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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: beltchem@slu.edu

Growth factors, hormones and

neurotransmitters have been implicated in the

regulation of stem cell fate. Since various

neural precursors express functional

neurotransmitter receptors, which include G

protein coupled receptors (GPCRs), it is

anticipated that they are involved in cell fate

decisions. We detected µ opioid receptor

(MOR-1) and κ opioid receptor (KOR-1)

expression and immunoreactivity in embryonic

stem (ES) cells and in retinoic acid induced ES

cell-derived, nestin-positive, neural progenitors.

Moreover, these GPCRs are functional as

[D-ala2,mephe

4,gly-ol

5] enkephalin, (DAMGO)

a MOR selective agonist and U69,593, a KOR

selective agonist induce a sustained activation

of extracellular signal-regulated kinase (ERK)

signaling throughout a 24 h treatment period in

undifferentiated, self-renewing ES cells. Both

opioids promote limited proliferation of

undifferentiated ES cells via the ERK/MAP

kinase signaling pathway. Importantly,

biochemical and immunofluorescence data

suggest that DAMGO and U69, 593 divert ES

cells from self-renewal and coax the cells to

differentiate. In retinoic acid-differentiated ES

cells, opioid-induced signaling features a

biphasic ERK activation profile and an opioid-

induced, ERK independent inhibition of

proliferation in these neural progenitors.

Collectively, the data suggest that opioids may

have opposite effects on ES cell self-renewal

and ES cell differentiation and ERK activation

is only required by the latter. Finally, opioid

modulation of ERK activity may play an

important role in ES cell fate decisions by

directing the cells to specific lineages.

The maintenance of ES cells in an

undifferentiated state in vitro is dependent on self-

renewing cell division in the presence of leukemia

inhibitory factor (LIF)1, which signals through

various receptor complexes (reviewed in 1-3, see

also 4, 5). Mouse ES cells can be induced to

differentiate into neural cells in the presence of

retinoic acid (RA) in vitro (reviewed in 6, 7).

During this induction, ES cells undergo a series of

steps that resemble key stages in the early mouse

embryo, supporting the hypothesis that the in vitro

pathway represents the normal developmental

pathway (8, 9).

Considerable effort has been recently

devoted to characterizing intrinsic, extrinsic

factors and signaling pathways regulating

proliferation and differentiation of stem cells. The

ERK/MAP kinase signaling pathway has been

implicated in both proliferation and differentiation

of many cell types, including stem cells (10). In

several studies ERK activation had a negative

influence on self-renewal/proliferation in murine

ES cells (4, 11-14). Moreover, LIF-dependent

activation of STAT3 was not mediated by ERK

activity (15). However, a dual role for the

Ras/ERK pathway was proposed for ES cells,

affecting both their division and differentiation

(16). Ras activation down-regulated levels of

Nanog, a protein that is normally expressed in

high amounts in self-renewing ES cells (17). A

functional role for ERK was proposed in the PI3K

dependent regulation of ES cell self-renewal (18).

The importance of ERK activation and its duration

for cell differentiation has been investigated in

various cells (19-22). Some evidence suggests that

RA-induced inhibition of LIF signaling in ES cells

is ERK independent (23). However, a specific

requirement of the ERK pathway was reported for

RA-dependent commitment of murine ES cells

http://www.jbc.org/cgi/doi/10.1074/jbc.M603862200The latest version is at JBC Papers in Press. Published on September 1, 2006 as Manuscript M603862200

Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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(24). Crosstalk may occur by non-cannonical

actions of RA with the MAP kinase

phosphorylation cascade. RA-induced

differentiation of ES cells may be achieved by

restricting nuclear entry of activated ERK (25).

ERK activation is required for regulation of cyclin

D1 levels, the increase of which parallels ES cell

differentiation (26, 27).

Neurotransmitters as well as growth factors

and hormones can influence cell division and

differentiation of self-renewing stem cells and

neural progenitors (NPs) via ERK in some cases

(28, 29). For example, stimulation of the

muscarinic acetylcholine receptor, a GPCR,

induces DNA synthesis in rat cortical

neuroepithelium progenitors via stimulation of

PI3K and ERK (30). The activated CB1 receptor

inhibits neuronal progenitor differentiation via

attenuation of ERK signaling in E17 cortical

cultures and in adult dentate gyrus (31). This

endocannabinoid system was also found to

promote astroglial differentiation by acting on

neural progenitor cells (32). Glutamate activates

NMDA receptors and promotes proliferation of

E15 precursors derived from the germinal zone of

the ventral telencephalon in vivo and in vitro (33).

Haloperidol acting via dopamine D2 receptors

increases the number of NPs, neurons and glia in

adult rat brain (34). G protein βγ subunits of

heterotrimeric G proteins are required for proper

mitotic-spindle orientation and asymmetric cell

fate decisions of cerebral cortical progenitors (35).

These studies were performed with brain derived

stem cells or NPs, but little is known about the

consequences of GPCR-ERK crosstalk in

blastocyst-derived ES cells or their NPs. Since the plasticity of uncommitted stem cells

has opened new perspectives in tissue

regeneration, recent research has been directed to

understand the signaling mechanisms that control

proliferation and/or differentiation of ES cells.

Here, we detected µ and κ ORs in ES cells and in

ES cell-derived NPs. More importantly, we found

that both opioids induced ES cell differentiation

via ERK and an ERK independent attenuation of

proliferation in RA-driven NPs

EXPERIMENTAL PROCEDURES

Mouse ES cells and their growth conditions: D3 (ATCC) and PRX-129/S6 # 7 ES cells

(Primogenix, Inc.) were used. PRX-129/S6 # 7 ES

cells were isolated from the inner cell mass of day

3.5 129/S6/SvEv mouse blastocyst. Cells have a

normal male karyotype and are specific pathogen-

free. They were injected into blastocysts and

produced chimeras at a high rate (first injection:

10/15 pups, no perinatal death). PRX-129/S6 # 7

cells were grown on mouse embryonic fibroblasts

(MEFs) in the presence of LIF, passage 10 cells

were propagated 2-3 times on gelatin-coated flasks

and these cells are currently used in the laboratory.

Quality control and characterization of the D3 ES

cells is documented by their depositor (36). D3

ES cells were propagated on irradiated STO cell

feeders (ATCC) for several passages before they

were transferred to gelatin-coated flasks.

D3 and PRX-129/S6 # 7 ES cells were

maintained in Dulbecco’s modified Eagles

medium (DMEM) containing 10% fetal calf

serum, LIF (100 ng/ml) and 0.1 mM 2-

mercaptoethanol (ME) on gelatin-coated dishes.

These growth conditions are known to prevent ES

cells from differentiating (6, 37, 38). Here, cells

grown under these conditions will be described as

“undifferentiated, self-renewing ES cells”.

The neural induction of ES cells entails the

“4-/4+ protocol” which has two phases (8, 39). In

the first phase, cells are cultured in non-adhesive,

bacterial grade Petri dishes in serum containing

media (without LIF and ME) in the absence of RA

for 4 days. During this phase, EBs of different

sizes appear. In the second phase, cells are grown

in RA (1 µM) and serum containing media for an

additional 4 days. Differentiation of these cells is

also a two-phase process: early differentiation,

during which nestin-positive cells develop and

their terminal differentiation is the final step in ES

cell growth. For this purpose, EBs are dissociated

and cultured on tissue culture plates in serum

containing DMEM for an additional 2-5 days

(short-term differentiation) or for 5-12 days (long-

term differentiation). The RA induced, short-term

differentiated cells are referred to as NPs.

Opioid treatments: ES cells or ES cell-

derived progenitors are maintained in their

corresponding growth media. Opioid agonists

(0.1-1 µM, DAMGO, a MOR selective agonist or

U69,593, a KOR selective agonist) were added for

various times (up to 24 h). In some experiments,

cells were pre-incubated with opioid antagonists (1

µM, 60 min, CTAP, MOR selective or nor-BNI,

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KOR selective) and then treated with the

corresponding opioid agonist (0.1 µM) for a given

time.

Measurement of ERK activity: For these

experiments, cells were grown in six-well plates.

In most cases, ES cells were maintained in serum-

containing media upon opioid treatment. In

contrast, ES cell-derived NPs were grown in

media deprived of serum for 24 h before treatment

with opioids. ERK phosphorylation was measured

by performing immunoblotting experiments with a

phospho-ERK Ab that recognizes the active form

of ERK (40-42). Cells were washed with PBS,

lysed with buffer: 20 mM HEPES, 10 mM EGTA,

40 mM β-glycerophosphate, 2.5 mM MgCl2, 2

mM sodium vanadate, 1% Nonidet-40, 1 mM

PMSF, 20 µg/ml aprotinin and 20 µg/ml leupeptin,

spun at 14,000 g and protein concentration of the

supernatants was determined. Cell lysates (10-20

µg protein/lane) were separated by 10% SDS-

PAGE. Proteins were blotted on Immobilon

membranes. Nonspecific sites were blocked with

5% milk in Tris-buffered saline + 0.2% Tween 20.

Blots were incubated with monoclonal phospho-

ERK Ab (1:2000, Cell Signaling) for at least 15 h

at 4°C, followed by incubation with a HRP

conjugated IgG (1:2000, Sigma) for 1 h at room

temperature. Bands were visualized with an ECL

chemiluminescence detection system (GE

Healthcare) and exposure to Classic Blue sensitive

X-ray film. Band intensities were determined by

densitometry with a Kodak DC120 digital camera,

Kodak ds 1D version 3.0.2 (Scientific Imaging

Systems) and NIH Image ImageJ version 1.32

sofware. ERK stimulation in opioid-treated cells

was expressed as fold change over basal levels in

control cells.

OR and nestin immunofluorescence microscopy: ES cells or ES cell-derived NPs were

grown in glass chamber slides (Nunc). The cells

were fixed in 4% PA for 20 min at room

temperature and permeabilized in 0.1%

Triton/PBS for 5 min. Cells were then incubated

in PBS containing 0.5% BSA and 0.1% Tween 20

for 30 min to reduce nonspecific binding, followed

by overnight incubation at 4°C with the following

rabbit polyclonal OR Abs: MOR-1 raised against

C-terminus (Neuromics, 1:2500); MOR-1 raised

against N-terminus (Santa Cruz, 1:50); KOR-1

(Santa Cruz, 1:50). In some cases, a mouse

monoclonal nestin Ab (Chemicon, 1:1000) was

added together with the OR Abs. After washing,

Alexa Fluor 594 (red) and/or Alexa Fluor 488

(green) conjugated secondary Abs (1:1000) were

applied for 1 h at room temperature. DAPI

(1:200) was added together with the secondary

Abs. The slides were treated with anti-fade

reagent (Molecular Probes) and examined for

immunofluorescence with NIKON-OPTIPHOT-2

or OLYMPUS AH3 microscopes with

simultaneous recording of dual fluorescence label

images.

Real-time quantitative (q)RT-PCR: Total

RNA from ES cells and RA-induced NPs was

isolated using Qiagen’s RNeasy Mini Kit (Qiagen,

Valencia, CA) according to the manufacturer’s

instructions. cDNA was generated from 1 µg total

RNA using the High Capacity cDNA Archive Kit

from Applied Biosystems (ABI, Foster City, CA)

with random primers as described by the

manufacturer. qRT-PCR was performed with

SYBR green chemistry on an ABI 7500

instrument. The primers were designed using

Primer Express (ABI) and supplied by Integrated

DNA Technologies (Coralville, IA). Primer

sequences for MOR-1 were: forward, 5’-

CCACTAGCACGCTGCCCTT-3’; reverse, 5’-

GCCACGTTCCCATCAGGTAG-3’. Primer

sequences for ΚOR-1 were: forward, 5’-

AGAGAGAGAAGCGGCAAGCA-3’; reverse,

5’- GCCAAGGCTCACTAACTCCAA-3’. The

cDNA templates for qRT-PCR were diluted 1:10

and the 50 µl SYBR-green reaction consisted of 1

x SYBR-green Master Mix (ABI), 300 nM

forward and reverse primers, and 5 µl of diluted

cDNA. Four replicates of each qRT-PCR reaction

were run on 96-well plates. The amplification

efficiencies of MOR-1 and KOR-1 were consistent

with those of the endogenous control, GAPDH.

Relative quantification measurements were made

as described (43) by using the comparative CT

Method (∆∆CT Method) in which the gene CT

values are normalized by subtracting GAPDH CT.

Immunoblotting with MOR-1 and KOR-1

specific Abs: For detection of receptor protein

levels in ES cells or ES cell-derived NPs,

immunoblotting with MOR-1 and KOR-1 specific

Abs was adopted. For this purpose, cells were

lysed in a modified RIPA buffer: 50 mM Tris-

HCl, pH 7.4; 1% NP40; 0.25% sodium

deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM

PMSF; 1 µg/ml leupeptin; 1 µg/ml aprotinin; 1

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mM Na3VO4; 1 mM NaF and samples containing

20-50 µg protein were loaded on 10 % SDS gels.

Receptor band(s) were detected using two rabbit

polyclonal MOR-1 Abs: C-terminus MOR-1 Ab

(1:2000, Neuromics) and N-terminus MOR-1 Ab

(1: 200, Santa Cruz). KOR-1 immunoreactivity

was detected with a rabbit polyclonal KOR-1

specific Ab (1:50, Santa Cruz). Horseradish

peroxidase linked IgGs were applied as secondary

Abs. Bands were visualized by

chemiluminescence detection as described for

ERK.

SOX-1 immunostaining and counting: D3

ES cells were grown in chamber slides, in

serum/LIF/ME containing media and were treated

with 1 µM DAMGO or U69, 593 for 24 h. In

some cases, cells were pretreated for 1 h either

with MOR antagonist (CTAP, 1 µM) or KOR

antagonist (nor-BNI, 1 µM) before addition of

opioid agonists for 24 h. Control and opioid-

treated ES cells were washed with PBS and fixed

with 2% PA for 10 min at room temperature.

Cells were then permeabilized with 0.4%

Triton/PBS for 5 min and incubated in PBS

containing 10% FCS and 0.4% Triton X-100 for

30 min to reduce nonspecific binding, followed by

incubation with chicken polyclonal SOX-1 Ab

(1:500, Chemicon) overnight at 4°C. After

washing, Alexa Fluor 594 (red) conjugated anti-

chicken secondary Ab (1:1000, Molecular Probes)

was applied for 1 h at room temperature. Cells

were counterstained with DAPI (1:200) to

visualize nuclei for cell counts. Chamber slides

were treated with anti-fade reagent (Molecular

Probes) and examined for immunofluorescence as

described above. NIH Image J version 1.32

sofware was used to count cells. The % of SOX-1

positive cells was estimated from the ratio

between the total number of cells (DAPI stained)

and the number of SOX-1 stained cells. Cells

from 3-5 fields per slide were counted and 3-4

slides per treatment group were used for the

counting. The total number of counted cells was

500-800/treatment group.

Cell proliferation assays: For these studies,

equal numbers of cells were seeded per well in

twelve-well dishes. Cell numbers were estimated

by serial dilutions and counting cells (volume of

10 µl) in several visual fields using a Bright-Line

Hemacytometer and light microscopy. Basal

levels or opioid-induced changes in DNA

synthesis were assessed by measuring the rate of

[methyl-3H] thymidine incorporation into cells.

Cells were grown in media ± opioids for 24 h. In

some experiments, the MEK-selective inhibitor

U0126 (1 µM) was present for 28 h, while opioid

agonists were added for 24 h and [methyl-3H]

thymidine (0.02 µCi/ml) was present for the last 4

h. [methyl-3H]Thymidine incorporation was

measured as described in our previous studies with

some modifications (44). Briefly, cells are washed

with PBS, followed by incubation with 5%

trichloroacetic acid at 4º C for 30 min. Then cells

were collected in 2% NaHCO3/0.1 N NaOH and

[methyl-3H] thymidine incorporation was

determined by liquid scintillation counting.

BrdU labeling of cells: D3 ES cells or RA-

induced NPs were grown until they reached about

50% confluency. They were then treated with

either 1 µM DAMGO or U69, 593 for 24 h. In

some cases, cells were pretreated with inhibitors or

opioid antagonists as described in the figure

legends. At the end of the treatment, media was

replaced with BrdU labeling medium (10 µM,

Molecular Probes, BrdU detection kit I) and cells

were incubated for 20-30 min. After several

washes, cultures were fixed with ethanol fixative

for 20 min at -20º C. For nestin co-labeling, cells

were incubated together with a monoclonal BrdU

Ab (1:10, Roche kit) and polyclonal nestin Ab

(1:1000, Covance) at 37° C for 30 min. For Sox-1

co-labeling, cells were first incubated with a

monoclonal BrdU Ab (1:10, Roche kit) at 37° C

for 30 min, then incubated in PBS containing 10%

FCS and 0.4% Triton X-100 for 30 min to reduce

nonspecific binding, followed by counter-staining

with a chicken Sox-1 polyclonal Ab (1:500,

Chemicon) overnight at 4° C. After washing, cells

were treated with fluorescein conjugated anti-

mouse IgG (green, 1:10, Roche kit) together with

Alexa Fluor 594 (red) conjugated anti-chicken or

anti-rabbit secondary Abs (1:1000, Molecular

Probes) for 1 h at room temperature. Slides were

examined for immunofluorescence as described

above.

RESULTS

Characterization of ES cells and ES cell-derived NPs: The state of differentiation of D3 or

PRX ES cells and RA-induced ES cell-derived

NPs was characterized by immunocytochemistry

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and immunoblotting. For this purpose, D3 and

PRX ES cells were maintained under

serum/LIF/ME induced self-renewal conditions or

they were differentiated in the presence of RA.

For the immunocytochemistry experiments,

cultures were stained with nestin (a neural

progenitor marker) Ab and DAPI (a nuclear

marker). As seen in double DAPI/nestin images

(Fig.1, D3 and PRX), there were no nestin (red)

stained cells among the undifferentiated ES cells,

whereas RA-induced NPs show heavy staining

with nestin (Fig.1, dD3 & dPRX).

Since the transcription factor Oct4 has been

demonstrated to be vital for the formation of self-

renewing pluripotent ES cells, cell lysates from

undifferentiated D3 ES cells or RA-induced NPs

were probed with an Oct4 Ab (Chemicon, 1:100)

as presented in Fig.1B. The densitometry data

from the Oct4 immunoblots suggest that there is

about a 50% reduction in Oct4 protein levels in D3

ES cell-derived NPs (3767 ± 85, n=3) in

comparison to the levels of this transcription factor

in undifferentiated ES cells (6954 ± 463, n=4).

MOR-1 and KOR-1 gene expression and

immunoreactivity was detected in ES cells and in ES cell-derived NPs: Three independent

approaches were taken to determine the presence

of MOR-1 and KOR-1 in undifferentiated D3 ES

cells and RA-induced ES cell-derived NPs: qRT-

PCR, immunoblot analysis of cell lysates and

immunofluorescence microscopy of cells at

different stages of development. In qRT-PCR

experiments, we found that in comparison with ES

cells, NPs have 1.37 and 2-fold increases in MOR-

1 and KOR-1 gene expression, respectively (n=3).

As shown in figure 2, immunoblotting performed

with polyclonal MOR-1 (C-terminus, Neuromics),

MOR-1 (N-terminus, Santa Cruz) and KOR-1

(Santa Cruz) specific Abs shows the presence of

major 50 and 55 kDA bands that correspond to the

expected molecular weights for MOR-1 and KOR-

1, respectively. In addition to the 50 kDa band,

the N-terminus MOR-1 Ab reveals the existence

of higher molecular weight bands (Fig. 2, lanes 6&

7), which were suggested to correspond to the

glycosylated form(s) of the receptor by several

groups (45-47). Both MOR-1 and KOR-1 bands

were detected in cell lysates, obtained from D3 ES

cells maintained under self-renewal conditions

(serum/LIF/ME). RA treatment does not appear to

induce significant changes in MOR-1 or KOR-1

gene expression or protein levels (Fig. 2) in D3

NPs. Brain homogenate was used as a positive

control and lysates from late passage immortalized

rat astrocytes were used as negative controls

because these cells lose detectable amounts of

ORs with passaging (42 and unpublished

observations).

Immunofluorescence microscopic analysis

further supports the occurrence of MOR-1 and

KOR-1 in D3 ES cells and NPs (Fig. 3). Once

again, the undifferentiated state of D3 ES cells is

confirmed by the absence of nestin (green) stained

cells (D3, Fig. 3E & G). In contrast, D3 ES cell-

derived NPs (dD3) show heavy nestin staining

(Fig. 3 F & H). The presence of MOR-1 (red)

immunoreactive cells was confirmed by applying

the two polyclonal MOR-1 Abs that were used for

the immunoblotting experiments (Fig. 2). It

appears that the N-terminus MOR-1 Ab detects the

receptor mainly on the cell surface of ES cells

(Fig. 3B) and NPs (Fig. 3D). Figures 3 E & F

show MOR-1 immunostaining using the C-

terminus MOR-1 Ab. KOR-1 immunoreactive

cells were detected in self-renewing D3 ES cells

(Fig. 3G) and in NPs (Fig. 3H).

Opioid regulation of ERK phosphorylation

in undifferentiated ES cells: Having found that

MOR-1 and KOR-1 are present in both

undifferentiated ES cells and in ES cell-derived

NPs, we sought to determine the functionality of

these receptors. Since MAP kinases have been

implicated in regulation of cell proliferation and

differentiation, we studied opioid regulation of

ERK activation in ES cells and ES cell-derived

NPs. MOR- or KOR-induced ERK activation was

measured by performing immunoblotting

experiments with a phospho-ERK Ab that

recognizes the active form of ERK.

Undifferentiated ES cells were grown in media

devoid of serum/LIF/ME for at least 24 h.

Thereupon, cells were treated with the µ selective

opioid agonist DAMGO (0.1 µM, 5 min) or the κ

selective opioid agonist U69,593 (0.1 µM, 5 min).

Both opioids stimulated ERK activity in D3 ES

cells by about 2-fold (Fig. 4). Similar data were

obtained with undifferentiated PRX ES cells (data

not shown). The corresponding, selective MOR

(CTAP) and KOR (nor-BNI) antagonists blocked

agonist induced stimulation of ERK

phosphorylation, supporting the notion that these

are OR-mediated effects.

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Opioid effects on ERK signaling were also

evaluated in cells grown in serum/LIF/ME

containing media, conditions that are expected to

maintain D3 ES cells in an undifferentiated, self-

renewing state. Under these conditions, both

DAMGO and U69,593 induced a sustained

activation of ERK signaling throughout the 24 h

treatment period (Fig. 5). For DAMGO, there was

2-fold or greater activation of ERK at all time

points (Fig. 5B). Maximal phosphorylation was

seen at 5 min and 12 h with each having greater

than 4-fold stimulation of ERK. The ERK

activation observed with U69,593 was also

sustained for all time points (Fig. 5C). This

activation was slightly less all overall, although

the greatest phosphorylation seen was over 6-fold

at 5 min.

Although serum induces high basal levels of

phosphorylated ERK in many cell types, ERK

basal levels were low in ES cells grown in serum

containing media (Fig. 5A). The low basal levels

of ERK phosphorylation in these cells support the

notion that ERK activity may not be required for

self-renewal of ES cells as recently reported (see

Introduction).

Opioids induce limited proliferation of

undifferentiated ES cells via the ERK pathway: To correlate the ERK data with cell proliferation,

we studied opioid regulation of ES cell growth.

In undifferentiated D3 ES cells, the findings are

that DAMGO, U69,593, and morphine all induced

limited proliferation. Specifically, DAMGO and

morphine caused a 40% rise in proliferation over

basal levels, whereas U69,593 caused a 30%

increase (Fig. 6). The mitogen, EGF (10 ng/ml)

induced about a 100% increase in ES cell

proliferation (N=3, P<0.05). EGF stimulates ES

cell proliferation via the ERK/MAP kinase

signaling pathway (48). Since ERK mediates

mitogen-induced cell proliferation, the MEK-

selective inhibitor U0126 was added to determine

the role of ERK in opioid enhanced cell

proliferation. Pretreatment of ES cells with U0126

blocked the opioid-induced increase of

proliferation seen in the absence of the inhibitor,

suggesting that opioids act through ERK/MAP

kinase to increase proliferation in D3 ES cells

(Fig. 6). In PRX ES cells, morphine also induced

a 30% increase in cell proliferation over basal

levels (data not shown). U0126 abolished this

increase, suggesting that ERK is responsible for

opioid-enhanced proliferation in undifferentiated

PRX ES cells as well.

Although basal levels of proliferation in the

presence and absence of U0126 were set at one to

simplify the estimation of opioid effects (controls

in Fig. 6), we found that U0126 pretreatment

enhanced basal levels of D3 ES cell proliferation

by about 40% (basal levels in dpms: 8726 ± 1168

vs basal in the presence of U0126: 12005 ± 1225).

This finding supports the notion that

undifferentiated ES cells (in the absence of

opioids) can proliferate even when ERK signaling

is blocked and suggest that the ERK/MAP kinase

signaling pathway may not be involved in ES cell

self-renewal.

Evidence for opioid-induced D3 ES cell

differentiation: To determine whether the opioid-

induced proliferation of ES cells is due to an

increase in self-renewal or asymmetric cell

division to form NPs, we examined the phenotype

of the opioid-induced “newly formed” cells.

Undifferentiated ES cells were treated with

opioids, labeled with BrdU, followed by staining

with nestin and BrdU Abs. The

immunofluorescence analysis raises the possibility

that DAMGO and U69, 593 induce the appearance

of nestin-positive cells that may be NPs derived

from ES cells (Fig. 7A). The representative

double images of control cells show mainly BrdU

(green) staining and only a few nestin (red) stained

cells.

To determine further the nature of opioid-

induced cells, we performed a quantitative

analysis of Sox-1 immunostained, control and

opioid-treated ES cells (Fig. 7 B-D). Sox-1 is a

selective marker of proliferating NPs; it belongs to

a family of transcription factors that are expressed

in ectodermal cells upon acquisition of neural

progenitor identity (49, 50). By counting cells (≥

1,000 cells/treatment group), we estimated that

only a few of the control cells (4 ± 1.1%, n=8)

were Sox-1 positive. Exposure of undifferentiated

ES cells to 1 µM MOR (Fig. 7B) or KOR (Fig.

7C) opioids for 24 h initiated the appearance of

about 7-8 fold more Sox-1 positive cells than in

control cells (Fig. 7D&E). The corresponding,

selective MOR (CTAP) and KOR (nor-BNI)

antagonists blocked agonist induced appearance of

Sox-1 positive cells, suggesting that this is an OR-

mediated process. CTAP (10 ± 2.6%, n=4) and

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nor-BNI (2 ± 0.5%, n=4), alone had no significant

effect on the number of Sox-1 labeled cells.

Opioid regulation of ERK phosphorylation in RA-induced ES cell-derived NPs: To

investigate opioid modulation of ERK activity

upon RA-induced differentiation of ES cells, RA-

induced EBs were dissociated and plated in serum

containing DMEM for an additional 2-5 days

(short-term differentiation) or for 5-12 days (long-

term differentiation). After growing in serum-

deprived media for 24 h, these cells were treated

with opioids for various times and ERK

phosphorylation was measured by immunoblotting

(Fig. 8). The results support the following

conclusions: a) basal levels of ERK

phosphorylation in long-term differentiated cells

were significantly higher (2.2±0.2, n=9, P < 0.05)

than basal levels of ERK activation in short-term

differentiated cells (NPs), suggesting that terminal

differentiation of these cells may require activated

ERK; b) MOR and KOR agonists elicit similar

effects on ERK activation in short- and long-term

differentiated cells (Figs. 8&9).

More detailed time course experiments with

DAMGO and U69, 593 were also conducted (Fig.

9). These data further confirm that the two opioids

induce a similar biphasic ERK activation profile

with peaks occurring at 2-15 min and at about 2-6

h in short-and long-term differentiated ES cells

(Fig. 9A-D). Thus, the opioid-induced sustained

ERK activation seen in undifferentiated ES cells

changes in differentiated cells to a biphasic ERK

activation profile.

Opioids inhibit NP proliferation: To

evaluate opioid effects on RA-induced NP

proliferation, we performed a quantitative analysis

of Sox-1/BrdU double labeled, control and opioid-

treated NPs (Fig. 10). Since Sox-1 is a selective

marker of proliferating NPs, we counted the BrdU

labeled cells among the Sox-1 positive cells and

thus estimated the changes in NP proliferation

upon treatment with opioids. The data presented

in figure 10 indicate that 1 µM DAMGO or

U69,593 decreased proliferation of NPs by 50-

60% in 24 h. The inhibitory effect of both opioid

agonists was reversed by the corresponding MOR

(CTAP) and KOR (NorBNI) antagonists,

indicating that DAMGO and U69,593 were acting

via their respective receptors. Interestingly,

norBNI alone induced a 27% increase (59 ± 4.7%,

n = 4, P = 0.0149) in cell proliferation over basal

levels. This finding may be due to the possibility

that NPs secrete endogenous κ opioid peptides that

may have inhibitory effects on basal levels of cell

division. Upon addition of KOR antagonist, the

effects of both endogenous and exogenous KOR

ligands were suppressed, resulting in an increase

in proliferation over basal levels. In contrast, in

the presence of CTAP alone, NP proliferation was

similar to basal levels (34 ± 5.4%, n=4, P = 0.168).

The involvement of the ERK signaling

pathway in opioid regulation of NP proliferation

was evaluated by treatment of the cells with the

MEK inhibitor, U0126 for 1 h before opioid

agonist exposure (Fig. 10). U0126 alone did not

significantly affect basal levels of NP proliferation

(44 ± 3.2%, n=4, P = 0.832 vs controls of 43 ±

4.1%, n=6). Interestingly, administration of

U0126 in the presence of DAMGO did not reverse

the inhibitory effect induced by the MOR agonist

alone suggesting that MOR regulation of NP

proliferation is independent of the ERK signaling

pathway. Furthermore, the inhibitory effect of

U69,593 was only partially reversed upon

blockade of the ERK signaling by U0126.

DISCUSSION

The analysis of our data reveals several

important findings: 1. MOR-1 and KOR-1 gene

expression and immunoreactivity was detected in

undifferentiated ES cells and in ES cell-derived

NPs. This is the first evidence of the occurrence of

MOR-1 and KOR-1 in blastocyst-derived ES cells.

2. MOR-1 and KOR-1 that occur in ES cells and

NPs, show functionality as established by the

detection of opioid-induced temporal regulation of

ERK/MAP kinase signaling. A sustained

activation of ERK was characteristic for the opioid

treated undifferentiated ES cells, whereas the RA-

induced NPs showed a biphasic profile of opioid

induced ERK activity. 3. The ERK signaling

pathway mediated µ- and κ-opioid-induced limited

proliferation in undifferentiated D3 ES cells, even

though in the absence of opioids, these cells may

not require ERK activation for their self-renewal.

More importantly, opioids induced ES cell

asymmetric division to generate nestin/SOX1-

positive NPs and reduced the self-renewal of ES

cells. Thus, a novel finding here is that opioid-

induced sustained activation of ERK may play a

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role in the initial differentiation of ES cells. 4.

Opioids attenuated proliferation of the population

of NPs that were generated upon differentiation of

ES cells with RA. This inhibitory effect is ERK

independent for DAMGO and partially for

U69,593 and correlated with MOR and KOR

agonist induction of a biphasic rather than a

sustained ERK activation profile in NPs.

In prior studies, KOR was found in P19

embryonic carcinoma cell line, which contains

pluripotent stem cells (51-53). Although these

cells are similar to ES cells derived from 4-5 day

old mouse embryos, they are transformed cells and

therefore capable of overexpressing genes not

expressed in their parent cell. RA promoted

expression of MOR gene, while KOR gene was

first suppressed and then reactivated in P19 cells

(54-56). KOR was also found on cell surface and

nuclear fractions of GTR1 ES cells (57-59). In

addition, MOR and δ opioid receptor (DOR), but

not KOR are expressed in adult hippocampal

progenitors and opioid peptides such as β-

endorphin can regulate proliferation of these

progenitor cells via ERK (60). Reduced ERK

signaling via MOR decreases proliferation, of

these progenitors, increases the number of in vitro

generated neurons and reduces the number of

astrocytes and oligodendrocytes. Finally, opiates

were found to inhibit neurogenesis in the adult rat

hippocampus (61).

We have characterized the mechanism of

ERK activation by ORs and demonstrated that

opioids either enhanced or inhibited DNA

synthesis in several types of primary cultures and

cell culture model systems (40-42, 44, 62, 63). In

most of these cases, opioids inhibited mitogen

stimulated proliferation as seen here (Fig. 10). It

was important to determine how opioids regulate

the ERK signaling pathways in ES cells and in ES

cell-derived NPs. As discussed in the

Introduction, several studies suggest that ERK

activation may not be required for ES cell self-

renewal (4, 11-14). Our thymidine incorporation

results further support this hypothesis. For

example, the finding that MEK inhibitor, U0126

alone enhanced basal levels of ES cell

proliferation by about 40% supports the notion

that undifferentiated ES cells can proliferate when

ERK signaling is blocked and suggest that ERK

activity may not be necessary for ES cell self-

renewal. Moreover, our U0126 data on basal

levels of ERK suggest that activation of this kinase

may have inhibitory effects on ES cell self-

renewal. In addition, under self-renewal

conditions (serum/LIF/ME), ES cells maintain low

basal ERK activity, data that further support the

lack of requirement for ERK activity by these cells

(Fig. 5). In contrast, the results also suggest that

opioid regulation of ES cell asymmetric cell

division in undifferentiated ES cells is ERK

dependent. Therefore, we propose that in

undifferentiated ES cells, two processes take

place: there is an ongoing ERK-independent,

symmetric cell division leading possibly to ES cell

self-renewal and an opioid-induced ERK-

dependent, asymmetric cell division leading to ES

cell differentiation (Fig. 11). Thus, both µ and κ

opioids promote ES asymmetric cell division at a

slightly more rapid rate than that of

serum/ME/LIF-induced self-renewal of ES cells.

The above findings raise the following

questions: If ERK signaling is not the regulator of

ES cell division/self-renewal than what is the

signaling pathway that modulates this process? It

was proposed that mouse ES cells utilize PI3K to

progress through the G1 phase and to avoid

differentiation (18, 27). What is the mechanism of

opioid induction of NP appearance? It has been

suggested that Sox proteins might contribute to the

transcriptional activation of the MOR gene and

that MOR could mediate some Sox regulated

developmental processes (64).

ERK signal duration impacts different

cellular responses in many cells and may produce

different outcomes in the same cells, such as

proliferation, differentiation or apoptosis (10, 26,

44, 65-68). In some cells, sustained but not

transient activation of ERK is required to initiate

proliferation (41, 65, 69, 70, McLennan, Kiss et

al., 2006 manuscript in preparation). Sustained

ERK activity appears to be required by many cells

to pass the G1 restriction point and to enter S

phase, in which cellular DNA is replicated (65,

71). In contrast, other studies have shown that

Ras/Raf mediated a transient EGF-induced ERK

activation that leads to proliferation, whereas

Rap/Raf mediated sustained activation of ERK is

required for PC12 cell differentiation (22, 72).

Our data suggest that the opioid induced sustained

activation of ERK triggers ES asymmetric cell

division and the “newly” formed cells appear to be

ES cell-derived NPs.

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The important role of the ERK signaling

pathway in neural differentiation has been well-

established. While some reports indicate that ERK

signaling inhibits differentiation of some types of

cells (73, 74), other studies support the idea that

the ERK signaling may be a positive regulator of

this process (75). A recent paper discusses the

existence of a biphasic regulation of ERK activity

during myogenic differentiation and suggests that

the signaling pathway(s) may play a dual role in

this multi-step process, wherein the late phase is

responsible for the formation of postmitotic

myotubes (76). A similar study shows that an

early stimulation and late inhibition of ERK

activity by IGF mediates the switch in IGF action

from inhibition to stimulation of skeletal muscle

cell differentiation (77). Finally, it was found that

the early stage of neuronal differentiation of

mouse ES cell line P19 triggered by aggregation of

the cells and RA treatment is accompanied by

biphasic activation of ERK signaling: a transient

phase and a second, sustained ERK activation that

is maintained until the appearance of neural

phenotype (78).

Here, we propose that opioid-induced

sustained activation of ERK may be a required

step for the development of ES cell-derived NPs

and opioid-induced biphasic ERK phosphorylation

may play a supporting role in the increased

differentiation rate during the generation and

maturation of NPs. This hypothesis is based on

the following findings: As seen in figure 7, basal

levels of ERK activity are higher in long-term

differentiated ES cells than in short-term

differentiated cells (NPs). In addition, µ- and κ-

opioids induce a moderate, biphasic activation of

ERK signaling in short- and long-term

differentiated ES cells. More importantly, we

found that the biphasic stimulation of ERK

phosphorylation by opioids accompanies the

opioid-induced inhibition of proliferation in NPs.

Although MOR regulation of NP proliferation was

independent of the ERK signaling pathway, the

inhibitory effect of U69,593 was only partially

reversed upon blockade of the ERK signaling by

U0126. This finding may be explained by the

involvement of dual signaling pathways in KOR

modulation of NP proliferation. In addition to

ERK signaling, p38/MAP kinase is a candidate for

a second signaling pathway that could be required

for KOR inhibition of NP proliferation.

The absence of a developmental phenotype in

opioid receptor knock out mice has been reported

(79). This is not uncommon given the functional

redundancies that cells display after targeted gene

disruption of critical signaling molecules such as

GPCRs. There are numerous accounts in the

literature regarding the absence of alteration of

function after the loss of GPCR gene expression

due to redundant or compensatory signaling

mechanisms (80, 81). In some instances,

developmental phenotypic changes are subtle after

gene disruption and only seen when the organism

is stressed as it is in its natural environment (82).

Alternatively, MOR and/or KOR may be

dispensable for development and the observations

made here may be only of relevance to maternal

opiate abuse.

In conclusion, our studies suggest that MOR-

1, KOR-1 and their exogenous ligands are able to

modulate ES cell proliferation and differentiation.

Thus, opioids and opioid-induced ERK signaling

may play an important role in ES cell fate

decisions by directing the cells to specific

lineages.

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FOOTNOTES

*Supported in part by grants from the National Institutes

of Health DA-05412 to CJC and American

Parapalegia Society and the Pediatrics Research Institute of St. Louis University School of Medicine to

MMB. We thank Dr. Michael Green and Akbar Siddiqui of the Department of Molecular Microbiology and

Immunology for their assistance with the qRT-PCR experiments. 1Abbreviations: Ab, antibody; BMP, bone morphogenic factor; DAMGO, [D-ala

2,mephe

4,gly-ol

5]

enkephalin; DMEM, Dulbecco’s Modified Eagle’s Medium; DOR, δ opioid receptor; EB, embryoid

body; EGF, epidermal growth factor; ES cells, embryonic stem cells; ERK, extracellular signal regulated

protein kinase; GPCR, G protein coupled receptor; ICM, inner cell mass; KOR, κ opioid receptors; LIF,

leukemia inhibitory factor; MAP, mitogen-activated protein; ME, 2-mercaptoethanol; MOR, µ opioid

receptor; nor-BNI, nor-binaltorphimine; NP, neural progenitor; OR, opioid receptor; PA,

paraformaldehyde; PI3K, phosphatidyl inositol 3 kinase; RA, retinoic acid.

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FIGURE LEGENDS

Fig. 1 Characterization of undifferentiated ES cells and ES cell-derived RA-induced NPs. (A)

Immunofluorescence microscopic analysis. D3 and PRX ES cells were maintained under

serum/LIF/ME induced self-renewal conditions (undifferentiated cells). Upon treatment with 1 µM RA,

cells undergo differentiation (dD3 and dPRX). Cells were grown in 8 well slide chambers for 2 days,

washed with PBS and fixed with 4% PA for 20 min at room temperature. Cells were then permeabilized

in 0.1% Triton/PBS for 5 min and incubated in PBS containing 0.5% BSA and 0.1% Tween 20 for 30 min

to reduce nonspecific binding, followed by incubation with mouse monoclonal nestin Ab (1:1000)

overnight at 4° C. After washing, Alexa Fluor 594 (red) conjugated anti-mouse secondary Ab (1:1000)

was applied for 1 h at room temperature. DAPI (1:200) was added together with the secondary Ab

Representative images show DAPI (blue), Nestin (red) and double DAPI/Nestin staining in short-term (2-

5 days) RA differentiated cells (dD3 and dPRX). Only DAPI staining is present on the double

DAPI/Nestin images from undifferentiated cells (D3 and PRX). Magnification X 20-40. (B)

Immunoblot analysis. D3 ES cells were maintained in serum/LIF/ME containing media or they were

differentiated with 1 µM RA (short-term differentiated D3 ES cells) and grown for additional 2-3 days

before use. Cell lysates (40 µg protein) were run on 10% SDS-PAGE and immunoblotting was

performed with an Oct4 Ab (Chemicon, 1:100). Shown is a representative immunoblot from 3-4

experiments.

Fig. 2 Immunoblot analysis of MOR-1 and KOR-1 in D3 ES cells and in NPs. D3 ES cells were

grown in serum/LIF/ME containing media (undifferentiated cells: D3 ES cells) or they were differentiated

with 1 µM RA (short-term differentiation: dD3 ES cells). Cell lysates (20-50 µg protein) were run on

10% SDS-PAGE and immunoblotting was carried with MOR-1 (C-terminus, Neuromics, 1:2000), MOR-

1 (N-terminus, Santa Cruz, 1:200) or KOR-1 (Santa Cruz, 1:50) Abs. Shown are representative

immunoblots from 4-8 experiments: C-terminus MOR-1 Ab: 1. D3 ES cells; 2. immortalized rat

astrocytes stably transfected with MOR-1; 3. rat brain membrane; 4. late passage immortalized rat

astrocytes (negative control); 5. dD3 ES cells; N-terminus MOR-1 Ab: 6. D3 ES cells; 7. dD3 ES cells;

KOR Ab: 1. D3 ES cells; 2. late passage immortalized rat astrocytes (negative control); 3. dD3 ES cells.

Fig. 3 Immunofluorescence microscopic detection of MOR-1 and KOR-1 in undifferentiated D3 ES cells and in RA-induced NPs. D3 ES cells were maintained in serum/LIF/ME induced self-renewal

conditions (undifferentiated cells, D3). Upon treatment with 1 µM RA they undergo differentiation

(dD3). Cells were fixed and permeabilized as in Fig. 1. Treatments were followed by overnight

incubation with polyclonal MOR-1 (C-terminus, 1:2500, A-D and N-terminus, 1:50, E&F) or KOR (1:50)

Abs at 4° C. Mouse monoclonal nestin Ab (1:1000) was added to E-H slides. After washing, Alexa

Fluor 594 (red) or Alexa Fluor 488 (green) conjugated secondary Abs (1:1000) were applied for 1 h at

room temperature. In some cases, DAPI (1:200) was added together with the secondary Ab (A-D).

Immunofluorescence in cells was examined as described in Experimental. Appropriate controls, such as

omission of primary Ab, have been run to confirm the specificity of the Abs (A&C). Representative

images show MOR-1 (B&E) or KOR-1 (G) (red) staining in undifferentiated (D3) cells or in short-term

differentiated ES cells (dD3, D). Double images of MOR-1/Nestin (F) or KOR-1/Nestin (H) staining in

short-term differentiated ES cells (dD3) are also presented. Magnification X 20-40.

Fig. 4 Opioid stimulation of ERK phosphorylation in ES cells. D3 ES cells grown in serum/LIF/ME-

free media were treated with vehicle, DAMGO (0.1 µM) or U69, 593 (0.1 µM ) for 5 min. In some

cases, cells were pretreated for 1 h either with MOR antagonist (CTAP, 1 µM) or KOR antagonist (nor-

BNI, 1 µM) before addition of opioid agonists for 5 min. ERK phosphorylation was measured by

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immunoblotting with a p-ERK Ab, directed against phospho Thr202/Tyr204. N = 3-4. *Significantly >

controls, #Significantly < agonist alone, P<0.05.

Fig. 5 Time course of opioid stimulation of ERK phosphorylation in undifferentiated ES cells. D3

ES cells grown in serum/LIF/ME containing media were treated at different time points with opioids (1

µM). (A) Representative gels of ERK phosphorylation. (B&C) Curves of quantified ERK

phosphorylation. N = 3-12. *Significantly > controls, P<0.05; **

Significantly > controls, P<0.01.

Fig. 6 Opioids induce limited proliferation in undifferentiated D3 ES cells. Proliferation was

measured by methyl-[3H] thymidine incorporation into ES cells. D3 ES cells grown in serum/LIF/ME

containing media were treated with either vehicle (Con), morphine (0.5 µM), DAMGO (1 µM), or

U69,593 (1 µM) for 24 h and 0.02 µCi/ml [methyl-3H] thymidine was added for the last 4 h. In some

experiments, the MEK-selective inhibitor U0126 (1 µM) was present for 28 h, while opioid agonists were

added for 24 h and [methyl-3H] thymidine was present for the last 4 h. N=3-5. **P<0.01.

Fig. 7 Evidence for opioid-induced neural differentiation of ES cells (A) BrdU and nestin

immunostaining. D3 ES cells grown in chamber slides and in serum/LIF/ME containing media were

treated with vehicle, DAMGO (1 µM) or U69,593 (1 µM) for 24 h. Then cells were incubated with BrdU

(10 µM, BrdU labeling and detection kit, Roche) in media at 37° C for 20 min and fixed in ethanol

fixative at -20° C for 20 min. After washing, monoclonal BrdU Ab (1:10, Roche kit) and polyclonal

nestin Ab (1:1000, Covance) were added together to the cells and then incubated at 37° C for 30 min.

After washing, fluorescein conjugated anti-mouse IgG (green, 1:10, Roche kit) and Alexa Fluor 594

conjugated anti-rabbit IgG (red, 1:1000) were applied at 37° C for 30 min. Representative double images

show BrdU (green) and nestin (red) stained cells. (B&C) Sox-1 immunostaining. Cells were grown and

treated with opioids as described in (A). In some cases, cells were pretreated for 1 h either with MOR

antagonist (CTAP, 1 µM, B) or KOR antagonist (nor-BNI, 1 µM, C) before addition of opioid agonists

for 24 h. After washing, cells were fixed with 2% PA for 10 min at room temperature. Cells were then

permeabilized in 0.4% Triton/PBS for 5 min and incubated in PBS containing 10% FCS and 0.4% Triton

X-100 for 30 min to reduce nonspecific binding, followed by incubation with chicken Sox-1 polyclonal

Ab (1:500, Chemicon) overnight at 4° C. After washing, Alexa Fluor 594 (red) conjugated anti-chicken

secondary Ab (1:1000, Molecular Probes) was applied for 1 h at room temperature. DAPI (1:200) was

added together with the secondary Ab. Representative double DAPI (blue)/Sox-1 (red) images are shown

in control and opioid-treated cells. N= 4-8. Magnification X 20-40. Bar graphs (D&E) show the

quantification of the Sox-1 counting data. The total number of cells was determined by counting DAPI

stained cells (≥ 1,000 cells/treatment group). Sox-1 positive cells are expressed as % of the total number

of cells. N=4-8. *Significantly > controls, #Significantly < agonist alone, P<0.0001

Fig. 8 Representative gels of opioid stimulation of ERK phosphorylation in RA-induced differentiated D3 ES cells. Upon differentiation of D3 ES cells with 1 µM RA, cells were grown for

additional 2-5 days (short-term differentiation) or for 5-12 days (long-term differentiation). Cells were

serum starved for 24 h and treated with DAMGO (0.1 µM) or U69, 593 (0.1 µM) for different time

intervals and ERK phosphorylation experiments were carried out. N = 3-11.

Fig. 9 Opioid stimulation of ERK phosphorylation in RA-induced D3 ES cell-derived short- and long-term differentiated cells. D3 ES cells were differentiated with 1 µM RA as in Fig. 8. (A&B)

Short-term differentiated cells were treated with either 1 µM DAMGO (A) or 1 µM U69,593 (B) for

different time intervals. N = 3-10. *Significantly different from controls, P<0.05. **

Significantly >

controls, P<0.001. (C&D) Long-term differentiated ES cells were treated with either 1 µM DAMGO (C)

or 1 µM U69,593 (D) for different time intervals. N = 3-11. *Significantly different from controls,

P<0.05. **

Significantly > controls, P<0.001.

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Fig.10 Opioids inhibit proliferation of ES cell-derived, RA differentiated NPs. Sox-1 and BrdU

immunocytochemistry. (A) D3 ES cell-derived, RA-induced NPs were grown in chamber slides in

DMEM containing FCS for 2 days. Cells were serum starved for 24 h and treated with vehicle, DAMGO

(1 µM) or U69,593 (1 µM) for an additional 24 h. In some cases, cells were pretreated for 1 h either with

MOR antagonist (CTAP, 1 µM), KOR antagonist (nor-BNI, 1 µM) or the MEK-selective inhibitor U0126

(1 µM), followed by opioid agonist treatment. Cells were then incubated with BrdU and co-labeled with

monoclonal BrdU and chicken Sox-1 polyclonal Abs as described in Experimental. Representative

double BrdU (green)/Sox-1 (red) images are shown in control and opioid-treated cells. Magnification X

20. (B) Quantification of BrdU/Sox-1 counting data. NIH ImageJ version 1.32 sofware was used to

count cells. Cells from > 5 fields per well were counted (>1,000 cells/treatment group). The total number

of NPs was estimated by counting Sox-1 stained cells. BrdU positive cells were expressed as % total

number of Sox-1 labeled NPs. N=3-7. *Significantly < controls, p ≤ 0.0004; #Significantly > agonist

alone, p ≤ 0.0281.

Fig. 11 Hypothetical model of ES cell division upon opioid treatment. (A) The symmetrical ES cell

division is ERK independent in the absence of opioids. (B) Opioids initiate asymmetrical cell division in

ES cells: ES cell self-renewal is ERK independent but may be reduced upon sustained activation of ERK;

the appearance of NPs is due to an ERK dependent cell division.

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Fig. 1

PRX

DAPI/ Nestin DAPI Nestin DAPI/ Nestin

dPRX dPRX dPRX

dD

D3 dD3 dD3 dD3

DAPI/ Nestin DAPI Nestin DAPI/ Nestin

A

B

ES NPs

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Fig. 2

MOR

50 kDa 1 2 3 4 5 6 7

98 kDa

KOR

1 2 3 kDa55

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Fig. 3

dD3 H

Nestin/ KOR

D3 G

Nestin/ KOR

D3 A D3 B dD3 C dD3 D

F D3

Nestin/ MOR

Nestin/ MOR

DAPI DAPI/ MOR DAPI DAPI/ MOR

E dD3

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Fig. 4

1.0

2.0

Control DAMGO CTAP+DAMGO U69,593 NorBNI+U69,593

**

# #ER

K P

ho

sph

ory

latio

n

(Fo

ld o

ve

r C

on

trol)

Control DAMGO

**

# #

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23

Fig. 5

Phosphory

lation

Min

ER

K P

hosphory

lation

DAMGO

**

** *

*

*

*

*

2 5 1

3

6

2 4 6 1

1

2

Time

1.0

4.0 2.0 3.0 5.0 6.0 7.0 U69,593

*

**

* * **

** * * * (F

old

over

Contr

ol) C

** **

1.

2.0 3.0 4.0 5.0 6.0

1.

1.0

1

3

6

4 6 1

1

2

15 30 60 4 6 12 18 24 Min Hours

B

2 5 1 3 6 2 4 6 12 18 24 Hours

5 15 30 60 Time

(Fold

over

Contr

ol)

ER

K

**

*

A

Con

trol

U 2

min

U 5

min

U 1

h

U 2

h

U 4

h

U 6

h

U69,593 (U)

DAMGO (D)

Con

trol

D 2

min

D 5

min

D 1

h

D 2

h

D 6

h

D 1

2h

D 1

8h

D 4

h

D 2

4h

U 1

2h

U 1

8h

U 2

4h

A

Con

trol

U 2

min

U 5

min

U 1

h

U 2

h

U 4

h

U 6

h

U69,593 (U)

DAMGO (D)

Con

trol

D 2

min

D 5

min

D 1

h

D 2

h

D 6

h

D 1

2h

D 1

8h

D 4

h

D 2

4h

U 1

2h

U 1

8h

U 2

4h

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Fig. 6

Morphine Con DAMGO U69

** ** **

U0126 absent

U0126 present

Ce

ll P

rolif

era

tion

(Fo

ld o

ve

r C

on

trol)

0.4

0.8

1.2

1.6

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Fig. 7

A

BrdU/ Nestin BrdU/ Nestin BrdU/ Nestin

Control

Control

DAMGO B

C

Control DAMGO

U69,593

CTAP +DAMGO

Control nor-BNI +U69,593

DAPI/ SOX-1 DAPI/ SOX-1 DAPI/ SOX-1

DAPI/ SOX-1 DAPI/ SOX-1 DAPI/ SOX-1

U69,593

0 10 20 30 40

% S

OX

-1 L

abelle

d C

ells

#

nor-BNI+ U69,593

0

10

20

30

% S

OX

-1 L

abelle

d C

ells

CTAP+ DAMGO

D

E

#

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Fig. 8

DAMGO(D)short-term long-term

long-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

long-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

DAMGO(D)short-term long-term

long-termshort-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

long-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

DAMGO(D)short-term long-term

long-termshort-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

DAMGO(D)short-term long-term

long-termshort-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

DAMGO(D)short-term long-term

DAMGO(D)short-term long-term

long-termshort-termshort-term

U69,593(U)

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

D 5

min

D 3

0m

in

D 2

4h

D 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

Contr

ol

U 5

min

U 3

0m

in

U 2

4h

U 1

h

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24 2 5 15 30 60 2 4 6 12 18

Time Min Hours

1.0 2.0 3.0 4.0 5.0

*

*

** *

ER

K Ph

osp

ho

ryla

tio

n

(Fo

ld o

ve

r C

on

tro

l)

C DAMGO

0.5 1.0 1.5 2.0 2.5 3.0

2 5 15 30 60 2 4 6 12 18 24

Time Min Hours

** ** **

* **

ER

K P

ho

sp

ho

ryla

tio

n

(Fo

ld o

ve

r C

on

tro

l)

A

** **

* ** DAMGO

ER

K Ph

osp

ho

ryla

tio

n

(Fo

ld o

ve

r C

on

tro

l)

D

0.5 1.0 1.5 2.0 2.5 3.0 3.5

** *

*

* U69,593

2 5 15 30 60 2 4 6 12 24

Time Min Hours

ER

K Ph

osp

ho

ryla

tio

n

(Fo

ld O

ver

Co

ntr

ol)

0.5 1.5 1.0 2.0 2.5 3.0 3.5 4.0

Time

2 5 15 30 60 2 4 6 12 18 24 Hours Min

U69,593

* ** *

*

*

B *

Fig. 9

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Fig. 10

Control

Control

DAMGO CTAP + DAMGO

U69,593 Nor-BNI + U69,593 U0126 + U69,593

U0126 + DAMGO

50

60

70

Control

DAMGO

CTAP +

DAMGO

U0126 +

DAMGO

*

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

10

20

30

40

50

60

70

Control

U69

norBNI

*

#

#*

*

40

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

10

20

30 *

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

Control

U69

norBNI +

U69,593

U0126 + U69,593

*

#

#*

*

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

50

60

70

Control

DAMGO

CTAP +

DAMGO

U0126 +

DAMGO

*

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

10

20

30

40

50

60

70

Control

U69

norBNI

*

#

#*

*

40

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

10

20

30 *

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

Control

U69

norBNI +

U69,593

U0126 + U69,593

*

#

#*

*

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

50

60

70

Control

DAMGO

CTAP +

DAMGO

U0126 +

DAMGO

*

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

10

20

30

40

50

60

70

Control

U69

norBNI

*

#

#*

*

40

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

10

20

30 *

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

Control

U69

norBNI +

U69,593

U0126 + U69,593

*

#

#*

*

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

50

60

70

Control

DAMGO

CTAP +

DAMGO

U0126 +

DAMGO

*

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

10

20

30

40

50

60

70

Control

U69

norBNI

*

#

#*

*

40

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

10

20

30 *

#

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

Control

U69

norBNI +

U69,593

U0126 + U69,593

*

#

#*

*

% B

rdU

posi

tive C

ells

% B

rdU

posi

tive C

ells

*

A B

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Fig. 11

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Coscia and Mariana M. BelchevaEunhae Kim, Amy L. Clark, Alexi Kiss, Jason W. Hahn, Robin Wesselschmidt, Carmine J.

progenitorsMu and kappa opioids induce the differentiation of embryonic stem cells to neural

published online September 1, 2006J. Biol. Chem. 

  10.1074/jbc.M603862200Access the most updated version of this article at doi:

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