(Received for publication, July 12, 1995; and in revised form, January 26, 1996)
From the
The linkage between G and morphogen-induced
promotion of F9 embryonic teratocarcinoma stem (F9 stem) cells to
primitive endoderm was explored using probes of the mitogen-activated
protein (MAP) kinase network. The morphogen-induced decline in
G
is shown to trigger activation of phospholipase C,
thereby activating protein kinase C, MAP kinase, and cell progression
to primitive endoderm. In the absence of retinoic acid,
reduction-of-function mutants (G
-deficient) display
the effects of morphogen, i.e. activation of phospholipase C,
protein kinase C, MAP kinase, and progression to primitive endoderm.
Gain-of-function mutants (expressing the Q205L activating-mutation of
G
) displayed no activation of phospholipase C,
protein kinase C, MAP kinase and no progression to primitive endoderm,
even in the presence of retinoic acid. Selective inhibitors of
protein kinase C, like the gain-of-function mutations, effectively
block morphogen-induced progression to primitive endoderm. Morphogen
triggers F9 stem cell progression by triggering G
loss and thereby activation of downstream elements, including
protein kinase C and MAP kinase.
Mouse F9 teratocarcinoma stem (F9 stem) cells provide a
plentiful source of embryonic cells whose differentiation can be
induced or influenced by exogenously added agents. F9 stem cells show
little spontaneous differentiation in culture and can be induced to
differentiate to primitive endoderm (PE) ()by physiological
concentrations of retinoic acid (RA)(1) . The new cell type PE
of F9 cells give rise to two different cell types, both of which are
characteristic of the extraembryonic endoderm lineage in the mouse
embryo; addition of dibutyryl cyclic AMP yields parietal endoderm (2) , whereas aggregation yields cells characteristic of
visceral endoderm(3) . This differentiation process can be
monitored by following the expression of several specific markers. The
production of tissue plasminogen activator (tPA), Type IV collagen,
laminin, and c-jun is induced with
differentiation(4, 5) , while the stage-specific
embryonic antigen disappears from the cell surface (6) and the
level of c-myc is dramatically decreased(7) . Because
of these distinct and versatile responses to differentiation, F9 cells
have been widely used as a model for studying the mechanism of
embryonic differentiation. Despite numerous studies, however, the
molecular mechanism of differentiation, particularly the nature of the
intracellular cascade leading to differentiation, remains poorly
understood.
The G-proteins are a family of membrane-associated
guanine nucleotide-binding proteins that transduce signals from cell
surface receptors to intracellular effectors. Members of this family
are heterotrimers composed of ,
, and
subunits(8) . The
subunit confers receptor and effector
specificity on the heterotrimer. When the G-protein is activated by
interaction with receptor, the
subunit exchanges bound GDP for
GTP. The intrinsic GTPase activity of the
subunit restores it to
the basal state in which GDP is bound. G
subunits,
too, modulate the activities of effectors units, including some
K
channels, PLC
(forms 2 and 3), pheromone action
in budding yeast, and some adenylyl cyclase (for a recent review, see (9) ). This form of signal transduction is basic to the
mechanisms that cells use in responding to hormones, neurotransmitters,
and growth factors.
RA induces F9 stem cell progression to PE and a
sharp decline in G(10) . A decrease in
G
by expression of antisense G
RNA
(reduction-of-function) mutant induces PE-like phenotype in the absence
of RA, while an increase in G
by expression of a
constitutively active (gain-of-function) mutant
G
Q205L is sufficient to block
differentiation(11) . Pertussis toxin-sensitive G-proteins,
like G
, have been implicated as potential mediators
both of inhibitory phospholipase C (PLC) signaling and of mitogenic
responses in several cell
lines(12, 13, 14, 15, 16, 17, 18, 19) .
Recently, suppression of G
in cultured cells and
adipocytes from transgenic mice has been shown to enhance PLC
signaling, providing a possible link between G
and
upstream regulators of the mitogen-activated protein kinase (MAPK)
network(20) . We tested this linkage, exploring the PLC
pathway.
We explored the relationship between G and
F9 stem cell progression to PE using stem cells, loss-of-function, and
gain-of-function mutants. G
is deficient in the F33
loss-of-function clones (Fig. 1), mimicking the loss of
G
in F9 stem cells induced to PE by RA(9) .
The gain-of-function clones express the Q205L mutant of
G
, which is constitutively activated (FQ). Owing to
expression of the Q205L mutant form, immunoreactive G
levels are increased in immunoblots of FQ clones, whereas
immunoreactive G
is essentially absent in the F33
clones. The levels of G
, G
, and
G
, in contrast, were quite similar in F33 and F9
clones (Fig. 1).
Figure 1:
Expression of G is
elevated in FQ clones and reduced in F33 clones, while expression of
G
, G
and G
are
similar among F9 stem cells, F33, and FQ clones. Proteins (100
µg/lane) were separated SDS-PAGE on 10% gels, transferred to
nitrocellulose membranes (Schleicher & Schuell), and probed with
primary antibodies to (1:200) G
, G
,
G
, and G
(10, 11) .
Blots were incubated with an alkaline phosphatase-conjugated second
antibody (1:1000), and developed with nitro blue tetrazolium chloride
and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine. The
vector stably transfected cells showed expression similar to that F9
stem cells (data not shown).
In order to determine to what extent PLC
activity was altered, IP mass accumulation was measured.
IP
levels were found to increase severalfold as F9 stem
cells (STEM) progressed to PE in response to RA (Fig. 2A). Both F9 stem cells and PE displayed elevated
IP
levels in response to stimulation of PLC by
5-hydroxytyramine (5HT; Fig. 2B), as well as to
bradykinin and
-adrenergic agonists (not shown).
Figure 2:
RA-induced decline in G
activates PLC and promotes F9 stem cell progression to PE. A,
basal PLC activity, measured by accumulation of IP
, is
increased in RA-induced PE cells and mimicked by F9 stem cells
deficient in G
(F33). B, activation of PLC,
measured by IP
accumulation, in response to 5HT
(10
M) is potentiated in RA-induced PE
cells, mimicked by F9 stem cells deficient in G
(F33), and abolished in stem cells expressing of Q205L
G
(FQ). Data are means ± S.E. (n = 4). By Student's test, significance of difference
is p < 0.05 (*) or < 0.01 (**) with respect to wild
type. C and D, progression of F9 stem cells to PE
either by RA or by deficiency of G
(F33) activates
PLC and increases IP
levels, as measured by metabolic
labeling (C) and quantified by liquid scintillation (D). The elution of IP
in indicated by the arrows. Metabolic labeling of the different clones was
equivalent quantitatively on a ``per cell'' basis, and
equivalent amounts of label were subjected to analysis by HPLC. E and F, deficiency in G
(F33), like the
action of RA on F9 stem cells, induced progression of stem cell to PE
phenotype, as analyzed by the PE-marker, tPA (E) or by
phase-contrast microscopy (F). One unit of tPA is arbitrarily
defined as that amount of tPA that results in a reaction rate of
10
A
min
. For phase-contrast microscopy, the cells
were fixed with 3% (w/v) paraformaldehyde and photographed with a Zeiss
Axiphot. The micrographs shown are representative of five independent
experiments for each clone or condition. Stem cells stably transfected
with vectors alone, showed activities similar to that F9 stem cells
throughout (data not shown).
We tested the
linkage further using loss-of-function mutants, stable transfectants
expressing RNA antisense to G and lacking
G
(F33). In the absence of morphogen,
loss-of-function mutants displayed elevated IP
levels and
progression to PE. Gain-of-function mutants, stably expressing the
Q205L activating mutant G
(FQ), displayed no
activation of PLC, no tPA production, and a stem-like phenotype in the
absence (Fig. 2) or presence (not shown) of RA. Analysis by HPLC
of the water-soluble inositol phosphates from cells metabolically
labeled with [
H]inositol confirmed the mass
assays of IP
levels for the different clones (Fig. 2C). Levels of IP
(arrows) were
elevated markedly in cells induced to PE by RA and in the F33
G
-deficient clones that progress to PE in the absence
of RA. Quantification of the label recovered as IP
from
HPLC of samples loaded with equivalent total counts is shown (Fig. 2D). In stem or FQ cells, in contrast, the
IP
levels were lower (Fig. 2C). Excepting
the changes in IP
levels, overall profiles of inositol
metabolites (Fig. 2C) and labeling of
inositol-containing lipids (not shown) were similar. Expression of the
PE-specific marker, tPA, and phase-contrast microscopy define
morphogen-induced progression to PE (Fig. 2, E and F, respectively). Stem cells stably transfected with vectors
alone showed activities similar to that ES cells throughout.
Calcium
mobilization in response to elevated IP levels was probed
by in vitro assays of calcium/calmodulin-dependent protein
kinase II (CaM kinase II, Fig. 3). Progression to PE by RA
elevated CaM kinase II activity in either the absence (Basal)
or the presence of activator calcium and calmodulin (+CaM). The in situ assay of CaM kinase II (26, 31) revealed a sharp increase in the
-form
(mass of 60 kDa) compared to
-form (50 kDa) of kinase activity (inset, Fig. 3). G
deficiency alone
(F33), like RA, increased CaM kinase II activity, well above that of PE
with the
-isoform activity predominant. Expression of a
constitutively active G
(FQ), in contrast, not only
blocks progression, but also reduces CaM kinase II activity to levels
below those of stem cells. Ionophore (A23187 or ionomycin)-induced
activation of CaM kinase II activity revealed that less than half of
the CaM kinase II was activated in stem cells, whereas >70% was
activated by RA treatment (PE) or loss of G
(F33,
data not shown).
Figure 3:
Expression of CaM kinase II activity is
elevated in PE, F33 cells, but not in F9 stem or FQ cells. Left, basal CaM kinase II activity was elevated in PE cells
and F9 stem cells deficient in G (F33). Inset, in situ CaM kinase II assay of basal activity
demonstrates an increase in the PE as well as F33 cells. Upper
band,
-subunit; lower band,
-subunit. Right, activation of CaM kinase II by Ca
/CaM
is potentiated in PE cells and mimicked by G
deficiency (F33). Stem cells stably transfected with vectors
lone, showed activities similar to that F9 stem cells (data not shown).
The data are mean values ± S.E. from three separate experiments.
By Student's test significance of difference is p <
0.05 (*) or < 0.01 (**) with respect to wild type. Ionophore (A23187
or ionomycin) was used as an independent means of activating CaM kinase
II. Maximal CaM kinase II activity was determined in the cells by
activation with the ionophore A23187 (1 µM, 5 min). Mean
values (± S.E., n = 3) for CaM kinase II
activities in the ionophore-treated cells were 8.21 ± 0.7, 23.15
± 2.1, 35.55 ± 2.2, and 3.03 ± 0.34 for STEM, PE,
F33, and FQ cells, respectively. Similar data were obtained with
ionomycin treatment (not shown).
PKC plays a prominent role in cell signaling,
growth and proliferation (32, 33) . The levels of DAG,
a metabolite of the PLC pathway and intracellular activator of PKC,
were explored in cells progressing to PE by RA or by loss of
G (F33). DAG content of stem cells nearly doubled
when induced to PE by RA (Fig. 4). Likewise, G
deficiency alone, in the absence of RA, elevated DAG content
nearly 2.5-fold. Expression of Q205L mutant G
displayed DAG levels similar to that of stem cells. 5HT elevated
DAG levels in stem, PE and G
-deficient cells, whereas
in cells expressing the Q205L mutant G
the response
to 5HT was essentially abolished.
Figure 4:
RA-induced progression to PE, like
G deficiency (F33), activates PLC and increases
intracellular DAG levels. Cells (10
) were grown to
confluence for 4 days. Cells were treated with and without 5HT
(10
M, 10 s at 37 °C), and then
extracted into chloroform. DAG was measured using a protein kinase
assay(27) , followed by thin-layer chromatography to separate
radioactivity incorporated into phosphatidic acid generated from the
reaction. The DAG mass was calculated from an authentic sn-1,2-DAG standard curve. The data are mean values ±
S.E. from three separate experiments. By Student's test
significance of difference is p < 0.01 (**) with respect to
wild type. Stem cells stably transfected with vectors alone, showed
activities similar to that F9 stem cells (data not
shown).
A critical linkage between PKC and
both G and stem cell progression was revealed by
measurement of PKC activity. In the absence of an exogenous activator
(PMA), PKC activity in PE was more than 10-fold greater than in F9 stem
cells not induced by RA (Fig. 5). Remarkably, total PKC activity
measured in the presence of PMA showed the same striking difference
following progression, i.e. PE display a 14-fold greater
amount of PKC activity than stem cells. The
G
-deficient clones (F33) that progress to PE in the
absence of RA mimicked the cells induced to PE by RA, displaying
elevated basal and total PKC activity. Expression of the constitutively
active Q205L mutant G
(FQ), in stark contrast,
resulted in an stem cell-like phenotype refractory to RA with levels of
PKC similar to those of F9 stem cells prior to progression. Immunoblots
of DEAE-cellulose-purified whole-cell extracts stained with antibodies
to PKC
are consistent with the activity measurements (Fig. 6A). Northern analysis of total cellular RNA
identified PKC
transcripts of 3.5 and 8.1 kilobases in PE and F33
clones. PKC
transcripts were not detected in Northern blots of RNA
from either F9 stem cells or FQ clones (Fig. 6B, top), with relative equivalent sample loading (Fig. 6C, bottom). PKC isoforms other than
PKC
do not appear to be major contributors to the changes in
activity, as PKC
protein is undetected (data not shown), and the
level of PKC
and PKC
transcripts is reduced, rather than
increased, in progression to PE(34) . Thus, stimulation by the
morphogen RA leads not only to an increase in DAG generation and
thereby PKC activation, but also to a sharp increase in expression of
the PKC
mRNA.
Figure 5:
RA-induced progression to PE and
deficiency of G (F33) elevate PKC activity. Left, PKC activity measured in the absence of PMA and
phosphatidylserine is increased in PE and F33 cells. Right,
total PKC activity, measured in response to PMA and phosphatidylserine,
is elevated greatly in PE and F33 cells. The data are mean values
± S.E. from three separate experiments. Stem cells stably
transfected with vectors alone, showed activities similar to that F9
stem cells (data not shown). By Student's test significance of
difference is p < 0.01 (**) with respect to wild
type.
Figure 6:
RA-induced progression to PE and
deficiency of G (F33) elevate the expression and mRNA
levels of PKC
. (A) Samples of DEAE-cellulose-purified
whole-cell extracts were subjected to SDS-PAGE and immunoblotting. The
protein blots were stained with antibodies to PKC
. B, top, Northern blots showing PKC
mRNA is increased in PE
and F33 cells, and below detection in stem and FQ cells. B, bottom, staining of ribosomal 18 and 28 S RNA by ethidium
bromide establishes relative equivalence in loading of RNA per lane in
each blot. The blots are representative of three independent
experiments for each condition.
MAPKs are members of a group of serine/threonine
kinases that are activated in response to growth factors, mitogenic
stimuli and neurotransmitters, that control cell proliferation and
differentiation (35) . Tyrosine kinase-encoded growth factor
receptors and G-coupled receptors (e.g. thrombin
receptor) are capable of activating MAPKs in response to
stimulation(36) . Phorbol esters, like PMA, also activate MAPK
in many cells. Immunoblotting of immunoprecipitated whole-cell extracts
with antibodies to p42 reveals an enhanced expression of MAPK in PE and
the F33 clones (Fig. 7A). In an effort to probe the
downstream events triggered by G
decline and PKC
activation, MAPK activity was assessed in cell extracts first subjected
to anion-exchange chromatography FPLC. MAPK activity resolves as two
peaks, the major p42 MAPK eluting in fractions 12-16 and a minor
p44 MAPK in fractions 17-19. In F9 stem cells, MAPK activity was
relatively low. Cells induced to PE by RA displayed a striking, 5-fold
increase in MAPK activity (Fig. 7B). The
G
-deficient F33 clones, like the PE cells, displayed
a striking increase in MAPK activity in the absence of RA. The FQ
clones expressing Q205L G
, in contrast, were shown to
have profiles of MAPK not unlike the untreated ES cells (Fig. 7C). Treatment of the cells with EGF provides a
positive control, demonstrating that RA-induced progression to PE is
accompanied by a substantial, though lesser activation of MAPK,
approximately 60% of that obtained by stimulation with EGF (Fig. 7D).
Figure 7:
RA-induced progression to PE and
deficiency in G (F33) activate MAPK. A,
immunoblotting of immunoprecipitated whole-cell extracts with
antibodies to MAPK p42. MAPK activity of F9 stem cells and PE (B), and FQ and F33 clones (C), as measured after
separation on Mono Q FPLC. Stem cells stably transfected with vectors
alone, showed activities similar to that F9 stem cells (data not
shown). D, serum-starved cells (18 h) were treated with mouse
EGF (50 ng/ml) for 5 min. Cell lysates were then prepared, resolved by
Mono Q FPLC, and assayed for MAPK activity. E, Western blot
analysis of Mono Q FPLC fractions with a monoclonal anti-MAP2 kinase
antibody reveals increased p42 protein in fractions from PE and F33
cells. Data are representative of three independent experiments for
each condition.
For PE and F33 clones, immunoblotting of
MAPK shows a modest increase in protein and an elution profile in which
the p42 isoform is most prominent (Fig. 7E), which,
together with the MAPK protein expression of the whole-cell extracts,
suggests that activation of MAPK is the basis for increased activity
both in F9 stem cells promoted to PE by RA and in
G-deficient cells that progress to PE in the absence
of RA. Activation of the upstream element regulating MAPK activity,
MEK, revealed a similar pattern in which progression to PE induced by
RA activated MEK to a significant but lesser level that EGF (Fig. 8).
Figure 8:
RA-induced progression to PE and
deficiency in G (F33) activate MEK. MEK activity of
F9 stem cells, PE and EGF-stimulated PE (A), and FQ and F33
clones (B), as measured after separation on Mono S FPLC. Cells
(5
10
) grown for 4 days were harvested and lysed.
To measure growth factor-stimulated MEK activities, the cells were
serum-starved for 18 h and incubated with mouse EGF (50 ng/ml) for 5
min. Cell extracts (1 ml) were fractionated by chromatography on a Mono
S FPLC column, developed with a 28-ml, linear 0-350 mM NaCl gradient, while collecting 1-ml fractions. The enzyme
activity was quantified by measurement of the phosphorylation of
inactive rMAPK. Endogenous MAPK elutes in fractions 1-5, whereas
MEK is contained in fractions 8-13. Fractions 1-7, which
include endogenous MAPK activity, are not displayed; only the region of
the chromatogram relevant to MEK is
shown(30) .
To probe events downstream of RA-induced
G loss mediating progression of stem cells to PE, the
effects of protein kinase inhibitors on F9 stem cell progression to PE
were explored (Table 1). F9 stem cells were treated with RA in
the absence and presence of selective inhibitors of PKC, protein kinase
A, or CaM kinase, after establishing the dose-response curve for
inhibition in F9 cells to be equivalent (not shown) to those in the
literature performed with other cell
lines(37, 38, 39, 40, 41) .
PKC-selective inhibitors calphostin C (37) (0.1
µM), bisindoylmaleimide (38) (0.1
µM), and H7 (39) (25 µM) effectively
blocked the ability of RA to activate MAPK and to promote F9 stem cell
progression to PE. In agreement with earlier observations that cyclic
AMP analogues and cholera toxin treatment do not influence progression
to PE, inhibitors of protein kinase A, such as KT5720 (40) and
HA1004 (39) at concentrations 20-fold in excess of their
IC
values were shown to be without effect on either
parameter. KN62(41) , a potent inhibitor of CaM kinase, blocked
CaM kinase activity (data not shown), but neither MAPK activation nor
progression to PE. CaM kinase II activity increases dramatically in
stem cells progressing to PE and the RA-induced change may well be
important to both calcium signaling and longer term features of gene
expression associated with the PE-phenotype. Clearly, RA triggers a
decline in G
, thereby activating PLC, PKC, MAPK, and
progression of F9 stem cells to PE.
To probe the linkage between
MAPK and RA-induced progression to PE, we employed phosphorothioate
oligodeoxynucleotides antisense to MAPK p42 (42) .
Oligodeoxynucleotides antisense, but not sense, to MAPK nearly
abolished immunoreactive MAPK in whole-cell blots of F9 cells (Fig. 9, top). Measurement of MAPK activity by the in situ assay confirmed the loss of MAPK (not shown).
Elimination of MAPK abolished the ability of RA to induce progression
of F9 stem cells to PE (Fig. 9, bottom), establishing
an obligate role of MAPK in the cascade from G to
stem cell progression.
Figure 9: Oligodeoxynucleotides antisense, but not sense, to MAPK abolish RA-induced progression of F9 stem cells to PE. F9 cells were incubated with 1.5 mM phosphorothioate oligodeoxynucleotides sense (S) and antisense (AS) to MAPK mRNA, using the sequences and conditions reported elsewhere(42) . Inset, the level of MAPK expression was determined from whole-cell extracts subjected to immune precipitation and immunoblotting. The ability of RA to induce progression was measured by quantification of the production of tPA, the hallmark of the PE phenotype.
RA-induced differentiation of a variety of
tumor cell types is accompanied by increased expression of PKC,
especially the -isoforms (34, 43) . The role of
G
in progression of these tumor cells in response to
RA is not known. Loss-of-function mutants mimic the loss of
G
and promote progression in the absence of RA
(present study). Gain-of-function mutants with constitutively active
G
show no activation of PLC, PKC, MAPK, and
progression in response to RA (present study). Like the recent
discovery of a critical role of G
in Caenorhabditis
elegans behavior(44) , the current study highlights a new
dimension of G-protein function in development, one in which a
G-protein (G
) triggers stem cell progression via
activation (derepression) of PLC, and thereby activation of PKC and the
MAPK regulatory network.