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Molecular and Cellular Biology, May 2000, p. 3004-3014, Vol. 20, No. 9
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cyclic AMP Signaling Functions as a Bimodal Switch
in Sympathoadrenal Cell Development in Cultured Primary Neural
Crest Cells
Matthew L.
Bilodeau,
Theresa
Boulineau,
Ronald L.
Hullinger, and
Ourania M.
Andrisani*
Department of Basic Medical Sciences, Purdue
University, West Lafayette, Indiana 47907
Received 3 August 1999/Returned for modification 28 September
1999/Accepted 17 February 2000
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ABSTRACT |
Cells of the vertebrate neural crest (crest cells) are an
invaluable model system to address cell fate specification. Crest cells
are amenable to tissue culture, and they differentiate to a variety of
neuronal and nonneuronal cell types. Earlier studies have determined
that bone morphogenetic proteins (BMP-2, -4, and -7) and agents that
elevate intracellular cyclic AMP (cAMP) stimulate the development of
the sympathoadrenal (SA, adrenergic) lineage in neural crest cultures.
To investigate whether interactive mechanisms between signaling
pathways influence crest cell differentiation, we characterized the
combinatorial effects of BMP-2 and cAMP-elevating agents on the
development of quail trunk neural crest cells in primary culture. We
report that the cAMP signaling pathway modulates both positive and
negative signals influencing the development of SA cells. Specifically,
we show that moderate activation of cAMP signaling promotes, in synergy
with BMP-2, SA cell development and the expression of the SA
lineage-determining gene Phox2a. By contrast, robust activation of cAMP
signaling opposes, even in the presence of BMP-2, SA cell development
and the expression of the SA lineage-determining ASH-1 and Phox2 genes.
We conclude that cAMP signaling acts as a bimodal regulator of SA cell
development in neural crest cultures.
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INTRODUCTION |
The neural crest is a stem cell-like
population of multipotent embryonic cells which separates from the
neuroepithelium as the neural tube forms, migrates to numerous and
diverse locations, and differentiates to diverse cell types (reviewed
in reference 35). Neural crest cells from the trunk
region of the embryo produce sensory and sympathetic neurons,
peripheral glial cells, adrenergic (chromaffin) cells of the adrenal
medulla, and melanocytes (reviewed in reference 40).
It is accepted that the developmental fate of most crest cells is
determined by the interplay of signals from the microenvironments
encountered during and after migration (reviewed in reference
3). Consistent with this model, sympathetic neurons
and adrenergic cells differentiate from fate-restricted sympathoadrenal
(SA) progenitor cells (5). These SA progenitor cells arise
from trunk-derived crest cells migrating to dorsal-lateral aspects of
the aorta, forming the sympathetic trunks (strands), and to the adrenal
gland primordia (2, 33). At other axial positions, SA
progenitor cells are precursors for vagal-derived crest cells
differentiating to subsets of enteric neurons and thyroid
parafollicular (calcitonin) cells and for sacral-derived crest cells
differentiating to parasympathetic neurons (reviewed in reference
4). Those cells committed to the SA lineage are distinguished by their at least transient expression of catecholamines (CA) and enzymes for CA biosynthesis, i.e., tyrosine-3-hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis, and
dopamine-
-hydroxylase (DBH), the enzyme converting dopamine to norepinephrine.
Expression of TH is tissue specific and dependent, in part, on a cyclic
AMP (cAMP)-responsive element (CRE) (11, 32, 39) which is
activated by CA and -adrenergic receptors in immortalized crest
cells (9). Similarly, expression of DBH is regulated by cAMP
and is dependent on a CRE (27, 65). In neural crest cultures, CA and elevated cAMP promote the development of the SA
lineage (9, 74). The developmental relevance of CA is supported in vivo by the presence of CA (26), -adrenergic
receptors, and CA biosynthetic enzymes (14) in early-stage
embryos, prior to the differentiation of neurons. As crest cells form
the sympathetic trunks, the notochord mesenchyme can synthesize CA from
l-DOPA (2, 34), and both notochord and ventral
neural tube accumulate and store CA (2, 34, 38, 50).
Furthermore, the induction of SA cell differentiation in vivo is
dependent on the presence of either notochord or neural tube (18,
64, 67).
Bone morphogenetic protein 2 (BMP-2), BMP-4, or BMP-7/OP-1 also
promotes development of the SA lineage in neural crest cultures (55, 68, 70). BMP-2, -4, and -7 are produced by endothelial cells of the aorta as crest cells form the sympathetic trunks and
adrenal gland primordia (55, 58), suggesting that these factors are relevant to SA cell development in vivo. Moreover, the
causal link of BMP-2 to SA cell development has been demonstrated by
expression of a constitutively active mutant of the BMP type I receptor
in cultured crest cells (69). Although the mechanism of
regulation has not been elucidated, BMPs induce the expression of genes
essential to SA lineage determination. For example, BMP-2 induces
expression of the basic helix-loop-helix transcription factor ASH-1
(58), a homolog of the Drosophila proneural
achaete-scute complex (29) with multiple roles in
vertebrate autonomic neurogenesis (reviewed in reference
17).
Targeted deletion of the ASH-1 gene in the mouse has demonstrated that
ASH-1 is essential for development of the SA lineage (7, 19, 25,
36). In primary cultures of neural crest stem cells
(63), ASH-1 maintains competence for neurogenesis (43) and promotes autonomic differentiation of committed
neuronal precursor cells (61). In addition, the closely
related paired-like homeodomain transcription factors Phox2a
(49) and Phox2b (52, 53) are essential for
development of the SA lineage. Phox2 proteins function synergistically
with the cAMP signaling pathway, via neighboring homeodomain and CRE
cis-acting elements, to directly regulate transcription of
both the TH and DBH genes (15, 31, 66, 71, 72). The Phox2
proteins, in addition to being necessary (42) and sufficient
(62) for SA cell development, likely promote survival of SA
progenitors by regulating the expression of the glial-derived
neurotrophic factor receptor c-Ret (10, 44, 49, 53).
Although expression of the Phox2a gene, but not the Phox2b gene, is
regulated by ASH-1 (25, 44), these genes display cross-regulation (52, 53, 62); i.e., the expression of one induces expression of the other. Moreover, Phox2b is necessary for the
maintenance, but not the induction, of ASH-1 (53).
The molecular mechanisms that regulate the expression of these SA
lineage-determining genes remain to be elucidated. Likewise, the
molecular mechanisms that integrate the function of the SA lineage-determining transcription factors with other transcription factors in directing the neurogenic program, the specification of
neurotransmitter identity, and the survival of the progenitor cell have
yet to be deciphered. Accordingly, the combinatorial effect of the
BMP-2 and cAMP signaling pathways on SA cell development and,
specifically, on the expression and function of the SA
lineage-determining factors remains to be explored.
Using primary cultures of avian neural crest cells, we explored
interactive mechanisms between the BMP-2 and cAMP signaling pathways.
Herein, we report that the cAMP signaling pathway is a dynamic
regulator of SA cell development. Moderate levels of cAMP signaling
exert an SA-promoting influence which functions in synergy with BMP-2
to dramatically stimulate SA cell development. By contrast, high levels
of cAMP signaling modulate a dominant antagonism that, even in the
presence of BMP-2, blocks SA cell development. Furthermore, we show
that the SA-promoting effect of cAMP signaling involves the induction
of the Phox2a gene and that the SA-antagonizing effect involves
inhibition of the expression of the SA lineage-determining genes
ASH-1, Phox2a, and Phox2b.
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MATERIALS AND METHODS |
Neural crest cultures.
Primary cultures of trunk neural
crest cells were prepared from Japanese quail (Coturnix
coturnix) embryos stage 12 to 13 (23) essentially as
described by Maxwell et al. (47). Eggs were incubated for
47.5 h at 37.5°C with 58% humidity. Embryos were separated from
the yolk and rinsed in Hanks' balanced salt solution buffered with 15 mM HEPES (pH 7.4). The neural tubes and associated neural crests were
dissected from embryos as explants of the trunk region including the
last five somites and extending through the unsegmented mesoderm to the
chordal neural hinge. Explants were incubated in pancreatin (6.25 mg/ml) until the neural tubes were freed from adjacent somites,
notochord, and surface ectoderm. Neural tubes were washed in growth
medium and plated in dishes coated with Vitrogen 100 (Collagen
Corporation). In primary culture, numerous crest cells emigrate from
the neural tubes onto the surface of the culture dish. After 42 h,
neural tubes were dissected free using tungsten needles and removed by washing from the plate with calcium- and magnesium-free
phosphate-buffered saline, pH 7.4 (CMF-PBS). Crest cells were harvested
in growth medium following a brief treatment with trypsin-EDTA.
Mass cultures.
Cells were suspended in growth medium and
seeded at a density of 320 cells/mm2 in tissue culture
dishes treated with bovine fibronectin (40 µg/ml; Sigma). Cells were
allowed to attach for 2 h, and then the seeding medium was
replaced with 2 ml of growth medium. Cells were fed by an exchange of 1 ml of growth medium on day 3 after passage into secondary culture and
every other day thereafter.
Clonal cultures.
Cells were suspended in growth medium at a
density of 100 cells/ml, and 1-ml aliquots were seeded into
35-mm-diameter tissue culture dishes treated with both Vitrogen 100 and
bovine fibronectin (20 µg/ml). After allowing 2 h for cells to
attach, the clonal cultures were maintained as described for mass cultures.
Growth medium and other reagents.
All cultures were grown at
37°C in a 5% CO2, humidified incubator. Growth medium
contained 75 ml of Dulbecco modified Eagle medium
Ham's F-12 (Life
Technologies), 15 ml of heat-inactivated horse serum (HyClone), 10 ml
of day 9 chick embryo extract (8), 10 mg of gentamicin
sulfate, 10 mg of kanamycin sulfate, 1 ml of 7.5% sodium bicarbonate,
1 ml of 0.2 M L-glutamine, and 1 ml of 100× stock vitamin
mix (47). Stock vitamin mix contained 1 mg of
6,7-dimethyl-5,6,7,8-tetrahydropterine, 100 mg of
L-ascorbic acid, and 5 mg of oxidized glutathione in 20 ml
of distilled water, pH 6.0 (45, 47).
BMP-2 was generously provided by Genetics Institute, Inc., and was
reconstituted as recommended by the provider. 8-Bromo-cAMP (8-Br-cAMP)
and norepinephrine were obtained from Sigma and reconstituted in water.
Forskolin, 3-isobutyl-1-methylxanthine (IBMX; Sigma), vinpocetine, Ro
20-1724, and MY-5445 were reconstituted in dimethyl sulfoxide. The
presence of 0.1% dimethyl sulfoxide had no observable effect on crest
cell growth or differentiation (data not shown). Stock solutions
(1,000×) for all reagents were maintained at 
80°C. Unless
otherwise noted, all experimental reagents were obtained from Biomol
(Plymouth Meeting, Pa.) and added to the cultures at the designated
feeding times.
Western blot analysis.
Cells from primary neural crest
cultures were subcultured in 24-well dishes and maintained as described
above. Cultures were harvested by triturating in 120 µl of
radioimmunoprecipitation buffer (150 mM NaCl, 2 mM EDTA, 1 mM sodium
orthovanadate, 10 µg of leupeptin and 25 µg of aprotinin per ml,
1.0% Triton X-100, 50 mM Tris [pH 7.6]) and sonicating on ice for
20 s. Total protein concentration was determined by the Bio-Rad
protein assay. Cell extract (20 µg) was boiled with a 1:5 dilution of
sample buffer (10% sodium dodecyl sulfate [SDS], 50% glycerol, 5%
2-mercaptoethanol, 0.4 M Tris [pH 6.8]), electrophoresed on an
SDS-10% polyacrylamide gel, and transferred to nitrocellulose.
Immunoblot analysis used a 1:5 dilution of supernatant containing the
monoclonal anti-TH antibody (13) (Developmental Studies
Hybridoma Bank, University of Iowa) and a 1:300 dilution of the rabbit
antiactin antibody (Sigma). Detection was with 1:10,000 and 1:2,000
dilutions of horseradish peroxidase-conjugated anti-mouse
immunoglobulin G (IgG) (Jackson Laboratory) and anti-rabbit IgG
(Vector) antibodies, respectively, using the Amersham ECL (enhanced
chemiluminescence) detection system.
Immunochemical and histochemical fluorescence.
TH-expressing
cells were visualized using indirect immunofluorescence. Neural crest
cultures were fixed for 20 min in 4% paraformaldehyde and
permeabilized for 30 min with PBT (0.2% bovine serum albumin and 0.1%
Triton X-100 in CMF-PBS [pH 7.4]). The monoclonal anti-TH antibody
(see "Western blot analysis" above) was applied as undiluted supernatant for 40 min followed by three washes with PBT. A 1:64 dilution of the fluorescein isothiocyanate-conjugated donkey anti-mouse IgG antibody (Sigma) was applied in the dark for 40 min followed by
washing once with PBT and twice with CMF-PBS. Cultures were rinsed with
distilled water and mounted using FluorSave reagent (Calbiochem) and a coverslip.
CA-containing cells were visualized by the procedure of Furness et al.
(
16), which produces a water-stable fluorophore.
Neural
crest cultures were washed twice with CMF-PBS followed
by incubation in
4% paraformaldehyde and 0.1% glutaraldehyde for
2 h at room
temperature. Cultures were washed once with CMF-PBS
and mounted using
glycerol and a
coverslip.
PKA activity.
Cells from primary neural crest cultures were
subcultured as described above except that a 6 × 105
cells were seeded in a 35-mm-diameter dish. Following 2 h for cell
attachment, seeding medium was replaced with 2 ml of growth medium and
the indicated cAMP-elevating agents. After 2 h, the cells were
harvested by scraping in extraction buffer (5 mM EDTA, 50 mM Tris [pH
7.5]) and lysed by briefly sonicating on ice. Protein kinase A (PKA)
activity was analyzed using a radiometric PKA assay system (Life
Technologies) as described by the manufacturer.
RNA and RT-PCR.
Total RNA was isolated from neural crest
cells subcultured in 35-mm-diameter plates using 1 ml of TRIZOL reagent
(Life Technologies) as described by the manufacturer. Reverse
transcription-PCR (RT-PCR) was performed with total RNA (1 µg) and
gene-specific primers using the Titan One Tube RT-PCR system (Roche
Biomolecular). Typically 30 cycles of amplification were conducted
using the following conditions: denaturation at 94°C, annealing at
55°C, and extension at 68°C, with 1 min at each step. Primers for
ASH-1 (5' sense oligonucleotide, 5'-AACCGAGTCAAGCTGGTGAA-3';
3' antisense oligonucleotide, 5'-TCAGAACCAGCTGGTGAA-3'),
Phox2a (5' sense oligonucleotide,
5'-CGTCCGCCTACGATTTCAACC-3'; 3' antisense oligonucleotide,
5'-TGATGGCCGATGGGTCCGAA-3'), Phox2b (5' sense
oligonucleotide, 5'-TCGAGCCTGGCTTCAGCGTAT-3'; 3' antisense oligonucleotide, 5'-TCAAACCGCCGTGGTCGGTG-3'), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5' sense
oligonucleotide, 5'-GTGAAAGTCGGAGTCAAC-3'; 3' antisense
oligonucleotide, 5'-TGGTGCACGATGCATTGC-3') were derived from
the sequenced chicken genes (J.-F. Brunet, personal communication; 28, 51). Primers for TH (5' sense oligonucleotide,
5'-CTGGAAGGAGGTGTACAGTA-3'; 3' antisense oligonucleotide,
5'-AGCAGCGTCAGGATCAAAGT-3') were derived from the sequenced
quail gene (12). To enhance photocopy reproducibility,
negative digital images were produced from photographs of ethidium
bromide-stained agarose gels by using Adobe Photoshop software.
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RESULTS |
Effects of BMP-2 and cAMP on SA cell development.
To
investigate whether the BMP-2 and cAMP signaling pathways exert a
combinatorial influence on SA cell development, we used primary
cultures of quail neural crest cells and assessed the expression of
phenotypic markers of the SA lineage, namely, TH and CA. We activated
the cAMP signaling pathway by using three distinct types of
cAMP-elevating agents: IBMX, a nonselective inhibitor of cyclic
nucleotide phosphodiesterases (PDE) (6); forskolin, an
activator of adenylate cyclase (37, 57); and 8-Br-cAMP, an
analog of cAMP with specificity for activating PKA and reduced
hydrolysis by PDE (24, 48, 56). These cAMP-elevating agents,
applied alone or in combination with BMP-2, resulted in complex effects
on the development of SA cells (Fig. 1
and 2) and on the overall growth of the
neural crest cultures (Table 1). Reported
herein, we measured SA cell development by quantifying the relative
expression levels of TH detected in Western blot assays (Fig. 1). In a
related study, results from this method of analysis were shown to
closely correlate with the proportion of TH-positive crest cells
detected by indirect immunofluorescence (data not shown). Additionally,
we qualitatively confirmed the effects on SA cell development by
monitoring the generation of TH-immunoreactive (TH-positive) and
CA-histofluorescent (CA-positive) cells (Fig. 2).
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FIG. 1.
Western blot analysis of TH protein expression in neural
crest cultures treated with cAMP-elevating agents and BMP-2. Cellular
extracts were prepared from cultures grown 7 days in the presence of
BMP-2 (10 ng/ml) and the cAMP-elevating agents IBMX (100 µM),
forskolin (10 µM), and 8-Br-cAMP (100 µM) as indicated. The 63-kDa
TH protein and 42-kDa actin protein, serving as an internal control,
are indicated by arrows. The histogram was derived by densitometric
scanning of blots from three independent cellular extract preparations.
The effects of IBMX and forskolin compared to the control, the
combination of BMP-2 with IBMX compared to treatment with either agent
alone, and the combination of BMP-2 with forskolin or 8-Br-cAMP are
significant at P < 0.05 (analysis of variance).
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FIG. 2.
Differentiation of TH- and CA-positive cells in neural
crest cultures. Crest cells were grown for 6 days in the presence of 10 ng of BMP-2 per ml and 100 µM IBMX as indicated. (A) Indirect
immunofluorescence to visualize TH-expressing cells; (B)
formaldehyde-induced fluorescence to visualize CA-containing cells
(bar = 100 µm).
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Consistent with results reported by Varley and Maxwell (
68),
treatment of the cultures with BMP-2 (10 ng/ml) dramatically
increased
both the expression of TH protein (Fig.
1, compare lanes
1 and 2) and
the development of TH-positive and CA-positive SA
cells (Fig.
2).
Treatment of the cultures with either 10 µM forskolin
or 100 µM
IBMX significantly increased the expression of TH protein
(Fig.
1,
lanes 3 and 5 compared to lane 1); however, treatment
with 100 µM
8-Br-cAMP produced no detectable increase in TH protein
expression
(lane 7). Interestingly, the combination of BMP-2 and
IBMX resulted in
a synergistic 2.7-fold increase in the expression
of TH protein (Fig.
1, lane 4 compared to lanes 2 and 3). In contrast
to the synergistic
effect of BMP-2 and IBMX, the combination of
BMP-2 with forskolin or
8-Br-cAMP decreased the expression of
TH protein by 37% and greater
than 93%, respectively (Fig.
1,
lanes 6 and 8 compared to lane
2).
The differential effects of the cAMP-elevating agents on neural crest
cultures were confirmed by microscopy using immunochemical
and
histochemical fluorescence assays (Fig.
2). The combination
of BMP-2
and IBMX stimulated an obvious increase in the development
of
TH-positive and CA-positive SA cells (Fig.
2). However, the
multilayer
nature of the cultures and the large number of TH-positive
and
CA-positive cells precluded our ability to accurately measure
these
effects. In contrast, 8-Br-cAMP had a profound negative
impact on the
development of SA cells, abolishing the generation
of TH-positive and
CA-positive cells, both in the absence and
in the presence of BMP-2
(data not
shown).
It is possible that the negative effect of 8-Br-cAMP on SA cell
development is related to its profound effect on the overall
growth of
the neural crest cultures (Table
1). By comparison,
treatment with IBMX
or forskolin, alone or in combination with
BMP-2, had a less pronounced
effect on overall growth (Table
1).
These data indicate that
cAMP-elevating agents differentially
influence SA cell development
(Fig.
1) and crest cell growth (Table
1).
To gain further insight into the developmental role of BMP-2 and IBMX,
we studied crest cell survival and development in cultures
initiated at
clonal density (Table
2). The total number of colonies
generated in the
assay was similar in all treatment groups, suggesting
that BMP-2 and
IBMX, alone or in combination, do not affect the
survival of colony
founder cells (Table
2). In agreement
with
results reported by Varley and Maxwell (
68), the
development
of CA-positive colonies was stimulated by treatment with
BMP-2.
IBMX alone generated a slight 0.3-fold increase relative to
BMP-2
in the proportion of CA-positive colonies. Interestingly, the
combination of BMP-2 and IBMX resulted in a dramatic increase
in the
proportion of CA-positive colonies, i.e., a 2.2-fold increase
relative
to BMP-2 and a 27-fold increase relative to IBMX. These
data provide
further evidence that BMP-2 and IBMX function in
synergy to promote the
development of SA cells.
IBMX is a nonselective PDE inhibitor and could act by inhibiting a PDE
other than the cAMP-specific (type III) PDE (
6).
Therefore,
we analyzed the effects of several selective PDE inhibitors
to confirm
that the cAMP signaling pathway was specifically promoting
SA cell
development (Fig.
3). The agents included
vinpocetine,
an inhibitor of the Ca
2+/calmodulin-dependent
(type I) PDE (
1,
21); Ro 20-1724, an
inhibitor of
cAMP-specific (type III) PDE (
30,
54,
59);
and MY-5445, an
inhibitor of the cGMP-specific (type V) PDE (
22).
In the
absence and presence of BMP-2, neither 100 µM vinpocetine
nor 30 µM
MY-5445 had a significant effect on TH protein expression
(Fig.
3A,
lanes 5 and 9 compared to lane 1; lanes 6 and 10 compared
to lane 2).
By contrast, the combination of BMP-2 and Ro 20-1724
produced a
synergistic 2.4-fold increase in the expression of
TH protein (Fig.
3A,
lane 8 compared to lanes 2 and 7) and stimulated
an obvious increase in
the development of SA cells (data not shown).
These increases were
comparable to the effects of BMP-2 and IBMX
(Fig.
2 and
3A, lane 4) and
support the conclusion that IBMX,
like Ro 20-1724, promotes SA cell
development by activating the
cAMP signaling pathway.
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FIG. 3.
Effects of PDE inhibitors on TH protein expression in
neural crest cultures. (A) Western blot analysis of TH protein
expression in cultures grown for 7 days in the presence of BMP-2 (10 ng/ml) and PDE inhibitor IBMX (100 µM), vinpocetine (100 µM), Ro
20-1724 (20 µM), or MY-5445 (30 µM) as indicated. The histogram was
derived by densitometric scanning of blots from three independent
cellular extract preparations. The effects of BMP-2 and Ro 20-1724 compared to treatment with either agent alone are significant at
P < 0.05 (analysis of variance). The effects of BMP-2
and either vinpocetine or MY-5445 are not significantly different from
treatment with only BMP-2. (B) Western blot analysis of TH protein
expression in cultures grown for 7 days in the presence of 10 ng of
BMP-2 per ml, 20 µM Ro 20-1724, 10 µM forskolin, and 100 µM
8-Br-cAMP as indicated.
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Furthermore, the combination of Ro 20-1724 and forskolin decreased the
expression of TH protein (Fig.
3B, lanes 4 and 6 compared
to lanes 3 and 5, respectively). Cotreatment with Ro 20-1724 and
forskolin
undoubtedly results in higher intracellular cAMP levels
than treatment
with either agent alone. Accordingly, these data
suggest that the
intracellular level of cAMP, which reflects the
level of activation of
the cAMP signaling pathway, is a criterion
for selecting the pathway's
function either to promote or to antagonize
SA cell
development.
Dose-dependent modulation of SA cell development by cAMP-elevating
agents.
To investigate the model that the specific function of the
cAMP pathway, i.e., as an SA-promoting or SA-antagonizing mechanism, is
determined by the level of cAMP signaling, we tested the ability of the
various cAMP-elevating agents to activate the cAMP signaling pathway.
Initially we used a radiometric PKA assay to test the ability of
cAMP-elevating agents to activate the endogenous PKA enzyme in neural
crest cultures (Table 3). Both forskolin
and 8-Br-cAMP stimulated marked activation, 6- and 32-fold,
respectively, of endogenous PKA. By contrast, IBMX or Ro 20-1724 stimulated only small increases in PKA activity. In related studies, we
used a nonradioactive assay to measure cAMP levels (Amersham Pharmacia) in cultures treated with combinations of BMP-2 and either IBMX or
forskolin. We observed that only forskolin, independent of the presence
of BMP-2, stimulated a detectable increase in intracellular cAMP (data
not shown). Additionally, we used a phospho-specific CREB (Ser133)
antibody (New England Biolabs) and Western blot assays to monitor the
activation of endogenous CREB following treatment of the cultures with
combinations of BMP-2 and either IBMX or forskolin. Only forskolin,
independent of the presence of BMP-2, induced a detectable increase in
CREB phosphorylation (data not shown). Taken together, these
observations indicate that the distinct effects of IBMX, forskolin, and
8-Br-cAMP on SA cell development (Fig. 1) are linked to their ability
to activate the cAMP signaling pathway to different degrees.
To further confirm this model, we measured the dose-dependent effects
of cAMP-elevating agents on BMP-2-induced TH protein
expression. As
shown in Fig.
4, low doses (0.1 to 1.0 µM) of forskolin
or 8-Br-cAMP dramatically enhanced BMP-2-induced TH
protein expression
(Fig.
4, lanes 6, 7, 10, and 11 compared to lane 1)
and produced
an obvious increase in the development of SA cells (data
not shown).
Moreover, the combinatorial effect of BMP-2 and low dose of
forskolin
or 8-Br-cAMP on the expression of TH protein was similar to
the
synergistic influence of BMP-2 and IBMX (Fig.
4, lanes 4 and 5).
Conversely, a high dose (100 µM) of forskolin or 8-Br-cAMP largely
blocked the stimulatory effect of BMP-2 on TH protein expression
(Fig.
4, lanes 9 and 13 compared to lane 1). These data are consistent
with
the model that moderate levels of cAMP signaling promote
SA cell
development, whereas high levels of cAMP signaling antagonize
SA cell
development.
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FIG. 4.
Low dose of forskolin or 8-Br-cAMP acts cooperatively
with BMP-2 to stimulate development of SA cells in neural crest cell
cultures. Shown is Western blot analysis of TH protein expression in
cultures grown 5 days in the presence of 10 ng of BMP-2 per ml and 0.1, 1.0, 10, or 100 µM cAMP-elevating agent as indicated. The histogram
was derived by densitometric scanning of blots from three independent
cellular extract preparations. The effects of BMP-2 and either low (0.1 to 1.0 µM) or high (100 µM) doses of forskolin or 8-Br-cAMP are
significant compared to treatment with BMP-2 alone (P < 0.05, analysis of variance). The effects of BMP-2 and low-dose
forskolin or 8-Br-cAMP are not significantly different from treatment
with BMP-2 and 1.0 to 100 µM IBMX.
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Early requirement of cAMP-elevating agents in antagonizing SA cell
development.
To further understand the mechanism by which the cAMP
signaling pathway antagonizes SA cell development, we tested the
temporal requirements for the inhibitory effect of high dose of
forskolin on the BMP-2-induced expression of TH protein. In parallel
assays, we also examined the inhibitory effect of high dose of
norepinephrine, a natural effector of the cAMP signaling pathway.
Importantly, 100 µM forskolin or 100 µM norepinephrine had a
significantly reduced impact on overall crest cell growth
(P < 0.05) (Table 4)
compared to 100 µM 8-Br-cAMP (Table 1), suggesting that the antagonism of SA cell development is not solely dependent on an inhibition of crest cell growth. As shown in Fig.
5, continuous treatment with either 100 µM forskolin or 100 µM norepinephrine for the entire culturing
period greatly reduced the ability of BMP-2 to stimulate TH protein
expression (compare lanes 1 and 2). Exposure of the cultures to a high
dose of forskolin or norepinephrine for at least the initial 24 h
of culturing was crucial for either agent to reduce the expression of
TH protein (Fig. 5, lane 7 compared to lane 1). Treatment with
forskolin beyond the first 24 h of culturing reduced TH protein
expression to levels comparable to those obtained by treatment for the
entire culturing period (Fig. 5A, lanes 4 to 6 compared to lane 2).
Norepinephrine required continuous exposure for a minimum of 72 h
to reduce TH protein expression (Fig. 5B, lanes 5 and 6 compared to
lane 1). Surprisingly, the addition of norepinephrine for only the
initial 48 h of culturing reproducibly increased TH protein
(n = 2; Fig. 5B, lane 4 compared to lane 1). The
mechanism of this effect has not been further examined.
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FIG. 5.
Temporal requirements of high doses of cAMP-elevating
agents to antagonize SA cell development in neural crest cultures,
determined by Western blot analysis of TH protein expression in 6-day
cultures treated with 100 µM forskolin (A) and 100 µM
norepinephrine (B).
|
|
Synergy of BMP-2 and cAMP signaling regulates the SA
lineage-determining gene Phox2a.
The SA-promoting and
SA-antagonizing influences of cAMP signaling (Fig. 4) suggest that the
cAMP signaling pathway regulates the expression of SA
lineage-determining genes, such as ASH-1 (7, 19, 25, 36),
Phox2a (49), and Phox2b (52, 53). To test this
hypothesis, we used RT-PCR to monitor the expression of the ASH-1,
Phox2a, and Phox2b genes under cAMP signaling conditions that either
promote or antagonize SA cell development (as discussed above). The TH
and GAPDH genes were monitored as controls for SA cell differentiation
and RNA loading, respectively.
In agreement with previous reports (
42,
58), BMP-2 increased
the expression of ASH-1, Phox2b, Phox2a, and TH (Fig.
6B
and D, lane 2 compared to lane 1).
Treatment of the cultures with
IBMX or 0.1 µM forskolin had no
detectable effect on the expression
of the SA lineage-determining genes
(Fig.
6B and D, lanes 3 and
5 compared to lane 1); however, IBMX did
produce an increase in
TH gene expression (Fig.
6D, lane 3).
Interestingly, the combination
of BMP-2 with IBMX or low dose of
forskolin, conditions that synergistically
promote SA cell development,
increased the expression of Phox2a
and TH (Fig.
6D, lanes 4 and 6 compared to lane 2), but these
conditions had no detectable effect on
ASH-1 or Phox2b (Fig.
6B,
lanes 4 and 6 compared to lane 2).
View larger version (56K):
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|
FIG. 6.
Synergy between the BMP-2 and cAMP signaling pathways
regulates the SA lineage-determining gene Phox2a in neural crest
cultures determined by RT-PCR of 1 µg of total RNA isolated from
neural crest cells after 2 (A and B) days or 3 (C and D) days in
culture. (A and C) Cycle-dependent amplification of TH and SA
lineage-determining genes expressed by neural crest cultures grown in
the presence of 10 ng of BMP-2 per ml. (B and D) Expression of TH and
SA lineage-determining genes in neural crest cultures treated with
combinations of 10 ng of BMP-2 per ml and the specified concentration
(micromolar) of cAMP-elevating agent as indicated. Representative
results of RT-PCR using 30 cycles of amplification to detect gene
expression are shown. Amplification of the GAPDH gene was used as an
internal control. Lane C is a negative control from which RNA was
omitted in the RT-PCR.
|
|
On the contrary, 100 µM forskolin, a condition that antagonizes SA
cell development, blocked the expression of SA lineage-determining
genes, inhibiting the BMP-2-induced expression of ASH-1, Phox2b,
Phox2a, and TH (Fig.
6B and D, compare lanes 2 and 8). Consistent
with
work by others (
17), these results support a conclusion
that
regulation of ASH-1, Phox2a, and Phox2b gene expression is
central to
the development of SA cells. Moreover, these results
identify possible
transcriptional targets of SA-promoting and
SA-antagonizing cAMP
signals. Thus, moderate levels of cAMP signaling
may stimulate SA cell
differentiation by inducing the expression
of Phox2a. High levels of
cAMP signaling may oppose SA cell differentiation
by blocking the
expression of the ASH-1 and Phox2
genes.
| |
DISCUSSION |
The ability of neural crest cells in primary culture to give rise
to an array of cell types provides a powerful model system to explore
molecular mechanisms that determine cell fate. Using primary cultures
of quail neural crest cells, we investigated the combinatorial effects
of the BMP-2 and cAMP signaling pathways on the development of the SA
lineage. Our findings indicate that the cAMP signaling pathway acts as
a bimodal switch on SA cell development, governing both positive and
negative signals which influence crest cell fate. We have shown that
moderate activation of the cAMP signaling pathway stimulates the
development of SA cells, whereas robust activation of the pathway
opposes SA cell development. Furthermore, our data indicate that the
cAMP signaling pathway influences the ability of BMP-2 to induce SA
cell development. Moderate levels of cAMP signaling act synergistically
with BMP-2 to further promote the development of SA cells. By contrast,
high levels of cAMP signaling oppose BMP-2 signaling and SA cell
development. We demonstrate that the combinatorial effect of BMP-2 and
cAMP signaling influences the expression of SA lineage-determining genes, namely, the transcription factors ASH-1, Phox2a, and Phox2b. Our
data show that the SA-promoting effect of cAMP signaling involves the
induction of Phox2a gene expression and that the SA-antagonizing effect
involves the inhibition of ASH-1 and Phox2 gene expression.
cAMP signaling promotes and antagonizes SA cell development.
Earlier studies examined the effects of cAMP-elevating agents on the in
vitro development of quail neural crest cells and reported seemingly
conflicting roles for the cAMP signaling pathway. Specifically,
8-Br-cAMP and IBMX were reported to inhibit the ability of
reconstituted basal membrane-like matrix to stimulate SA cell
development (46). By contrast, forskolin and -adrenergic ligands were shown to regulate the expression of the quail TH gene and
promote the development of SA cells (9). More recent investigations also demonstrated that BMP-2, -4, and -7/OP-1
dramatically stimulate SA cell development (55, 68, 70) and
established the causal link of a BMP ligand on the generation of SA
cells (69).
Based on these investigations and the model proposing that multiple
signaling cues direct crest cell fate (
3), we sought
first
to resolve the role of cAMP signaling in SA cell development
and second
to investigate the combinatorial effects the BMP-2
and cAMP signaling
pathways on SA cell development. Using primary
cultures of quail neural
crest cells, we examined SA cell development
in cultures treated with
combinations of BMP-2 and either IBMX,
forskolin, or 8-Br-cAMP. We used
Western blot assays of TH protein,
a phenotypic marker of the SA
lineage, to measure SA cell development
(Fig.
1). We observed that the
various cAMP-elevating agents had
distinct abilities to stimulate the
expression of TH protein.
IBMX and forskolin increased the expression
of TH protein, whereas
8-Br-cAMP had no detectable effect. Of even more
interest, the
various cAMP-elevating agents differentially influenced
BMP-2-induced
TH protein expression. IBMX acted in synergy with BMP-2
to increase
the expression of TH protein, whereas forskolin and
8-Br-cAMP
greatly reduced the ability of BMP-2 to stimulate TH protein
expression.
We reasoned that these differences might be due to the molecular
mechanism by which each agent elevates intracellular cAMP.
Specifically, forskolin directly stimulates de novo cAMP synthesis
by
activating the catalytic enzyme adenylate cyclase (
37,
57).
The 8-Br-cAMP analog mimics the effects of endogenous cAMP by
specifically activating PKA but exhibits a significantly reduced
rate
of degradation by the cAMP-specific PDE (
24,
48,
56).
By
contrast, IBMX increases cAMP levels by nonselectively inhibiting
a
variety of cyclic nucleotide PDEs, prolonging the intracellular
half-life of endogenous cyclic nucleotides (
6). Thus,
agents
efficiently increasing the intracellular concentration of cAMP,
i.e., forskolin and 8-Br-cAMP, are expected to produce a higher
steady-state activation of the cAMP signaling pathway compared
to an
agent that passively sustains the levels of endogenous cAMP,
i.e.,
IBMX. In support of this proposal, Table
3 shows that 8-Br-cAMP
is the
most potent activator of the cAMP signaling
pathway.
Although both BMP-2 and IBMX independently produced obvious increases
in the development of TH-positive and CA-positive SA
cells, their
combined effects were striking (Fig.
2). Data obtained
from analyzing
clonal cultures of crest cells (Table
2) further
corroborate the
dramatic, synergistic influence of BMP-2 and IBMX
on the development of
SA cells. Despite the common usage of IBMX
in cAMP signaling studies,
IBMX is a nonspecific inhibitor of
PDE. Our studies confirmed that the
effects of IBMX were specifically
dependent on the cAMP signaling
pathway by comparing the influence
of several selective PDE inhibitors
on TH protein expression (Fig.
3A). Importantly, we demonstrated that
the cAMP-specific (type
III) PDE inhibitor Ro 20-1724 (
30,
54,
59) dramatically
increased the expression of TH protein (Fig.
3A)
and the development
of SA cells (data not shown). In contrast,
selective inhibition
of either the
Ca
2+/calmodulin-dependent (type I) or the cGMP-specific
(type V) PDE
had no significant effect. We further tested the effects
of cotreating
neural crest cultures with Ro 20-1724 and either
forskolin or
8-Br-cAMP (Fig.
3B). Independent of the presence of BMP-2,
the
combination of forskolin and PDE inhibitor decreased TH protein
expression. Undoubtedly, the combination of Ro 20-1724 and forskolin
results in higher intracellular cAMP levels than treatment with
either
agent alone. Accordingly, these data suggest that the specific
function
of the cAMP pathway to either promote or antagonize SA
cell development
is determined by the intracellular concentration
of cAMP and, in turn,
the activation level of the cAMP signaling
pathway.
cAMP signaling levels regulate SA cell development.
To
investigate this model, we tested the ability of the cAMP-elevating
agents to activate the endogenous PKA enzyme (Table 3). IBMX or Ro
20-1724 produced small, detectable increases in PKA activity, whereas
forskolin or 8-Br-cAMP markedly stimulated PKA activity. Importantly,
the differential activation of PKA correlates with the ability of these
cAMP-elevating agents to either promote or antagonize SA cell development.
Additionally, we related the effects of BMP-2, IBMX, and forskolin to
changes in both intracellular cAMP levels and CREB phosphorylation.
In
experiments not shown, we found that the standard growth conditions
required for neural crest cultures resulted in extremely high
baselines, rendering both assays inadequate for the detection
of
changes in the respective components of the cAMP signaling
pathway.
Crest cells do not survive well in the absence of serum
and/or chick
embryo extract; culturing crest cells for 18 h in
a 1:200 dilution
of standard culture medium enabled us to maintain
a reasonable degree
of cell survival and to marginally reduce
the assay baselines. Under
these conditions, 100 µM forskolin
produced a sustained elevation in
intracellular cAMP and induced
the phosphorylation of CREB (data not
shown); however, such changes
were not detectable in treatments with
IBMX or 0.1 µM forskolin.
Likewise, BMP-2 alone or in combination
with a cAMP-elevating
agent produced no detectable effects on cAMP
levels or CREB phosphorylation.
In summary, the high content of serum
and embryo extract in the
medium required for the maintenance of crest
cells in vitro precluded
the quantitative measurement of small changes
in cAMP levels and
CREB phosphorylation produced by treatment with IBMX
or a low
dose of forskolin. However, taken with results of our PKA
experiments
(Table
3), these observations fully support the view that
the
various cAMP-elevating agents exhibit different capacities for
activating cAMP signaling
mechanisms.
From the analyses described above, we cannot conclude whether BMP-2
regulates cAMP signaling in crest cells. Studies on chondrogenesis
(
41) and renal branching (
20) have revealed that
BMP-2 stimulates
the activation of PKA and the phosphorylation of CREB
by a mechanism
not yet understood. Investigations using primary rat
neural crest
cultures observed that the combination of 10 ng of BMP-2
per ml
and 5 µM forskolin weakly induced TH protein expression, but
the
combination of 0.1 ng of BMP-2 per ml and 5 µM forskolin
stimulated
a robust induction of TH protein (
42). If BMP-2
does regulate
the cAMP signaling pathway in crest cells, then indeed
this decreased
dosage of BMP-2 is akin, in part, to a reduced level of
cAMP signaling
promoting SA cell development. Additionally, we are
puzzled by
the biphasic nature of BMP-2 (and BMP-4) dosage on SA cell
development
(
55,
68) and by our data indicating that 10 µM
forskolin is
converted by BMP-2 from an SA-promoting agent to an
SA-antagonizing
agent (Fig.
1). A finding that BMP-2 activates the cAMP
signaling
pathway in crest cells would provide a logical explanation
for
the attenuated effect of high dose of BMP-2 on SA cell development
(
55,
68) and the SA-antagonizing conversion of 10 µM
forskolin
by BMP-2 (Fig.
1).
To confirm by an another method that the cAMP signaling pathway
functions to either promote or antagonize SA cell development
as a
consequence of the level of its activation, we examined the
dose-dependent effects of the various cAMP-elevating agents on
the
BMP-2-induced expression of TH protein (Fig.
4). These data
conclusively demonstrate that TH protein expression is promoted
by low
levels of cAMP signaling, induced by 0.1 to 1.0 µM forskolin
or
8-Br-cAMP, and that TH protein expression is antagonized by
high levels
of cAMP signaling, induced by 100 µM forskolin or
8-Br-cAMP.
Accordingly, our investigations support a mechanism
whereby cAMP
signaling functions as a bimodal switch on SA cell
development.
The antagonistic effect of the cAMP signaling pathway is temporally
defined.
To verify that the observed antagonistic effect of
cAMP-elevating agents on SA cell development is authentic, we performed two types of analyses. First, we demonstrated that this negative effect
displays specific temporal requirements (Fig. 5A). Importantly, the
inhibitory capacity of 100 µM forskolin is fully established within
the first 48 h of culturing and is essentially lost if it is
applied after the initial 24 h of culturing. This suggests that
cAMP signaling mechanisms interfere with pathways required for crest
cells to commit to the SA lineage.
Second, we demonstrated that this negative effect is reproduced by the
addition of a natural effector of the cAMP signaling
pathway (Fig.
5B).
Similar to 100 µM forskolin, 100 µM norepinephrine
has an
inhibitory capacity that is lost if it is applied after
the initial
24 h of culturing. The major difference between the
two agents is
that the negative effect of norepinephrine on SA
cell development
requires the continuous treatment of the cultures
for at least 72 h. Treatment with norepinephrine for only the
initial 48 h of
culturing increases TH protein expression. Based
on earlier studies
(
60,
73,
74), we interpret this transient
positive effect to
involve the norepinephrine transporter and
intracellular calcium which,
in turn, may induce the expression
of necessary molecular links, i.e.,
adrenergic autoreceptors,
for norepinephrine to antagonize SA cell
development. In conclusion,
the brief but specific temporal
requirements of forskolin as well
as the ability of a natural cAMP
signaling effector such as norepinephrine
to inhibit TH protein
expression further validate the significance
of a mechanism whereby
cAMP signaling opposes SA cell
development.
Integrating BMP-2 and cAMP signaling pathways in SA cell
development.
The stimulatory and inhibitory capacities of cAMP
signaling (Fig. 4) suggested that the pathway regulates the expression
of genes necessary for crest cell commitment to the SA lineage, i.e., genes acting upstream from the expression of TH and DBH. Gene knockout
studies in the mouse have demonstrated that the ASH-1 gene (7, 19,
25, 36) and the Phox2 genes, Phox2a (49) and Phox2b
(52, 53), are essential for development of SA progenitor cells. Accordingly, we investigated whether the cAMP signaling mechanisms regulate the expression of these SA lineage-determining genes.
Importantly, we showed by RT-PCR that the combinatorial effect of BMP-2
and moderate levels of cAMP signaling, i.e., IBMX
or 0.1 µM
forskolin, is exerted not only on the increased expression
of TH but
also on the expression of the SA lineage-determining
gene Phox2a (Fig.
6D). This moderate level of cAMP signaling does
not affect induction of
the ASH-1 or Phox2b gene (Fig.
6B). Together
with the recent reports
that the Phox2 proteins are necessary
(
42) and sufficient
(
62) for development of the SA lineage,
these data suggest
that the BMP-2 and cAMP signaling pathways
converge on Phox2a gene
expression to promote SA cell differentiation.
Additionally, we
demonstrated that a high level of cAMP signaling,
i.e., 100 µM
forskolin, blocks the expression of both TH and the
SA
lineage-determining genes (Fig.
6B and D). These observations
lend
further support to the crucial role of the ASH-1 and Phox2
genes for
development of the SA lineage (
17) and suggest that
the cAMP
signaling pathway antagonizes SA cell differentiation
by regulating
other pathways mediating the expression of the SA
lineage-determining
genes.
In conclusion, our results demonstrate the dynamic role of the cAMP
signaling pathway in the development of SA cells, activating
either a
mechanism that promotes phenotypic specification or a
mechanism that
opposes crest cell commitment to the SA lineage.
Although the nature of
each mechanism remains to be elucidated,
Fig.
7 depicts a model for how intracellular
cAMP levels govern
the bimodal switch on SA cell development. Thus,
moderate levels
of intracellular cAMP promote the expression of Phox2a,
which,
in turn, drives differentiation (
62), and probably
survival
(
10,
44,
49), of the SA progenitor cell. In
contrast, high
levels of intracellular cAMP activate a pathway that
opposes BMP-2
signaling and inhibits the induction of genes necessary
for SA
lineage determination, i.e., the ASH-1 and Phox2 genes.
View larger version (22K):
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|
FIG. 7.
Model for the BMP-2 and cAMP signaling pathways in SA
cell development. The diagram illustrates the possible molecular
mechanisms regulating the observed synergy and antagonism between the
BMP-2 and cAMP signaling pathways. Arrows indicate direct and indirect
relationships. Differentiation of the SA lineage requires activation of
the ASH-1 and Phox2 genes, which, in turn, regulate the neurogenic
program, neurotransmitter identity, and progenitor survival
(17). BMP-2 in combination with moderate intracellular
levels of cAMP promotes differentiation of SA progenitor cells by
inducing expression of ASH-1 and Phox2 genes. Conversely, high
intracellular levels of cAMP activate a yet to be delineated mechanism
antagonistic to development of the SA lineage which opposes BMP-2
signaling and the consequent expression of SA lineage-determining
genes.
|
|
| |
ACKNOWLEDGMENTS |
We express gratitude to G. Maxwell and J. Varley, University of
Connecticut Health Center, for sharing their techniques and culture
conditions for primary neural crest cells; the Genetics Institute for
providing BMP-2; J.-F. Brunet for communicating the partial sequences
of chicken Phox2a and Phox2b; P. Robinson for sharing the
epifluorescence microscope and digital imaging equipment; and the
Poultry Research Center for maintaining the Japanese quail flock.
This work is supported by grants from the Whitehall Foundation and NIH
DK 44533 to O.M.A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Basic Medical Sciences, 1246 Lynn Hall, Purdue University, West
Lafayette, IN 47907-1246. Phone: (765) 494-8131. Fax: (765) 494-0781. E-mail: oma{at}vet.purdue.edu.
| |
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Molecular and Cellular Biology, May 2000, p. 3004-3014, Vol. 20, No. 9
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
