1 Department of Experimental Medicine, University of L'Aquila, Via Vetoio,
Coppito II, 67100 L'Aquila, Italy
2 Department of Histology and Embryology, University of Rome `La Sapienza', Via
Scarpa 14, 00161 Rome, Italy
3 Department of Neuroscience, Section of Pharmacology and Medical Oncology,
University of Rome Tor Vergata, Via di Tor Vergata 135, 00133 Rome,
Italy
Author for correspondence (e-mail:
zani{at}univaq.it)
Accepted 28 June 2002
![]() |
Summary |
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Here, by monitoring the signalling pathways triggered by TPA, we
demonstrate that PKC mediates these effects by inducing transient
activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation
of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all
referred to as MAPKs). Activation of MAPKs following ectopic expression of
constitutively active PKC
, but not its dominant-negative form, is also
demonstrated.
We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.
Key words: PKC, MAPKs, RD
![]() |
Introduction |
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Unlike normal myoblasts, which respond to both the proliferative and
differentiative action of insulin and IGFs
(Coolican et al., 1997;
Florini et al., 1991
), RD
cells do not differentiate in response to IGF. Recent reports have suggested
that elevated cyclin D1 and CDK activities contribute to the inability of RD
cells to arrest growth when cultured in mitogendeprived medium
(Knudsen et al., 1998
).
Nevertheless, RD cells treated with PKC activators, such as the tumor promoter
TPA, progressively acquire a more elongated shape, which is the typical
myogenic morphology, and become unresponsive to mitogens and undergo growth
arrest and myogenic differentiation
(Aguanno et al., 1990
). This
suggests that activated PKC interferes with the transduction of mitogenic
signals, thereby leaving myogenic transcription factors free to initiate
muscle differentiation. Thus, knowledge of the downstream pathways of PKC in
RD cells might help the search for agonists, other than tumor promoters,
capable of reverting cells back to their non-transformed phenotype.
Growth factor signal transduction pathways involve several kinases,
including those of the MAPK and P13K/Akt kinase families
(Garrington and Johnson,
1999). Within the MAPK family, ERKs, JNKs and p38 have been
implicated in relaying extracellular signals to the nucleus. MAPK pathways, by
regulating transcription factory activity
(Hill and Treisman, 1995
;
Treisman, 1996
), mediate
specific responses, including proliferation, differentiation, apoptosis and
stress (Minden and Karin,
1997
; Pang et al.,
1995
; Racke et al.,
1997
; Robinson and Cobb,
1997
; Traverse et al.,
1992
). In a typical pathway, Ras activation initiates a protein
kinase cascade that leads to MAPK activation through the intervening protein
kinases Raf and MEKs.
Although downstream effectors of MAPKs that elicit specific responses have
yet to be fully identified, recent reports have suggested that MEF2 is a
substrate of activated p38 kinase, a transcriptional target for signalling
pathways that control skeletal myogenesis
(Francisco and Eric, 1999;
Zhao et al., 1999
). In this
regard, p38 has been reported to induce muscle differentiation in both L8 and
C2C12 (Cuenda and Cohen, 1999
;
Zetser et al., 1999
), whereas
P13K/Akt is thought to be involved in differentiation and hypertrophy
(Bodine et al., 2001
;
Jiang et al., 1998
;
Rommel et al., 2001
) of L6
myogenic lines. Moreover, ERKs are activated in C2C12 when myogenesis is
promoted by the removal of mitogens
(Gredinger et al., 1998
).
Recently, a pivotal role for p38 has been documented in the pathological
myogenic differentiation of a number of RMS lines, including RD cells
transfected with the p38 upstream kinase MKK6
(Puri et al., 2000
). In
addition, evidence has emerged indicating that crosstalk between MAPKs, such
as the antagonistic effect of JNKs and p38 in the L6 and C2C12 myogenic cell
lines, controls myogenesis (Meriane et
al., 2000
).
The involvement of both PKC-dependent and PKC-independent pathways of
Raf/MEK activation in response to an agonist has been reported as a possible
mechanism of MAPK activation (Qiu and
Leslie, 1994; Schonwasser et
al., 1998
). The involvement of PKCs in Raf activation has been
mainly studied in cells that activate ERKs in response to TPA
(El Shemerly et al., 1997
;
Kaneki et al., 1999
;
Miranti et al., 1999
;
Racke et al., 1997
). In fact,
Raf and MEK phosphorylation have been observed in cells co-transfected with
PKC
and ß, which provides strong evidence for the involvement of
PKCs in the regulation of these pathways
(Marquardt et al., 1994
;
Sozeri et al., 1992
).
Furthermore, MEK-1 activation is mediated by conventional, novel and atypical
PKC isoforms in a Raf-dependent and -independent manner
(Schonwasser et al.,
1998
).
In this study, we report that activation of the myogenic program of RD
cells is dependent on PKC-mediated ERK, JNK and p38 activation. The use
of MAPK inhibitors and agonists allowed us to dissect TPA-mediated kinase
activation and thereby correlate a cell response with specific kinase
pathways. The results obtained show an anti-mitogenic role for activated JNKs
and a role for activated p38 in the expression of muscle-specific genes.
Moreover, ERK activation may be involved in the maintenance of the RD
myogenic-related morphology.
![]() |
Materials and Methods |
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For growth analysis, RD cells were harvested in trypsin-EDTA and counted in a hemocytometer chamber.
Subcellular fractionation, SDS-PAGE and immunoblotting
Total extracts were prepared by scraping cells in 2% SDS containing 2 mM
pheny1 methy1 sulphony1 fluoride (PMSF), 10 µg/ml antipain, leupeptin and
trypsin inhibitor, 10 mM sodium fluoride and 1 mM sodium orthovanadate and
sonicating them for 30 seconds. Nuclear and membrane fractions were obtained
by lysing cells in 10 mM Tris-HCl pH 7.5 containing protease and phosphatase
inhibitors, homogenized by 20 strokes in a Dounce homogenizer and centrifuging
them at 900 g. The nuclei-containing pellets were resuspended in 2%
SDS containing proteases and phosphatases inhibitors. The 900 g
supernatants were centrifuged at 100,000 g to sediment the enriched
membrane fractions. An aliquot of total lysates and nuclear and membrane
fractions were used to evaluate the amount of proteins
(Lowry et al., 1951). Equal
amounts of total lysates or membrane and nuclear fractions (100 µg) were
separated by 10% SDS-PAGE (Laemmli,
1970
) and transferred to a nitrocellulose membrane (Hybond C Extra
Amersham) following standard procedures
(Towbin et al., 1979
). 8%
SDS-PAGE was used for myosin detection. Immunoblottings were performed with
the following antibodies: 1:1000 of anti-phospho-ERKs, anti-phospho-JNKs and
anti-phospho-p38 (New England Biolabs, NEB), all of which recognise the
phosphorylated/activated forms of these kinases; 1:250 antibodies which
recognize the total ERKs, JNKs and p38 (Santa Cruz); 1:10 MF20 (supernatant of
hybridoma kindly provided by D. Fischman, Cornell University); 1:1000
anti-PKC
and ß1 (Transduction Laboratory); 1:500 anti-PKC
as previously described (Aquino et al.,
1990
); 1:500 anti-PCNA and 1:250 anti-hemagglutinin (Santa Cruz).
Peroxidase-conjugated anti-mouse (1:3000) or anti-rabbit IgG (1:1500)
purchased from Amersham or from Santa Cruz was used for ECL (Amersham)
detection.
As the anti-phospho-MAPK antibodies detect each kinase only when dually phosphorylated, an increase in phosphorylation is an index of their activation. Densitometric analysis of bands, relative to both total and phosphorylated proteins, provided quantification (phospho-MAPK:total-MAPK) of TPA-induced ERK, JNK and p38 activation expressed as a fold increase over the control value, which was arbitrarily set at 1.
Immunofluorescence and FACS analysis
For the immunofluorescence study, untreated and treated cells were fixed
with 4% paraformaldehyde (SIGMA) for 15 minutes at room temperature, washed
three times, permeabilized in 0.2% Triton X-100 in PBS for 20 minutes and
saturated for 1 hour with 1% of BSA (immunoglobulin free, SIGMA) in PBS.
Undiluted MF20 was incubated for 1 hour and immunocomplexes were detected
using (1:50) FITC-conjugated rabbit anti-mouse IgG (Zymed). For FACS analysis,
untreated and treated cells were washed, and the pellets were resuspended in
50% FBS in PBS. Cells were fixed overnight after three parts of 70% cold
ethanol had been added. Fixed cells were washed twice and resuspended in PBS
additioned with 50 µg/ml of propidium iodide and 100 U/ml of DNAse-free
RNAse (SIGMA). Cell cycle analysis of cells stained with propidium iodide was
performed using a Coulter Epics XL flow cytometer (Beckman Coulter).
Transfection
One day after plating (3.5x105 cells/ml), cells were
transiently transfected with 0.5 µg of constitutively active
PKC-coding vector (A25E) or a dominant-negative version of PKC
(K368R), kindly provided by Dr Baier (University of Innsbruck)
(Baier-Bitterlich et al., 1996
)
or with 0.5 µg of constitutively active MKK2 (KW71A), kindly provided by Dr
Ahn (Howard Hughes Medical Institute)
(Mansour et al., 1996
). In the
present paper, MKK2 is named MEK2, according to the current nomenclature
(Cohen, 1997
). For the in vivo
c-Jun and Elk1 trans-reporting system (Stratagene), cells were transfected
with the following plasmids: (i) 0.1 µg of pFA-c-Jun or pFA-Elk1 activator
plasmids, which express proteins that consist of the DNA-binding domain of
yeast GAL4 and the activation domains of c-Jun or Elk1; (ii) 1 µg of
pFR-Luc plasmid, which contains a synthetic promoter with five tandem repeats
of the GAL4-binding sites controlling expression of the luciferase gene. In
this assay the fusion activators, which are phosphorylated and activated by
JNKs and ERKs, transactivate the luciferase promoter. Thus, the extent of
luciferase activity reflects the activation of a specific kinase and the
corresponding signal transduction pathway. Lipofectamine Plus reagent was used
as the transfectant according to the manufacturer's instructions (GIBCO BRL).
One day after transfection, cells were treated with TPA, or left untreated,
for 1 day. Total lysates from transfected cells were processed either for
SDS-PAGE and immunoblotting or assayed for luciferase activity according to
the manufacturer's instructions (Promega).
![]() |
Results |
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Analysis of MAPK activation (Fig. 1) shows, in control RD cells (C0), a high level of phospho-ERKs and phospho-p38 but a low level of phospho-JNKs. In culture, a drastic decrease in phospho-ERKs and phospho-JNKs and a significant increase in phospho-p38 occur over time (C2-C5, Fig. 1). Short TPA treatment (30 minutes to 7 hours) induces a rapid and sustained increase in both phospho-ERKs (2.3) and p38 (2.4) in comparison with the levels of control cells, whereas the increment in phospho-JNK/p46, though more marked (8.6), is less persistent.
|
After prolonged treatments (2-5 days), neither basal (C2, C5) nor TPA-induced phospho-JNKs are detectable, whereas a decrease in ERK phosphorylation of control cells causes more evident phospho-ERK stimulation (3.2-4.3), which is in contrast to the decrease in p38 stimulation following an increase in the basal phosphorylation level in control cells. Moreover, TPA does not alter the expression level of total kinases and the fold increases in ERK, JNK and p38 phosphorylation provide a quantification of kinase activation (see Materials and Methods). The ongoing activation of MAPKs is also demonstrated by a marked increase in the phospho-ATF2 and phospho-c-jun, the downstream targets of MAPKs (data not shown). These results demonstrate that TPA induces the concomitant activation of three distinct MAPK cascades.
PKC-dependent MAPK kinase induction
We previously suggested that the effects of TPA on RD cell differentiation
may be caused by the activation of PKC rather than other isoforms,
(
and ß1) (Bouche et al.,
1995
). In this paper, we demonstrate that there is a significant
and rapid (30 minutes) TPA-induced selective PKC
membrane translocation
that is not accompanied by either any expression of PKC
or an increase
in PKCß1 membrane translocation (Fig.
2A).
|
PKC-induced activation of ERKs, JNKs and p38 was then studied in
transiently transfected RD cells with a constitutively active mutant form of
PKC
(A25E) and with a control vector (CMV) by analysing the
phosphorylated forms of ERKs, JNKs and p38 after immunoblottings of total
lysates 24 hours after transfection. The results in
Fig. 2B show that the increase
in PKC
expression (two-fold) in A25E-transfected cells over the level
of control vector CMV-transfected cells induces a significant increase in ERK
phosphorylation (1.7-fold) and an even more marked increase in the levels of
phosphorylated JNKs (2.2-fold for p46 and 7.3-fold for p54) and p38
(7.3-fold). It is noteworthy that a marked phosphorylation of JNK2/p54 is
present in PKC
-transfected cells. Moreover, the extent of kinase
activation in PKC
-transfected cells is consistent with that observed
after 30 minutes to 7 hours of TPA treatment
(Fig. 1).
To further demonstrate the role of PKC in MAPK activation, we tested
the effect of both the wild-type (WT PKC
) and dominant-negative mutant
of PKC
(DN PKC
) by using a c-Jun or Elk1 trans-reporting system
designed for the assessment of in vivo MAPK activation (see Materials and
Methods). In these experiments, RD cells were co-transfected with WT
PKC
or DN PKC
with activator plasmid (pFA-c-Jun or pFA-Elk1) and
a reporter plasmid pFR-Luc and were left untreated or were treated with TPA.
The results in Fig. 2C show
that Elk1- and c-Jun-driven luciferase activity (respectively, Elk-Luc and
Jun-Luc) are induced by TPA in both control vector- (CMV+TPA) (4.7- and
2.1-fold respectively) and PKC
-transfected cells (WT PKC
+ TPA)
(4.2- and 3-fold respectively). By contrast, the ectopic expression of the
DN-mutant form of PKC
decreases TPA-induced luciferase activity (DN
PKC
+TPA) by about 61% for Elk-Luc and 38% for Jun-Luc. Taken together,
these results indicate that activation of PKC
by TPA mediates MAPK
pathway activation.
Evidence that PKC is an upstream effector of MAPK activation was
provided by the use of PKC inhibitor Ro320432 at a concentration (60 nM),
which selectively inhibits the PKC
and ß isoforms
(Wilkinson et al., 1993
). To
analyse MAPK phosphorylation, RD cells were pre-treated with Ro320432 for 6
hours and were then left untreated or were treated with TPA for 30 minutes.
Immunoblotting analysis shows that Ro320432 does not affect the level of
phosphorylated MAPKs in untreated cells but completely prevents TPA-induced
phosphorylation of ERKs, JNKs and p38 (Fig.
3A).
|
Likewise, to assess PKC dependence on growth arrest and myogenic
differentiation, growth curve (0-6 days) and late sarcomeric myosin heavy
chain (MHC) (6 days) accumulation were analysed in RD cells pre-treated with
Ro320432 and then treated with TPA. The results, shown in
Fig. 3B, clearly demonstrate
that PKC inhibition prevents TPA-mediated growth arrest (Ro+TPA) without
affecting the level of control cell proliferation (Ro).
Fig. 3C shows that Ro320432
inhibits the TPA-induced sarcomeric MHC accumulation. It is noteworthy that,
since PKCß activation is not altered by TPA treatment, the results
obtained by using Ro320432 can be ascribed to the selective inhibition of
PKC
. Taken together, these results demonstrate that PKC
is an
upstream effector of both TPA-induced MAPK activation and myogenic phenotype
expression in RD cells.
MEK1/2 inhibition induces downregulation of both ERK and JNK pathways
and correlates with morphological changes
Sustained ERK activation after prolonged treatment (2-5 days) with TPA may
involve ERKs in the induction of late biological responses to TPA
(Bennett and Tonks, 1997). We
investigated the MEK1/2 inhibitor, U0126, which was used effectively to
investigate the role of ERKs in regulating cellular responses owing to the
fact that it inhibits the MEK/ERK pathway by preventing the activation of
MEK1/2 and by blocking activated MEK1/2
(Favata et al., 1998
). We
initially determined whether the simple inhibition of ERKs leads to an
alteration in the basal and TPA-induced phosphorylation levels of p38 and
JNKs. A time-course experiment was performed with 10 µM U0126, the minimal
concentration required to obtain maximal inhibition of ERK phosphorylation in
a dose-response experiment (data not shown).
Thus, the pattern of MAPK activation was analysed after immunoblotting of RD cells (Fig. 4A) pre-treated with 10 µM U0126 and left untreated (U) or treated with TPA for 30 minutes, 2 and 5 days (U+TPA). Within 30 minutes, both in the presence and in the absence of TPA, a marked reduction in ERK and, unexpectedly, JNK phosphorylation is observed in U0126-treated cells (Fig. 4A). By contrast, p38 phosphorylation is instead increased after 30 minutes of U0126 treatment. After 2 and 5 days of treatment, the U0126-mediated inhibition of ERK phosphorylation is still present, both in the absence and presence of TPA, although to a lesser extent, whereas JNK phosphorylation is undetectable in both treated and untreated cells. After 2 and 5 days, phospho-p38 does not significantly change in U0126-treated cells, either with or without TPA, but does increase in control cells (C; Fig. 4A) as already seen in the experiment shown in Fig. 1. It is noteworthy that p38 phosphorylation is rapidly (30 minutes) stimulated by U0126, which suggests that, upon MEK inhibition, either p38 is the only active MAPK or the activation of p38 is inversely correlated with the inactive state of ERKs or JNKs.
|
To rule out the possibility of a time-dependent inactivation of U0126 in the culture medium, we tested the 30 minute and 5 day conditioned U0126-containing media for their ability to inhibit ERK phosphorylation within 30 minutes of incubation of parallel RD cultures. Immunoblotting analysis shows that both these conditioned media retain their capacity to prevent ERK phosphorylation (Fig. 4B).
Furthermore, prolonged U0126 treatment (3-6 days) induces drastic morphological changes in both untreated and TPA-treated cells, appear as round-shaped (Fig. 5), although no cell detachment is observed even after 15 days of treatment. These results show that inhibition, by U0126, of the MEK/ERK pathway in RD cells parallels inhibition of JNK phosphorylation and is accompanied by a drastic morphological change.
|
Interestingly, the U0126-mediated JNK downregulation suggests that MEK1/2
are upstream activator kinases of JNKs, just as U937 cells have been reported
to be (Franklin and Kraft,
1995). To verify this hypothesis, RD cells were transfected with
constitutively active HA-tagged-MEK2 or empty vector; subsequent
immunoblotting analysis of total lysates shows a significant increase in the
level of both phospho-JNK and phospho-ERK in MEK2-transfected cells (MEK2)
when compared with control cells (CMV)
(Fig. 6A).
|
To assess the in vivo activation of the JNK pathway, parallel RD cell cultures were co-transfected with either constitutively active HA-tagged-MEK2 (CA MEK2) or control vector (CMV) and with both pFA-c-Jun activator plasmid and a reporter plasmid pFR-Luc. A dramatic increase in luciferase activity is detected in MEK2-transfected cells when compared with control vector-transfected cells (Fig. 6B).
Taken together, these results demonstrate that, in RD cells, MEK2 is an upstream activator kinase of JNK.
Selective JNK activation as well as ERK and JNK modulation induce
growth arrest
The concomitant deletion of MAPK activation, growth arrest and
myogenic-specific marker expression induced by the PKC inhibitor
(Fig. 3) led us to investigate
whether ERK, JNK and p38 pathways play distinct roles in these effects by
using a selective MAPK agonist or inhibitor. To study possible changes in
growth potential of RD cells, we used anisomycin, which is reported to act as
a true signaling agonist of JNKs and p38
(Hazzalin et al., 1998),
U0126, shown here to inhibit ERKs and JNKs
(Fig. 4), and SB203580, which
inhibits p38.
We first performed a time-course experiment to investigate whether
long-lasting anisomycin treatment induces persistent JNK activation without
altering cell viability. Ten ng/ml of anisomycin is the dose that induces
maximal JNK activation and is ineffective on ERKs either in the presence or
absence of TPA (data not shown) (Cano et
al., 1994). For the time-course experiment, cells were left
untreated or were treated, for different periods of time, with 10 ng/ml of
anisomycin, added 1 hour before TPA treatment. Immunoblots of total lysate
show that anisomycin persistently stimulates (30 minutes-5 days)
phosphorylation of p38 and JNKs, though it does this to a lesser extent at 5
days (Fig. 7A). Moreover, 3 day
anisomycin pre-treatment does not impair the activation of myosin expression
during chase in the presence of TPA (Fig.
7B) even at doses as high as 50 ng/ml. Notably,
anisomycin-pre-treated cells synthesize more myosin than control cells during
chase in the presence of TPA (Fig.
7B). These results rule out the possibility that prolonged
anisomycin treatment affects cell viability.
|
We therefore investigated whether cell proliferation is affected by
prolonged JNK or p38 activation by treating cells with anisomycin, in the
presence of the p38 inhibitor SB203580, to exclude the contribution of p38.
2.5 µM of SB203580 is the minimal concentration that does not affect either
ERK and JNK activation or, as recently reported, PKB/Akt
(Lali et al., 2000) (data not
shown). Cells were treated and the number of cells was counted after 2 to 6
days of treatment. Fig. 8A
shows that anisomycin induces drastic growth arrest, which is not modified by
SB203580. Similarly, TPA, which concomitantly activates ERKs, JNKs and p38,
also induces growth arrest, which is not modified by SB203580 even after 4
days of treatment (Fig. 8B).
However, a 30% growth increase, after 6 days of treatment, occurs in
SB203580-treated cells with or without TPA. These results led us to
hypothesize that the growth arrest pathway is triggered by highly activated
JNKs, rather than p38, to counterbalance the mitogenic effects of a threshold
level of active ERKs.
|
To verify this hypothesis, we used U0126, which, by downregulating ERK and JNK pathways (Fig. 4A), drastically alters the critical MAPK pathways balance, which is, in turn, likely to be the cause of the transduction of mitogenic signals. Cells were treated with U0126 for different periods of time, and the number of cells was counted after 2 to 6 days of culture. Fig. 8C shows that U0126 induces drastic growth arrest, which is not reversed by p38 inhibition for up to 4 days of culture, whereas a 38% growth recovery occurs between days 4 and 6 of SB203580 treatment.
Moreover, in order to investigate whether the decrease in cell numbers
(Fig. 8A-C) observed during the
various treatments was a result of withdrawal from the cell cycle, we analysed
the nuclear distribution of proliferating cell nuclear antigen (PCNA), which
is known to be downregulated in growth arresting cells
(Mercer et al., 1991). The
distribution of RD cells in the cell cycle by flow cytometric analysis was
also analysed. Immunoblots of nuclear extracts from untreated cells and from
cells treated with TPA, U0126 and anisomycin for 3 days
(Fig. 8D) shows a consistent
reduction in nuclear PCNA in TPA-treated cells, whereas nuclear PCNA in U0126-
and anisomycin-treated cells is undetectable. Furthermore, the results of the
flow cytometric analysis point to G1 arrest in TPA-, anisomycin- and
U0126-treated cells between days 1 and 4 of treatment
(Table 1).
|
Taken together, these results indicate that JNK activation or complete ERK and JNK downregulation are sufficient to induce growth arrest, whereas p38 activity is likely to contribute to maintaining a steady state in RD cells already tending towards myogenic differentiation.
Opposite roles of p38 and sustained JNK activation in myogenic
differentiation
The differentiated phenotype of myogenic lines, including RD, has recently
been ascribed to selective p38 activation
(Puri et al., 2000;
Wu et al., 2000a
). In this
study, since activated p38 was detected in the absence (U0126) or in the
presence of different levels of activated ERKs and JNKs (TPA) or in the
presence of highly and persistently activated JNKs (anisomycin), we
investigated whether activated p38 is necessary and sufficient to induce
myogenic phenotype expression independently of other activated MAPKs. For this
purpose, analysis of sarcomeric MHC expression was performed by immunoblotting
of lysates (Fig. 9A) and by
immunofluorescence (Fig. 9B) in
RD cells treated with TPA, U0126, in the presence and in the absence of
SB203580, and with TPA in the presence of anisomycin for 6 days. As shown in
Fig. 9A, both U0126-treated and
TPA-treated cells accumulate more sarcomeric MHC (U) than control
proliferating RD cells (C); moreover, U0126 potentiates the effect of TPA on
MHC expression (U+TPA). By contrast, both SB203580 and anisomycin inhibit
TPA-mediated accumulation of sarcomeric MHC (SB+TPA, AN+TPA), and SB203580
also inhibits U0126-mediated MHC accumulation (U+SB).
|
Immunofluorescence analysis confirmed the results of the immunoblotting and shows that filamentous fluorescence is present in TPA-treated cells, whereas unassembled diffused myosin staining is observed in U0126-treated round cells both in the presence and in the absence of TPA (Fig. 9B). It is noteworthy that myosin accumulation in U0126-treated cells rules out any toxic effect of this inhibitor, which suggests that U0126-treated cells are metabolically active.
These data indicate that p38 plays a pivotal role in the activation of the myogenic program and that persistently activated JNK might antagonise the differentiating effects of activated p38. To further verify the latter hypothesis, we treated U0126-pre-treated RD cells, in which both JNKs and ERKs are completely downregulated, with anisomycin for 30 minutes, 3 and 5 days, to reactivate JNK in the absence of activated ERKs. We then analysed the myosin expression and the level of phospho-active-JNKs by immunoblotting experiments. The results in Fig. 10 show that TPA- and U0126-mediated myosin expression is strongly inhibited by anisomycin at day 3 as well as at day 5. Interestingly, U0126 induces MHC accumulation earlier (3 days) than TPA treatment does, whereas anisomycin alone (AN) does not induce MHC expression in spite of sustained p38 activation (Fig. 7). As expected, besides activating p38, anisomycin potently and persistently (from 30 minutes to 5 days) re-activates JNK in U0126-pre-treated cells at all times of treatment (Fig. 10).
|
These results suggest that JNKs counteract the p38-induced myogenic program, thereby indicating that p38 activation is required but is not sufficient to induce the expression of myogenic markers when sustained activation of JNKs occurs.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TPA-mediated PKC- activation induces MAPK pathways
Analysis of MAPK pathways in proliferating RD cells reveals a high level of
activated ERKs and a low level of activated JNKs. These findings, which are in
keeping with data in the literature, suggest that activated ERKs correlate
with proliferation, whereas activated JNKs correlate with cellular responses
to stress (Iordanov et al.,
1998; Rosette and Karin,
1996
). Surprisingly, though myogenic phenotype is induced by p38
activation (Puri et al., 2000
;
Wu et al., 2000a
), the
consistent increase in phospho-p38 in cultured control RD cells fails to
induce myogenesis, suggesting that p38 downstream pathways are inhibited. TPA,
which promotes the myogenesis process in RD cells, induces a rapid and
sustained increase in phospho-active ERKs and p38 and a transient activation
in JNKs. Concomitant activation of the three MAPKs is not a common event in
other myogenic cell lines or in other cell types, although we have previously
observed similar results in an inflammatory-like response induced by
TNF-
treatment in Sertoli cells (De
Cesaris et al., 1998
; De
Cesaris et al., 1999
). It has also been reported that TPA-induced
macrophagic differentiation of U937 leukemic cells requires ERK, JNK and p38
activation (Franklin and Kraft,
1997
).
In this study, we demonstrate that PKC is an upstream kinase of MAPK
cascades, which regulate growth arrest and myogenic differentiation. Notably,
the PKC inhibitor Ro320432 prevents MAPK phosphorylation as well as growth
arrest and myogenic differentiation. The involvement of PKC
in the
activation of the three MAPK pathways is strongly supported by transient
transfection experiments. In fact, ectopic expression of the constitutively
active form of PKC
, but not its dominant-negative form, induces ERK,
JNK and p38 activation. In conclusion, these data suggest that PKC
is
an essential activator of the three MAPK cascades which, in turn, play
relevant roles in growth arrest and myogenic differentiation in RD cells
(Fig. 11).
|
JNKs activation is controlled by MEK2 and is involved in growth
arrest
Although it has been demonstrated that normal and pathological myogenesis
is dependent on p38 activation, the pathways inducing growth arrest, which
enable myogenic tumor cells to activate myogenic-specific programs, require
further investigation.
The use of MAPK inhibitors allowed us to dissect the TPA-mediated kinase activation and, hence, to show that ERKs and JNKs are involved in the regulation of withdrawal from the cell cycle and that p38 is required for the initiation of the myogenic differentiation program. The MEK1/2 inhibitor, U0126, which drastically and persistently inhibits ERK and, unexpectedly, JNK activation, induces rapid (1 day) and drastic growth arrest. This is demonstrated by a decreased growth potential (2-6 days), an increased number of G1-arrested cells as shown by FACS analysis, as well as by a drastic decrease in nuclear PCNA. These effects are not modified after prolonged U0126 treatment, although the extent of ERK inhibition decreased slightly between days 2 and 5, and this decrease is not due to the instability of the drug in the culture medium. Moreover, the inhibition of JNK activation is not a result of a non-specific effect of U0126, since cells transfected with a constitutively active form of MEK2 express a much higher level of activated JNKs than control cells transfected with empty vector do.
Besides preventing ERK and JNK activation, U0126 also induces an increase in activated p38; thus, growth inhibition may be due either to the downregulation of ERKs and/or JNKs or to the activation of p38. By using the p38 inhibitor SB203580 together with the MEK inhibitor, we demonstrate that inhibition of p38 can only revert growth arrest after prolonged incubation times (6 days). Similarly, in TPA-treated cells, in which the three MAPKs are activated, p38 inhibition does not reverse growth arrest before 6 days. These data point to a role of p38 in the maintenance, rather than in the induction, of growth arrest.
Since there is no specific JNK inhibitor with which to gain an insight into the role of JNKs in growth arrest, we used anisomycin, a potent JNK and p38 agonist. The fact that anisomycin does not affect ERK activity permits us to study the induction of strong JNK activation in the absence of ERK modulation. We found that persistent activation of JNKs in anisomycin-treated RD cells induces growth arrest even in the presence of the p38 inhibitor, as shown by the reduction in growth potential and in nuclear PCNA, as well as by an increased number of G1-arrested cells.
All these data indicate that a critical level of activated ERKs sustains deregulated growth of RD cells (control cells) and that a high level of activated JNKs is required to counteract the proliferative action of ERKs (TPA-treated cells). When the ERK pathway is abrogated (U0126-treated cells), the JNK pathway is concomitantly downregulated, and growth arrest occurs following the lack of proliferative action of ERKs. We conclude that growth arrest of RD cells comes from two alternative pathways triggered either by downregulation of ERKs or by activation of JNKs, which thus confer a growth arresting function on JNKs (Fig. 11).
Crosstalk between MAPKs modulates myogenic-related morphology and
differentiation
Interestingly, the MEK inhibitor induces a round as opposed to the typical
spindle-shaped morphology, without detachment or apoptotic events; this
suggests that activated ERK or JNK may play a role in maintaining
myogenic-related morphology. It has recently been reported that in the C2C12
myogenic cell line, MAPK-specific phosphatase (MKP-1) overexpression
downregulates ERK activity sufficiently to rescue myogenic-specific gene
expression but prevents mature myotube formation. These results point to a
role for activated ERKs during early and late myogenic differentiation
(Bennett and Tonks, 1997).
Furthermore, the role of activated ERKs in late myogenesis has been confirmed
by other studies in which the MEK inhibitor PD98059 prevents the myoblast
fusion process without affecting the expression of muscle-specific genes in
C2C12 cells (Gredinger et al.,
1998
). By comparing the data from those studies with our results,
which show that the MEK inhibitor prevents the acquisition of a
myogenic-related morphology, we suggest that a critical level of activated
ERKs, which probably affects cytoskeleton or sarcomeric organization, may be
responsible for the morphological phenotype of RD cells. Indeed, the recovery
of ERK phosphorylation, during U0126 treatments, may reflect a requirement of
the ERK pathway during the late myogenic process. However, we cannot rule out
the possibility that the acquisition of a myogenic-related morphology requires
functional interaction between the three MAPKs.
Surprisingly, myosin expression is detected in MEK-inhibitor-treated cells
in spite of their round morphology. The finding that myogenic differentiation
of RD cells occurs both after TPA-mediated ERK and JNK activation and after
their downregulation, by the MEK inhibitor, seems contradictory. It is
notworthy that both treatments induce an increase in p38 activation.
Similarly, p38 activation, following ERK downregulation by the MEK inhibitor
PD98059, has already been reported in non-myogenic cell lines, but has been
found to be associated with apoptosis
(Berra et al., 1998).
Furthermore, growth arrest, an obligatory step for differentiation as well as
for the myogenic process, is dramatically impaired in RD cells, probably
because of the predominance of ERK-mediated mitogenic signals. Thus, an
explanation for our contradictory data comes from the finding that, in RD
cells, the attainment of the myogenic process can occur either when the ERK
pathway is abrogated or when it is antagonised by JNKs. Growth arrest is
necessary but not sufficient to induce the myogenic phenotype, although both
these events can be dissociated. In fact, the p38 inhibitor SB203580 prevents
myosin accumulation in both growth arrested TPA- and U0126-treated cells.
Moreover, this finding clearly demonstrates that p38 is responsible for
promoting the myogenic program, which is in agreement with data in the
literature, thereby showing that p38 mediates myogenic-specific gene
expression (Wu et al.,
2000a
).
Interestingly, no myosin expression is found in cells treated with TPA and
anisomycin, the latter inducing long-lived p38 and JNK activation. The failure
of TPA to induce myogenic differentiation in RD cells in the presence of
anisomycin may be because of persistent JNK activation, which can inhibit the
p38-mediated myogenic pathway (Fig.
11). JNKs activate c-Jun, which may antagonise MyoD function by
impairing myogenic differentiation (Bengal
et al., 1992). In addition, a role for JNKs has recently been
proposed in the loss of Myf5 nuclear localization, an event which occurs early
in the myogenic process (Meriane et al.,
2000
). It is noteworthy that during anisomycin removal, cells
progressively re-acquire the ability to activate the TPA-mediated myogenic
program, which suggests that the removal of the JNK agonist permits
myogenic-specific gene expression. In addition, RD cells pretreated with a
high anisomycin concentration (50 ng/ml) synthesized, during chase, more
myosin that cells treated with lower doses. This result supports the finding
that growth inhibition, caused by persistently active JNK, although inhibitory
for myogenic-specific gene expression, does not irreversibly impair the
myogenic process but allows RD cells to be more responsive to differentiation
signals induced by TPA. Moreover, since transient JNK activation, following
TPA treatment, does not impair myosin expression, it could be postulated that
transient activation of JNKs does not interfere with the p38-induced myogenic
program whereas persistent activation does. The pathway that induces myogenic
markers in RD cells might, therefore, be dependent on a balance between JNK
and p38 activities. In agreement with this, the U0126-induced myosin
expression is also drastically inhibited by anisomycin treatment, which
persistently re-activates JNKs even after long incubation times, thus
supporting the inhibitory role of JNK on the myogenic gene expression
program.
The authors of one recent noteworthy paper found that p38 plays a role in
both growth arrest and myogenic phenotype expression in RD cell lines stably
transfected with the p38 upstream kinase MKK6
(Puri et al., 2000). The
partial discrepancy between those results and ours, concerning the role of p38
in growth arrest, may depend on the different experimental approaches used to
induce myogenic differentiation in RD cells. The forced expression of a
constitutively active isoform of MKK6 kinase, when used to study downstream
kinase activation, may impair the temporal sequence of responses related to
kinase modulatory events. By contrast, in this study, treatment with agonists
that activate endogenous kinase cascades expanded the temporal scale of the
differentiation process, thereby allowing a more detailed characterization of
events.
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interestingly, here we demonstrate that the coordinated activation of ERKs,
JNKs and p38, all playing distinct roles in growth arrest and expression of
myogenic-specific markers in RD cells, is controlled by activated PKC.
Knowledge of pathways that induce growth arrest in RD cells provides important
information concerning the role of PKC
in the balance between
proliferation and differentiation in the pathological myogenic process.
Moreover, our data point both to a crucial role for MAPK activation length and
to a drastic change in the scenario of activated kinases that induce RD cells
to attain the growth arrest state alone or to move on towards myogenic
differentiation. We believe that it may be possible to exploit these results
for future studies of novel therapeutic approaches.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
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References |
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