JOURNAL OF NEUROCHEMISTRY

| 2015

doi: 10.1111/jnc.13085

*College of Pharmacy, CHA University, Seongnam, Korea †Creative Research Initiatives Center for Nuclear Receptor Signals and Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, Korea

Abstract The orphan nuclear receptor estrogen-related receptor gamma (ERRc) is highly expressed in the nervous system during embryogenesis and in adult brains, but its physiological role in neuronal development remains unknown. In this study, we evaluated the relevance of ERRc in regulating dopaminergic (DAergic) phenotype and the corresponding signaling pathway. We used retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA induced neurite outgrowth of SH-SY5Y cells with an increase in DAergic neuron-like properties, including up-regulation of tyrosine hydroxylase, dopamine transporter, and vesicular monoamine transporter 2. ERRc, but not ERRa, was up-regulated by RA, and participated in RA effect on SH-SY5Y cells. ERRc over-expression enhanced mature DAergic neuronal phenotype with neurite

outgrowth as with RA treatment; and RA-induced increase in DAergic phenotype was attenuated by silencing ERRc expression. ERRc appears to have a crucial role in morphological and functional regulation of cells that is selective for DAergic neurons. Polo-like kinase 2 was up-regulated in ERRc-over-expressing SH-SY5Y cells, which was involved in phosphorylation of glycogen synthase kinase 3b and resulting downstream activation of nuclear factor of activated T cells. The likely involvement of ERRc in regulating the DAergic neuronal phenotype makes this orphan nuclear receptor a novel target for understanding DAergic neuronal differentiation. Keywords: dopamine transporter, dopaminergic neuron, ERRc, GSK3b, neurite outgrowth, tyrosine hydroxylase. J. Neurochem. (2015) 10.1111/jnc.13085

The estrogen-related receptors (ERRs) – ERRa (ERR1, NR3B1), ERRb (ERR2, NR3B2), and ERRc (ERR3, NR3B3) – belong to the NR3B subgroup of the orphan nuclear receptors. Although ERRs possess high sequence identity to estrogen receptors (Halberg et al. 2009), 17bestradiol, an estrogen ligand, does not directly bind to or activate the transcriptional activity of ERRs. Instead, ERRs are constitutively active nuclear receptors, and their transcriptional activities are primarily controlled by their expression levels. The expression patterns of the three ERRs are tissue specific. ERRb is expressed in the developing placenta and undifferentiated trophoblast stem cell line, and its postnatal expression is highly restricted (Pettersson et al. 1996; Bonnelye et al. 1997a; Heard et al. 2000). On the other hand, both ERRa and ERRc mRNAs are detected during development and in the adult, and show similar tissue distribution patterns with high expression levels in the heart, brain, and kidney (Bonnelye et al. 1997b). Because ERRs

are highly expressed in tissues with metabolic demands (Rangwala et al. 2010), the functional role of ERRs has been largely focused on its regulation of energy homeostasis, cell proliferation, and cancer metabolism (Bianco et al. 2012; Deblois et al. 2013). Although enhanced ERRc expression is also observed in the nervous system during development in humans (Hermans-Borgmeyer et al. 2000) as well as in adult brain, its biological role in the nervous system is largely unknown. Received January 8, 2015; revised manuscript received February 17, 2015; accepted February 26, 2015. Address correspondence and reprint requests to Hyun Jin Choi, College of Pharmacy, CHA University, Seongnam-si, Gyeonggi-do 463836, Korea. E-mail: [email protected] Abbreviations used: DAergic, dopaminergic; DAT, dopamine transporter; ERRc, estrogen-related receptor gamma; GSK3b, glycogen synthase kinase 3b; NFAT, nuclear factor of activated T cells; PLK2, polo-like kinase 2; RA, retinoic acid; TH, tyrosine hydroxylase.

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Dopaminergic (DAergic) neurons in the ventral midbrain are critical for cognitive and motor behavior, and are associated with various central nervous system disorders. The development of midbrain DAergic neurons is a complex process which is controlled by cooperative action of multiple signaling molecules including transcription factors (Rossler et al. 2010; Jacobs et al. 2011; Luo 2012). Fibroblast growth factor 8 (FGF8), sonic hedgehog (SHH), LIM homeobox transcription factor 1 (Lmx1), Msh homeobox 1 (MSx1), Nuclear receptor related 1 protein (Nurr1), and Pituitary homeobox 3 (Pitx3) have been demonstrated to play essential roles in the development of DAergic neurons. It is of interest that ERRc expression is most prominent in the nervous system during development. In the embryo, ERRc transcripts are detected as early as day 10.5 post-coitum (E10.5), and significant expression occurs at E12.5 (Susens et al. 2000), which overlaps with the period when substantia nigra pars compacta (SNpc) DAergic neurons are born (between E10 and E13). In addition, abundant ERRc transcripts are also detected in the isocortex, olfactory system, cranial nerve nuclei, and major parts of the extrapyramidal motor system of the adult brain (Hentschke et al. 2002). Because ERRc is constitutively active in the absence of endogenous ligands and its relative concentration is responsible for transcriptional activity of ERRc (Chao et al. 2006), ERRc activity might be increased and play a role in neural development and in the maintenance of the differentiated properties in the brain. It is of interest that there are strong connections between developmental factors and essential neuronal functions including regulation of neuronal survival and death (Alavian et al. 2008, 2014). For example, there is evidence that inherent vulnerability of SNpc DAergic neurons in the brain of Parkinson’s disease patients could be a consequence of insufficiency in certain key transcription factors such as engrailed and Nurr1 (Alavian et al. 2014). They are necessary for regulation of not only mesencephalic DAergic neuronal development but also lifelong maintenance of these neurons. This evidence suggests that deregulation of transcription factors that are crucial for embryonic specification and differentiation of DAergic neurons could also contribute to the demise of DAergic neurons during Parkinson’s disease progression. The aim of this study was to evaluate whether nuclear receptor ERRc contributes to the determination of the DAergic phenotype. For this study, we used all-trans-retinoic acid (RA) to differentiate human neuroblastoma SH-SY5Y cells. RA is generated during early development in a specific region of the embryo and plays a prominent role in regulating the transition of proliferating precursor cells to post-mitotic differentiated cells (Joshi et al. 2006). Evidences show that RA plays an essential role in nervous system development (e.g., in neuronal patterning, survival, neurite outgrowth) in many cultured embryonic neurons and human neuroblastoma cells, and in in vivo studies (Engberg et al. 2010; Jacobs

et al. 2011). In fact, a highly specific DAergic phenotype is observed in SH-SY5Y after differentiation with RA; RA treatment not only induces the expression of the DA-synthesizing enzyme tyrosine hydroxylase (TH) but also results in a molecular phenotype profile that is characteristic for DAergic cells (Korecka et al. 2013). Using RA-induced differentiated SH-SY5Y cells, we first evaluated whether ERRc is regulated by RA or plays a role in RA-induced morphological changes and increase in DArgic properties, and we identified the mechanism whereby ERRc regulates DAergic neuronal phenotype. Because RAinduced differentiation of SH-SY5Y cells has been considered to mimic the phenotype of DAergic neurons in many studies, the present study provided a: (i) better understanding of the effects of ERRc signaling on neurobiological aspects related to DAergic neuronal differentiation, and (ii) new target for controlling the DAergic phenotype.

Materials and methods Antibodies and reagents Dulbecco’s modified Eagle’s medium (DMEM), a-minimum essential medium, fetal bovine serum (FBS), penicillin/streptomycin, and B27 were obtained from Gibco (Grand Island, NY, USA). All-trans RA was purchased from Sigma-Aldrich (St. Louis, MO, USA), polo-like kinase (PLK) inhibitor BI2536 was from Selleck Chemicals (Houston, TX, USA) and Hoechst dye 33258 was obtained from Molecular Probes (Eugene, OR, USA), respectively. Basic FGF (bFGF) and FGF4 were purchased from R&D systems (Minneapolis, MI, USA). ERRc siRNA was supplied by Thermo Scientific (Waltham, MA, USA), and PLK2 siRNA and cytosolic nuclear factor of activated T cells 1 (NFATc1) siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies used in this study were as follows: anti-ERRc and anti-ERRa (all from PPMX, Tokyo, Japan); anti-dopamine transporter (DAT), anti-TH, anti-neuronspecific enolase (NSE), anti-vesicular monoamine transporter 2 (VMAT2), anti-PLK2, anti-a-tubulin, anti-Lamin B, anti-glycogen synthase kinase 3b (GSK3b), anti-NFATc1, anti-c-myc, horseradish peroxide-conjugated anti-mouse IgG, anti-rabbit, anti-goat, and anti-rat IgG (all from Santa Cruz Biotechnology); Alexa Fluorâ conjugated anti-rabbit, anti-mouse, anti-goat, and anti-rat IgG (all from Invitrogen, Carlsbad, CA, USA); anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH), and anti-p-GSK3b, (all from Cell Signaling Technology, Danvers, MA, USA); anti-TH (from Millipore, Billerica, MA, USA); anti-a-tubulin and anti-a-actin (all from Sigma-Aldrich). Anti-dopamine-b-hydroxylase (DBH) was kindly provided by K.S. Kim (Harvard Medical School). All other chemicals were of reagent grade and were purchased from Sigma-Aldrich. Culture and differentiation of cells SH-SY5Y, SK-N-BE(2), SN56, and PC12 cells were purchased from ATCC (Rockville, MD, USA) and grown in DMEM containing 10% FBS, 100 IU/L penicillin, and 10 lg/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were placed on polystyrene culture plates or dishes, and after

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13085

ERRc regulates dopaminergic differentiation

24 h, cells were fed fresh medium in subsequent experiments. To induce differentiation into neuronal cells, SH-SY5Y cells were treated with 20 lM RA in 1% FBS containing DMEM for 3–5 days. SK-N-BE(2) cells were differentiated with 10 lM RA and 50 lM forskolin in 1% FBS containing DMEM for 2 days. SN56 cells were treated with 1 mM dibutyryl-cAMP (db-AMP) and 1 lM RA without FBS in DMEM for 2 days, and PC12 cells were treated with 50 lg/mL nerve growth factor (NGF) in 0.1% FBS containing DMEM for 5 days. Differentiated neuronal properties were evaluated using specific differentiation markers [microtubuleassociated protein 2 (MAP2), DBH, and NSE]. Human bonemarrow mesenchymal stem cells (hBM-MSC) (Lonza; Walkersville, MD, USA) were grown in a-minimum essential medium, containing 10% FBS, 100 IU/L penicillin, and 10 lg/mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2. The differentiation of the hBM-MSC into DAergic neurons was achieved using the method of Trzaska and Rameshwar, et al., with some modifications (Trzaska and Rameshwar 2011). Transient transfection SH-SY5Y cells were transfected with pcDNA3.1-Flag-ERRc, pGL3-ERRc luciferase, pcDNA3.1-Flag-NFATc1 (a kind gift from K.Y. Lee, Chonnam National University), pGL3-NFAT minimal promoter (a kind gift from B.Y. Kang, Chonnam National University), pcDNA3.1-empty vector, control siRNA, ERRc siRNA, NFAT siRNA, and PLK2 siRNA using the polyethyleneimine reagent (Polysciences, Inc., Warrington, PA, USA) according to the manufacturer’s instructions. Western blot analysis To prepare for the western blot analysis, cells were washed for 20 min in ice with phosphate-buffered saline (PBS) and lysed in ristocetin-induced platelet agglutination buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-Cl, pH 7.5) containing a complete protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Cellular lysates were centrifuged at 21,130 g for 20 min at 4°C. Supernatant proteins were collected and quantified using the Bradford protein assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated with specific primary antibodies and subsequently with horseradish peroxide-conjugated secondary antibodies. Specific proteins were visualized using enhanced chemiluminescent detection kits (Millipore) and analyzed by a luminescent image analyzer LAS-4000 (Fujifilm, Tokyo, Japan). The relative intensities of each band were measured using ImageJ (NIH, Bethesda, MD, USA). Alpha-actin, a-tubulin, and GAPDH were used as loading controls. Cytosolic and nuclear fractionation For cytosolic/nuclear fractionation, cells were washed with ice-cold PBS, harvested, and lysed in 0.5% Triton X-100 buffer using a complete protease inhibitor cocktail and phosphatase inhibitor cocktail for 20 min on ice. Cellular lysates were centrifuged at 15,871 g for 15 min at 4°C. The supernatant included the cytosolic proteins. The collected pellet was resuspended in 0.5% SDS containing 0.5% Triton X-100 buffer and then incubated for

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20 min in ice. After centrifugation at 15,871 g for 15 min at 4°C, supernatant proteins (nuclear fraction) were collected and quantified using the Bradford protein assay reagent. Lamin B was used as the nuclear fraction marker. Reverse transcription polymerase chain reaction Total RNA from cells was extracted using the Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s protocol. Reverse transcription polymerase chain reaction (RT-PCR) was performed using the Maxime RT-PreMix Kit, Maxime PCR PreMix Kit (iNtRON Biotechnology, Seongnam, Korea) and specific primers. PCR was performed at 94°C for 30 s, 60°C for 40 s, and 72°C for 1 min for 30 cycles. PCR products were separated by 1% agarose gel electrophoresis. Gels were stained using a Gel GreenTM nucleic acid prestaining kit (Biotium, Inc., Hayward, CA, USA) and analyzed by a luminescent image analyzer LAS-4000 containing a SYBR filter (Fujifilm). ERRc primers (50 -GACTTGACTCGCCACCTCTC-30 , 50 -GTGGTACCCAGAAGCGAT GT-30 ); b-actin primers (50 -GTCATCACCATTGGCAATGAG-30 , 50 -CGTCATACTCCTGCTTGCTG-30 ). Reporter gene assay A dual-luciferase reporter assay system (Promega, Madison, WI, USA) was used to determine ERRc and NFAT luciferase activities. SH-SY5Y cells were transiently transfected with pGL3-ERRc, pGL3-NFATc1, and pRL-TK plasmids using polyethyleneimine according to the manufacturer’s protocol. After drug treatment, Firefly and Renilla luciferase activities in the cell lysates were determined using a luminometer (Microlumat Plus LB96V; Berthold Technologies, Bad Wildbad, Germany). Immunofluorescence confocal microscopy SH-SY5Y cells were plated on poly-D-lysine-coated cover slips in 12well culture plates. After individual experiments, cells were washed twice with PBS and fixed for 10 min in cold PBS with 4% paraformaldehyde and 4% sucrose, pH 7.4. After washing twice in PBS containing 1% bovine serum albumin (BSA), cells were permeabilized with 0.25% Triton X-100 for 10 min and blocked with 1% BSA, and 1% normal goat serum in PBS for 1 h at 15–25°C. Cells were incubated overnight with primary antibodies at 4°C. The primary antibodies used in this study were as follows: rat anti-DAT (1 : 100), rabbit anti-VMAT2 (1 : 100), mouse anti-ERRc (1 : 400), mouse anti-Flag (1 : 400), and mouse anti-a-tubulin (1 : 400). After washing twice with 1% BSA in PBS, cells were incubated with Alexa Fluorâ conjugated secondary antibodies for 1 h at 15–25°C, and Hoechst dye 33258 was added for the last 5 min. Samples were mounted with Fluoromount/Plus mounting media (Diagnostic Biosystems, Pleasanton, CA, USA) for imaging. Images were obtained using a TCS SP5 AOBS/Tandem laser confocal scanning microscope (Leica GmBh, Wetzlar, Germany) at the Korea Basic Science Institute, Gwangju Center. High-performance liquid chromatography-electrochemical detector analysis DA and DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured by high-performance liquid chromatography-electrochemical detector analysis (HPLCECD) analysis as described in our earlier study (Kim et al. 2014).

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Data analysis Data were analyzed and expressed as mean  SEM. Comparisons were made using one-way ANOVA, followed by Newman–Keuls post hoc test. p < 0.05 was considered statistically significant.

Results

cence was mainly localized along the cell membrane (Fig. 1g). DA synthesis is an important function of mature DAergic neuron. Data from HPLC-ECD analysis showed that intracellular DA and DA metabolites DOPAC and HVA were significantly increased in ERRc-over-expressing SH-SY5Y cells (Fig. 1i). Interestingly, ERRc agonist GSK4716 (Kim et al. 2009b) also induced up-regulation of DAT, TH, and NSE (Figure S1). All these results indicate that ERRc is involved in the differentiation of SH-SY5Y cells to the DAergic neuronal phenotype.

ERRc regulates morphological and biochemical characteristics of SH-SY5Y cells to DAergic neuronal phenotype In the present study, we treated SH-SY5Y cells with RA (20 lM) in low serum conditions (1% FBS in DMEM) for 3– 5 days (Biedler et al. 1978), and characterized DAergic neuron-like phenotypes by evaluating morphological changes and expression levels of DA neurotransmission-related genes. As shown in Fig. 1(a), significant neurite extension was observed in RA-treated SH-SY5Y cells. In addition, the expression levels of the DAergic neuron-specific marker DAT, TH, VMAT2, and differentiated neuronal marker NSE were all significantly increased after RA treatment, suggestive of the enhanced DAergic phenotype (Fig. 1b–d). TH is a ratelimiting enzyme in DA synthesis, and DAT and VMAT2 are involved in the process of DA neurotransmission. DAT is normally localized at the terminal region of mature DAergic neurons. In this study, DAT was not only up-regulated (Fig. 1b), but also highly localized at the ends of the neurite in RA-treated SH-SY5Y cells (Fig. 1c). On the basis of morphological and biochemical changes in RA-treated SH-SY5Y cells, we explored whether orphan nuclear receptor ERRc plays a role in the regulation of DAergic phenotype of SH-SY5Y cells. We transfected SHSY5Y cells with ERRc and evaluated the subsequent morphological and biochemical characteristics of these cells. Neurite extension was observed in ERRc-over-expressing SHSY5Y cells (Fig. 1e). Similar to findings with RA-treated cells, the expression levels of DAT, TH, and VMAT2 were all elevated in ERRc-over-expressing SH-SY5Y cells (Fig. 1f– h). Immunofluorescence images showed that DAT fluores-

ERRc is up-regulated by RA in SH-SY5Y cells Because ERRc-over-expressing SH-SY5Y cells showed similar morphological and biochemical changes as those seen in RA-treated cells (Fig. 1), we explored whether ERRc expression was regulated during the RA-induced differentiation of SH-SY5Y cells. A significant increase in ERRc expression was detected after RA treatment: ERRc mRNA (Fig. 2a), transcriptional activity (Fig. 2b), and protein expression (Fig. 2c and d) were all significantly increased after RA treatment. We next investigated whether upregulation of ERRc was a critical step in the process of RA-induced differentiation in SH-SY5Y cells by evaluating the effect of ERRc knockdown on RA-induced differentiation. ERRc silencing using both shERRc adenovirus (Fig. 2e) and ERRc siRNA (Fig. 2f) significantly attenuated the RA-induced up-regulation of DAT, a terminal DAergic differentiation marker. Because ERRa is also present during neurogenesis and in the adult brain with a similar tissue distribution pattern as that of ERRc (Bonnelye et al. 1997b), we evaluated whether ERRa was also up-regulated by RA in SH-SY5Y cells. Unlike ERRc, there were no significant changes in the expression levels of ERRa after RA treatment (Fig. 2g). Moreover, ERRc was also up-regulated during differentiation of hBM-MSC into DAergic neuron (Fig. 2h). Taken together, these results suggest that ERRc plays a crucial role as a positive regulator in RA-induced DAergic differentiation of SH-SY5Y cells.

Fig. 1 Retinoic acid (RA) and estrogen-related receptor gamma (ERRc) induce dopaminergic (DAergic) differentiation in SH-SY5Y cells. (a–d) SH-SY5Y cells were cultured in the presence or absence of 20 lM RA for up to 5 days. (a) Morphological images were obtained by phase-contrast microscope. Scale bar = 100 lm. (b) Cells were treated with 20 lM RA for the indicated durations, and the protein levels of dopamine transporter (DAT), tyrosine hydroxylase (TH), and neuron-specific enolase (NSE) were analyzed by western blotting. Data were representative of three independent experiments and were quantified by densitometric analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (c and d) DAT (c, green fluorescence) and vesicular monoamine transporter 2 (VMAT2) (d, red fluorescence) protein expression and localization in SH-SY5Y cells were analyzed by immunofluorescent confocal microscopic analysis. Alphatubulin (c, red fluorescence) staining reveals the cell morphology and

Hoechst staining (d, blue fluorescence) represents the nuclei. Scale bar = 20 lm (c), 10 lm (d). (e–h) ERRc was over-expressed in SHSY5Y cells by transient transfection of Flag-ERRc cDNA plasmid. (e) The phase-contrast images were obtained after ERRc transfection. Scale bar = 100 lm. (f) DAT, TH, and ERRc protein levels were analyzed by western blot analysis. Alpha-actin was used as a loading control. Data were representative of three independent experiments and were quantified by densitometric analysis. (g and h) DAT (g, green fluorescence) and VMAT2 (h, purple fluorescence) protein expression and localization in SH-SY5Y cells were analyzed by immunofluorescent confocal microscopic analysis. ERRc was stained with red fluorescence. Scale bar = 10 lm. (i) DA and DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in control and ERRcover-expressing SH-SY5Y cells were measured using the HPLC-ECD system. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001.

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 10.1111/jnc.13085

ERRc regulates dopaminergic differentiation

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ERRc increases GSK3b phosphorylation and the nuclear accumulation of NFATc1 GSK3 and its downstream regulators are known to play key roles in many fundamental processes during neural development (Hur and Zhou 2010). Therefore, we evalu-

ated whether GSK3 signaling was responsible for the ERRc-induced increase in DAergic phenotype of SHSY5Y cells. As shown in Fig. 3(a) and (b), phosphorylation of GSK3b at the serine 9 (Ser9) position (p-GSK3b) was significantly increased both in RA-treated (Fig. 3a)

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and ERRc-over-expressing (Fig. 3b) SH-SY5Y cells. In addition, the RA-induced phosphorylation of GSK3b was attenuated by ERRc knockdown using siRNA of ERRc (Fig. 3c). These data suggest the involvement of GSK3b phosphorylation in the ERRc-mediated differentiation of SH-SY5Y cells. Transcription factors such as b-catenin, cAMP response element-binding protein, and NFATc are well-known signaling molecules that link GSK3b and neurite outgrowth. Phosphorylation of NFATc by GSK3b inhibits DNA-binding activity of NFATc via exclusion from the nucleus (Neal and Clipstone 2001; Sheridan et al. 2002). Therefore, GSK3b phosphorylation, suggestive of GSK3b inactivation, could activate NFATc1mediated gene transcription. In Fig. 4(a)–(d), the increase in nuclear NFATc1 and NFAT-mediated transcriptional activity were shown in RA-treated (Fig. 4a and b) and ERRc-over-expressing (Fig. 4c and d) SH-SY5Y cells, respectively. To verify whether NFATc1 signaling was directly involved in neurite outgrowth in SH-SY5Y cells, cells were transfected with NFATc1 and then morphological characteristics were evaluated. As shown in Fig. 4(e) and (f), significant increase in neurite length and up-regulation of MAP2 were detected in NFATc1over-expressing SH-SY5Y cells. In cells silencing NFATc1 using siRNA, ERRc-induced up-regulation of MAP2 was not detected (Fig. 4g). Together, GSK3b and downstream NFATc1 signaling were regulated by ERRc and contributed to ERRc-induced neurite outgrowth in SH-SY5Y cells. PLK2 is involved in ERRc-induced GSK3b phosphorylation Recent evidence has shown that ERRc can regulate the transcription of differentiation-inducible genes, including PLK2 (Park et al. 2007). It is known that PLK2 is not expressed in proliferating tissues, but expressed in the brain (Seeburg et al. 2005). Although its function is not fully understood, there is evidence that PLK2 is strongly upregulated in NGF-treated PC12 cells (Dijkmans et al. 2008), where PLK2 is essential for NGF-induced differentiation (Draghetti et al. 2009). Thus, to verify whether PLK2 is an ERRc transcription target, we compared PLK2 expression between control and ERRc-over-expressing SHSY5Y cells. As shown in Fig. 5(a), PLK2 protein level was significantly increased by ERRc over-expression. Interestingly, PLK2 over-expression caused a dramatic increase in the phosphorylation of GSK3b (Ser9) (Fig. 5b). The crucial role of PLK2 in ERRc-induced GSK3b phosphorylation was supported by the result showing that PLK inhibitor BI2536 attenuated increase in p-GSK3b in PLK2-overexpressing cells (Fig. 5c). The role of PLK2 in ERRcinduced GSK3b phosphorylation was verified by the results shown in Fig. 5(d); PLK2 knockdown attenuated the increase in the level of p-GSK3b in ERRc-over-expressing cells. In addition, DAT up-regulations in PLK2- or ERRc-

over-expressing SH-SY5Y cells were all attenuated by the presence of BI2536, respectively (Fig. 5e and f). All these results indicate that PLK2 is a transcriptional target of ERRc and mediates GSK3b phosphorylation and the regulation of DAergic phenotype of SH-SY5Y cells. ERRc is selectively up-regulated in RA-treated SH-SY5Y cells and involved in neurite outgrowth of DAergic cells We explored next whether the role of ERRc in neuronal differentiation was selective for DAergic neurons, or common to other type of neurons. To answer this, we used several neuronal cell lines with different characteristics: DA-producing SH-SY5Y cells, norepinephrine-producing SK-N-BE(2) cells, epinephrine-producing PC12 cells, and cholinergic SN56 cells. We first evaluated whether ERRc was up-regulated by a differentiation-inducing stimulus in those cells. Differentiation-inducing conditions for each cell line were described in Materials and Methods. Differentiation of each cell line under the respective conditions was verified by detecting morphological changes such as neurite extension, and the up-regulation of differentiated neuronal markers including MAP2, DBH, and NSE. As shown previously, neurite extension and MAP2 up-regulation were detected in RA-treated SHSY5Y cells with a significant increase in ERRc expression (Fig. 6a). On the other hand, no significant changes in ERRc expression levels were detected by differentiation of SK-N-BE(2) (Fig. 6b), PC12 (Fig. 6c), or SN56 cells (Fig. 6d). To verify the selective contribution of ERRc to neurite outgrowth of DAergic cells, we compared the effects of ERRc over-expression on cell morphology and MAP2 expression between SH-SY5Y, SK-N-BE (2), PC12, and SN56 cells. As shown in Fig. 7, ERRc over-expression significantly induced neurite outgrowth and MAP2 up-regulation in SH-SY5Y cells (Fig. 7a), but not in SK-N-BE(2) (Fig. 7b), PC12 (Fig. 7c), or SN56 cells (Fig. 7d). In addition, ERRc-induced GSK3b phosphorylation was only detected in SHSY5Y cells (Fig. 7e). Together, our data indicate that ERRc might be selectively involved in the enhancement of the DAergic neuron phenotype.

Discussion RA is frequently used to differentiate neuroblastoma SHSY5Y cells to obtain DA neuron-like properties (Agholme et al. 2010), which provides a valuable clue for understanding DA neuronal development during neurogenesis. In the present study, we showed for the first time that ERRc is involved in RA-induced DAergic neuronal differentiation (i.e., highly differentiated cell morphology and up-regulation of DAergic neuron-specific proteins). It is of interest that the contribution of ERRc to neuronal differentiation

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Fig. 2 Retinoic acid (RA) increases estrogen-related receptor gamma (ERRc) expression in SH-SY5Y cells. (a–d) SH-SY5Y cells were treated with 20 lM RA for indicated durations. (a) ERRc mRNA levels were measured via RT-PCR analysis with b-actin as a control. Data were representative of three independent experiments and were quantified by densitometric analysis. (b) ERRc luciferase activity was determined using a reporter gene assay. ERRc luciferase activity was normalized to Renilla luciferase activity in each sample. Data (mean  SEM) were representative of at least three independent experiments and expressed as the fold-induction relative to untreated cells (at time zero). (c) ERRc protein levels were analyzed by western blotting. (d) ERRc (green fluorescence) expression and localization in SH-SY5Y cells were analyzed by immunofluorescent confocal microscopic analysis. Hoechst staining (blue fluorescence) represents the nuclei. Scale bar = 20 lm. (e and f) Cells were infected with shERRcGFP or US-shRNA-GFP adenovirus (e) or transfected with control

siRNA or ERRc siRNA (f). After 48 h, cells were treated with 20 lM RA for 3 days. DAT and ERRc protein levels were analyzed by western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e) and a-actin (f) were used as loading controls. All data were representative of three independent experiments and were quantified by densitometric analysis. (g) SH-SY5Y cells were treated with 20 lM RA for 5 days, and ERRa protein levels were analyzed by western blotting. GAPDH was used as a loading control. (h) Differentiation of the hBM-MSC into dopaminergic (DAergic) neuron was performed as described in Materials and Methods. Morphological changes were evaluated under phase-contrast microscope, and tyrosine hydroxylase (TH) and ERRc protein levels were analyzed by western blotting. GAPDH was used as a loading control. Scale bar = 100 lm. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant.

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(c) Fig. 3 Estrogen-related receptor gamma (ERRc) is involved in retinoic acid (RA)induced glycogen synthase kinase 3b (GSK3b) phosphorylation. (a and b) Phospho-GSK3b (p-GSK3b, Ser9) and GSK3b protein levels were analyzed by western blot analysis in SH-SY5Y cells treated with 20 lM RA for the indicated durations (a) and in cells transiently transfected with Flag-ERRc (b). All data were representative of three independent experiments and were quantified by densitometric analysis. (c) Cells were transfected with siRNA against ERRc, treated with 20 lM RA for 3 days, and evaluated p-GSK3b levels. Data were representative of three independent experiments and were quantified by densitometric analysis. Statistical significance: *p < 0.05 and **p < 0.01.

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seems to be selective for DAergic neurons by virtue of the fact that: (i) ERRc was up-regulated during the RAinduced differentiation of DAergic SH-SY5Y cells, but not during the differentiation of norepinephrinergic SK-N-BE (2) (Fig. 6b), epinephrinergic PC12 (Fig. 6c), and cholinergic SN56 cells (Fig. 6d); and (ii) unlike the impact on SH-SY5Y cells (Fig. 1 and 6a), the over-expression of ERRc did not induce morphological changes to highly differentiated cells or up-regulation of the differentiated neuronal markers in SK-N-BE(2) (Fig. 7b), PC12 (Fig. 7c), or SN56 cells (Fig. 7d). The selective effect of ERRc on DAergic neuronal differentiation in SH-SY5Y cells might be related to the differences in intracellular signaling events caused by ERRc. ERRc induces GSK3b phosphorylation in SH-SY5Y cells, but not in SK-N-BE(2), PC12, or SN56 cells (Fig. 7e). Although the precise molecular mechanisms underlying this selective effect of ERRc on GSK3b phosphorylation in SH-SY5Y cells are not clear at this time, and it is unknown whether this selectivity would be detected in the developing brain, it is interesting that GSK3b phosphorylation increases neuronal differentiation in ventral mesencephalon precursor cells and modulates the generation of DAergic neurons (Castelo-Branco et al. 2004). In addition, among the three ERRs, ERRc was strongly up-regulated by RA in SH-SY5Y cells (Fig. 2a–d). In spite of evidence demonstrating that ERRa and ERRc are detected both during development and in the adult brain and show similar tissue distribution patterns with high expression in the brain (Bonnelye et al. 1997b), our present study indicates that ERRa expression was RA-independent (Fig. 2g). These observations are corroborated by the fact

that three main DAergic regions (i.e., retrorubral field, SNpc, ventral tegmental area) exhibit strong hybridization signals for ERRc transcripts (Lorke et al. 2000), which is suggestive of the crucial role of ERRc in the development and function of DAergic neurons. Neurite outgrowth and axonal elongation are key steps for neuronal maturation (Polleux and Snider 2010). In the present study, neurite outgrowth was detected with enhanced DAergic properties in ERRc-over-expressing SH-SY5Y cells (Fig. 1); ERRc induced (i) an up-regulation of DAT, TH, and VMAT2; (ii) membrane localization of DAT; and (iii) an increase in DA synthesis. All these data suggest that ERRc induces DAergic neuronal differentiation and enhances the DAergic phenotype. In addition, cell proliferation was significantly inhibited and cell cycle arrest was induced in RA-treated as well as ERRc-over-expressing SH-SY5Y cells (Figure S2). Because regulating the switch between proliferation and differentiation is fundamental to neurogenesis (Janesick et al. 2015), this evidence is supportive of the crucial role of ERRc in neuronal differentiation. Nurr1 is critical for the development and maintenance of midbrain DAergic neurons (Kadkhodaei et al. 2009), therefore, we cannot eliminate the possible involvement of Nurr1 in the RA-induced increase in ERRc, or in the ERRc-induced differentiation of SH-SY5Y cells. In the embryo, ERRc transcripts are detected as early as E10.5, similar to the developmental stage at which Nurr1 is turned on (Saucedo-Cardenas et al. 1998; Susens et al. 2000), suggesting possible cross-talk between ERRc and Nurr1 during DAergic neuron development. Interestingly, Lammi et al. (Lammi et al. 2007) reported transcriptional interference between NR3B and NR4A receptors (including Nurr1,

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Fig. 4 Cytosolic nuclear factor of activated T cells 1 (NFATc1) nuclear accumulation is induced by estrogen-related receptor gamma (ERRc) and mediates neurite outgrowth of SH-SY5Y cells. (a–d) SH-SY5Y cells were treated with 20 lM retinoic acid (RA) for 3 days (a and b) or transiently transfected with Flag-ERRc (c and d). (a and c) Cytosolic and nuclear fractions were obtained as described in Materials and Methods. ERRc and NFATc1 protein levels were analyzed by western blot analysis. Lamin B and a-tubulin were used as a nuclear fraction marker and a cytosolic marker, respectively. All data were representative of three independent experiments and were quantified by densitometric analysis. (b and d) NFAT-mediated transcriptional

activity was normalized to Renilla luciferase activity in each sample. Data (mean  SEM) were representative of at least three independent experiments and expressed as the fold-induction relative to pcDNAtransfected cells. (e and f) Cells were transiently transfected with NFATc1, and evaluated morphological changes using phase-contrast image analysis (e, Scale bar = 100 lm) and microtubule-associated protein 2 (MAP2) expression levels by western blotting (f). (g) Cells were transfected with siRNA against NFATc1 with or without ERRc cDNA plasmid, and nuclear NFATc1 or total MAP2 expression levels were measured by western blotting. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001.

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Fig. 5 Polo-like kinase 2 (PLK2) mediates estrogen-related receptor gamma (ERRc)-induced glycogen synthase kinase 3b (GSK3b) phosphorylation. (a) SH-SY5Y cells were transfected with Flag-ERRc, and PLK2 protein expression levels were analyzed by western blotting. (b and c) Cells were transfected with PLK2, and evaluated pGSK3b (Ser9) and GSK3b levels by western blotting in the presence or absence with PLK inhibitor BI2536 (1 and 5 nM). (d) Cells were transfected with siRNA

against PLK2 with or without Flag-ERRc, and evaluated phosphoGSK3b (p-GSK3b) (Ser9) and GSK3b levels by western blotting. (e and f) Cells over-expressing PLK2 (e) or ERRc (f) were treated with BI2536 (1 and 5 nM), and dopamine transporter (DAT) expression levels were analyzed by western blotting. All data in Fig. 5 were representative of three independent experiments and were quantified by densitometric analysis. Statistical significance: *p < 0.05, and **p < 0.01.

NGF1-B, Nor1, etc.). Our preliminary studies also showed that ERRc repressed Nurr1 expression in SH-SY5Y cells (data not shown). Although RA-induced ERRc up-regulation as well as ERRc-induced DAergic differentiation seem to be Nurr1-independent, but further studies are required to identify how these two nuclear receptors interact during nervous system development and the physiological significance of this interaction. GSK3b plays a key role in the regulation of metabolic enzymes and transcription factors, and modulates diverse cellular processes, including the coordination of the proliferation and differentiation of progenitor cells during brain development (Zhang et al. 2010). The expression of GSK3b is ubiquitous in the nervous system, but the highest level is observed during development (Mukai et al. 2002). In the present study, GSK3b phosphorylation at Ser9 was significantly increased in SH-SY5Y cells overexpressing ERRc (Fig. 3b). Studies have shown that GSK3b phosphorylation is increased at the later stages of development, which promotes neural differentiation (Kim et al. 2009a). Most GSK3b substrates are transcription

factors, including cAMP response element-binding protein, the NFAT family, b-catenin, and c-Jun (de Groot et al. 1993; Bullock and Habener 1998; Aragon et al. 2011). Among the GSK3b substrates, NFAT is well-known transcription factor involved in neurite outgrowth. NFAT was initially identified in immune cells but is abundantly expressed in neurons. When NFATc is dephosphorylated, it translocates to the nucleus and complexes with nuclear partner NFATn, which results in transcriptional activation. Here, we show the crucial involvement of GSK3b/NFAT signaling in the ERRc-induced differentiation of SH-SY5Y cells by the fact that: (i) the nuclear-level and transcriptional activity of NFATc are increased by ERRc (Fig. 4c and d); (ii) NFATc over-expression induces neurite outgrowth and enhances MAP2 expression (Fig. 4e and f); and (iii) NFATc knockdown attenuates ERRc-induced upregulation of MAP2 (Fig. 4g). The crucial contribution of GSK3b/NFATc signaling to ERRc-induced DAergic neuronal differentiation is corroborated by previous studies showing that the NFATc protein is present in cells throughout the dorsal striatum and nucleus accumbens

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Fig. 6 Retinoic acid (RA)-induced up-regulation of estrogen-related receptor gamma (ERRc) is selective for SH-SY5Y cells. (a–d) SHSY5Y (a), SK-N-BE(2) (b), PC12 (c), and SN56 (d) cells were differentiated as described in Materials and Methods. After differentiation, morphological analysis was performed by phase-contrast microscope (Scale bar = 100 lm), and microtubule-associated protein 2 (MAP2), dopamine-b-hydroxylase (DBH), neuron-specific enolase

(NSE), and ERRc protein levels were analyzed by western blotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a-actin were used as loading controls. All western blotting data were representative of three independent experiments and were quantified by densitometric analysis. Statistical significance: *p < 0.05, **p < 0.01, and NS, not significant.

(Bradley et al. 2005), and GSK3b/NFAT-mediated gene transcription is a downstream event in neurotrophin-induced axon growth. Because ERRc is a transcription factor, we evaluated whether the regulatory effect of ERRc on DAergic neuronal phenotype depends on its transcriptional activity. In the present study, we show that PLK2 is up-regulated in SHSY5Y cells over-expressing ERRc (Fig. 5a). PLK2 is a multi-functional serine/threonine kinase that regulates the cell cycle and the cellular response to stress (Winkles and Alberts 2005). PLK2 has been suggested as a novel target of ERRc in cancer cell lines (Park et al. 2007), but there are no reports in the case of neuronal cells. Based on studies demonstrating that (i) PLK2 mRNA is detected in the human brain (Liby et al. 2001) but not in proliferating tissues (Simmons et al. 1992), (ii) its expression level in the brain is related to synaptic plasticity (Knecht et al. 1999), and (iii) PLK2 is related to the particular posttranslational modification contributing to normal a-synuclein physiology in DAergic neurons (Lou et al. 2010), our present study reveals the possibility that ERRc-induced PLK2 up-regulation plays a role not only in neurogenesis but also in adult brain function. These lines of evidence

support our suggestion that ERRc regulation and its downstream signaling events could be exclusive targets for understanding the mechanism of DAergic neuronal differentiation as well as pathogenesis of DAergic neuron dysfunction. The present study shows that (i) ERRc is up-regulated during the RA-induced differentiation of SH-SY5Y cells; (ii) ERRc enhances DAergic phenotypes such as DAT, TH, and VMAT2 expression, as well as neurite outgrowth; (iii) ERRc up-regulates PLK2, which induces GSK3b phosphorylation and NFAT nuclear accumulation and is responsible for differentiation, and (iv) ERRc selectively contributes to DAergic neuronal differentiation. All these results provide the first insights into the role of ERRc in the brain, which leads to a better understanding of the effects of ERRc signaling on the neurobiological aspects related to DAergic neuronal cell differentiation.

Acknowledgments and conflict of interest disclosure This work was supported by grant #2012R1A1A3011420 from the Basic Science Research Program of the National Research Foun-

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Fig. 7 Estrogen-related receptor gamma (ERRc) induces neurite outgrowth in SH-SY5Y cells, but not in SK-N-BE(2), PC12, or SN56 cells. (a–e) ERRc was transfected to SH-SY5Y (a, e), SK-N-BE(2) (b, e), PC12 (c, e), and SN56 cells (d, e). (a-d) After 2 days of transfection, morphological analysis was performed by phase-contrast microscope (Scale bar = 100 lm), and microtubule-associated protein 2 (MAP2), dopamine-b-hydroxylase (DBH), neuron-specific enolase (NSE), and ERRc protein levels were analyzed by western blotting. Glyceralde-

dation of Korea (NRF), which is funded by the Ministry of Education, Science and Technology. None of the authors has any conflicts of interest to disclose. All experiments were conducted in compliance with the ARRIVE guidelines.

Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. ERRc agonist GSK4716 increases TH and DAT expression. Figure S2. RA and ERRc inhibit cellular proliferation and induce G1/S cell cycle arrest.

hyde-3-phosphate dehydrogenase (GAPDH) and a-actin were used as loading controls. All western blotting data were representative of three independent experiments and were quantified by densitometric analysis. (e) Protein expression levels of glycogen synthase kinase 3b (GSK3b) and phosph-GSK3b (p-GSK3b) (Ser9) were analyzed by western blotting. Statistical significance: **p < 0.01, ***p < 0.001, and NS, not significant.

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