcoal/dextran-treated fetal bovine serum (FBS), and 1 nM estradiol.

Determination of Cell Viability A separate set of experiments was carried out to determine whether simvastatin, GGPP, and FPP affect the number of viable cells during the same time interval as studies evaluating invasiveness. The cells were seeded at a density of 15 000 cells/well in 96-well plates and cultured at 378C in a humidified atmosphere of 95% air/5% CO2 in phenol red-free, serum-free DMEM with 1 nM estradiol without additives (control) or with simvastatin (1– 30 lM), GGPP (30 lM), and/or FPP (30 lM) (Sigma Chemical Co.). The determination of the number of viable cells was performed after 24-h treatment using a CellTiter-Blue Cell Viability Assay (MTS; Promega). This assay involves the reduction of resazurin to resorufin by metabolically active cells, resulting in the generation of a fluorescent product at the excitation wavelength of 579 nm and the emission wavelength of 584 nm that is proportional to the number of living cells. Fluorescence was determined using a microplate reader (Fluostar Omega; BMG Labtech).

Adhesion Assay HES cells were plated in 96-well plates (10,000 cells/well) in phenol redfree DMEM with 1% antibiotic, 3% charcoal/dextran-treated FBS, and 1 nM estradiol, and incubated for 24 h. Then the media were changed, and the cells were cultured for an additional 48 h at 378C in a humidified atmosphere of 95% air/5% CO2 without (control) or with simvastatin (1–30 lM) (Sigma Chemical Co.). Subsequently, the number of attached cells, determined by quantification of viable cells, was estimated using a cell viability assay (MTS assay, described below); the cells were then washed with PBS (three times), trypsinized, reconstituted in phenol red-free DMEM with 1% antibiotic, 10% charcoal/ dextran-treated FBS, and 1 nM estradiol, and incubated for 2 h. The unattached cells were washed out using PBS (three times), and the number of reattached cells was evaluated one more time by the MTS assay. The proportion of reattached cells was expressed as a percentage of control. The experiment was repeated three times (eight replicates for each experiment).

Total RNA Isolation and Quantitative Real-Time PCR Human endometrial stromal cells were plated on 24-well plates (140 000 cells/well) and cultured at 378C in a humidified atmosphere of 95% air/5% CO2 for 24 h in DMEM with 10% charcoal/dextran-treated FBS and 1 nM estradiol. Then the media were changed for phenol red-free, serum-free DMEM with 1 nM estradiol for the next 24 h. Subsequently, the cells were incubated without additives (control), with simvastatin (30 lM), and/or GGPP (30 lM) for 24 or 48 h (Sigma Chemical Co.). Total RNA was extracted using KingFisher 96 instrument (Thermo Electron Corporation) and MagMAX-96 Total RNA Isolation Kit (Ambion). Reverse transcription of total RNA to cDNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative real-time PCR reactions were performed in triplicate using the ABI 7300 RealTime PCR System (Applied Biosystems) and 2X SYBR Green PCR Master Mix (Applied Biosystems). The relative amount of target mRNA was expressed as a ratio normalized to 18S ribosomal RNA (18S). The primer sequences were as presented in Table 1. Serial, separate cDNA dilutions were included in each

Invasion Assay Twenty-four-well plates with transwell inserts (6.5 mm diameter) with 8.0 lm pore size polycarbonate membrane (Transwell Permeable Supports; Corning) were used for the assay. The membranes of precooled inserts were coated using 40 ll of Matrigel (ECM gel, growth factor reduced, without phenol red from Engelberth-Holm-Swarm mouse sarcoma; Sigma-Aldrich) diluted to a final protein concentration 1.2 mg/ml with cold phenol red-free culture media without FBS [20]. The Matrigel layers were left to dry in the

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laminar hood for 2 h and then rehydrated by adding warm (378C) phenol redfree, serum-free DMEM and incubating in a cell culture incubator for 30 min [21, 22]. The cells were trypsinized, washed and suspended in phenol red-free, serum-free DMEM with 1 nM estradiol, and transferred to the transwell inserts (50 000 cells/transwell insert). The lower chambers of the wells were filled with phenol red-free DMEM with 1 nM estradiol and 10% charcoal/dextran-treated FBS, which were used as a source of chemoattractants [20–22] (Fig. 1). Subsequently, the cells were cultured for 24 h at 378C in a humidified atmosphere of 95% air/5% CO2 without (control) or with simvastatin (1–30 lM), GGPP (30 lM), and/or FPP (30 lM) (Sigma Chemical Co.). The abovementioned concentrations were selected based on our previous study evaluating the effect of simvastatin on apoptosis and cytoskeleton of HES cells [12]. The noninvading cells were scraped off from the top of the transwell inserts using a cotton swab. Invading cells were fixed in 4% paraformaldehyde (for 30 min), washed in PBS, stained in crystal violet (for 60 min), and washed several times in PBS. The number of invading cells was assessed under light microscope (magnification 103) [21–23]. The mean number of invading cells was calculated from four replicates and analyzed as a percentage of the control. The experiment was repeated eight times.

SIMVASTATIN DECREASES ENDOMETRIAL INVASIVENESS TABLE 1. Primers used for real-time PCR. Sequencea

Source

F: 5 0 -AAATAGCCTTTGCCATCACTG-3 0 R: 5 0 -ACGTTCCACCTCATCCTC-3 0 F: 5 0 -GCAGATCTCAGGAGTGACAGG-3 0 R: 5 0 -GATGGCACCCATTTACACCTA-3 0 Catalog no. QT00060025 F: 5 0 -CAAGAGGATCCAGTATGAGAT-3 0 R: 5 0 -GGCCGTGTAGATAAACTCTAT-3 0 F: 5 0 -AACCGTGATGGCACCCGCTATGTC-3 0 R: 5 0 -GGACCAGAGGTTGTGTTTGCTCCA-3 0

Invitrogen

Primer 18S MMP2 MMP3 TIMP2 CD44 a

Invitrogen Qiagen Invitrogen Invitrogen

F, forward; R, reverse.

real time-PCR run to generate standard curves. Data were analyzed using 7300 Real-Time PCR System Sequence Detection Software Version 1.4 (Applied Biosystems).

Statistical Analysis Statistical analysis was carried out using JMP statistical program (SAS). Comparisons between groups were performed by analysis of variance followed by post-hoc comparisons using the Tukey-Kramer HSD test. Results are presented as mean 6 SEM; P , 0.05 was considered statistically significant.

Effects of Simvastatin on Human Endometrial Stromal Cells Adhesiveness In three separate experiments using HES cells from three different subjects, exposure to simvastatin reduced the ability of the cells to adhere to fibronectin. Simvastatin at concentration of 1, 10, and 30 lM decreased the number of attached cells by 46% (P , 0.0001), 51% (P , 0.0001), and 55% (P , 0.0001) (Fig. 2), respectively. Effect of Simvastatin and Substrates of Isoprenylation on Human Endometrial Stromal Cells Invasiveness In eight separate experiments using HES cells from eight different subjects, exposure to simvastatin reduced HES cells invasiveness. Simvastatin at concentration of 10 and 30 lM

FIG. 3. A) Effect of simvastatin (1–30 lM) on invasiveness of HES cells. B) Effects of GGPP (30 lM) and FPP (30 lM) on invasiveness of HES cells in the presence and absence of simvastatin. Each bar represents the mean 6 SD; means with no letters in common are significantly different (P , 0.05).

decreased the number of invading cells by 28% (P ¼ 0.01) and by 61% (P , 0.0001), respectively. Simvastatin at a concentration of 1 lM had no statistically significant effect (Fig. 3A). Furthermore, to test the role of isoprenylation in the invasive capacity of HES cells, substrates for isoprenylation— 30 lM FPP and 30 lM GGPP—were added to the cultures in the absence and presence of 30 lM simvastatin. FPP and GGPP alone did not affect HES cell invasiveness. Addition of GGPP, but not FPP, abrogated the inhibitory effect of simvastatin (P ¼ 0.0071) (Fig. 3B). Effect of Simvastatin, FPP, and GGPP on Number of Viable Cells Because the reduced number of invading cells may be due, at least in part, to a toxic effect of simvastatin, FPP, and/or GGPP, parallel experiments were performed to evaluate the number of viable cells (MTS assay). In all the experiments, 24h exposure to simvastatin, GGPP, and FPP did not significantly alter the number of viable cells (data not shown).

FIG. 2. Effect of simvastatin (1–30 lM) on adhesiveness of HES cells. Each bar represents the mean 6 SD; means with no letters in common are significantly different (P , 0.05).

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RESULTS

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TIMP2 (Fig. 5). Simvastatin decreased the MMP2/TIMP2 ratio by 43% (P , 0.0001) and the MMP3/TIMP2 ratio by 56% (P , 0.0001).

Effects of Simvastatin and GGPP on MMP2, MMP3, TIMP2, and CD44 Gene Expression To study potential molecular mechanisms responsible for the inhibitory effect of simvastatin on invasiveness of HES cells, subsequent experiments evaluated the effects of simvastatin on mRNA levels of selected genes encoding for proteins relevant to modulation of invasiveness, that is, MMP2, MMP3, TIMP2, and CD44, as determined by quantitative real-time PCR (Fig. 4, A–D). Because GGPP, but not FPP, abrogated the inhibitory actions of simvastatin on cell invasiveness, Figures 4 and 5 also summarize the effects of GGPP in the absence and presence of simvastatin. Simvastatin (10 lM) had no significant effect on mRNA level of any of the tested genes after 24 h (data not shown), however, after 48 h, it induced significant reduction of mRNA level of MMP2 by 30% (P ¼ 0.001), MMP3 by 46% (P , 0.0001), and CD44 by 30% (P ¼ 0.03). Addition of 30 lM GGPP to 10 lM simvastatin abrogated the inhibitory effect of statin on MMP2, MMP3, and CD44 gene expression. Simvastatin also modestly, but statistically significantly, up-regulated TIMP2 gene expression by 24% (P ¼ 0.008). The net effect of changes in MMP2, MMP3, and TIMP2 mRNA expression is also presented as a ratio of MMPs to

DISCUSSION Current treatment options for women with endometriosis are limited and frequently involve either hormonal manipulation and/or surgery. For many women, the side effects of medical therapy can be as unpleasant as the symptoms of endometriosis and surgical treatment is generally noncurative with a high rate of recurrence [24–33]. Therefore, development of better, more effective treatment strategies for women with endometriosis remains a high priority for research and will likely require a more precise understanding of the disease etiology. To this end, we have investigated the impact of simvastatin, a cholesterollowering drug with potent anti-inflammatory and antioxidant effects, on endometrial stromal cell adhesion and invasion. The present study has provided insight into not only the mechanisms by which this agent inhibits development of experimental endometriosis, but also provides additional support for pursuing simvastatin as a potential therapy for women with this disease. Specifically, we have demonstrated that: 1) simvastatin decreases adhesiveness of HES cells in 4

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FIG. 4. Effect of simvastatin (10 lM) and/or GGPP (30 lM) on mRNA expression of MMP2 (A), MMP3 (B), CD44 (C), and TIMP2 (D). Expression of genes of interest was normalized to the expression of 18S. Each bar represents the mean 6 SD; means with no letters in common are significantly different (P , 0.05).

SIMVASTATIN DECREASES ENDOMETRIAL INVASIVENESS

FIG. 5. Effect of simvastatin (10 lM) and/or GGPP (30 lM) on the ratio of mRNA expression of MMP2/TIMP2 and MMP3/TIMP2. Expression of genes of interest was normalized to expression of 18S. Each bar represents the mean 6 SD; means with no letters in common are significantly different (P , 0.05).

vitro; 2) simvastatin decreases invasiveness of HES cells in vitro in a concentration-dependent fashion; 3) the inhibitory effect of simvastatin on invasiveness is abrogated in the presence of GGPP, but not FPP; 4) simvastatin down-regulates MMP2, MMP3, and CD44, and up-regulates TIMP2 gene expression; and 5) addition of GGPP to simvastatin abrogates the inhibitory effect of statin on MMP2, MMP3, and CD44 gene expression. To our knowledge, this is the first report evaluating the effects of simvastatin on the invasive capacity of HES cells. The present observations provide a novel mechanistic explanation for the findings of our recent in vivo study whereby simvastatin induced a dose-dependent reduction in the number and size of endometriotic implants in a nude mouse model of human endometriosis [13]. The invasion of the cells across the basement membrane consists of several events: initial attachment-adhesion, degradation of the basement membrane, and finally migration across the membrane. The simvastatininduced decline in the number of invading HES cells cannot be attributed to a decreased number of cells in culture because during the 24-h culture simvastatin had no effect on the total number of viable cells, as determined by the MTS assay. However, cell motility and hence invasiveness may be affected by the changes in cellular cytoskeleton. Indeed, previously, we have demonstrated that within 24 h, simvastatin alters the morphology of HES cells disrupting the cytoskeleton by disorganizing F-actin fibers, altering cell shape, and inducing cell shrinkage [12]. Statins, by interfering with isoprenylation (i.e., farnesylation and geranylgeranylation), cause the alteration of the actin cytoskeleton [34]. Small GTPases—Rho, Rac, and Cdc42—play an important role in the maintenance and rearrangement of the cytoskeleton and cellular polarity [35–38]. In particular, Rho activation is involved in signaling pathways stimulating actin stress fiber formation [39], Rac plays a role in the generation of lamellipodia, and Cdc42 is important in the formation of actin spikes and filopodia [40]. Moreover, increased expression of RhoA and Rho-associated coiled-coil-forming protein kinase-I (ROCK-I) and ROCK-II was observed in endometriotic stromal cells, suggesting a role of the activation of Rho-ROCK-mediated signaling pathway in 5

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the pathogenesis of endometriosis-associated fibrosis [41]. Statins reduce GGPP and hence decrease geranylgeranylation of Rho, Rac, and Cdc42, leading to an accumulation of these proteins in their inactive form in the cytoplasm and causing detrimental changes in the cell cytoskeleton that leads to a loss of attachment. Furthermore, even though simvastatin had no effect on the total number of viable cells, simvastatin may profoundly alter cellular function by initiation of early apoptotic events such as activation of executioner caspases 3/ 7; such activation of caspases 3/7 was detected after 24 h exposure to simvastatin [12]. Because simvastatin inhibits the first step of the mevalonate pathway, the effects of simvastatin may result from a reduction in the level(s) of any or all the downstream products of this pathway. Among the most important products of mevalonate pathway are the substrates of isoprenylation: FPP and GGPP. The present study demonstrates that the effects of simvastatin on cell invasiveness are most likely due to reduction of GGPP and not FPP. In the presence of GGPP, simvastatin had no effect on cell invasiveness, while in the presence of FPP simvastatininduced inhibition remained unaffected. These studies clearly indicate the importance of geranylgeranylation, but not farnesylation, in stromal cells invasion in our model and, potentially, in the early events of endometriosis development. These findings are consistent with studies in other biological systems, whereby inhibition of geranylgeranylation resulted in suppression of invasiveness of various neoplastic cells, including breast and thyroid cancer cells [42, 43]. Another relevant finding of this study is the observation that simvastatin modulates the relative abundance of mRNAs of MMP2, MMP3, TIMP2, and CD44 by reducing the abundance of mRNAs for MMPs and CD44, which promote adhesiveness and invasiveness, while increasing mRNA for TIMP2, which counteracts invasiveness induced by MMPs. Comparable effects of statins on reduction of several MMPs, including MMP2 and MMP3, have been previously reported in several other in vitro systems in various cell types [44, 45]. Furthermore, a recent clinical trial in patients with chronic heart failure has demonstrated that statin therapy led to a significant decrease in the circulating levels of MMPs and an increase of TIMP2 [46]. While the net effect of actions of MMPs and TIMPs is crucial for dynamic tissue remodeling throughout the reproductive cycle [47], the potential impact of statins on physiologic changes of the endometrium should be address in future in vivo studies. Far less is known regarding the effects of statins on CD44; however, consistent with the present report, a recent study on breast tumor cells has also demonstrated a statin-induced reduction of CD44 expression [48]. The present observations of the effects of simvastatin on MMPs, TIMP2, and CD44 provide additional, albeit indirect, evidence in support of anti-invasive effects of simvastatin on HES cells. Notably, changes in the abundance of the above mRNAs may not explain the effects of simvastatin on the invasiveness assay as performed in this study: simvastatin inhibited invasiveness within 24 h, while the effects on mRNAs were detected after 48 h. Consequently, we postulate that simvastatin may affect invasive capacity of HES cells by several independent mechanisms. Because GGPP reversed not only the effects of simvastatin on invasiveness assay but also on the abundance of relevant mRNAs, the common feature of these mechanisms appears to be the importance of statininduced inhibition of geranylgeranylation. In summary, the findings of this study provide new evidence in support of the concept that simvastatin may exert beneficial and protective effects against the development of endometriosis by reducing the invasive capacity of HES cells.

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