JBC Papers in Press. Published on September 1, 2006 as Manuscript M603862200 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M603862200 MU AND KAPPA OPIOIDS INDUCE THE DIFFERENTIATION OF EMBRYONIC STEM CELLS TO NEURAL PROGENITORS* Eunhae Kim‡, Amy L. Clark‡, Alexi Kiss‡, Jason W. Hahn‡, Robin Wesselschmidt‌†, Carmine J. Coscia‡ and Mariana M. Belcheva‡ From the ‡E. A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, 63104 and †Primogenix, Inc., Los Angeles, CA, 90033 Running title: Opioids induce ES cell differentiation Address correspondence to: Mariana M. Belcheva, Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO, 63104; Tel. 314-977-9256; Fax. 314-977-9205; E-mail: [email protected] Growth factors, hormones and The maintenance of ES cells in an neurotransmitters have been implicated in the undifferentiated state in vitro is dependent on self- regulation of stem cell fate. Since various renewing cell division in the presence of leukemia neural precursors express functional inhibitory factor (LIF)1, which signals through neurotransmitter receptors, which include G various receptor complexes (reviewed in 1-3, see protein coupled receptors (GPCRs), it is also 4, 5). Mouse ES cells can be induced to D o w anticipated that they are involved in cell fate differentiate into neural cells in the presence of n lo decisions. We detected µ opioid receptor retinoic acid (RA) in vitro (reviewed in 6, 7). ad e (MOR-1) and κ opioid receptor (KOR-1) During this induction, ES cells undergo a series of d fro expression and immunoreactivity in embryonic steps that resemble key stages in the early mouse m h stem (ES) cells and in retinoic acid induced ES embryo, supporting the hypothesis that the in vitro ttp cell-derived, nestin-positive, neural progenitors. pathway represents the normal developmental ://w w Moreover, these GPCRs are functional as pathway (8, 9). w [D-ala2,mephe4,gly-ol5] enkephalin, (DAMGO) Considerable effort has been recently .jbc .o a MOR selective agonist and U69,593, a KOR devoted to characterizing intrinsic, extrinsic rg selective agonist induce a sustained activation factors and signaling pathways regulating by/ g of extracellular signal-regulated kinase (ERK) proliferation and differentiation of stem cells. The ue s signaling throughout a 24 h treatment period in ERK/MAP kinase signaling pathway has been t o n undifferentiated, self-renewing ES cells. Both implicated in both proliferation and differentiation D e c opioids promote limited proliferation of of many cell types, including stem cells (10). In em b undifferentiated ES cells via the ERK/MAP several studies ERK activation had a negative e r 2 kinase signaling pathway. Importantly, influence on self-renewal/proliferation in murine 4 , 2 biochemical and immunofluorescence data ES cells (4, 11-14). Moreover, LIF-dependent 01 8 suggest that DAMGO and U69, 593 divert ES activation of STAT3 was not mediated by ERK cells from self-renewal and coax the cells to activity (15). However, a dual role for the differentiate. In retinoic acid-differentiated ES Ras/ERK pathway was proposed for ES cells, cells, opioid-induced signaling features a affecting both their division and differentiation biphasic ERK activation profile and an opioid- (16). Ras activation down-regulated levels of induced, ERK independent inhibition of Nanog, a protein that is normally expressed in proliferation in these neural progenitors. high amounts in self-renewing ES cells (17). A Collectively, the data suggest that opioids may functional role for ERK was proposed in the PI3K have opposite effects on ES cell self-renewal dependent regulation of ES cell self-renewal (18). and ES cell differentiation and ERK activation The importance of ERK activation and its duration is only required by the latter. Finally, opioid for cell differentiation has been investigated in modulation of ERK activity may play an various cells (19-22). Some evidence suggests that important role in ES cell fate decisions by RA-induced inhibition of LIF signaling in ES cells directing the cells to specific lineages. is ERK independent (23). However, a specific requirement of the ERK pathway was reported for RA-dependent commitment of murine ES cells 1 Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc. (24). Crosstalk may occur by non-cannonical (Primogenix, Inc.) were used. PRX-129/S6 # 7 ES actions of RA with the MAP kinase cells were isolated from the inner cell mass of day phosphorylation cascade. RA-induced 3.5 129/S6/SvEv mouse blastocyst. Cells have a differentiation of ES cells may be achieved by normal male karyotype and are specific pathogen- restricting nuclear entry of activated ERK (25). free. They were injected into blastocysts and ERK activation is required for regulation of cyclin produced chimeras at a high rate (first injection: D1 levels, the increase of which parallels ES cell 10/15 pups, no perinatal death). PRX-129/S6 # 7 differentiation (26, 27). cells were grown on mouse embryonic fibroblasts Neurotransmitters as well as growth factors (MEFs) in the presence of LIF, passage 10 cells and hormones can influence cell division and were propagated 2-3 times on gelatin-coated flasks differentiation of self-renewing stem cells and and these cells are currently used in the laboratory. neural progenitors (NPs) via ERK in some cases Quality control and characterization of the D3 ES (28, 29). For example, stimulation of the cells is documented by their depositor (36). D3 muscarinic acetylcholine receptor, a GPCR, ES cells were propagated on irradiated STO cell induces DNA synthesis in rat cortical feeders (ATCC) for several passages before they neuroepithelium progenitors via stimulation of were transferred to gelatin-coated flasks. PI3K and ERK (30). The activated CB1 receptor D3 and PRX-129/S6 # 7 ES cells were D inhibits neuronal progenitor differentiation via maintained in Dulbecco’s modified Eagles o w n attenuation of ERK signaling in E17 cortical medium (DMEM) containing 10% fetal calf lo a cultures and in adult dentate gyrus (31). This serum, LIF (100 ng/ml) and 0.1 mM 2- de d endocannabinoid system was also found to mercaptoethanol (ME) on gelatin-coated dishes. fro m promote astroglial differentiation by acting on These growth conditions are known to prevent ES h neural progenitor cells (32). Glutamate activates cells from differentiating (6, 37, 38). Here, cells ttp NMDA receptors and promotes proliferation of grown under these conditions will be described as ://w w w E15 precursors derived from the germinal zone of “undifferentiated, self-renewing ES cells”. .jb the ventral telencephalon in vivo and in vitro (33). The neural induction of ES cells entails the c.o Haloperidol acting via dopamine D2 receptors “4-/4+ protocol” which has two phases (8, 39). In brg/ increases the number of NPs, neurons and glia in the first phase, cells are cultured in non-adhesive, y g u adult rat brain (34). G protein βγ subunits of bacterial grade Petri dishes in serum containing es t o heterotrimeric G proteins are required for proper media (without LIF and ME) in the absence of RA n D mitotic-spindle orientation and asymmetric cell for 4 days. During this phase, EBs of different e c e fate decisions of cerebral cortical progenitors (35). sizes appear. In the second phase, cells are grown m b e These studies were performed with brain derived in RA (1 µM) and serum containing media for an r 2 4 stem cells or NPs, but little is known about the additional 4 days. Differentiation of these cells is , 2 0 consequences of GPCR-ERK crosstalk in also a two-phase process: early differentiation, 18 blastocyst-derived ES cells or their NPs. during which nestin-positive cells develop and Since the plasticity of uncommitted stem cells their terminal differentiation is the final step in ES has opened new perspectives in tissue cell growth. For this purpose, EBs are dissociated regeneration, recent research has been directed to and cultured on tissue culture plates in serum understand the signaling mechanisms that control containing DMEM for an additional 2-5 days proliferation and/or differentiation of ES cells. (short-term differentiation) or for 5-12 days (long- Here, we detected µ and κ ORs in ES cells and in term differentiation). The RA induced, short-term ES cell-derived NPs. More importantly, we found differentiated cells are referred to as NPs. that both opioids induced ES cell differentiation Opioid treatments: ES cells or ES cell- via ERK and an ERK independent attenuation of derived progenitors are maintained in their proliferation in RA-driven NPs corresponding growth media. Opioid agonists (0.1-1 µM, DAMGO, a MOR selective agonist or EXPERIMENTAL PROCEDURES U69,593, a KOR selective agonist) were added for various times (up to 24 h). In some experiments, Mouse ES cells and their growth conditions: cells were pre-incubated with opioid antagonists (1 D3 (ATCC) and PRX-129/S6 # 7 ES cells µM, 60 min, CTAP, MOR selective or nor-BNI, 2 KOR selective) and then treated with the added together with the OR Abs. After washing, corresponding opioid agonist (0.1 µM) for a given Alexa Fluor 594 (red) and/or Alexa Fluor 488 time. (green) conjugated secondary Abs (1:1000) were Measurement of ERK activity: For these applied for 1 h at room temperature. DAPI experiments, cells were grown in six-well plates. (1:200) was added together with the secondary In most cases, ES cells were maintained in serum- Abs. The slides were treated with anti-fade containing media upon opioid treatment. In reagent (Molecular Probes) and examined for contrast, ES cell-derived NPs were grown in immunofluorescence with NIKON-OPTIPHOT-2 media deprived of serum for 24 h before treatment or OLYMPUS AH3 microscopes with with opioids. ERK phosphorylation was measured simultaneous recording of dual fluorescence label by performing immunoblotting experiments with a images. phospho-ERK Ab that recognizes the active form Real-time quantitative (q)RT-PCR: Total of ERK (40-42). Cells were washed with PBS, RNA from ES cells and RA-induced NPs was lysed with buffer: 20 mM HEPES, 10 mM EGTA, isolated using Qiagen’s RNeasy Mini Kit (Qiagen, 40 mM β-glycerophosphate, 2.5 mM MgCl , 2 Valencia, CA) according to the manufacturer’s 2 mM sodium vanadate, 1% Nonidet-40, 1 mM instructions. cDNA was generated from 1 µg total PMSF, 20 µg/ml aprotinin and 20 µg/ml leupeptin, RNA using the High Capacity cDNA Archive Kit D spun at 14,000 g and protein concentration of the from Applied Biosystems (ABI, Foster City, CA) o w supernatants was determined. Cell lysates (10-20 with random primers as described by the nlo a µg protein/lane) were separated by 10% SDS- manufacturer. qRT-PCR was performed with de d PAGE. Proteins were blotted on Immobilon SYBR green chemistry on an ABI 7500 fro m membranes. Nonspecific sites were blocked with instrument. The primers were designed using h 5% milk in Tris-buffered saline + 0.2% Tween 20. Primer Express (ABI) and supplied by Integrated ttp Blots were incubated with monoclonal phospho- DNA Technologies (Coralville, IA). Primer ://w w w ERK Ab (1:2000, Cell Signaling) for at least 15 h sequences for MOR-1 were: forward, 5’- .jb at 4(cid:176) C, followed by incubation with a HRP CCACTAGCACGCTGCCCTT-3’; reverse, 5’- c.o conjugated IgG (1:2000, Sigma) for 1 h at room GCCACGTTCCCATCAGGTAG-3’. Primer brg/ temperature. Bands were visualized with an ECL sequences for ΚOR-1 were: forward, 5’- y g u chemiluminescence detection system (GE AGAGAGAGAAGCGGCAAGCA-3’; reverse, es t o Healthcare) and exposure to Classic Blue sensitive 5’- GCCAAGGCTCACTAACTCCAA-3’. The n D X-ray film. Band intensities were determined by cDNA templates for qRT-PCR were diluted 1:10 ec e densitometry with a Kodak DC120 digital camera, and the 50 µl SYBR-green reaction consisted of 1 m b e Kodak ds 1D version 3.0.2 (Scientific Imaging x SYBR-green Master Mix (ABI), 300 nM r 2 4 Systems) and NIH Image ImageJ version 1.32 forward and reverse primers, and 5 µl of diluted , 2 0 sofware. ERK stimulation in opioid-treated cells cDNA. Four replicates of each qRT-PCR reaction 18 was expressed as fold change over basal levels in were run on 96-well plates. The amplification control cells. efficiencies of MOR-1 and KOR-1 were consistent OR and nestin immunofluorescence with those of the endogenous control, GAPDH. microscopy: ES cells or ES cell-derived NPs were Relative quantification measurements were made grown in glass chamber slides (Nunc). The cells as described (43) by using the comparative C T were fixed in 4% PA for 20 min at room Method (∆∆C Method) in which the gene C T T temperature and permeabilized in 0.1% values are normalized by subtracting GAPDH C . T Triton/PBS for 5 min. Cells were then incubated Immunoblotting with MOR-1 and KOR-1 in PBS containing 0.5% BSA and 0.1% Tween 20 specific Abs: For detection of receptor protein for 30 min to reduce nonspecific binding, followed levels in ES cells or ES cell-derived NPs, by overnight incubation at 4(cid:176) C with the following immunoblotting with MOR-1 and KOR-1 specific rabbit polyclonal OR Abs: MOR-1 raised against Abs was adopted. For this purpose, cells were C-terminus (Neuromics, 1:2500); MOR-1 raised lysed in a modified RIPA buffer: 50 mM Tris- against N-terminus (Santa Cruz, 1:50); KOR-1 HCl, pH 7.4; 1% NP40; 0.25% sodium (Santa Cruz, 1:50). In some cases, a mouse deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM monoclonal nestin Ab (Chemicon, 1:1000) was PMSF; 1 m g/ml leupeptin; 1 m g/ml aprotinin; 1 3 mM Na VO ; 1 mM NaF and samples containing synthesis were assessed by measuring the rate of 3 4 20-50 µg protein were loaded on 10 % SDS gels. [methyl-3H] thymidine incorporation into cells. Receptor band(s) were detected using two rabbit Cells were grown in media ± opioids for 24 h. In polyclonal MOR-1 Abs: C-terminus MOR-1 Ab some experiments, the MEK-selective inhibitor (1:2000, Neuromics) and N-terminus MOR-1 Ab U0126 (1 µM) was present for 28 h, while opioid (1: 200, Santa Cruz). KOR-1 immunoreactivity agonists were added for 24 h and [methyl-3H] was detected with a rabbit polyclonal KOR-1 thymidine (0.02 µCi/ml) was present for the last 4 specific Ab (1:50, Santa Cruz). Horseradish h. [methyl-3H]Thymidine incorporation was peroxidase linked IgGs were applied as secondary measured as described in our previous studies with Abs. Bands were visualized by some modifications (44). Briefly, cells are washed chemiluminescence detection as described for with PBS, followed by incubation with 5% ERK. trichloroacetic acid at 4º C for 30 min. Then cells SOX-1 immunostaining and counting: D3 were collected in 2% NaHCO /0.1 N NaOH and 3 ES cells were grown in chamber slides, in [methyl-3H] thymidine incorporation was serum/LIF/ME containing media and were treated determined by liquid scintillation counting. with 1 µM DAMGO or U69, 593 for 24 h. In BrdU labeling of cells: D3 ES cells or RA- some cases, cells were pretreated for 1 h either induced NPs were grown until they reached about D with MOR antagonist (CTAP, 1 µM) or KOR 50% confluency. They were then treated with o w n antagonist (nor-BNI, 1 µM) before addition of either 1 µM DAMGO or U69, 593 for 24 h. In lo a opioid agonists for 24 h. Control and opioid- some cases, cells were pretreated with inhibitors or de d treated ES cells were washed with PBS and fixed opioid antagonists as described in the figure fro m with 2% PA for 10 min at room temperature. legends. At the end of the treatment, media was h Cells were then permeabilized with 0.4% replaced with BrdU labeling medium (10 µM, ttp Triton/PBS for 5 min and incubated in PBS Molecular Probes, BrdU detection kit I) and cells ://w w w containing 10% FCS and 0.4% Triton X-100 for were incubated for 20-30 min. After several .jb 30 min to reduce nonspecific binding, followed by washes, cultures were fixed with ethanol fixative c.o incubation with chicken polyclonal SOX-1 Ab for 20 min at -20º C. For nestin co-labeling, cells brg/ (1:500, Chemicon) overnight at 4(cid:176) C. After were incubated together with a monoclonal BrdU y g u washing, Alexa Fluor 594 (red) conjugated anti- Ab (1:10, Roche kit) and polyclonal nestin Ab es chicken secondary Ab (1:1000, Molecular Probes) (1:1000, Covance) at 37(cid:176) C for 30 min. For Sox-1 t on D was applied for 1 h at room temperature. Cells co-labeling, cells were first incubated with a ec e were counterstained with DAPI (1:200) to monoclonal BrdU Ab (1:10, Roche kit) at 37(cid:176) C mb e visualize nuclei for cell counts. Chamber slides for 30 min, then incubated in PBS containing 10% r 2 4 were treated with anti-fade reagent (Molecular FCS and 0.4% Triton X-100 for 30 min to reduce , 2 0 Probes) and examined for immunofluorescence as nonspecific binding, followed by counter-staining 18 described above. NIH Image J version 1.32 with a chicken Sox-1 polyclonal Ab (1:500, sofware was used to count cells. The % of SOX-1 Chemicon) overnight at 4(cid:176) C. After washing, cells positive cells was estimated from the ratio were treated with fluorescein conjugated anti- between the total number of cells (DAPI stained) mouse IgG (green, 1:10, Roche kit) together with and the number of SOX-1 stained cells. Cells Alexa Fluor 594 (red) conjugated anti-chicken or from 3-5 fields per slide were counted and 3-4 anti-rabbit secondary Abs (1:1000, Molecular slides per treatment group were used for the Probes) for 1 h at room temperature. Slides were counting. The total number of counted cells was examined for immunofluorescence as described 500-800/treatment group. above. Cell proliferation assays: For these studies, equal numbers of cells were seeded per well in RESULTS twelve-well dishes. Cell numbers were estimated by serial dilutions and counting cells (volume of Characterization of ES cells and ES cell- 10 µl) in several visual fields using a Bright-Line derived NPs: The state of differentiation of D3 or Hemacytometer and light microscopy. Basal PRX ES cells and RA-induced ES cell-derived levels or opioid-induced changes in DNA NPs was characterized by immunocytochemistry 4 and immunoblotting. For this purpose, D3 and gene expression or protein levels (Fig. 2) in D3 PRX ES cells were maintained under NPs. Brain homogenate was used as a positive serum/LIF/ME induced self-renewal conditions or control and lysates from late passage immortalized they were differentiated in the presence of RA. rat astrocytes were used as negative controls For the immunocytochemistry experiments, because these cells lose detectable amounts of cultures were stained with nestin (a neural ORs with passaging (42 and unpublished progenitor marker) Ab and DAPI (a nuclear observations). marker). As seen in double DAPI/nestin images Immunofluorescence microscopic analysis (Fig.1, D3 and PRX), there were no nestin (red) further supports the occurrence of MOR-1 and stained cells among the undifferentiated ES cells, KOR-1 in D3 ES cells and NPs (Fig. 3). Once whereas RA-induced NPs show heavy staining again, the undifferentiated state of D3 ES cells is with nestin (Fig.1, dD3 & dPRX). confirmed by the absence of nestin (green) stained Since the transcription factor Oct4 has been cells (D3, Fig. 3E & G). In contrast, D3 ES cell- demonstrated to be vital for the formation of self- derived NPs (dD3) show heavy nestin staining renewing pluripotent ES cells, cell lysates from (Fig. 3 F & H). The presence of MOR-1 (red) undifferentiated D3 ES cells or RA-induced NPs immunoreactive cells was confirmed by applying were probed with an Oct4 Ab (Chemicon, 1:100) the two polyclonal MOR-1 Abs that were used for D as presented in Fig.1B. The densitometry data the immunoblotting experiments (Fig. 2). It o w n from the Oct4 immunoblots suggest that there is appears that the N-terminus MOR-1 Ab detects the lo a about a 50% reduction in Oct4 protein levels in D3 receptor mainly on the cell surface of ES cells de d ES cell-derived NPs (3767 ± 85, n=3) in (Fig. 3B) and NPs (Fig. 3D). Figures 3 E & F fro m comparison to the levels of this transcription factor show MOR-1 immunostaining using the C- h in undifferentiated ES cells (6954 ± 463, n=4). terminus MOR-1 Ab. KOR-1 immunoreactive ttp MOR-1 and KOR-1 gene expression and cells were detected in self-renewing D3 ES cells ://w w w immunoreactivity was detected in ES cells and (Fig. 3G) and in NPs (Fig. 3H). .jb in ES cell-derived NPs: Three independent Opioid regulation of ERK phosphorylation c.o approaches were taken to determine the presence in undifferentiated ES cells: Having found that brg/ of MOR-1 and KOR-1 in undifferentiated D3 ES MOR-1 and KOR-1 are present in both y g u cells and RA-induced ES cell-derived NPs: qRT- undifferentiated ES cells and in ES cell-derived es t o PCR, immunoblot analysis of cell lysates and NPs, we sought to determine the functionality of n D immunofluorescence microscopy of cells at these receptors. Since MAP kinases have been e c e different stages of development. In qRT-PCR implicated in regulation of cell proliferation and m b e experiments, we found that in comparison with ES differentiation, we studied opioid regulation of r 2 4 cells, NPs have 1.37 and 2-fold increases in MOR- ERK activation in ES cells and ES cell-derived , 2 0 1 and KOR-1 gene expression, respectively (n=3). NPs. MOR- or KOR-induced ERK activation was 18 As shown in figure 2, immunoblotting performed measured by performing immunoblotting with polyclonal MOR-1 (C-terminus, Neuromics), experiments with a phospho-ERK Ab that MOR-1 (N-terminus, Santa Cruz) and KOR-1 recognizes the active form of ERK. (Santa Cruz) specific Abs shows the presence of Undifferentiated ES cells were grown in media major 50 and 55 kDA bands that correspond to the devoid of serum/LIF/ME for at least 24 h. expected molecular weights for MOR-1 and KOR- Thereupon, cells were treated with the µ selective 1, respectively. In addition to the 50 kDa band, opioid agonist DAMGO (0.1 m M, 5 min) or the κ the N-terminus MOR-1 Ab reveals the existence selective opioid agonist U69,593 (0.1 m M, 5 min). of higher molecular weight bands (Fig. 2, lanes 6& Both opioids stimulated ERK activity in D3 ES 7), which were suggested to correspond to the cells by about 2-fold (Fig. 4). Similar data were glycosylated form(s) of the receptor by several obtained with undifferentiated PRX ES cells (data groups (45-47). Both MOR-1 and KOR-1 bands not shown). The corresponding, selective MOR were detected in cell lysates, obtained from D3 ES (CTAP) and KOR (nor-BNI) antagonists blocked cells maintained under self-renewal conditions agonist induced stimulation of ERK (serum/LIF/ME). RA treatment does not appear to phosphorylation, supporting the notion that these induce significant changes in MOR-1 or KOR-1 are OR-mediated effects. 5 Opioid effects on ERK signaling were also opioid-enhanced proliferation in undifferentiated evaluated in cells grown in serum/LIF/ME PRX ES cells as well. containing media, conditions that are expected to Although basal levels of proliferation in the maintain D3 ES cells in an undifferentiated, self- presence and absence of U0126 were set at one to renewing state. Under these conditions, both simplify the estimation of opioid effects (controls DAMGO and U69,593 induced a sustained in Fig. 6), we found that U0126 pretreatment activation of ERK signaling throughout the 24 h enhanced basal levels of D3 ES cell proliferation treatment period (Fig. 5). For DAMGO, there was by about 40% (basal levels in dpms: 8726 ± 1168 2-fold or greater activation of ERK at all time vs basal in the presence of U0126: 12005 ± 1225). points (Fig. 5B). Maximal phosphorylation was This finding supports the notion that seen at 5 min and 12 h with each having greater undifferentiated ES cells (in the absence of than 4-fold stimulation of ERK. The ERK opioids) can proliferate even when ERK signaling activation observed with U69,593 was also is blocked and suggest that the ERK/MAP kinase sustained for all time points (Fig. 5C). This signaling pathway may not be involved in ES cell activation was slightly less all overall, although self-renewal. the greatest phosphorylation seen was over 6-fold Evidence for opioid-induced D3 ES cell at 5 min. differentiation: To determine whether the opioid- D Although serum induces high basal levels of induced proliferation of ES cells is due to an o w n phosphorylated ERK in many cell types, ERK increase in self-renewal or asymmetric cell lo a basal levels were low in ES cells grown in serum division to form NPs, we examined the phenotype de d containing media (Fig. 5A). The low basal levels of the opioid-induced “newly formed” cells. fro m of ERK phosphorylation in these cells support the Undifferentiated ES cells were treated with h notion that ERK activity may not be required for opioids, labeled with BrdU, followed by staining ttp self-renewal of ES cells as recently reported (see with nestin and BrdU Abs. The ://w w w Introduction). immunofluorescence analysis raises the possibility .jb Opioids induce limited proliferation of that DAMGO and U69, 593 induce the appearance c.o undifferentiated ES cells via the ERK pathway: of nestin-positive cells that may be NPs derived brg/ To correlate the ERK data with cell proliferation, from ES cells (Fig. 7A). The representative y g u we studied opioid regulation of ES cell growth. double images of control cells show mainly BrdU es t o In undifferentiated D3 ES cells, the findings are (green) staining and only a few nestin (red) stained n D that DAMGO, U69,593, and morphine all induced cells. e c e limited proliferation. Specifically, DAMGO and To determine further the nature of opioid- m b e morphine caused a 40% rise in proliferation over induced cells, we performed a quantitative r 2 4 basal levels, whereas U69,593 caused a 30% analysis of Sox-1 immunostained, control and , 2 0 increase (Fig. 6). The mitogen, EGF (10 ng/ml) opioid-treated ES cells (Fig. 7 B-D). Sox-1 is a 18 induced about a 100% increase in ES cell selective marker of proliferating NPs; it belongs to proliferation (N=3, P<0.05). EGF stimulates ES a family of transcription factors that are expressed cell proliferation via the ERK/MAP kinase in ectodermal cells upon acquisition of neural signaling pathway (48). Since ERK mediates progenitor identity (49, 50). By counting cells (≥ mitogen-induced cell proliferation, the MEK- 1,000 cells/treatment group), we estimated that selective inhibitor U0126 was added to determine only a few of the control cells (4 ± 1.1%, n=8) the role of ERK in opioid enhanced cell were Sox-1 positive. Exposure of undifferentiated proliferation. Pretreatment of ES cells with U0126 ES cells to 1 µM MOR (Fig. 7B) or KOR (Fig. blocked the opioid-induced increase of 7C) opioids for 24 h initiated the appearance of proliferation seen in the absence of the inhibitor, about 7-8 fold more Sox-1 positive cells than in suggesting that opioids act through ERK/MAP control cells (Fig. 7D&E). The corresponding, kinase to increase proliferation in D3 ES cells selective MOR (CTAP) and KOR (nor-BNI) (Fig. 6). In PRX ES cells, morphine also induced antagonists blocked agonist induced appearance of a 30% increase in cell proliferation over basal Sox-1 positive cells, suggesting that this is an OR- levels (data not shown). U0126 abolished this mediated process. CTAP (10 ± 2.6%, n=4) and increase, suggesting that ERK is responsible for 6 nor-BNI (2 ± 0.5%, n=4), alone had no significant levels. This finding may be due to the possibility effect on the number of Sox-1 labeled cells. that NPs secrete endogenous κ opioid peptides that Opioid regulation of ERK phosphorylation may have inhibitory effects on basal levels of cell in RA-induced ES cell-derived NPs: To division. Upon addition of KOR antagonist, the investigate opioid modulation of ERK activity effects of both endogenous and exogenous KOR upon RA-induced differentiation of ES cells, RA- ligands were suppressed, resulting in an increase induced EBs were dissociated and plated in serum in proliferation over basal levels. In contrast, in containing DMEM for an additional 2-5 days the presence of CTAP alone, NP proliferation was (short-term differentiation) or for 5-12 days (long- similar to basal levels (34 ± 5.4%, n=4, P = 0.168). term differentiation). After growing in serum- The involvement of the ERK signaling deprived media for 24 h, these cells were treated pathway in opioid regulation of NP proliferation with opioids for various times and ERK was evaluated by treatment of the cells with the phosphorylation was measured by immunoblotting MEK inhibitor, U0126 for 1 h before opioid (Fig. 8). The results support the following agonist exposure (Fig. 10). U0126 alone did not conclusions: a) basal levels of ERK significantly affect basal levels of NP proliferation phosphorylation in long-term differentiated cells (44 ± 3.2%, n=4, P = 0.832 vs controls of 43 ± were significantly higher (2.2±0.2, n=9, P < 0.05) 4.1%, n=6). Interestingly, administration of D than basal levels of ERK activation in short-term U0126 in the presence of DAMGO did not reverse o w n differentiated cells (NPs), suggesting that terminal the inhibitory effect induced by the MOR agonist lo a differentiation of these cells may require activated alone suggesting that MOR regulation of NP de d ERK; b) MOR and KOR agonists elicit similar proliferation is independent of the ERK signaling fro m effects on ERK activation in short- and long-term pathway. Furthermore, the inhibitory effect of h differentiated cells (Figs. 8&9). U69,593 was only partially reversed upon ttp More detailed time course experiments with blockade of the ERK signaling by U0126. ://w w w DAMGO and U69, 593 were also conducted (Fig. .jb 9). These data further confirm that the two opioids c.o induce a similar biphasic ERK activation profile DISCUSSION brg/ with peaks occurring at 2-15 min and at about 2-6 y g u h in short-and long-term differentiated ES cells The analysis of our data reveals several es (Fig. 9A-D). Thus, the opioid-induced sustained important findings: 1. MOR-1 and KOR-1 gene t on D ERK activation seen in undifferentiated ES cells expression and immunoreactivity was detected in e c e changes in differentiated cells to a biphasic ERK undifferentiated ES cells and in ES cell-derived m b e activation profile. NPs. This is the first evidence of the occurrence of r 2 4 Opioids inhibit NP proliferation: To MOR-1 and KOR-1 in blastocyst-derived ES cells. , 2 0 evaluate opioid effects on RA-induced NP 2. MOR-1 and KOR-1 that occur in ES cells and 18 proliferation, we performed a quantitative analysis NPs, show functionality as established by the of Sox-1/BrdU double labeled, control and opioid- detection of opioid-induced temporal regulation of treated NPs (Fig. 10). Since Sox-1 is a selective ERK/MAP kinase signaling. A sustained marker of proliferating NPs, we counted the BrdU activation of ERK was characteristic for the opioid labeled cells among the Sox-1 positive cells and treated undifferentiated ES cells, whereas the RA- thus estimated the changes in NP proliferation induced NPs showed a biphasic profile of opioid upon treatment with opioids. The data presented induced ERK activity. 3. The ERK signaling in figure 10 indicate that 1 µM DAMGO or pathway mediated µ- and κ-opioid-induced limited U69,593 decreased proliferation of NPs by 50- proliferation in undifferentiated D3 ES cells, even 60% in 24 h. The inhibitory effect of both opioid though in the absence of opioids, these cells may agonists was reversed by the corresponding MOR not require ERK activation for their self-renewal. (CTAP) and KOR (NorBNI) antagonists, More importantly, opioids induced ES cell indicating that DAMGO and U69,593 were acting asymmetric division to generate nestin/SOX1- via their respective receptors. Interestingly, positive NPs and reduced the self-renewal of ES norBNI alone induced a 27% increase (59 ± 4.7%, cells. Thus, a novel finding here is that opioid- n = 4, P = 0.0149) in cell proliferation over basal induced sustained activation of ERK may play a 7 role in the initial differentiation of ES cells. 4. levels of ERK suggest that activation of this kinase Opioids attenuated proliferation of the population may have inhibitory effects on ES cell self- of NPs that were generated upon differentiation of renewal. In addition, under self-renewal ES cells with RA. This inhibitory effect is ERK conditions (serum/LIF/ME), ES cells maintain low independent for DAMGO and partially for basal ERK activity, data that further support the U69,593 and correlated with MOR and KOR lack of requirement for ERK activity by these cells agonist induction of a biphasic rather than a (Fig. 5). In contrast, the results also suggest that sustained ERK activation profile in NPs. opioid regulation of ES cell asymmetric cell In prior studies, KOR was found in P19 division in undifferentiated ES cells is ERK embryonic carcinoma cell line, which contains dependent. Therefore, we propose that in pluripotent stem cells (51-53). Although these undifferentiated ES cells, two processes take cells are similar to ES cells derived from 4-5 day place: there is an ongoing ERK-independent, old mouse embryos, they are transformed cells and symmetric cell division leading possibly to ES cell therefore capable of overexpressing genes not self-renewal and an opioid-induced ERK- expressed in their parent cell. RA promoted dependent, asymmetric cell division leading to ES expression of MOR gene, while KOR gene was cell differentiation (Fig. 11). Thus, both µ and κ first suppressed and then reactivated in P19 cells opioids promote ES asymmetric cell division at a D (54-56). KOR was also found on cell surface and slightly more rapid rate than that of o w n nuclear fractions of GTR1 ES cells (57-59). In serum/ME/LIF-induced self-renewal of ES cells. lo a addition, MOR and δ opioid receptor (DOR), but The above findings raise the following de d not KOR are expressed in adult hippocampal questions: If ERK signaling is not the regulator of fro m progenitors and opioid peptides such as β- ES cell division/self-renewal than what is the h endorphin can regulate proliferation of these signaling pathway that modulates this process? It ttp progenitor cells via ERK (60). Reduced ERK was proposed that mouse ES cells utilize PI3K to ://w w w signaling via MOR decreases proliferation, of progress through the G1 phase and to avoid .jb these progenitors, increases the number of in vitro differentiation (18, 27). What is the mechanism of c.o generated neurons and reduces the number of opioid induction of NP appearance? It has been brg/ astrocytes and oligodendrocytes. Finally, opiates suggested that Sox proteins might contribute to the y g u were found to inhibit neurogenesis in the adult rat transcriptional activation of the MOR gene and es t o hippocampus (61). that MOR could mediate some Sox regulated n D We have characterized the mechanism of developmental processes (64). e c e ERK activation by ORs and demonstrated that ERK signal duration impacts different m b e opioids either enhanced or inhibited DNA cellular responses in many cells and may produce r 2 4 synthesis in several types of primary cultures and different outcomes in the same cells, such as , 2 0 cell culture model systems (40-42, 44, 62, 63). In proliferation, differentiation or apoptosis (10, 26, 18 most of these cases, opioids inhibited mitogen 44, 65-68). In some cells, sustained but not stimulated proliferation as seen here (Fig. 10). It transient activation of ERK is required to initiate was important to determine how opioids regulate proliferation (41, 65, 69, 70, McLennan, Kiss et the ERK signaling pathways in ES cells and in ES al., 2006 manuscript in preparation). Sustained cell-derived NPs. As discussed in the ERK activity appears to be required by many cells Introduction, several studies suggest that ERK to pass the G1 restriction point and to enter S activation may not be required for ES cell self- phase, in which cellular DNA is replicated (65, renewal (4, 11-14). Our thymidine incorporation 71). In contrast, other studies have shown that results further support this hypothesis. For Ras/Raf mediated a transient EGF-induced ERK example, the finding that MEK inhibitor, U0126 activation that leads to proliferation, whereas alone enhanced basal levels of ES cell Rap/Raf mediated sustained activation of ERK is proliferation by about 40% supports the notion required for PC12 cell differentiation (22, 72). that undifferentiated ES cells can proliferate when Our data suggest that the opioid induced sustained ERK signaling is blocked and suggest that ERK activation of ERK triggers ES asymmetric cell activity may not be necessary for ES cell self- division and the “newly” formed cells appear to be renewal. Moreover, our U0126 data on basal ES cell-derived NPs. 8 The important role of the ERK signaling phosphorylation by opioids accompanies the pathway in neural differentiation has been well- opioid-induced inhibition of proliferation in NPs. established. While some reports indicate that ERK Although MOR regulation of NP proliferation was signaling inhibits differentiation of some types of independent of the ERK signaling pathway, the cells (73, 74), other studies support the idea that inhibitory effect of U69,593 was only partially the ERK signaling may be a positive regulator of reversed upon blockade of the ERK signaling by this process (75). A recent paper discusses the U0126. This finding may be explained by the existence of a biphasic regulation of ERK activity involvement of dual signaling pathways in KOR during myogenic differentiation and suggests that modulation of NP proliferation. In addition to the signaling pathway(s) may play a dual role in ERK signaling, p38/MAP kinase is a candidate for this multi-step process, wherein the late phase is a second signaling pathway that could be required responsible for the formation of postmitotic for KOR inhibition of NP proliferation. myotubes (76). A similar study shows that an The absence of a developmental phenotype in early stimulation and late inhibition of ERK opioid receptor knock out mice has been reported activity by IGF mediates the switch in IGF action (79). This is not uncommon given the functional from inhibition to stimulation of skeletal muscle redundancies that cells display after targeted gene cell differentiation (77). Finally, it was found that disruption of critical signaling molecules such as D the early stage of neuronal differentiation of GPCRs. There are numerous accounts in the o w n mouse ES cell line P19 triggered by aggregation of literature regarding the absence of alteration of lo a the cells and RA treatment is accompanied by function after the loss of GPCR gene expression de d biphasic activation of ERK signaling: a transient due to redundant or compensatory signaling fro m phase and a second, sustained ERK activation that mechanisms (80, 81). In some instances, h is maintained until the appearance of neural developmental phenotypic changes are subtle after ttp phenotype (78). gene disruption and only seen when the organism ://w w w Here, we propose that opioid-induced is stressed as it is in its natural environment (82). .jb sustained activation of ERK may be a required Alternatively, MOR and/or KOR may be c.o step for the development of ES cell-derived NPs dispensable for development and the observations brg/ and opioid-induced biphasic ERK phosphorylation made here may be only of relevance to maternal y g u may play a supporting role in the increased opiate abuse. es t o differentiation rate during the generation and In conclusion, our studies suggest that MOR- n D maturation of NPs. This hypothesis is based on 1, KOR-1 and their exogenous ligands are able to e c e the following findings: As seen in figure 7, basal modulate ES cell proliferation and differentiation. m b e levels of ERK activity are higher in long-term Thus, opioids and opioid-induced ERK signaling r 2 4 differentiated ES cells than in short-term may play an important role in ES cell fate , 2 0 differentiated cells (NPs). In addition, µ- and κ- decisions by directing the cells to specific 18 opioids induce a moderate, biphasic activation of lineages. ERK signaling in short- and long-term differentiated ES cells. More importantly, we found that the biphasic stimulation of ERK 9 REFERENCES 1. Stavridis, M. P., and Smith, A. G. (2003) Biochem. Soc. Trans 31, 45-49. 2. Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K., and Dalton, S. (2005) Development 132, 885-896. 3. Wobus, A. M., and Boheler, K. R. (2005) Physiol. Rev. 85, 635-678. 4. 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