Molecular and Cellular Endocrinology 407 (2015) 37–45

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

The mechanism and significance of synergistic induction of the expression of plasminogen activator inhibitor-1 by glucocorticoid and transforming growth factor beta in human ovarian cancer cells Xiao-yu Pan a,1, Yan Wang b,1, Jie Su b, Gao-xiang Huang b, Dong-mei Cao b, Shen Qu a,2, Jian Lu b,2,* a b

Department of Endocrinology, Shanghai 10th People’s Hospital, Tongji University, Shanghai 200072, China Department of Pathophysiology, The Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China

A R T I C L E

I N F O

Article history: Received 5 December 2014 Received in revised form 5 March 2015 Accepted 6 March 2015 Available online 11 March 2015 Keywords: Plasminogen activator inhibitor-1 Dexamethasone Transforming growth factor beta Phosphorylation Ovarian cancer

A B S T R A C T

Plasminogen activator inhibitor-1 (PAI-1) plays a key role in tissue remodeling and tumor development by suppression of plasminogen activator function. Glucocorticoids (GCs) and transforming growth factor beta (TGF-β) signal pathways cross-talk to regulate gene expression, but the mechanism is poorly understood. Here we investigated the mechanism and significance of co-regulation of PAI-1 by TGF-β and dexamethasone (DEX), a synthetic glucocorticoid in ovarian cancer cells. We found that TGF-β and DEX showed rapidly synergistic induction of PAI-1 expression, which contributed to the early pro-adhesion effects. The synergistic induction effect was accomplished by several signal pathways, including GC receptor (GR) pathway and TGF-β-activated p38MAPK, ERK and Smad3 pathways. TGF-β-activated p38MAPK and ERK pathways cross-talked with GR pathway to augment the expression of PAI-1 through enhancing DEX-induced GR phosphorylation at Ser211 in ovarian cancer cells. These findings reveal possible novel mechanisms by which TGF-β pathways cooperatively cross-talk with GR pathway to regulate gene expression. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Glucocorticoids (GCs) and transforming growth factor-β (TGFβ) play important roles in regulation of cell growth, differentiation, extracellular matrix (ECM) formation, immune function, inflammation and tumor metastasis (Baschant and Tuckermann, 2010; Derynck et al., 2001; Gordon and Blobe, 2008; Phuc Le et al., 2005; Sheen et al., 2013). Synthetic GCs are widely prescribed therapeutics for the treatment of numerous inflammatory disorders and cancers. Increasing evidences have indicated that TGF-β and GCs signaling pathways cross-talk positively and negatively in regulating

Abbreviations: PAI-1, plasminogen activator inhibitor-1; TGF-β, transforming growth factor beta; GC, glucocorticoid; DEX, dexamethasone; GR, glucocorticoid receptor; GRE, glucocorticoid response element; ECM, extracellular matrix; pGRS211, phosphorylation of GR at Ser211; MAPK, mitogen-activated protein kinase; ERK, extra-cellular-signal regulated kinase; OSE, ovarian surface epithelium; siRNA, small interfere RNA; Smad3, mothers against decapentaplegic homolog 3; GAPDH, glyceraldehydes-3-phosphate dehydrogenase. * Corresponding author. Department of Pathophysiology, the Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China. Tel.: +86 021 81871020; fax: +86 021 81871018. E-mail address: [email protected] (J. Lu). 1 X-Y Pan and Y Wang contributed equally to this work. 2 Jian Lu and Shen Qu are co-corresponding authors. http://dx.doi.org/10.1016/j.mce.2015.03.005 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.

a variety of physiologic and pathologic processes through regulation of gene expression, although the molecular mechanisms involved remain to be established (Beck et al., 1993; Derynck et al., 2001; Kanatani et al., 1996; Kassel and Herrlich, 2007; Periyasamy and Sanchez, 2002; Pierce et al., 1989; Takuma et al., 2003). The effect of GCs is mediated by the GC receptor (GR). As a transcription factor, liganded/activated GR regulates gene expression through either a direct interaction with GC response element (GRE) in the promoter region of target genes or through interference of other transcription factors to inhibit their transcriptional activity (Baschant and Tuckermann, 2010; Kassel and Herrlich, 2007; Phuc Le et al., 2005). The signaling and transcriptional activity of GR can be modulated by various post-transcriptional modifications including phosphorylation (Anbalagan et al., 2012; Davies et al., 2008; Housley and Pratt, 1983). GR is subject to hormone-dependent and -independent phosphorylation on several serines (such as S113, S141, S203, S211, S226, and S404) in its N terminus. The role of phosphorylation in regulating the transcriptional function of GR varies with different phospho-related sites within GR, different target genes and cell types (Beck et al., 2009; Galliher-Beckley and Cidlowski, 2009; Housley and Pratt, 1983). The phosphorylations of GR are regulated by protein kinases and phosphatases. Several cell-specific kinases such as cyclin-dependent kinases (CDKs), glycogen synthase kinase 3β (GSK3β), casein kinase II and mitogen-activated protein kinases

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(MAPKs) have been reported to phosphorylate GR (Beck et al., 2009; Bouazza et al., 2012; Galliher-Beckley and Cidlowski, 2009; Kino et al., 2007; Krstic et al., 1997; Miller et al., 2005, 2007; Wang et al., 2007). In the classical TGF-β pathway, TGF-β binding induces activation of its two types of trans-membrane serine/threonine kinase receptors which then phosphorylate and activate cytoplasmic proteins called Smads (Smad2/3), which form heteromeric Smad complexes with Smad4, and subsequently translocate to the nucleus. Once in the nucleus, the Smad complexes control gene transcription directly or in cooperation with other transcription factors (Gordon and Blobe, 2008; Sheen et al., 2013). TGF-β also activates non-Smad pathways, including PI3K-AKT, Rho-ROCK and members of the MAPK family, such as p38 MAPK, ERK and JNK (Derynck and Zhang, 2003; Gordon and Blobe, 2008; Massague, 2008; Sheen et al., 2013; Zhang, 2009). TGF-β activated non-Smad pathways can interact with Smad pathway to regulate gene expression (Derynck and Zhang, 2003; Dziembowska et al., 2007; Ohshima and Shimotohno, 2003; Vasilaki et al., 2010). TGF-β and GC modulate each other’s activities (Almawi and Irani-Hakime, 1998; AyanlarBatuman et al., 1991; Chen et al., 2010; Li et al., 2006; Oursler et al., 1993; Peltier et al., 2003). For example TGF-β increases GC binding and signaling in macrophages (Peltier et al., 2003). Our previous work demonstrated that GC up-regulates the expression of type II TGF-β receptor (TβRII) and enhances TGF-β signaling in prostate cancer PC3 cells (Li et al., 2006) and ovarian cancer HO-8910 cells (Chen et al., 2010). However, whether TGF-β cross-talk with GR signaling pathways through altering GR phosphorylation to regulate gene expression has not been investigated before. Plasminogen activator inhibitor-1(PAI-1) plays a key role in hemostatic balance, tissue remodeling, tumor invasion, angiogenesis and reproduction by virtue of suppression of plasminogen activator function (Durand et al., 2004; Ghosh and Vaughan, 2012; Rerolle et al., 2000; Zorio et al., 2008). PAI-1 is a target gene of TGF-β, and PAI-1 promoter contains binding elements of the Smads (Smad3 and Smad4) (Dennler et al., 1998; Lund et al., 1987). GC can also upregulate the expression of PAI-1 (Bruzdzinski et al., 1993; Halleux et al., 1999). GC can cross-talk with TGF-β to regulate the expression of PAI-1 in different kinds of cells (Hamilton et al., 1993; Kimura et al., 2011; Ma et al., 2002; Song et al., 1999; Wickert et al., 2007). For example, previous studies demonstrated that liganded GR represses TGF-β/Smad3 transactivation of the PAI-1 gene in Hep3B human hepatoma cells by directly targeting the transcriptional activation function of Smad3 (Song et al., 1999). Conversely, GC and TGF-β were found to co-induce PAI-1 expression in human trophoblast cell (Ma et al., 2002), human monocytes (Hamilton et al., 1993) and human proximal tubular epithelial cells (Kimura et al., 2011). However, the mechanism by which TGF-β and GC/GR pathways interact to co-induce the expression of PAI-1 is unclear. Epithelial ovarian cancer is the leading cause of cancer-related death in women diagnosed with gynecologic malignancies. At least 85% of ovarian cancers come from the human ovarian surface epithelium (OSE). OSE can not only secrete TGF-β1 (Peng, 2003) but also express 11β-hydroxysteroid dehydrogenase-1 (HSD1) which converts cortisone to the receptor active cortisol (Rae et al., 2004). Ovulation-associated inflammation factors, such as IL-1, could stimulate activity of HSD1, thereby producing GCs locally besides circulating GCs (Rae et al., 2004). PAI-1 is expressed in ovarian tissue, and plays an important role in ovulation and ovulation-associated wound healing (Liu, 1988, 2004; Ny et al., 1985). Moreover, high PAI-1 expression levels have been found to be associated with malignancy and metastasis of epithelial ovarian cancer, therefore PAI-1 is considered as an independent factor for overall survival and a strong predictor of metastasis in ovarian cancer (Cai et al., 2007; Koensgen et al., 2006). Our previous studies found that DEX and TGF-β1 cooperatively promote cell adhesion to extracellular matrix (ECM) in ovarian cancer

cells, which enhances resistance of cells to chemotherapeutics (Chen et al., 2010). Since PAI-1 is an endogenous target gene of both TGFβ1 and GC, and plays important roles in ECM formation and cell adherence by inhibiting the plasmin-mediated ECM degradation system (Durand et al., 2004; Ghosh and Vaughan, 2012; Zorio et al., 2008), it is interesting to explore whether TGF-β signaling pathways cross-talk with GR to regulate the expression of PAI-1, thereby contributing the synergistic pro-adhesion effect of DEX and TGFβ1 in ovarian cancer cells. In this study, we found that there is a rapid synergistic induction effect of GC and TGF-β1 on the expression of PAI-1. Then we further investigated its mechanism and significance in ovarian cancer cells. 2. Materials and methods 2.1. Cell culture Human ovarian cancer cell line HO-8910 was established and kindly provided by Dr. Xu Shenhua (Zhejiang Cancer Research Institute, Zhejiang Cancer Hospital, China) at passage number 26 (Mou et al., 1994). SKOV3 cell line was obtained from National Infrastruture of Cell Line Resourse (Beijing, China) at passage number 30. The two cell lines were cultured in RPMI 1640 medium (Gibco, USA), supplemented with 10% newborn bovine serum (NBS; PAA Laboratories, Canada) at 37 °C under a humidified atmosphere of air containing 5% CO2. Cells (less than 60 passages) were grown to ~70% confluence, and rinsed thrice with PBS, then cultured in medium containing 5% Dextran-coated charcoal (DCC) treated NBS to avoid possible interference by serum steroids, and the cells were incubated with 100 nM DEX (Sigma, Aldrich Saint Louis, Missouri, USA) or 10 ng/ ml recombinant human TGF-β1 (PeproTech Inc., Rocky Hill, USA) or both of them for different periods of time. Control cells were incubated with ethanol (1‰). P38MAPK inhibitor SB203580 (10 μM), ERK inhibitor PD98059 (20 μM), and RU486 (1 μM) (Sigma-Aldrich, Saint Louis, Missouri, USA) were added 1 h before the administration of DEX and/or TGFβ1. 2.2. RNA extraction and quantitative real-time RT-PCR analysis Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 2 μg of total RNA was reverse transcribed using Reverse Transcription Reagents (Fermentas, Lithuania, USA) in accordance with the manufacturer’s instructions. The mRNA levels of the indicated genes were analyzed in triplicate using SYBR Green PCR Master Mix (Toyobo, Japan) on Mastercycler ep realplex (Eppendorf, Germany). The primer sequences used in the PCR reactions were: 5′GCCTCCAAAGACCGAAATGTG-3′ (forward) and 5′-GTCGTTGATGATG AATCTGGCTC-3′ (reverse) for human PAI-1, and 5′-TAGCCCA GGATGCCCTTTAGT-3′ (forward), 5′-CCCCCAATGTATCCGTTGTG-3′ (reverse) for human GAPDH. Thermal cycling conditions consisted of an initial denaturing step (95 °C, 2 min) followed by 40 cycles of denaturing (95 °C, 20 s), annealing (60 °C, 20 s), and extending (72 °C, 45 s). The specified mode of reaction was controlled with the melting curve. The mRNA levels were normalized to GAPDH (internal control) using the formula ΔCT = CT target-CT reference. The differential expression signal was calculated as ΔΔCt = ΔCt (gene of DEX or/and TGFβ1 treated group) − ΔCt (gene of untreated group) and expressed as relative fold of change using the formula: 2ΔΔCT. 2.3. Western blot analysis Whole cells were prepared in SDS lysis buffer and protein extracts were equally loaded on SDS–polyacrylamide gel, and transferred to nitrocellulose membrane (Millipore, Ireland). The membranes were blocked in TBST (tris-buffered saline with Tween20) containing 5% nonfat milk and probed with specific anti-phospho

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GR(Ser211), anti-phospho-p38MAPK, anti-p38MAPK, anti-phosphoERK1/2 (T202/Y204), anti-ERK1/2 (1:1000 dilution, Cell Signaling Technology, USA), anti-GR (1:1000 dilution, Santa Cruz, USA) or antiPAI-1 (1:500 dilution, Santa Cruz, USA) overnight at 4 °C. The membranes were washed three times and incubated with horseradish perioxidase (HRP)-conjugated secondary antibodies (1:5000 dilution, Rockland Immunochemicals, USA). All blots were reprobed with anti-β-actin antibody (1:10,000 dilution, Sigma-Aldrich Chemicals, USA) to confirm equal loading among samples. Detection was visualized using enhanced chemiluminescence (ECL) assay kit (Thermo Scientific, USA) following the manufacturer’s recommended protocol. 2.4. SiRNA transfections The siRNA targeting human PAI-1 (PAI-1 siRNA 5′-AAGCAGCU AUGGGAUUCAAGA-3′) (Fang et al., 2012) and human Smad3 (Smad3 siRNA : 5′-CUGUGUGAGUUCGCCUUCA-3′) (Kobayashi et al., 2006) were manufactured by GenePharma Co., Ltd (Shanghai, China). Control siRNA (Con siRNA: 5′-UUCUCCGAACGUGUCACGU-3′) was designed and manufactured by GenePharma Co., Ltd. Ovarian cancer HO-8910 and SKOV3 cells at approximately 30–50% confluence were transfected with each siRNA (10 nM) using INTERFERinTM (Polyplus Transfection SA, Boulevard Sébastien Brant, ILLKIRCH Cedex, France) according to the manufacturer’s instruction for 36 h prior to further experiments. 2.5. Cell adhesion assay Cell adhesion ability was determined by cell adhesion assay (Chen et al., 2010). Cells were transfected with PAI-1 siRNA for 36 hours and then incubated in 5% DCC-treated media containing with or without 100 nM DEX and 10 ng/ml TGF-β1 for another 8 hours. Then cells were digested into single cell suspension and 8 × 104 cells were seeded into 96-well plates and incubated at 37 °C for 60 min. The plates were gently washed thrice with PBS to remove the unattached cells. The remaining cells in the 96-well plates were determined by MTT assay. 2.6. Statistical analysis Data are expressed as mean ± S.D. of at least three determinations. Statistical significance between experimental groups was analyzed by ANOVA. A value of P < 0.05 was considered to be statistically significant.

Fig. 1. Effects of DEX or/and TGF-β1 on the expression of PAI-1 in ovarian cancer cells. HO-8910 (A) and SKOV3 (B) cells were incubated with DEX (100 nM), TGF-β1 (10 ng/ml) or both for the indicated time periods, and PAI-1 mRNA was measured by real-time PCR. HO-8910 (C) and SKOV3 (D) cells were incubated with DEX (100 nM), TGF-β1 (10 ng/ml) or both for 6 hours, and PAI-1 protein expression was analyzed by western blot. GAPDH was used as a normalization control for real-time PCR and β-actin as a loading control for western blot. Data shown are representative of at least three separate experiments. *P < 0.05, **P < 0.01 vs. DEX treatment of the same time points; #P < 0.05, ##P < 0.01 vs. TGF-β1 treatment of the same time points.

3. Results 3.1. DEX and TGF-β1 synergistically up-regulate the expression of PAI-1 in ovarian cancer cell lines We first examined the expression of PAI-1 in HO-8910 cells treated with 100 nM DEX or/and 10 ng/ml TGF-β1 at different time (0–96 h) intervals. Real-time PCR analysis revealed that TGF-β1 upregulated the expression of PAI-1 mRNA in a time-dependent manner. Maximal stimulation by TGF-β1 was observed at 4 h after TGF-β1 treatment (approximately 3.7-fold relative to control). DEX alone also increased PAI-1 mRNA approximately 1.5-fold relative to control at all time points. Of note co-treatment with DEX and TGFβ1 showed a rapid and synergistic effect on the induction of PAI-1 with the maximum induction of PAI-1 mRNA approximately 12.7fold compared to control at 4 h. After that the synergistic effect gradually declined and disappeared at 24 h. Although the combined effect of DEX and TGF-β1 was observed again at late period (72 h), it was rather an additive effect than a synergistic effect (Fig. 1A). Similar rapid synergistic effect of DEX and TGF-β1 on induction of PAI-1 mRNA was also seen in other ovarian cancer SKOV3

cells after the combined stimulation for 2–4 h (Fig. 1B). Western blot analysis confirmed that PAI-1 protein levels were also markedly increased in HO-8910 (Fig. 1C) and SKOV3 (Fig. 1D) cells after co-treatment with DEX and TGF-β1 for 6 h compared with those after a single stimulation of DEX or TGF-β1. 3.2. DEX or/and TGF-β1 do not enhance PAI-1 mRNA stability in HO8910 cells The synergistic induction of the PAI-1 mRNA by DEX and TGFβ1 could be a result of an increase in PAI-1 gene transcription, or PAI-1 mRNA stability or both. To elucidate further the mechanism responsible for the increases of PAI-1 mRNA, we investigated the effects of DEX or/and TGF-β1 on PAI-1 mRNA stability. Changes in PAI-1 mRNA level were determined after treatment of 100 nM DEX or/and 10 ng/ml TGF-β1 for 4 h in the absence or presence of actinomycin D (Act D) (5 μg/ml), a transcription inhibitor. As shown in Fig. 2, Act D resulted in gradual decrease of PAI-1 mRNA. DEX or/and TGF-β1 could not delay the decay of PAI-1 mRNA from 1 to 7 hours. These results indicate that DEX or/and TGF-β1 cannot

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Fig. 3. GR activation but not expression affects the synergistic effect of PAI-1 mRNA induced by DEX and TGF-β1 in ovarian cancer cells. HO-8910 (A) and SKOV3 (B) cells were pretreated with or without GR antagonist RU486 for 1 hour, and then stimulated with DEX (100 nM), TGFβ1 (10 ng/ml) or both for 4 hour. The expression of PAI-1 mRNA was measured by real-time PCR. HO-8910 (C) and SKOV3 (D) cells were incubated with DEX (100 nM), or/and TGFβ1 (10 ng/ml) for the indicated time periods, and the protein level of GR was analyzed by western blot. GAPDH was used as a normalization control for real-time PCR and β-actin as a loading control for western blot. Data shown are representative of at least three separate experiments. *P < 0.05;**P < 0.01 for the treatment without vs. with RU486 in the presence of DEX, TGFβ1, or DEX + TGFβ1.

Fig. 2. Effects of DEX or/and TGF-β1 on PAI-1 mRNA stability in HO-8910 cells. HO-8910 cells were either pretreated with DEX (100 nM) (A), TGFβ1 (10 ng/ml) (B), or both (C) for 4 hours, then cells were treated with actinomycin D (5 μg/ml) and total RNA samples were collected at the indicated time points (0, 0.5, 1, 2, and 4 h) after treatment with actinomycin, and analyzed by real-time PCR. PAI-1 mRNA levels were normalized against GAPDH and plotted as a percentage of PAI-1 mRNA vs. time. Each time point represents the means ± SD of one experiment performed in triplicate. Control group (con) is pretreated with corresponding vehicle.

enhance the stability of PAI-1 mRNA and that the synergistically upregulated effect on the expression of PAI-1 in ovarian cancer cells occurs at transcriptional level. 3.3. Synergistic effect of DEX and TGF-β1 on induction of PAI-1 is not due to increase of GR expression Next we investigated the effect of RU486, an antagonist of GR on the up-regulation of PAI-1 by DEX or/and TGF-β1 in ovarian cancer cells. As shown in Fig. 3A. RU486 not only blocked the expression of PAI-1 mRNA induced by DEX alone, but also markedly decreased co-induced level of PAI-1 mRNA by both DEX and TGF-β1 to the level induced by TGF-β1 alone (from 11.9- to 4.1-fold) at 4 h in HO-8910 cells. Similar results were observed in SKOV3 cells (Fig. 3B). Since RU486 did not affect the TGF-β1induced expression of PAI-1, we supposed that the function of GR is enhanced in ovarian cancer cells during co-treatment of DEX and TGF-β1. In order to test whether the synergistic effect occurs via increasing the expression of GR in ovarian cancer cells, we then examined the effects of DEX or/and TGF-β1 on the expression of GR. The results showed that the level of GR proteins did not change after co-treatment of HO-8910 cells and SKOV3 cells with DEX and

TGF-β1 for 2–6 h compared to treatment of DEX or TGF-β1 alone (Fig. 3C and D). 3.4. TGF-β1 rapidly enhances DEX-dependent phosphorylation of GR at serine 211 Phosphorylation of GR at serine 211 (pGR-S211) is considered as a transcriptionally active form of GR and its level appears proportional to the activation status of GR (Beck et al., 2009; Galliher-Beckley and Cidlowski, 2009; Krstic et al., 1997; Miller et al., 2005). It has been reported that GCs induced phosphorylation of GR at serine 211 and enhanced sensitivity to GC treatment in human lymphoid cells by activation of p38MAPK (Miller et al., 2005, 2007). In order to know whether increased level of pGR-S211 contribute to the synergistic effect of DEX and TGF-β1 on the induction of PAI1, we first observed the effect of DEX alone on the phosphorylation of GR at serine 211 in HO-8910 cells. The result showed that 100 nM DEX alone rapidly increased the phosphorylation of GR at serine 211 at 15 min and this effect was sustained for at least 48 h (Fig. 4A). Next, we examined the effect of TGF-β1 alone or in combination with DEX on the level of pGR-S211 in ovarian cells. We noticed that in HO-8910 cells TGF-β1 alone had no influence on the level of pGR-S211, but it rapidly increased DEX-dependent phosphorylation of GR at serine 211. As shown in Fig. 4B, the levels of pGRS211 significantly increased when co-treated HO-8910 cells with DEX and TGF-β1 for 60 min and reached the peak at 90 min as compared with that in cells treated with DEX alone. An enhanced level of pGR-S211 was still seen in HO-8910 cells co-treated with DEX and TGF-β1 for 2 h, but not observed at 4 h (data not shown). Similar results were also found in SKOV3 cells (Fig. 4C), indicating TGF-β1 enhances DEX-dependent phosphorylation of GR at serine 211, which is a rapid and transient event.

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3.5. The TGF-β1-activated ERK is involved in the rapid increase of pGR-S211 and the synergistic induction of PAI-1 by DEX and TGF-β1 It is known that several serine/threonine kinases phosphorylate human GR at serine 211 and modulate transcriptional activity of GR, such as ERK and p38MAPK, which can be activated by TGF-β1. We therefore investigated whether TGF-β/ERK pathway was involved in the rapid synergistic induction effect of PAI-1 as well as the phosphorylation of GR at serine 211 by DEX and TGF-β1. We first examined the activation of ERK after TGF-β1 treatment by evaluating its phosphorylation level. As shown in Fig. 5A and B, TGF-β1 rapidly increased the phosphorylated ERK, and the effect only sustained for 2 h in ovarian cancer cells, indicating that TGFβ1 activates ERK rapidly and transiently. Inhibiting the ERK signaling pathway with specific inhibitor PD98059 (20 μM) not only repressed TGF-β1-enhanced the phosphorylation of GR at serine 211 (Fig. 5C and D), but also significantly reduced PAI-1 mRNA expression co-induced by TGF-β1 and DEX (Fig. 5E and F) in ovarian cancer cells. These results indicate that TGF-β1-activated ERK is involved in enhancing phosphorylation of GR at serine 211 and accounts in part for the rapid synergistic induction effect of PAI-1 by DEX and TGF-β1.

3.6. The TGF-β1-activated p38MAPK also contributes to the rapid increase of pGR-S211 and the synergistic induction of PAI-1 by DEX and TGF-β1 We further investigated whether p38MAPK is involved in TGFβ1-enhanced phosphorylation of GR at serine 211 as well as the rapid synergistic induction effect of PAI-1 by DEX and TGF-β1. The results showed that TGF-β1 rapidly activated p38 MAPK in HO-8910 (Fig. 6A) and SKOV3 (Fig. 6B) cells. Inhibiting p38 MAPK with specific inhibitor, SB203580 (10 nM) not only repressed TGF-β1-enhanced phosphorylation of GR at serine 211 (Fig. 6C and D), but also significantly reduced PAI-1 mRNA expression co-induced by TGF-β1 and DEX (from 9.9-fold to 5.6-fold in HO-8910 cells, P < 0.01, from 12.9-fold to 8.6-fold in SKOV3 cells, P < 0.05) (Fig. 6E and F). These results indicated that rapid activation of p38 MAPK by TGF-β1 is involved in enhancing DEX-dependent phosphorylation of GR at serine 211 and synergistic induction effect of PAI-1 by DEX and TGFβ1. Furthermore, inhibition of the activation of p38MAPK using SB203580 also significantly decreased PAI-1 mRNA level induced by TGF-β1 alone, indicating that activated p38MAPK pathway also partially contributes to TGF-β1-induced expression of PAI-1 in ovarian cancer cells.

3.7. TGF-β1/Smad3 pathway partially mediates the synergistic induction of PAI-1 by DEX and TGF-β1

Fig. 4. Effect of TGF-β1 on DEX-dependent GR phosphorylation at serine 211 in ovarian cancer cells. (A) HO-8910 cells were incubated with DEX (100 nM) for the indicated time periods, and the protein levels of pGR-S211 and total GR were analyzed by western blot. HO-8910 (B) and SKOV3 (C) cells were incubated with DEX (100 nM), TGF-β1 (10 ng/ml) or both for the times indicated. The protein level of pGR-S211 and total GR was analyzed by western blot, and quantified by densitometric analysis (ImageJ software, NIH). The results were expressed as fold over control and representative of at least three independent experiments. **P < 0.01 vs. control, # P < 0.05, ##P < 0.01 vs. DEX.

TGF-β1 activated Smad3 has been reported to mediate the expression of PAI-1 mRNA by forming heteromeric Smad complexes with Smad4 and binding elements in PAI-1 promoter (Dennler et al., 1998; Lund et al., 1987). Therefore we further investigated the role of Smad3 in the synergistic induction of PAI-1 by DEX and TGF-β1. As shown in Fig. 7A, knockdown of the expression of Smad3 in SKOV3 cells with small interfere RNA (Smad3 siRNA) was confirmed by western blot. Compared with SKOV3 cells transfected with control siRNA, inhibiting the expression of Smad3 with Smad3 siRNA resulted in 35% inhibition in TGF-β1 induced PAI-1 mRNA and 28% inhibition in co-induced PAI-1 mRNA by DEX and TGF-β1, respectively (Fig. 7B). These results indicate that TGF-β1/Smad3 pathway at least partially mediats the expression of PAI-1 induced by TGF-β1 alone or combined with DEX.

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Fig. 5. Effect of TGF-β1-activated ERK on the expression of pGR-S211 and PAI-1 induced by DEX and TGF-β1 in ovarian cancer cells. HO-8910 (A) and SKOV3 (B) cells were incubated with TGF-β1 (10 ng/ml) for different times as indicated. The protein level of pERK1/2 and total ERK1/2 was measured by western blot. HO-8910 (C) and SKOV3 cells (D) were pretreated with or without ERK1/2 inhibitor PD98059 for 1 hour and then further stimulated for 1.5 hours with DEX (100 nM), TGF-β1 (10 ng/ml) or both. The protein level of pGR-S211 and total GR was measured by western blot. HO-8910 (E) and SKOV3 cells (F) were pretreated with or without PD98059 for 1 hour and then further stimulated for 4 hours with DEX (100 nM), TGF-β1 (10 ng/ml) or both. The expression of PAI-1 mRNA was analyzed by real-time PCR and normalized to housekeeping gene GAPDH. Data shown are representative of at least three separate experiments.*P < 0.05 for treatment without vs. with PD98059.

Fig. 6. Effect of TGF-β1-activated p38MAPK on the expression of pGR-S211 and PAI-1 induced by DEX and TGF-β1 in ovarian cancer cells. HO-8910 (A) and SKOV3 (B) cells were incubated with TGF-β1 (10 ng/ml) for different times as indicated. The protein level of p-p38 and total p38 was measured by western blot. HO-8910 (C) and SKOV3 cells (D) were pretreated with or without p38MAPK inhibitor SB203580 for 1 hour and then further stimulated for 1.5 hours with DEX (100 nM), TGF-β1 (10 ng/ml) or both. The protein level of pGR-S211 and total GR was measured by western blot. HO-8910 (E) and SKOV3 cells (F) were pretreated with or without SB203580 for 1 hour and then further stimulated for 4 hours with DEX (100 nM), TGF-β1 (10 ng/ml) or both. The expression of PAI-1 mRNA was analyzed by real-time PCR and normalized to housekeeping gene GAPDH. Data shown are representative of at least three separate experiments. *P < 0.05;**P < 0.01 for the treatment without vs. with SB203580.

3.8. Synergistic induction of PAI-1 by DEX and TGF-β1 contributes to the pro-adhesion effect in ovarian cancer cells Our previous studies found that DEX and TGF-β1 cooperatively promoted cell adhesion to ECM in ovarian cancer cells (Chen et al., 2010). Since PAI-1 plays an important role in the accumulation of ECM and promoting cell adherence through inhibition of plasmindependent ECM degradation (Durand et al., 2004; Ghosh and Vaughan, 2012; Zorio et al., 2008), we therefore investigated the

relationship between the up-regulated PAI-1 expression by DEX and TGF-β1 and pro-adhesion effect of both two agents. As shown in Fig. 8A, the adhesion ability of SKOV3 cells co-treated with 100 nM DEX and 10 ng/ml TGF-β1 for 8 h was increased by 1.6-fold compared to that of the control cells (P < 0.05). Inhibiting the expression of PAI-1 in SKOV3 cells with 10 nM PAI-1 small interfere RNA (PAI-1siRNA) not only strongly inhibited the expression of PAI-1 protein, but also significantly weaken the pro-adhesion effect of both the agents. Similar results were also observed in HO-8910 cells

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Fig. 7. Effect of Smad3 knock-down on synergistic PAI-1 mRNA induction by DEX and TGF-β1 in ovarian cancer cells. SKOV3 (A) and HO-8910 (B) cells were transfected with Smad3 siRNA or control siRNA (Con siRNA) for 36 hours, then transfected cells were treated with TGF-β1 (10 ng/ml) alone or combined with DEX (100 nM) for another 4 hours. At the end of culture, Smad3 knockdown was monitored at the protein level by western blot and the mRNA expression of PAI-1 was measured by real-time PCR. GAPDH was used as a normalization control for real-time PCR and β-actin as a loading control for western blot. Data shown are representative of at least three separate experiments. #P < 0.05; ##P < 0.01 for Smad3 siRNA vs. Con siRNA treated with TGF-β1 or TGF-β1 + DEX.

(Fig. 8B). These results indicate that synergistic induction of PAI-1 is involved in early cooperative pro-adhesion effect of DEX and TGFβ1 in ovarian cancer cells. 4. Discussion In this study we demonstrated that TGF-β1 and DEX, either individually or in combination, up-regulate the expression of PAI-1. Co-treatment with DEX and TGF-β1 showed a rapid and synergistic effect with the maximum induction of PAI-1 mRNA at 4 h. The further study showed that the synergistic induction effect occurred at the transcriptional level and was largely blocked by RU486, an antagonist of GR, suggesting that the function of GR is enhanced in ovarian cancer cells during co-treatment of DEX and TGFβ1. However, the synergistic effect did not occur via increasing the expression of GR because we did not find significant change of GR

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protein in cells treated with both DEX and TGF-β1 for 2–6 h in ovarian cancer cells. Phosphorylation of GR at serine 211 (pGR-S211) is hormonedependent and -independent, and is considered as a transcriptionally active form of GR in several human cells (Beck et al., 2009; Galliher-Beckley and Cidlowski, 2009; Krstic et al., 1997; Miller et al., 2005). For example, phosphorylation of GR at Ser211 caused by GCincreased p38 MAPK activity was required for GC-induced apoptosis in lymphoid cells (Miller et al., 2005, 2007). In ovarian cancer cells we demonstrated that DEX increased pGR-S211 level, which sustained for at least 48 h. Moreover, we found for the first time that TGF-β1 could enhance DEX-dependent pGR-S211 level. The event is rapid and transient, in agreement with synergistic induction effect of PAI-1 by DEX and TGF-β1. We further found that TGF-β1 rapidly activated ERK and p38MAPK. Inhibiting activation of ERK and p38MAPK with their specific inhibitors not only markedly repressed TGF-β1 enhanced pGR-S211 level, but also largely reduced the synergistic induction effect of PAI-1 mRNA by DEX and TGF-β1, indicating rapidly enhanced GR phosphorylation at serine 211 by TGF-β1 is related to the rapid synergistic induction effect of PAI-1. We further found that TGF-β1 activated Smad3 pathway also partially mediated both TGF-β1-induced PAI-1 mRNA and the co-induced PAI-1 mRNA by the two agents. How the phosphorylation of GR at serine 211 affects GR function and signaling is still unclear. It has been reported that the increased transcriptional activity of the pGR-S211 is in part due to a conformational change within the GR and increases its recruitment to GRE-containing promoters, therefore promoting GC sensitivity in several cell types (Beck et al., 2009; Galliher-Beckley and Cidlowski, 2009). However no specific functional GRE has been identified in human PAI-1 gene until now (Wickert et al., 2007). Therefore it is unknown whether direct binding of pGR-S211 to the PAI-1 gene is responsible for the synergistic induction of PAI-1 mRNA by DEX and TGF-β1 in ovarian cancer cells. We also did not find cotreatment of cells with DEX and TGF-β1 significantly changed the transcriptional activity of Smad3 on the expression of PAI-1. Further work is required in order to fully understand the effect of pGRS211 status on the transcription of PAI-1 in ovarian cancer cells. It has been reported that the levels of PAI-1 are associated with malignancy and metastasis of epithelial ovarian cancer (Cai et al., 2007; Koensgen et al., 2006; Kwaan et al., 2013). The promotion of tumor metastasis may not directly be a result of enhanced ovarian cancer invasion, but through inhibition of apoptosis, promotion of cell proliferation, survival, and angiogenesis (Kwaan et al., 2013).

Fig. 8. Effect of PAI-1 knock-down on pro-adhesion induced by DEX and TGF-β1 in ovarian cancer cells. SKOV3 (A) and HO-8910 (B) cells were transfected with PAI-1 siRNA or control siRNA(Con siRNA) for 36 hours, then transfected cells were treated with or without DEX (100 nM) and TGF-β1 (10 ng/ml) for another 8 hours. Single cell suspension prepared and 8 × 104 cells were seeded into a 96-well plate. Sixty minutes later, cells were washed thrice with PBS, and the number of the remaining cells attached was determined by MTT assay. PAI-1 knockdown was monitored at the protein level by western blot and β-actin was used as a normalization control. Data shown are representative of at least three separate experiments.*P < 0.05, **P < 0.01 vs. control siRNA, #P < 0.05 vs. control siRNA with DEX and TGF-β1 treatment.

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Fig. 9. A model whereby the synergistic induction of PAI-1 expression by TGF-β and DEX. The synergistic induction of PAI-1 expression by DEX and TGF-β1 is accomplished by several pathways, including TGF-β1-activated p38MAPK, ERK and Smad3 as well as GR pathways. Enhancement of DEX-dependent phosphorylation of GR at serine 211 by TGF-β1 activated-p38MAPK and -ERK pathways may also contribute to the induction of PAI-1 expression in ovarian cancer cells.

Our previous studies found that DEX and TGF-β1 cooperatively promoted cell adhesion to ECM in ovarian cancer cells, which enhanced cell resistance to chemotherapeutics (Chen et al., 2010). In this study we further demonstrated that up-regulation of PAI-1 by DEX and TGF-β1 contributed to early pro-adhesion to ECM. Since enhanced cell adhesion was reported to be associated with cell survival and resistance to chemotherapeutics in ovarian cancer cells, while synthetic GCs, such as DEX, are routinely administered before, during and after epithelial cell tumor-chemotherapy to mitigate nausea and allergic reactions, it should be of important clinical relevance when enhancing cell adhesion by DEX and TGF-β1 interferes with the effect of chemotherapeutics. Up-regulation of PAI-1 in OSE by TGF-β1 and GC/GR may also play an important role in ovulation and ovulationassociated wound healing by promoting thrombin and ECM formation. The physiological significance needs to be further studied. Overall, in the present study we found that the rapidly synergistic effects on the induction of PAI-1 by DEX and TGF-β1 contributed to their early pro-adhesion effects in ovarian cancer cells. The synergistic induction of PAI-1 expression is accomplished by GR pathway and TGF-β1-activated p38MAPK (also involved in the effect of up-regulation of PAI-1 by TGF-β1 alone), ERK and Smad3 pathways. TGF-β1-activated p38MAPK and ERK pathways crosstalk with GR pathway to induce the expression of PAI-1 through enhancing the phosphorylation of GR at serine 211 in ovarian cancer cells (Fig. 9). These findings revealed a possible novel mechanism by which TGF-β pathways cooperatively cross-talk with GR pathway to regulate gene expression. Acknowledgments This work was supported by grant (No. 31301164 and No. 91029722) from the National Natural Science Foundation of China. We acknowledge Dr. Liang-nian Song (Department of Medicine, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center) for critical reading of the manuscript. References Almawi, W.Y., Irani-Hakime, N., 1998. The antiproliferative effect of glucocorticoids: is it related to induction of TGF-beta? Nephrol. Dial. Transplant. 13, 2450–2452. Anbalagan, M., Huderson, B., Murphy, L., Rowan, B.G., 2012. Post-translational modifications of nuclear receptors and human disease. Nucl. Recept. Signal. 10, e001. AyanlarBatuman, O., Ferrero, A.P., Diaz, A., Jimenez, S.A., 1991. Regulation of transforming growth factor-beta 1 gene expression by glucocorticoids in normal human T lymphocytes. J. Clin. Invest. 88, 1574–1580. Baschant, U., Tuckermann, J., 2010. The role of the glucocorticoid receptor in inflammation and immunity. J. Steroid Biochem. Mol. Biol. 120, 69–75.

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