ORIGINAL ARTICLE: ENDOMETRIOSIS
Dienogest enhances autophagy induction in endometriotic cells by impairing activation of AKT, ERK1/2, and mTOR JongYeob Choi, Ph.D.,a MinWha Jo, M.S.,b EunYoung Lee, M.S.,a Dong-Yun Lee, M.D., Ph.D.,a and DooSeok Choi, M.D., Ph.D.a a
Infertility Clinic, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine; and b Center for Clinical Research, Samsung Biomedical Research Institute, Seoul, South Korea
Objective: To elucidate the therapeutic mechanisms of progestin and the effects of progesterone and progestin (dienogest) on autophagy induction and regulation in endometriotic cells, specifically the effects of progesterone and dienogest on the phosphoinositide-3/protein kinase B (PI3K-AKT) and mitogen-activated protein kinase kinases 1 and 2 (MEK1/2)/extracellularsignal-regulated kinase 1/2 (ERK1/2) pathways, which activate mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Design: In vitro study using human endometriotic cyst stromal cells (ECSCs). Setting: University medical center. Patient(s): Fifteen patients with ovarian endometrioma. Intervention(s): ECSCs treated with progesterone or dienogest. Main Outcome Measure(s): Autophagy as measured by the expression of the microtubule-associated protein light chain 3-II (LC3-II) and autophagosome formation, and levels of AKT, ERK1/2, and mTOR activity to quantify the phosphorylation of AKT, ERK1/2, and S6K (the downstream target of mTOR). Result(s): Progesterone treatment had not statistically significant effect on LC3-II expression, autophagosome formation, or phosphorylation of AKT, ERK1/2, or S6K in estrogen-treated ECSCs. However, dienogest treatment up-regulated LC3-II expression and stimulated autophagosome formation. These effects were accompanied by decreased activation of AKT, ERK1/2, and S6K. Furthermore, incubation of ECSCs with AKT and ERK1/2 inhibitors, which mimicked dienogest-mediated inhibition of AKT and ERK1/2 activity, suppressed S6K activity, followed by an increase in LC3-II expression. In addition, cotreatment with dienogest and 3-methyladenine (autophagy inhibitor) decreased the levels of apoptosis of ECSCs compared with the single treatment with dienogest. Conclusion(s): Our results suggest that dienogest treatment of endometriotic cells suppresses AKT and ERK1/2 activity, thereby in turn inhibiting mTOR, inducing autophagy, and promotUse your smartphone ing apoptosis. (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive to scan this QR code Medicine.) and connect to the Key Words: AKT, autophagy, dienogest, endometriosis, ERK1/2 Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/choij-dienogest-enhances-autophagy-induction/
E
ndometriosis is defined by the presence of endometrium-like tissue outside the uterine cavity,
primarily on the ovaries and pelvic peritoneum. Endometriosis is one of the most common causes of chronic pelvic
Received March 17, 2015; revised and accepted May 14, 2015. J.Y.C. has nothing to disclose. M.W.J. has nothing to disclose. E.Y.L. has nothing to disclose. D.-Y.L. has nothing to disclose. D.S.C. has nothing to disclose. Supported by a grant from the Samsung Biomedical Research Institute. Reprint requests: DooSeok Choi, M.D., Ph.D., Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.020 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
pain, dysmenorrhea, and infertility (1, 2) and affects about 5% to 15% of all women of reproductive age and 20% to 50% of all infertile women (3, 4). The typical characteristics of endometriosis are increased production of estradiol, which stimulates the proliferation of endometriotic tissue, and perturbations in the progesterone response in a phenomenon known as progesterone resistance (5–7). Therefore, current medical therapies focus either on 1
ORIGINAL ARTICLE: ENDOMETRIOSIS lowering estradiol levels or stimulating the progesterone response (2, 8). The most effective medical treatment options for endometriosis are gonadotropin-releasing hormone (GnRH) analogues. However, although GnRH agonists provide effective pain relief and suppress the progression of endometriotic implants by inducing hypoestrogenism (9), these treatments are not suitable for long-term applications because of their severe hypoestrogenic effects. On the other hand, progestins, which are synthetic versions of progesterone, reduce serum estrogen levels by preventing ovulation without causing hypoestrogenism (10). Progestins also exert progestogenic effects on estrogen-primed endometrium (11). Therefore, progestin therapy is a practical option for long-term treatment of endometriosis because it is associated with fewer adverse effects. However, the exact mechanism(s) by which progestin acts on endometriotic cells have yet to be elucidated. Apoptosis is a form of programmed cell death. Accumulating evidence indicates that reduced apoptosis in refluxed endometrial cells might enhance their survival at ectopic sites, thereby promoting the establishment of endometriosis (12, 13). However, apoptosis may not be the only mechanism of endometrial cell death. Autophagy, a nonapoptotic form of programmed cell death, is an intracellular bulk degradation system in which a portion of the cytoplasm is enveloped in double-membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation (14, 15). It is also known to play an important role in promoting cell death by promoting caspasedependent apoptosis in some normal (16, 17) and cancer cells (18–20). We recently showed that the induction of autophagy exerts a proapoptotic effect on normal human endometrial cells (21). However, autophagy is suppressed in endometriotic cells due to derepression of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy, which results in decreased endometriotic cell apoptosis (22). These findings implicate a direct role for mTORmediated suppression of autophagy in the pathogenesis of endometriosis. Previous studies have shown that the class I phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT) and the mitogen-activated protein kinase 1/2 (MEK1/2)/ extracellular signal-regulated kinase 1/2 (ERK1/2) pathways negatively regulate autophagy induction by activating mTOR (23, 24), suggesting that inhibition of AKT and ERK1/ 2 could promote autophagy by preventing mTOR activation. In addition, progestin has been demonstrated to inhibit the PI3K-AKT (25) and MEK1/2-ERK1/2 (26) pathways in endometrial and breast cancer cells, respectively. Therefore, we hypothesized that progestin enhances autophagy induction by inhibiting AKT and ERK1/2, resulting in mTOR inactivation. However, it is not yet known whether progestin regulates AKT and ERK1/2 activity in endometriotic cells, and it is also unknown whether these pathways influence autophagy in endometriotic cells via mTOR signaling. In this study we evaluated the effects of progestin (dienogest) on autophagy in endometriotic cells. Specifically, we determined whether the PI3K-AKT pathway and the MEK1/2-ERK1/2 pathway 2
are involved in progestin-mediated autophagy induction via mTOR signaling.
MATERIALS AND METHODS Human Endometriotic Cyst Stromal Cell Isolation Ectopic endometriotic tissue samples (n ¼ 15) were obtained from proliferating ovarian endometriotic cysts (endometrioma). No study participant had taken oral contraceptives or hormone agents for at least 3 months before surgery. The average age of the participants from whom ectopic endometrial tissue was obtained was 29.5 5.4 years. Proliferating endometriotic cyst stromal cells (ECSCs) were dissociated and purified from ovarian endometriotic tissue essentially as described elsewhere (27) but with minor modifications. Briefly, tissue samples were minced with a sterile surgical blade and digested in phosphate-buffered saline (PBS) containing 2 mg/mL of type IV collagenase (Sigma-Aldrich) at 37 C for 60 minutes with agitation. Stromal cells were separated from the epithelial glands by use of 70-mm pore filters followed by 45 mm-pore nylon mesh. Filtered cells were plated in T75 flasks and allowed to adhere for approximately 30 minutes. The flasks were then washed with PBS to remove blood cells and debris. The stromal cells were cultured in Dulbecco's modified Eagle's/F12 medium (DMEM/F12; GIBCO BRL) supplemented with 10% charcoal-stripped fetal bovine serum (FBS; GIBCOBRL), 100 U/mL penicillin, and 100 mg/mL streptomycin (GIBCO BRL). The cells were maintained in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every other day. Upon reaching confluence, the cells were subcultured in 24-well plates (1 mL/well). Endometrial stromal cell suspension purity was determined by immunostaining with vimentin stromal cell-specific antibodies. This study was approved by the ethics committee of Samsung Medical Center, and written informed consent was obtained from all participants.
In Vitro Experiments Subcultured ECSCs were seeded at 1 106 cells/mL in polyL-lysine-coated nonfluorescent thin-bottom glass culture dishes (MatTek). The cells were incubated at 37 C in 5% CO2 in DMEM/F12 supplemented with 10% charcoalstripped FBS, glutamine, N-2-hydroxyethylpiperazine-N0 2-ethanesulfonic acid (HEPES), 100 U/mL penicillin, and 100 mg/mL streptomycin. Upon reaching 70% to 80% confluence, the cultures were serum starved in serum-free Earle's balanced salt solution (EBSS) medium (SigmaAldrich). To evaluate the effects of progesterone and progestin on autophagy in endometriotic cells, the cells were cultured in EBSS medium before the hormone treatment. After 24 hours of culture, estradiol (10 nM; Sigma-Aldrich) alone, estradiol (10 nM) þ progesterone (1 mM; Sigma-Aldrich), or estradiol (10 nM) þ dienogest (10 mM; Abcam) were added for 72 hours. In this experiment, we added same amount of dimethyl sulfoxide (DMSO; Sigma-Aldrich) in the culture media of estrogen and/or progesterone-treated groups to minimize VOL. - NO. - / - 2015
Fertility and Sterility® experimental error by different solvents because the estrogen and progesterone were dissolved in water whereas dienogest was dissolved in DMSO. For the pharmacologic inhibition experiments, ECSCs were cultured in EBSS medium supplemented with estradiol (10 nM) for 72 hours. At 1 hour before analysis, an AKT inhibitor (5 mM AKT inhibitor VIII; Sigma-Aldrich) or an ERK1/2 inhibitor (30 mM U0126; Sigma-Aldrich) was added in the culture medium to inhibit AKT or ERK activity, respectively. For the autophagy experiments, ECSCs were cultured in EBSS supplemented with dienogest (10 mM) for 72 hours. Next, 3-methyladenine (10 mM 3-MA; Sigma-Aldrich) was added to the culture medium 6 hours before analysis to inhibit the induction of autophagy. The treatments were stopped by removing the medium, and ECSCs were either harvested by scraping to generate protein extracts or fixed for subsequent immunofluorescence and electron microscopy analyses. Autophagy and apoptosis of ECSCs were evaluated using acridine orange and annexinV/propidium iodide staining, respectively.
Western Blot Analysis During the induction of autophagy, microtubule-associated protein light chain 3 (LC3) is converted from LC3-I to LC3II, and LC3-II then localizes to isolated membranes and autophagosomes (28, 29). Because the expression of LC3-II has been shown to correlate with autophagosome number (30), we measured the levels of LC3-II by Western blot analysis as a readout for endometrial cell autophagy. The amount of autophagy is also reflected by the expression of p62, a selective target that undergoes autophagic degradation (31). Both AKT and ERK1/2 activities were determined by quantifying the phosphorylated (i.e., active) forms of AKT and ERK1/2, respectively. The activity of the mTOR pathway was also determined by measuring the phosphorylation of ribosomal protein S6 kinase (S6K). Because S6K is a direct substrate of mTOR, the phosphorylation status of S6K can be used as an indicator of the activity of the mTOR pathway (32). Endometrial cell apoptosis was determined by measuring the levels of cleaved caspase-3, which is the final mediator of apoptosis (33). Cultured ECSCs were lysed with ice-cold radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich). For complete solubilization of cellular proteins, lysates were incubated on ice for 30 minutes and then clarified by centrifugation at 13,000 g at 4 C for 10 minutes. Whole-cell lysates (20 mg/lane) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). After blocking with 5% skim milk, the membranes were incubated with rabbit polyclonal antibodies against LC3 (1:1,000; Cell Signaling Technology), cleaved caspase-3 (1:1,000; Cell Signaling Technology), total and phosphorylated AKT (Ser473) (1:1,000 and 1:1,000, respectively; Cell Signaling Technology), total and phosphorylated ERK1/2 (Thr202/ Tyr204) (1:1,000 each; Cell Signaling Technology) or total and phosphorylated S6K (Ser235/236) (1:1,000 each; Cell Signaling Technology) overnight at 4 C. The membranes VOL. - NO. - / - 2015
were washed three times for 15 minutes in wash buffer (PBS containing 0.1% Tween 20) and were incubated with the appropriate secondary antibodies (1:5,000; Santa Cruz Biotechnology) at room temperature for 1 hour. After three 15-minute washes in wash buffer at room temperature, immunoreactive bands were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). The bands were quantified using the National Institutes of Health ImageJ software (NIH Image Processing and Analysis in Java). The expression levels of LC3-II and cleaved caspase-3 were normalized to b-actin, and the expression levels of phosphorylated AKT, ERK1/2, and S6K were normalized to those of total AKT, ERK1/2, and S6K, respectively.
Immunofluorescence Endometriotic stromal cells were cultured on sterile glass coverslips and fixed with 4% paraformaldehyde. Nonspecific binding sites were blocked with 0.1% bovine serum albumin in PBS, and the fixed cells were then incubated with anti-LC3 rabbit polyclonal antibodies (1:200), antiphosphorylated AKT mouse polyclonal antibodies (1:100), or anti-phosphorylated ERK1/2 mouse polyclonal antibodies (1:100) in PBS. After this, cells were incubated with Alexa Fluor 488-conjugated and 568-conjugated secondary antibodies (1:5,000; Vector Laboratories). The slides were mounted in mounting medium (Vector Laboratories), and nuclei were counterstained with 40 ,6-diamidino-2phenylindole. Images were captured with a confocal microscope (Bio-Rad Laboratories).
Transmission Electron Microscopy To allow observation autophagosomes on the ultrastructural level, endometriotic stromal cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 45 minutes at 4 C, rinsed in cacodylate buffer, postfixed in 1% OsO4 in cacodylate buffer, dehydrated, and embedded in Eponate. Ultra-thin sections were briefly contrasted with uranyl acetate and images were then captured with a transmission electron microscope (Hitachi 7100; Hitachi High Technologies).
Detection and Quantification of Acidic Vesicular Organelles with Acridine Orange Autophagy, a cellular process in which cytoplasmic proteins are sequestered in proteolytic compartments, is characterized by the formation of numerous acidic vesicles that are referred to as acidic vesicular organelles (34, 35). Acidic vesicular organelles were detected and quantified in luteal cells by vital staining with acridine orange. In an acridine orangestained cell, the cytoplasm and nucleolus fluoresce bright green and dim red, respectively, whereas acidic compartments fluoresce bright red (36, 37). Moreover, the red fluorescence intensity is proportional to the acidity and/or volume of the cellular compartment. This staining enables the measurement of changes in acidity and/or the fractional volume of acidic cellular compartments. 3
ORIGINAL ARTICLE: ENDOMETRIOSIS Briefly, luteal cells were stained with acridine orange (1 mg/mL final concentration) for 15 minutes and then harvested by treatment with trypsin-EDTA. Cells were collected in phenol red-free growth medium. The levels of green (510–530 nm) and red (650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light were measured with a FACSAria flow cytometer (BD Bioscience). Data analysis was conducted using CellQuest software (BD Bioscience).
Assessment of Human Endometriotic Stromal Cell Apoptosis Apoptotic cell percentages were determined with an annexinV/fluorescein isothiocyanate apoptosis detection kit (BD Bioscience) according to the manufacturer's protocol. Briefly, after drug treatment, 1 105 cells were pelleted and washed once with PBS. The cells were then resuspended in 100 mL binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM potassium chloride, 1 mM MgCl2, and 2 mM calcium chloride). Subsequently, 5 mL annexin-V and propidium iodide were added, and the cells were incubated for 15 minutes at room temperature in the dark. Next, 400 mL binding buffer was added, and cells were analyzed on a FACSAria flow cytometer (BD Bioscience). At least 10,000 cells were analyzed per treatment. The data were analyzed using CellQuest software (BD Bioscience).
Statistical Analysis Relative protein expression levels and the proportions of autophagic and apoptotic cells are reported as mean standard error (SE). Statistical analysis was performed using analysis of variance (ANOVA). Statistically significant differences between the treatment groups were identified using Duncan's multiplerange test. P< .05 was considered statistically significant.
RESULTS Dienogest Enhances Autophagy Induction and Inhibits AKT, ERK1/2, and mTOR Signaling in Endometriotic Stromal Cells To investigate whether progesterone and/or dienogest induce autophagy in ECSCs cultured with estrogen, the levels of LC3II and p62 were investigated via Western blotting. As shown in Figure 1A and B, the addition of progesterone had no effect on the expression level of LC3-II or p62 in ECSCs cultured with estrogen. In contrast, the expression levels of these proteins were statistically significantly increased by the addition of dienogest. To further characterize autophagic activity in ECSCs, immunofluorescence staining was performed to examine the subcellular localization of endogenous LC3. In ECSCs cultured with estrogen alone, only a few punctate LC3-II structures were detected throughout the cytoplasm (see Fig. 1C, left). Although the number of punctate LC3-II structures did not increase with the addition of progesterone (see Fig. 1C, middle), these structures did accumulate upon the addition of dienogest (see Fig. 1C, right). To confirm these observations on the ultrastructural level, autophagosome formation was analyzed by transmission elec4
tron microscopy. Autophagic structures are characterized by multiple autophagosomes, which are double-membraned vacuoles containing engulfed cytoplasmic material. In ECSCs cultured with estrogen, autophagic vacuoles indicative of autophagy induction accumulated after the addition of dienogest. In contrast, these vacuoles did not accumulate after the addition of progesterone (see Fig. 1D). Acridine orange-based flow cytometry assays yielded similar results; specifically, progestintreated ECSCs exhibited a higher proportion of autophagic cells (24.3-fold higher) compared with ECSCs cultured with estrogen alone (see Fig. 1E and F). However, the proportion of autophagic cells in ECSCs cultured with estrogen alone did not increase after the addition of progesterone. Subsequently, the phosphorylation levels of AKT, ERK1/ 2, and S6K in ECSCs cultured with estrogen were assessed after the addition of progesterone or dienogest to determine whether the PI3K-AKT pathway, the MEK1/2-ERK1/2 pathway, and/or mTOR regulate dienogest-mediated autophagy in endometriotic stromal cells. In ECSCs cultured with estrogen, progesterone treatment did not affect the phosphorylation of AKT, ERK1/2, or S6K. In contrast, each of these signaling molecules exhibited significantly decreased phosphorylation upon treatment with dienogest (see Fig. 1G and H).
Dienogest Induces Endometriotic Stromal Cell Autophagy by Inhibiting AKT, Leading to Inactivation of mTOR To determine whether inhibition of AKT activity affects mTOR activity and autophagy induction in ECSCs, we examined the effects of inhibiting AKT on the phosphorylation of S6K and the expression level of LC3-II. As shown in Figure 2A and B, dienogest treatment statistically significantly decreased AKT and S6K phosphorylation (P< .05). In contrast, LC3-II expression statistically significantly increased after dienogest treatment (P< .05). Similarly, treatment with an AKT inhibitor also statistically significantly decreased AKT and S6K phosphorylation, which was accompanied by increased LC3-II expression (P< .05). To confirm these findings, we examined the subcellular localizations of endogenous phosphorylated AKT and endogenous LC3 using immunofluorescence staining. Phosphorylated AKT and LC3 were readily detected via red and green fluorescence, respectively, in cultured ECSCs (see Fig. 2C). In ECSCs cultured with estrogen, the addition of dienogest or an AKT inhibitor decreased phosphorylated AKT staining (see Fig. 2C, p-AKT, middle). Moreover, punctate LC3-II structures were observed to accumulate in the cytoplasm (see Fig. 2C, LC3, middle).
Dienogest-mediated Autophagy is Associated with mTOR-mediated Down-regulation of ERK Activity To determine whether reduced ERK1/2 activity inhibits mTOR, leading to the induction of autophagy in dienogest-treated ECSCs, we next examined the effects of inhibiting ERK1/2 on the expression levels of phosphorylated S6K and LC3-II. VOL. - NO. - / - 2015
Fertility and Sterility®
FIGURE 1
Effects of progesterone and dienogest on autophagy induction and the activation (phosphorylation) of protein kinase B (AKT), extracellular-signalregulated kinase 1/2 (ERK), and S6K in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblots of microtubule-associated protein light chain 3 (LC3) and p62 in ECSCs. (B) Densitometric quantification of the levels of LC3-II and p62 in ECSCs. Experiments were repeated four times, and data are expressed as mean standard error (SE). *Statistically significant differences (P
Dienogest enhances autophagy induction in endometriotic cells by impairing activation of AKT, ERK1/2, and mTOR JongYeob Choi, Ph.D.,a MinWha Jo, M.S.,b EunYoung Lee, M.S.,a Dong-Yun Lee, M.D., Ph.D.,a and DooSeok Choi, M.D., Ph.D.a a
Infertility Clinic, Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine; and b Center for Clinical Research, Samsung Biomedical Research Institute, Seoul, South Korea
Objective: To elucidate the therapeutic mechanisms of progestin and the effects of progesterone and progestin (dienogest) on autophagy induction and regulation in endometriotic cells, specifically the effects of progesterone and dienogest on the phosphoinositide-3/protein kinase B (PI3K-AKT) and mitogen-activated protein kinase kinases 1 and 2 (MEK1/2)/extracellularsignal-regulated kinase 1/2 (ERK1/2) pathways, which activate mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Design: In vitro study using human endometriotic cyst stromal cells (ECSCs). Setting: University medical center. Patient(s): Fifteen patients with ovarian endometrioma. Intervention(s): ECSCs treated with progesterone or dienogest. Main Outcome Measure(s): Autophagy as measured by the expression of the microtubule-associated protein light chain 3-II (LC3-II) and autophagosome formation, and levels of AKT, ERK1/2, and mTOR activity to quantify the phosphorylation of AKT, ERK1/2, and S6K (the downstream target of mTOR). Result(s): Progesterone treatment had not statistically significant effect on LC3-II expression, autophagosome formation, or phosphorylation of AKT, ERK1/2, or S6K in estrogen-treated ECSCs. However, dienogest treatment up-regulated LC3-II expression and stimulated autophagosome formation. These effects were accompanied by decreased activation of AKT, ERK1/2, and S6K. Furthermore, incubation of ECSCs with AKT and ERK1/2 inhibitors, which mimicked dienogest-mediated inhibition of AKT and ERK1/2 activity, suppressed S6K activity, followed by an increase in LC3-II expression. In addition, cotreatment with dienogest and 3-methyladenine (autophagy inhibitor) decreased the levels of apoptosis of ECSCs compared with the single treatment with dienogest. Conclusion(s): Our results suggest that dienogest treatment of endometriotic cells suppresses AKT and ERK1/2 activity, thereby in turn inhibiting mTOR, inducing autophagy, and promotUse your smartphone ing apoptosis. (Fertil SterilÒ 2015;-:-–-. Ó2015 by American Society for Reproductive to scan this QR code Medicine.) and connect to the Key Words: AKT, autophagy, dienogest, endometriosis, ERK1/2 Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/choij-dienogest-enhances-autophagy-induction/
E
ndometriosis is defined by the presence of endometrium-like tissue outside the uterine cavity,
primarily on the ovaries and pelvic peritoneum. Endometriosis is one of the most common causes of chronic pelvic
Received March 17, 2015; revised and accepted May 14, 2015. J.Y.C. has nothing to disclose. M.W.J. has nothing to disclose. E.Y.L. has nothing to disclose. D.-Y.L. has nothing to disclose. D.S.C. has nothing to disclose. Supported by a grant from the Samsung Biomedical Research Institute. Reprint requests: DooSeok Choi, M.D., Ph.D., Department of Obstetrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.05.020 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
pain, dysmenorrhea, and infertility (1, 2) and affects about 5% to 15% of all women of reproductive age and 20% to 50% of all infertile women (3, 4). The typical characteristics of endometriosis are increased production of estradiol, which stimulates the proliferation of endometriotic tissue, and perturbations in the progesterone response in a phenomenon known as progesterone resistance (5–7). Therefore, current medical therapies focus either on 1
ORIGINAL ARTICLE: ENDOMETRIOSIS lowering estradiol levels or stimulating the progesterone response (2, 8). The most effective medical treatment options for endometriosis are gonadotropin-releasing hormone (GnRH) analogues. However, although GnRH agonists provide effective pain relief and suppress the progression of endometriotic implants by inducing hypoestrogenism (9), these treatments are not suitable for long-term applications because of their severe hypoestrogenic effects. On the other hand, progestins, which are synthetic versions of progesterone, reduce serum estrogen levels by preventing ovulation without causing hypoestrogenism (10). Progestins also exert progestogenic effects on estrogen-primed endometrium (11). Therefore, progestin therapy is a practical option for long-term treatment of endometriosis because it is associated with fewer adverse effects. However, the exact mechanism(s) by which progestin acts on endometriotic cells have yet to be elucidated. Apoptosis is a form of programmed cell death. Accumulating evidence indicates that reduced apoptosis in refluxed endometrial cells might enhance their survival at ectopic sites, thereby promoting the establishment of endometriosis (12, 13). However, apoptosis may not be the only mechanism of endometrial cell death. Autophagy, a nonapoptotic form of programmed cell death, is an intracellular bulk degradation system in which a portion of the cytoplasm is enveloped in double-membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation (14, 15). It is also known to play an important role in promoting cell death by promoting caspasedependent apoptosis in some normal (16, 17) and cancer cells (18–20). We recently showed that the induction of autophagy exerts a proapoptotic effect on normal human endometrial cells (21). However, autophagy is suppressed in endometriotic cells due to derepression of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy, which results in decreased endometriotic cell apoptosis (22). These findings implicate a direct role for mTORmediated suppression of autophagy in the pathogenesis of endometriosis. Previous studies have shown that the class I phosphoinositide-3 kinase (PI3K)/protein kinase B (AKT) and the mitogen-activated protein kinase 1/2 (MEK1/2)/ extracellular signal-regulated kinase 1/2 (ERK1/2) pathways negatively regulate autophagy induction by activating mTOR (23, 24), suggesting that inhibition of AKT and ERK1/ 2 could promote autophagy by preventing mTOR activation. In addition, progestin has been demonstrated to inhibit the PI3K-AKT (25) and MEK1/2-ERK1/2 (26) pathways in endometrial and breast cancer cells, respectively. Therefore, we hypothesized that progestin enhances autophagy induction by inhibiting AKT and ERK1/2, resulting in mTOR inactivation. However, it is not yet known whether progestin regulates AKT and ERK1/2 activity in endometriotic cells, and it is also unknown whether these pathways influence autophagy in endometriotic cells via mTOR signaling. In this study we evaluated the effects of progestin (dienogest) on autophagy in endometriotic cells. Specifically, we determined whether the PI3K-AKT pathway and the MEK1/2-ERK1/2 pathway 2
are involved in progestin-mediated autophagy induction via mTOR signaling.
MATERIALS AND METHODS Human Endometriotic Cyst Stromal Cell Isolation Ectopic endometriotic tissue samples (n ¼ 15) were obtained from proliferating ovarian endometriotic cysts (endometrioma). No study participant had taken oral contraceptives or hormone agents for at least 3 months before surgery. The average age of the participants from whom ectopic endometrial tissue was obtained was 29.5 5.4 years. Proliferating endometriotic cyst stromal cells (ECSCs) were dissociated and purified from ovarian endometriotic tissue essentially as described elsewhere (27) but with minor modifications. Briefly, tissue samples were minced with a sterile surgical blade and digested in phosphate-buffered saline (PBS) containing 2 mg/mL of type IV collagenase (Sigma-Aldrich) at 37 C for 60 minutes with agitation. Stromal cells were separated from the epithelial glands by use of 70-mm pore filters followed by 45 mm-pore nylon mesh. Filtered cells were plated in T75 flasks and allowed to adhere for approximately 30 minutes. The flasks were then washed with PBS to remove blood cells and debris. The stromal cells were cultured in Dulbecco's modified Eagle's/F12 medium (DMEM/F12; GIBCO BRL) supplemented with 10% charcoal-stripped fetal bovine serum (FBS; GIBCOBRL), 100 U/mL penicillin, and 100 mg/mL streptomycin (GIBCO BRL). The cells were maintained in a humidified atmosphere with 5% CO2 at 37 C, and the medium was changed every other day. Upon reaching confluence, the cells were subcultured in 24-well plates (1 mL/well). Endometrial stromal cell suspension purity was determined by immunostaining with vimentin stromal cell-specific antibodies. This study was approved by the ethics committee of Samsung Medical Center, and written informed consent was obtained from all participants.
In Vitro Experiments Subcultured ECSCs were seeded at 1 106 cells/mL in polyL-lysine-coated nonfluorescent thin-bottom glass culture dishes (MatTek). The cells were incubated at 37 C in 5% CO2 in DMEM/F12 supplemented with 10% charcoalstripped FBS, glutamine, N-2-hydroxyethylpiperazine-N0 2-ethanesulfonic acid (HEPES), 100 U/mL penicillin, and 100 mg/mL streptomycin. Upon reaching 70% to 80% confluence, the cultures were serum starved in serum-free Earle's balanced salt solution (EBSS) medium (SigmaAldrich). To evaluate the effects of progesterone and progestin on autophagy in endometriotic cells, the cells were cultured in EBSS medium before the hormone treatment. After 24 hours of culture, estradiol (10 nM; Sigma-Aldrich) alone, estradiol (10 nM) þ progesterone (1 mM; Sigma-Aldrich), or estradiol (10 nM) þ dienogest (10 mM; Abcam) were added for 72 hours. In this experiment, we added same amount of dimethyl sulfoxide (DMSO; Sigma-Aldrich) in the culture media of estrogen and/or progesterone-treated groups to minimize VOL. - NO. - / - 2015
Fertility and Sterility® experimental error by different solvents because the estrogen and progesterone were dissolved in water whereas dienogest was dissolved in DMSO. For the pharmacologic inhibition experiments, ECSCs were cultured in EBSS medium supplemented with estradiol (10 nM) for 72 hours. At 1 hour before analysis, an AKT inhibitor (5 mM AKT inhibitor VIII; Sigma-Aldrich) or an ERK1/2 inhibitor (30 mM U0126; Sigma-Aldrich) was added in the culture medium to inhibit AKT or ERK activity, respectively. For the autophagy experiments, ECSCs were cultured in EBSS supplemented with dienogest (10 mM) for 72 hours. Next, 3-methyladenine (10 mM 3-MA; Sigma-Aldrich) was added to the culture medium 6 hours before analysis to inhibit the induction of autophagy. The treatments were stopped by removing the medium, and ECSCs were either harvested by scraping to generate protein extracts or fixed for subsequent immunofluorescence and electron microscopy analyses. Autophagy and apoptosis of ECSCs were evaluated using acridine orange and annexinV/propidium iodide staining, respectively.
Western Blot Analysis During the induction of autophagy, microtubule-associated protein light chain 3 (LC3) is converted from LC3-I to LC3II, and LC3-II then localizes to isolated membranes and autophagosomes (28, 29). Because the expression of LC3-II has been shown to correlate with autophagosome number (30), we measured the levels of LC3-II by Western blot analysis as a readout for endometrial cell autophagy. The amount of autophagy is also reflected by the expression of p62, a selective target that undergoes autophagic degradation (31). Both AKT and ERK1/2 activities were determined by quantifying the phosphorylated (i.e., active) forms of AKT and ERK1/2, respectively. The activity of the mTOR pathway was also determined by measuring the phosphorylation of ribosomal protein S6 kinase (S6K). Because S6K is a direct substrate of mTOR, the phosphorylation status of S6K can be used as an indicator of the activity of the mTOR pathway (32). Endometrial cell apoptosis was determined by measuring the levels of cleaved caspase-3, which is the final mediator of apoptosis (33). Cultured ECSCs were lysed with ice-cold radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich). For complete solubilization of cellular proteins, lysates were incubated on ice for 30 minutes and then clarified by centrifugation at 13,000 g at 4 C for 10 minutes. Whole-cell lysates (20 mg/lane) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). After blocking with 5% skim milk, the membranes were incubated with rabbit polyclonal antibodies against LC3 (1:1,000; Cell Signaling Technology), cleaved caspase-3 (1:1,000; Cell Signaling Technology), total and phosphorylated AKT (Ser473) (1:1,000 and 1:1,000, respectively; Cell Signaling Technology), total and phosphorylated ERK1/2 (Thr202/ Tyr204) (1:1,000 each; Cell Signaling Technology) or total and phosphorylated S6K (Ser235/236) (1:1,000 each; Cell Signaling Technology) overnight at 4 C. The membranes VOL. - NO. - / - 2015
were washed three times for 15 minutes in wash buffer (PBS containing 0.1% Tween 20) and were incubated with the appropriate secondary antibodies (1:5,000; Santa Cruz Biotechnology) at room temperature for 1 hour. After three 15-minute washes in wash buffer at room temperature, immunoreactive bands were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech). The bands were quantified using the National Institutes of Health ImageJ software (NIH Image Processing and Analysis in Java). The expression levels of LC3-II and cleaved caspase-3 were normalized to b-actin, and the expression levels of phosphorylated AKT, ERK1/2, and S6K were normalized to those of total AKT, ERK1/2, and S6K, respectively.
Immunofluorescence Endometriotic stromal cells were cultured on sterile glass coverslips and fixed with 4% paraformaldehyde. Nonspecific binding sites were blocked with 0.1% bovine serum albumin in PBS, and the fixed cells were then incubated with anti-LC3 rabbit polyclonal antibodies (1:200), antiphosphorylated AKT mouse polyclonal antibodies (1:100), or anti-phosphorylated ERK1/2 mouse polyclonal antibodies (1:100) in PBS. After this, cells were incubated with Alexa Fluor 488-conjugated and 568-conjugated secondary antibodies (1:5,000; Vector Laboratories). The slides were mounted in mounting medium (Vector Laboratories), and nuclei were counterstained with 40 ,6-diamidino-2phenylindole. Images were captured with a confocal microscope (Bio-Rad Laboratories).
Transmission Electron Microscopy To allow observation autophagosomes on the ultrastructural level, endometriotic stromal cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 45 minutes at 4 C, rinsed in cacodylate buffer, postfixed in 1% OsO4 in cacodylate buffer, dehydrated, and embedded in Eponate. Ultra-thin sections were briefly contrasted with uranyl acetate and images were then captured with a transmission electron microscope (Hitachi 7100; Hitachi High Technologies).
Detection and Quantification of Acidic Vesicular Organelles with Acridine Orange Autophagy, a cellular process in which cytoplasmic proteins are sequestered in proteolytic compartments, is characterized by the formation of numerous acidic vesicles that are referred to as acidic vesicular organelles (34, 35). Acidic vesicular organelles were detected and quantified in luteal cells by vital staining with acridine orange. In an acridine orangestained cell, the cytoplasm and nucleolus fluoresce bright green and dim red, respectively, whereas acidic compartments fluoresce bright red (36, 37). Moreover, the red fluorescence intensity is proportional to the acidity and/or volume of the cellular compartment. This staining enables the measurement of changes in acidity and/or the fractional volume of acidic cellular compartments. 3
ORIGINAL ARTICLE: ENDOMETRIOSIS Briefly, luteal cells were stained with acridine orange (1 mg/mL final concentration) for 15 minutes and then harvested by treatment with trypsin-EDTA. Cells were collected in phenol red-free growth medium. The levels of green (510–530 nm) and red (650 nm) fluorescence emission from 10,000 cells illuminated with blue (488 nm) excitation light were measured with a FACSAria flow cytometer (BD Bioscience). Data analysis was conducted using CellQuest software (BD Bioscience).
Assessment of Human Endometriotic Stromal Cell Apoptosis Apoptotic cell percentages were determined with an annexinV/fluorescein isothiocyanate apoptosis detection kit (BD Bioscience) according to the manufacturer's protocol. Briefly, after drug treatment, 1 105 cells were pelleted and washed once with PBS. The cells were then resuspended in 100 mL binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM potassium chloride, 1 mM MgCl2, and 2 mM calcium chloride). Subsequently, 5 mL annexin-V and propidium iodide were added, and the cells were incubated for 15 minutes at room temperature in the dark. Next, 400 mL binding buffer was added, and cells were analyzed on a FACSAria flow cytometer (BD Bioscience). At least 10,000 cells were analyzed per treatment. The data were analyzed using CellQuest software (BD Bioscience).
Statistical Analysis Relative protein expression levels and the proportions of autophagic and apoptotic cells are reported as mean standard error (SE). Statistical analysis was performed using analysis of variance (ANOVA). Statistically significant differences between the treatment groups were identified using Duncan's multiplerange test. P< .05 was considered statistically significant.
RESULTS Dienogest Enhances Autophagy Induction and Inhibits AKT, ERK1/2, and mTOR Signaling in Endometriotic Stromal Cells To investigate whether progesterone and/or dienogest induce autophagy in ECSCs cultured with estrogen, the levels of LC3II and p62 were investigated via Western blotting. As shown in Figure 1A and B, the addition of progesterone had no effect on the expression level of LC3-II or p62 in ECSCs cultured with estrogen. In contrast, the expression levels of these proteins were statistically significantly increased by the addition of dienogest. To further characterize autophagic activity in ECSCs, immunofluorescence staining was performed to examine the subcellular localization of endogenous LC3. In ECSCs cultured with estrogen alone, only a few punctate LC3-II structures were detected throughout the cytoplasm (see Fig. 1C, left). Although the number of punctate LC3-II structures did not increase with the addition of progesterone (see Fig. 1C, middle), these structures did accumulate upon the addition of dienogest (see Fig. 1C, right). To confirm these observations on the ultrastructural level, autophagosome formation was analyzed by transmission elec4
tron microscopy. Autophagic structures are characterized by multiple autophagosomes, which are double-membraned vacuoles containing engulfed cytoplasmic material. In ECSCs cultured with estrogen, autophagic vacuoles indicative of autophagy induction accumulated after the addition of dienogest. In contrast, these vacuoles did not accumulate after the addition of progesterone (see Fig. 1D). Acridine orange-based flow cytometry assays yielded similar results; specifically, progestintreated ECSCs exhibited a higher proportion of autophagic cells (24.3-fold higher) compared with ECSCs cultured with estrogen alone (see Fig. 1E and F). However, the proportion of autophagic cells in ECSCs cultured with estrogen alone did not increase after the addition of progesterone. Subsequently, the phosphorylation levels of AKT, ERK1/ 2, and S6K in ECSCs cultured with estrogen were assessed after the addition of progesterone or dienogest to determine whether the PI3K-AKT pathway, the MEK1/2-ERK1/2 pathway, and/or mTOR regulate dienogest-mediated autophagy in endometriotic stromal cells. In ECSCs cultured with estrogen, progesterone treatment did not affect the phosphorylation of AKT, ERK1/2, or S6K. In contrast, each of these signaling molecules exhibited significantly decreased phosphorylation upon treatment with dienogest (see Fig. 1G and H).
Dienogest Induces Endometriotic Stromal Cell Autophagy by Inhibiting AKT, Leading to Inactivation of mTOR To determine whether inhibition of AKT activity affects mTOR activity and autophagy induction in ECSCs, we examined the effects of inhibiting AKT on the phosphorylation of S6K and the expression level of LC3-II. As shown in Figure 2A and B, dienogest treatment statistically significantly decreased AKT and S6K phosphorylation (P< .05). In contrast, LC3-II expression statistically significantly increased after dienogest treatment (P< .05). Similarly, treatment with an AKT inhibitor also statistically significantly decreased AKT and S6K phosphorylation, which was accompanied by increased LC3-II expression (P< .05). To confirm these findings, we examined the subcellular localizations of endogenous phosphorylated AKT and endogenous LC3 using immunofluorescence staining. Phosphorylated AKT and LC3 were readily detected via red and green fluorescence, respectively, in cultured ECSCs (see Fig. 2C). In ECSCs cultured with estrogen, the addition of dienogest or an AKT inhibitor decreased phosphorylated AKT staining (see Fig. 2C, p-AKT, middle). Moreover, punctate LC3-II structures were observed to accumulate in the cytoplasm (see Fig. 2C, LC3, middle).
Dienogest-mediated Autophagy is Associated with mTOR-mediated Down-regulation of ERK Activity To determine whether reduced ERK1/2 activity inhibits mTOR, leading to the induction of autophagy in dienogest-treated ECSCs, we next examined the effects of inhibiting ERK1/2 on the expression levels of phosphorylated S6K and LC3-II. VOL. - NO. - / - 2015
Fertility and Sterility®
FIGURE 1
Effects of progesterone and dienogest on autophagy induction and the activation (phosphorylation) of protein kinase B (AKT), extracellular-signalregulated kinase 1/2 (ERK), and S6K in endometriotic cyst stromal cells (ECSCs). (A) Representative immunoblots of microtubule-associated protein light chain 3 (LC3) and p62 in ECSCs. (B) Densitometric quantification of the levels of LC3-II and p62 in ECSCs. Experiments were repeated four times, and data are expressed as mean standard error (SE). *Statistically significant differences (P
