Activation of RAF-1 through Ras and Protein Kinase C
Mediates
1
,25(OH)2-Vitamin D3 Regulation of the
Mitogen-activated Protein Kinase Pathway in Muscle Cells*
Claudia Graciela
Buitrago,
Verónica González
Pardo,
Ana
R.
de Boland, and
Ricardo
Boland
From the Departamento de Biología, Bioquímica and
Farmacia, Universidad Nacional del Sur, San Juan 670, 8000 Bahía Blanca, Argentina
Received for publication, June 10, 2002, and in revised form, October 7, 2002
 |
ABSTRACT |
We have previously shown that stimulation of
proliferation of avian embryonic muscle cells (myoblasts) by
1
,25(OH)2-vitamin D3
(1
,25(OH)2D3) is mediated by activation of
the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how
1
,25(OH)2D3 up-regulates the MAPK cascade,
we have investigated whether the hormone acts upstream through
stimulation of Raf-1 and the signaling mechanism by which this
effect might take place. Treatment of chick myoblasts with
1
,25(OH)2D3 (1 nM) caused a fast
increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a
specific Ras inhibitor peptide that involves Ras in
1
,25(OH)2D3 stimulation of Raf-1.
1
,25(OH)2D3 rapidly induced tyrosine
de-phosphorylation of Ras-GTPase-activating protein, suggesting that
inhibition of Ras-GTP hydrolysis is part of the mechanism by which
1
,25(OH)2D3 activates Ras in myoblasts. The
protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I,
and Ro 318220 blocked 1
,25(OH)2D3-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of
Raf-1 also involves PKC. In addition, transfection of muscle cells with
an antisense oligodeoxynucleotide against PKC
mRNA suppressed
serine phosphorylation by 1
,25(OH)2D3. The
increase in MAPK activity and tyrosine phosphorylation caused by
1
,25(OH)2D3 could be abolished by Ras
inhibitor peptide, compound PD 98059, which prevents the activation of
MEK by Raf-1, or incubation of cell lysates before
1
,25(OH)2D3 exposure with an anti-Raf-1
antibody. In conclusion, these results demonstrate for the first time
in a 1
,25(OH)2D3 target cell that activation
of Raf-1 via Ras and PKC
-dependent serine
phosphorylation plays a central role in hormone stimulation of the
MAPK-signaling pathway leading to muscle cell proliferation.
 |
INTRODUCTION |
Raf-1 was discovered as the first member of a
cytoplasmatic family of serine/threonine kinases and plays a crucial
role in the activation of the classical cytoplasmatic-signaling cascade that is involved in the regulation of cellular proliferation, differentiation, and apoptosis (1). Activation of Raf-1 downstream of
protein-tyrosine kinase receptors is mediated by the Ras GTP-binding proteins, which are required for stimulation of Raf-1 kinase activity (2, 3). Raf-1 then phosphorylates and activates the mitogen-activated protein kinase kinase, also known as
MEK1 (4), initiating a
protein kinase cascade that follows with the phosphorylation and
activation of the extracellular signal-regulated mitogen-activated
protein (MAP) kinase isoforms ERK1 and ERK2 (5). Upon activation, MAP
kinase (MAPK) is translocated from the cytoplasm into the nucleus (6),
where it phosphorylates transcription factors (7) and culminates in
proliferation or differentiation of a variety of cell types.
With an estimated molecular mass of 74 kDa, Raf-1 possesses three
conserved regions, CR1, CR2, and CR3, that are embedded in variable
regions. The CR1 and CR2 domains are part of the regulatory N-terminal
half of the Raf-1 protein, whereas CR3 forms the C-terminal kinase
domain (1). The molecular mechanism of Raf-1 activation, however, is
still unclear. Although Raf-1 binds directly to the effector domain of
activated GTP-bound Ras proteins, this interaction does not appear to
stimulate Raf-1 kinase activity (8). Rather, it seems that the role of
the Ras-Raf interaction is to recruit Raf-1 to the plasma membrane,
where it can be activated by membrane lipids or other protein kinases,
the nature of which remains to be determined (9, 10). Phosphorylation
of Raf on both tyrosine and serine/threonine residues is required for
enzymatic activity (11). It has been shown that the Src
protein-tyrosine kinase can activate Raf by phosphorylation on Tyr-340
and Tyr-341 (12, 13). However, mutant Raf proteins in which Tyr-340 and
Tyr-341 have been changed to Asp can still be activated after membrane association, indicating that an additional mechanism(s) of Raf activation exists (13). Among the mechanisms involved, there is
evidence for the operation of both protein kinase C
(PKC)-dependent and PKC-independent pathways of Raf
activation in response to agonists (14).
The 13 members of the PKC family can be grouped into three major
classes of Ca2+-dependent classical PKCs,
Ca2+-independent, novel PKCs, and Ca2+- and
lipid-independent, atypical PKCs. There is a fourth PKC subgroup
consisting of PKCµ (15). Previous reports showed that classical and
novel PKCs activate the MAP kinase pathway at the level of Raf-1,
whereas atypical PKCs activate MEK by an independent mechanism (16,
17). One of the classical PKCs, PKC
can directly activate Raf-1 by
serine phosphorylation of Raf-1 (18).
The steroid hormone 1
,25-dihydroxy-vitamin D3
(1
,25(OH)2D3) triggers responses in muscle
cells both through a nuclear receptor-mediated mechanism that promotes
gene transcription (19) and a fast non-genomic mode of action
independent of new RNA and protein synthesis (20, 21). In previous work
we have demonstrated that the steroid hormone rapidly stimulates in
skeletal muscle cells the phosphorylation and activity of the MAP
kinase isoforms ERK1 and ERK2 and have implicated the MAPK cascade in
hormone control of myoblast proliferation (22). Moreover, initial
investigations on the mechanisms underlying 1
,25(OH)2D3 stimulation of the muscle cell
(myoblast) MAPK pathway revealed that PKC and Ca2+ are two
upstream activators mediating the hormone effect (23). In addition, it
has been shown that 1
,25(OH)2D3 enhancement
of myoblast proliferation correlates to increased PKC
expression, whereas decreased PKC
levels are observed during the subsequent activation of muscle cell differentiation by the hormone (24). Furthermore, inhibition of PKC
expression by using antisense oligonucleotide technology resulted in a significant decrease of
culture cell density and DNA synthesis, clearly showing that this
isozyme is involved in signaling cascades that promote muscle cell
proliferation (25).
The upstream-signaling pathway that leads to activation of the
Ras/Raf-1/MAPK (ERK1/2) cascade by
1
,25(OH)2D3 remains incompletely understood,
and direct evidence on the participation of any of the PKC isoforms is
lacking. In view of the information discussed above, we have
investigated the role of Ras as well as PKC
in Raf 1 activation.
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MATERIALS AND METHODS |
Chemicals--
1
,25(OH)2D3 was kindly
provided by Hoffmann-La Roche. Dulbecco's modified Eagle's medium
(DMEM), fetal bovine serum, specific Ras inhibitory peptide
(VPPPVPPRRR), and protein A-Sepharose were from Sigma. Lipofectin was
from Invitrogen. Sense and antisense oligodeoxynucleotides were
synthesized by the DNAgency (Malvern, PA). Rabbit polyclonal
anti-phosphoserine antibody was obtained from Upstate Biotechnology
Inc. (Lake Placid, NY). Anti-Raf-1 monoclonal antibody and
anti-(phospho)-active MAP kinase antibody (reactive against p42 and p44
isoforms) were from Promega (Madison, WI). Anti-Ras-GTPase-activating
protein (GAP) antibody and donkey anti-goat IgG antibody were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibody goat
anti-rabbit horseradish peroxidase-conjugated IgG and the Super Signal
CL-HRP substrate system for enhanced chemiluminescence (ECL) were
obtained from Amersham Biosciences. [
-32P]ATP (3,000 Ci/mmol) was from PerkinElmer Life Sciences. The compounds PD 98059, Ro
318220, calphostin C, and bisindolylmaleimide I were from Calbiochem.
All other reagents were of analytical grade.
Cell Culture--
Chick skeletal muscle cells were obtained from
13-day-old chick embryo breast muscles by stirring in Earle's balanced
salt solution containing 0.06% trypsin for 30 min essentially as
previously described (26). The freed cells were collected by
centrifugation, and the pellet was resuspended in DMEM supplemented
with 10% fetal bovine serum and antibiotic-antimycotic solution. The
suspension was dispersed by pipette, filtered through nylon mesh, and
"preplated" on gelatin-coated Petri dishes to remove contaminating
fibroblasts. The unadsorbed cells were seeded at an appropriate density
(120,000 cells/cm2) in Petri dishes (100-mm diameter) and
cultured at 37 °C under a humidified atmosphere (air 95%, 5%
CO2). Under these conditions, myoblasts divide within the
first 48 h and at day 4 become differentiated into myotubes
expressing both biochemical and morphological characteristics of adult
skeletal muscle fibers (27). Cells cultured for 2 days (proliferative stage) were used for treatments.
Immunoprecipitation--
After
1
,25(OH)2D3 or vehicle (ethanol, < 0.01%)
treatment, muscle cells were lysed (15 min at 4 °C) in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EGTA, 25 mM NaF, 0.2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 0.25% sodium deoxycholate, and
1% Nonidet P-40 and homogenized by sonication for 15 s. Insoluble
material was pelleted in a microcentrifuge at 12,000 × g for 15 min. The protein content of the clear lysates was
determined according to Lowry et al. (28). Aliquots
(500-700 µg of protein) were incubated overnight at 4 °C with
anti-Raf-1 or anti-Ras-GAP antibodies followed by precipitation of the
complexes with protein A conjugated with Sepharose. The immune
complexes were washed 4 times with cold immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate,
1% Triton X-100m and 1% Nonidet P-40) and a final wash in cold
phosphate-buffered saline.
Blockade with Antibodies--
Insoluble material of lysate
proteins was pelleted in a microcentrifuge at 12,000 × g for 15 min. The protein content of the clear lysates was
determined according to Lowry et al. (28). Aliquots (500 µg of protein) were incubated with anti-Raf-1 or anti-goat IgG
antibody on an ice bath with shaking for 10 min. Then the lysates were
exposed to 1 nM 1
,25(OH)2D3 or
vehicle (ethanol < 0.01%) for 1 min. The treatments were stopped
by adding Laemmli sample buffer (29).
SDS-PAGE and Immunoblotting--
Immunoprecipitated proteins (or
lysate proteins) dissolved in Laemmli sample buffer were separated on
SDS-polyacrylamide (8%) gels (29) and electrotransferred to
polyvinylidene difluoride membranes. The membranes were blocked for
1 h at room temperature in TBST (50 mM Tris-HCl, pH
7.4, 200 mM NaCl, 1% Tween 20) containing 5% dry milk.
Membranes were subjected to immunoblotting using anti-PKC
,
anti-phosphoserine, or anti-phosphotyrosine antibodies. Next the
membranes were washed 3 times in TBST, incubated in TBST containing 1%
dry milk with a 1:10,000 dilution of peroxidase-conjugated anti-rabbit
or anti-mouse secondary antibodies for 1 h at room temperature,
and washed 3 additional times with TBST. The membranes were then
visualized using an enhanced chemiluminescent technique (ECL, Amersham
Biosciences) according to the manufacturer's instructions. Images were
obtained with a model GS-700 Imaging Densitomer from Bio-Rad by
scanning at 600 dots per inch and printing at the same resolution. Bands were quantified using the Molecular Analyst program
(Bio-Rad).
To strip the membranes for reprobing with anti-Raf-1, anti Ras-GAP, or
anti-ERK1/2 antibodies, the membranes were washed for 10 min in TBST
and then incubated in stripping buffer (62.5 mM Tris-HCl,
pH 6.8, 2% SDS, and 50 mM mercaptoethanol) for 30 min at
50 °C. The membranes were again blocked and blotted as described above.
Measurement of MAP Kinase Activity--
Muscle cells were
pretreated with PD 98059 (10 µM, 10 min) or Ras inhibitor
peptide (25 µM, 10 min) and then exposed for 1 min with 1 nM 1
,25(OH)2D3 or vehicle (< 0.01% ethanol) at 37 °C. Lysates were prepared followed by
immunoprecipitation of MAP kinase (p42 and p44) as described above.
After 3 washes with immunoprecipitation buffer and 2 washes with kinase
buffer (10 mM Tris-HCl, pH 7.2, 5 mM
MgCl2, 1 mM MnCl2, 1 mM
dithiothreitol, 0,1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and
20 µg/ml aprotinin), the immune complexes were incubated at 37 °C for 10 min in kinase buffer (50 µl/sample) containing myelin basic protein as an exogenous substrate for MAP kinase (20 µg/assay), 25 µM ATP, and [
-32P]ATP (2.5 µCi/assay).
To terminate the reaction, the phosphorylated product was separated
from free isotope on ion exchange phosphocellulose filters (Whatman
P-81). Papers were immersed immediately into ice-cold 75 mM
H3PO4, washed (1 × 5 min, 3 × 20 min), and counted in a scintillation counter.
Cell Transfection--
Transfection with oligodeoxynucleotides
(ODNs) using Lipofectin was performed according to the manufacturer's
instructions. ODNs were incubated with Lipofectin in DMEM for 15 min at
room temperature. Plates of subconfluent cells were washed to remove serum before the addition of ODN-Lipofectin mixtures, and incubation was performed for 4 h at 37 °C. The ODN solution was removed, DMEM was added, and the plates were placed into a metabolic incubator for an additional period of 20 h. Control treatments included DMEM
or Lipofectin only. Dose- and time-response studies for Lipofectin and
ODNs were previously performed to establish optimum conditions for the
effective blockade of PKC
expression (25). The following ODN
sequences with phosphorothioate linkages throughout the entire ODN
molecule were used: antisense-PKC
(AS), 5'-CATGGTYCCCCCCAACCACC-3', Y = T or C (antisense sequence against 20 nucleotides upstream of the AUG codon); sense PKC
(S), 5'-GGTGGTTGGGGGGRACCATG-3', R = A or G (25). Each antisense oligonucleotide was used at a final
concentration of 5 µM. These antisense sequences showed no homology to any DNA in the GenBankTM except PKC
(human, rat, mouse, and rabbit species). Cell death in cultures under
control and treatment (ODNs) conditions was measured by trypan blue staining.
Statistical Analysis--
Statistical significance of the data
was evaluated using Student's t test (30), and probability
values below 0.05 (p < 0.05) were considered
significant. Results are expressed as the means ± S.D. from
the indicated set of experiments.
 |
RESULTS AND DISCUSSION |
To understand how the steroid hormone
1
,25(OH)2D3 controls the MAP kinase cascade
in skeletal muscle cells, it is essential to identify the molecules
that participate in the cellular sequence of events involved in the
signaling pathway of this steroid hormone. As in other cell types,
Raf-1 and MEK belong to the MAP kinase cascade that leads to muscle
cell proliferation (31, 32). As a major step in this direction, we
report here for the first time that
1
,25(OH)2D3 stimulation of the MAPK (ERK1/2)
pathway in skeletal muscle cells involves at least in part rapid
activation of Raf-1 and provide information on the mechanism of action
by which this hormone-regulated event takes place.
The complex process of Raf activation is still incompletely understood.
Existing data suggest that activation of Raf-1 engages multiple factors
and steps (9, 10), and phosphorylation of Raf-1 on Ser 338 and Tyr-341
is a critical step in this process (11). To evaluate whether the
serine-threonine kinase Raf-1 is part of the
1
,25(OH)2D3-signaling mechanism in chick
muscle cells, we first investigated the effect of the steroid hormone on Raf-1 serine phosphorylation. To that end, muscle cells were exposed
to 1 nM 1,25(OH)2D3 (0.5-5 min),
and cell lysates were immunoprecipitated with a highly specific
anti-Raf-1 monoclonal antibody followed by immunoblotting with
anti-phosphoserine antibody. As shown in Fig.
1, 1
,25(OH)2D3
caused a time-dependent increase in Raf-1 phosphorylation
in muscle cells. The stimulation of Raf-1 serine phosphorylation could
be detected already at 30 s, increased 1-fold over basal at
60 s, and reached a maximum after 2 min of hormone exposure
(3-fold).

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Fig. 1.
Time course of
1 ,25(OH)2D3
stimulation of Raf-1 serine phosphorylation. Chick skeletal
muscle cells were incubated in the presence of
1 ,25(OH)2D3 (1 nM) or vehicle
(ethanol <0.01%) for the indicated times. Immunoprecipitation of
Raf-1 and immunoblotting with an anti-phosphoserine
(anti-P-serine) antibody were carried out in cell lysates as
detailed under "Materials and Methods." A,
representative immunoblot. B, quantification by scanning
volumetric densitometry of blots from three independent experiments
performed in duplicate; averages ± S.D. are given. *,
p < 0.01, with respect to the control.
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It has been reported that the small G protein Ras is the direct
upstream activator of Raf, which in its GTP-bound activated form binds
to the Ras binding domain (Raf-RBD) and recruits the inactive
cytoplasmic Raf to the plasma membrane for activation (33). This
binding induces a conformational change of Raf-1 that yields an opened
structure suitable for phosphorylation by membrane-associated kinases.
To study Ras-dependent changes in Raf activation induced by
1
,25(OH)2D3, muscle cells were preincubated for 2 h with a specific Ras inhibitor peptide, which blocks the association of Grb2 and Sos1 and, thus, the nucleotide exchange of Ras,
avoiding its activation (34), followed by hormone treatment (1 nM, 2 min). As shown in Fig.
2, Ras inhibition abolished Raf-1 serine
phosphorylation by 1
,25(OH)2D3,
demonstrating that Ras is necessary for
1
,25(OH)2D3-Raf-1 activation in these cells. At the concentration used (25 µM), it is likely that
sufficient amounts of the inhibitor peptide entered the cells by
endocytosis to reach the Kd (25 nM) for
its binding to Grb2 (35).

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Fig. 2.
1 ,25(OH)2D3-induced
serine phosphorylation (P) of Raf-1 is suppressed by
Ras inhibitor peptide. Chick skeletal muscle cells were
preincubated for 2 h with a specific Ras inhibitor peptide (25 µM) and then exposed to 1 nM
1 ,25(OH)2D3 for 2 min. After cell lysis,
comparable aliquots were immunoprecipitated with an anti-Raf-1 antibody
followed by Western blotting with anti-phosphoserine antibody as
described under "Materials and Methods." A,
representative immunoblot. B, quantification by scanning
volumetric densitometry of blots of three independent experiments
performed in duplicate; averages ± S.D. are given. *,
p < 0.01, with respect to basal; **, p < 0.05 with respect to 1 ,25(OH)2D3
stimulation.
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Ras proteins play a central role in control of cell proliferation,
differentiation, and other cellular functions (36). They function by
cycling between inactive GDP- and active GTP-bound forms. This
molecular switch is mainly regulated by guanine nucleotide exchange
factors, which catalyze an exchange of GTP for GDP, and by GAPs, which
activate the intrinsic GTPase activity of Ras and, thus, convert
Ras-GTP to Ras-GDP (37). It has been reported that tyrosine
phosphorylation of Ras-GAPs on Tyr-460 allows rise of its GTPase
activity (38), promoting Ras inactivation. Therefore, the tyrosine
phosphorylation of Ras-GAP would promote Ras inactivation by
Ras-GAP-induced hydrolysis of Ras-GTP bound. We next explored the
effect of 1
,25(OH)2D3 on Ras-GAP tyrosine
phosphorylation. As shown in Fig. 3,
muscle cells treated with 1 nM
1
,25(OH)2D3 showed a rapid and transient
tyrosine de-phosphorylation of Ras-GAP, which was maximal at 0.5 min
(
80%) and returned to basal level upon 10 min of hormone exposure.
This result suggests that inhibition of Ras-GTP hydrolysis is part of
the mechanism by which 1
,25(OH)2D3 activates
Ras in skeletal muscle cells.

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Fig. 3.
Transient inactivation of Ras-GAP by tyrosine
dephosphorylation (P) in response to
1 ,25(OH)2D3.
Lysates from chick muscle cells exposed to 1 nM
1 ,25(OH)2D3 for the indicated times were
immunoprecipitated with anti-Ras-GAP antibody followed by immunoblot
analysis with anti-phosphotyrosine antibody as described under
"Materials and Methods." A representative immunoblot from three
independent experiments is shown. The blotted membranes were re-probed
with anti-Ras-GAP antibody to evaluate the equivalence of Ras-GAP
content among the different experimental conditions (bottom
panel).
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PKC, discovered as a serine/threonine kinase (39), mediates
intracellular responses to a variety of agonists including
1
,25(OH)2D3 (40). The direct phosphorylation
of Raf-1 by PKC isoforms has been suggested as an activation mechanism
of PKC on the Raf-1/ERK1/2-signaling pathway (16, 18). In previous
investigations we have demonstrated that stimulation of the
extracellular signal-regulated mitogen-activated protein kinases ERK1
and ERK2 by 1
,25(OH)2D3 is mediated by PKC and Ca2+ (23). These data prompted us to study whether PKC
participates in Raf-1 serine phosphorylation induced by the hormone. To
that end, muscle cells were pretreated with the PKC inhibitors
calphostin C or bisindolylmaleimide I and stimulated with
1
,25(OH)2D3 (1 nM, 2 min)
followed by immunoprecipitation of cell lysates with anti-Raf-1
antibody and then immunoblotting with anti-phosphoserine antibody. As
shown in Fig. 4, Raf-1 serine
phosphorylation was suppressed by either PKC inhibitor. Moreover, Ro
318220, a PKC specific inhibitor that blocks all PKC isoforms
completely, abolished Raf-1 serine phosphorylation (Fig.
5) and further confirmed that hormone
activation of Raf-1 is a PKC-dependent event. It has been shown that PKC
can directly phosphorylate and activate Raf-1 in
other cell types (18). PKC
belongs to the family of conventional protein kinases that are Ca2+-dependent. It is
well recognized that when skeletal muscle cells are subjected to
1
,25(OH)2D3 stimulation, a rapid increase of intracellular Ca2+ (22), inositol trisphosphate, and
diacylglycerol (43) and activation of PKC
(44) occurs. To
demonstrate that PKC
is involved in Raf-1 activation, we used
antisense technology to block PKC
protein expression. Cultured
muscle cells were transfected with an antisense oligodeoxynucleotide
against 20 nucleotides upstream of the AUG codon of PKC
mRNA to
suppress expression of its encoded protein. Under these conditions,
phosphorylation of Raf-1 induced by
1
,25(OH)2D3 was fully abolished (Fig.
6A, upper panel).
Suppression of PKC
expression was verified by immunoblotting of cell
lysates with anti-PKC
antibody (Fig. 6A, bottom
panel). Although we cannot rule out the possibility that other PKC
isoforms may also contribute to Raf-1 serine phosphorylation, our
results clearly show that PKC
is also a component of the mitogenic
pathway leading to Raf-1 activation in response to
1
,25(OH)2D3 stimulation.

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Fig. 4.
PKC inhibitors calphostin C and
bisindolylmaleimide I suppress muscle cell Raf-1 serine phosphorylation
(P) induced by
1 ,25(OH)2D3.
Muscle cells were treated with 1 nM
1 ,25(OH)2D3 for 2 min in the absence or
presence of calphostin C (100 nM) or bisindolylmaleimide I
(30 nM). The cells were then lysed and immunoprecipitated
with anti-Raf-1 antibody followed by Western blotting with
anti-phosphoserine antibody as described under "Materials and
Methods." A, representative immunoblot. B,
quantification by scanning volumetric densitometry of blots from three
independent experiments performed in duplicate; averages ± S.D.
are given. *, p < 0.01 with respect to basal and
1 ,25(OH)2D3 stimulation in the presence of
PKC inhibitors.
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Fig. 5.
The PKC inhibitor Ro 318220 blocks the
activation of Raf-1 induced by
1,25(OH)2D3. Muscle cells were
treated with 1 nM 1,25(OH)2D3 for 2 min in the absence or presence of Ro 318220 (200 nM). Then
cells were lysed and immunoprecipitated with anti Raf-1 antibody
followed by Western blotting with anti-phosphoserine
(anti-P-serine) antibody as described under "Materials and
Methods." A, representative immunoblot. B,
quantification by scanning volumetric densitometry of blots from three
independent experiments performed in duplicate; averages ± S.D.
are given. *, p < 0.01, with respect to basal; **,
p < 0.05 with respect to
1 ,25(OH)2D3 stimulation.
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Fig. 6.
Effects of an antisense oligodeoxynucleotide
against PKC mRNA on
1, 25(OH)2D3-induced
Raf-1 serine phosphorylation. Muscle cells were transfected
with a sense (ODN-S) or an antisense (ODN-AS)
oligodeoxynucleotide against PKC mRNA or Lipofectin
(LPF) to have a basal expression control. 48 h later
the cells were exposed to 1 nM
1 ,25(OH)2D3 for 2 min. Raf-1 was
immunoprecipitated from cell lysates and then immunoblotted with
anti-phosphoserine (anti-P-serine) antibody. A,
top panel, representative blot showing changes in Raf-1
serine phosphorylation; bottom panel, representative blot
showing the amount of PKC expressed in the cells. B,
quantification by scanning volumetric densitometry of top blot from
four independent experiments performed in duplicate; averages ± S.D. are given. p < 0.01 (*) and p < 0.05 (**), with respect to the corresponding control.
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We finally evaluated the effects of Ras, Raf-1, and MEK inhibition on
MAP kinase activity and tyrosine phosphorylation changes induced by
1
,25(OH)2D3. To measure MAPK activity, the
enzyme from cell lysates exposed for 1 min to the hormone (1 nM) was immunoprecipitated with an anti-(phospho)-active
MAPK antibody, which recognizes both the p42 and p44 active isoforms,
and then incubated with [
-32P]ATP and myelin basic
protein, as exogenous MAPK substrate. As previously shown (22),
1
,25(OH)2D3 caused a 3-fold increase in MAP
kinase activity (Fig. 7). The hormone
effect was abolished by either the Ras inhibitor peptide or compound PD
98059, which prevents the activation of the dual MAPK kinase MEK by
Raf-1 (45). These results are in agreement with previous observations
showing that PD 98059 prevents the stimulation of skeletal muscle cell proliferation by 1
,25(OH)2D3 (22). When
muscle cell lysates were preincubated with anti-Raf-1 antibody and then
exposed to 1
,25(OH)2D3 (1 nM, 1 min), hormone-induced MAPK tyrosine phosphorylation was also abolished
(Fig. 8). Furthermore, suppression of
PKC
expression in cells transfected with the antisense
oligodeoxynucleotide against PKC
mRNA abolished by 70% the
phosphorylation of MAPK induced by
1
,25(OH)2D3 (Fig.
9). These results stress the relevance of
PKC
, Ras, Raf-1, and MEK in the
1
,25(OH)2D3-signaling pathway, which results
in MAP kinase stimulation in muscle cells.

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Fig. 7.
Stimulation of MAP kinase activity by
1 ,25(OH)2D3 is
mediated by Ras and MEK. Cells were incubated for 1 min with
1 ,25(OH)2D3 (1 nM) in the
absence or presence of a MEK-specific inhibitor (PD 98059, 10 µM) or a Ras inhibitory peptide (25 µM).
Immunoprecipitation of MAP kinase and assay of its activity using
[ -32P]ATP and myelin basic protein as exogenous
substrate were carried out in cell lysates as described under
"Materials and Methods." Results are the average of three
independent experiments performed in triplicate ± S.D. *,
p < 0.01, with respect to basal and
1 ,25(OH)2D3 stimulation in the presence of
inhibitors.
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Fig. 8.
1 ,25(OH)2D3
stimulation of MAP kinase tyrosine phosphorylation (P)
is suppressed by antibody inhibition of Raf-1. After cell
lysis, comparable aliquots of lysate protein (500 µg) were incubated
with anti-Raf antibody (2 µg) or anti-goat IgG antibody (2 µg, as a
control of the basal levels of MAP kinase) on an ice bath for 10 min.
Then the lysates were exposed to 1 ,25(OH)2D3
(1 nM) or vehicle ethanol (<0.01%) for 1 min. Proteins
were resolved by SDS-PAGE followed by immunoblotting with
anti-(phospho)-active MAP kinase as described under "Materials and
Methods." A, representative immunoblot. B,
quantification by scanning volumetric densitometry of blots from three
independent experiments performed in duplicate; averages ± S.D.
are given. *, p < 0.01 with respect to basal and
1 ,25(OH)2D3 stimulation in the presence of
anti-Raf antibody.
|
|

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Fig. 9.
Effects of an antisense oligodeoxynucleotide
against PKC mRNA on
1 ,25(OH)2D3-induced
MAPK phosphorylation. Muscle cells were transfected
(T) with a sense (ODN-S) or an antisense
(ODN-AS) oligodeoxynucleotide against PKC mRNA.
48 h later the cells were exposed to 1 nM
1 ,25(OH)2D3 for 2 min and then lysed.
Proteins were resolved by SDS-PAGE followed by immunoblotting with
anti-(phospho)-active MAP kinase as described under "Materials and
Methods." Top panel, representative blot showing changes
in phospho-MAPK. Bottom panel, quantification by scanning
volumetric densitometry of the top blot from three independent
experiments performed in duplicate; averages ± S.D. are given.
p < 0.01 (*) and p < 0.05 (**), with
respect to the corresponding control.
|
|
With regard to the initiation of the
1
,25(OH)2D3 signal that leads to activation
of the MAP kinase pathway, new lines of evidence indicate that steroid
hormone intracellular receptors mediate rapid, non-transcriptional
stimulation of MAPK via interaction with upstream components of the
cascade (46). More specifically, we recently reported that activation
of MAPK by 1
,25(OH)2D3 in muscle cells is
preceded by rapid tyrosine phosphorylation of the vitamin D receptor
followed by its association with Src (47). Subsequent interaction with
the Shc-Grb2-Sos-Ras complex may then occur as observed in
keratinocytes (48). However, the involvement of a novel
1
,25(OH)2D3 membrane receptor, whose
existence in various hormone target cell types has received
experimental support (41, 42, 49), cannot be excluded. In
summary, our results demonstrate for the first time in a
1
,25(OH)2D3 target cell that activation of
Raf-1 via Ras (through inhibition of Ras-GAP activity by tyrosine
dephosphorylation) and PKC
-dependent serine
phosphorylation play a central role in hormone stimulation of the
MAPK-signaling pathway (depicted in the schematic diagram of Fig.
10).

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|
Fig. 10.
Schematic diagram indicating the chain of
events leading to
1 ,25(OH)2D3 activation
of MAP kinase in skeletal muscle cells. By acting at the plasma
membrane, 1 ,25(OH)2D3 inhibits Ras-GAP
activity by tyrosine dephosphorylation (YP), which elicits
Ras activation. The hormone also stimulates PKC . Activated Ras along
with PKC lead to serine phosphorylation and stimulation of Raf-1
followed by MEK and MAP kinase activation.
|
|
 |
FOOTNOTES |
*
This research was supported by grants from the Agencia
Nacional de Promoción Cientifica y Tecnológica, Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET), and Universidad Nacional del Sur, Argentina.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Depto.
Biología, Bioquímica y Farmacia, Universidad Nacional
del Sur, San Juan 670, 8000 Bahía Blanca, Argentina. Tel.:
54-291-4595101 (ext. 2430); Fax: 54-291-4595130; E-mail:
rboland@criba.edu.ar.
Published, JBC Papers in Press, November 1, 2002, DOI 10.1074/jbc.M205732200
 |
ABBREVIATIONS |
The abbreviations used are:
MEK, mitogen-activated protein (MAP) kinase (MAPK) kinase;
ERK, extracellular signal-regulated kinase;
PKC, protein kinase C;
1
, 25(OH)2D3, 1
,25-dihydroxyvitamin
D3;
DMEM, Dulbecco's modified Eagle's medium;
GAP, GTPase-activating protein;
ODN, oligodeoxynucleotide.
 |
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