doi: 10.1242/10.1242/jcs.00037
PKC
-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells
Annunziata Mauro*,1,
Carmela Ciccarelli*,1,
Paola De Cesaris1,
Arianna Scoglio2,
Marina Bouché2,
Mario Molinaro2,
Angelo Aquino3 and
Bianca Maria Zani1,
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
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Fig. 1. TPA induces phosphorylation/activation of ERKs, JNKs and p38. The time
course of ERK, JNK and p38 phosphorylations in RD cells, either untreated (C0,
C2d and C5d) or treated with 10-7 M TPA for different times (30
minutes to 5 days). Immunoblots of total lysate were performed using
antibodies against phospho-active forms of MAPKs. Each blot was re-probed with
antibodies that recognize total proteins. 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 arbitrarily set at 1. The data shown
are representative of three independent experiments.
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Fig. 3. The PKC inhibitor abrogates MAPK activation, growth arrest and myosin
expression. (A) Immunoblots of total lysates from RD cells, either untreated
(C) or treated with TPA for 30 minutes, pre-treated (6 hours) with 60 nM PKC
inhibitor Ro320432 (Ro) in the presence and absence of TPA, using the
antibodies described in the legend for Fig.
1. (B) Growth curve of RD cells either untreated (C) or treated
with TPA for different times (0, 1, 3 and 6 days, TPA) and after pre-treatment
with the PKC inhibitor (Ro, Ro + TPA). Each value represents the
mean±s.e.m. of three samples. (C) Immunoblots of total lysate from
cells, either untreated (C) or treated with TPA for 6 days (TPA) and
pre-treated with Ro320432, in the absence or presence of TPA, (Ro, Ro + TPA)
using anti-myosin heavy chain (MHC) antibody. The data shown are
representative of three independent experiments.
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Fig. 4. Effect of U0126 on ERK, JNK and p38 phosphorylations. (A) Immunoblots of
total lysates from untreated cells (C), cells treated with TPA (TPA) and 10
µM U0126 in the absence (U) or presence of TPA (U + TPA) for 30 minutes, 2
and 5 days, using anti-phospho-active ERK and JNK antibodies. For
normalization, filters were re-probed with antibodies that recognize total
proteins. (B) Immunoblots of total lysate from cells incubated for 30 minutes
with control medium (C30 min, C5d) or with U0126-containing conditioned medium
derived from cells treated with U0126 for 30 minutes (U 30 min) and 5 days (U
5d), using anti-phospho-active ERK antibody. The data shown are representative
of four independent experiments.
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Fig. 5. Effects of U0126 on the morphology of RD cells. Phase contrast morphology
of RD cells either untreated (C) or treated with TPA for 6 days in the absence
(TPA) and in the presence of U0126 (U + TPA).
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Fig. 6. MEK2-dependent JNK activation. (A) Immunoblots of RD cells transfected with
the constitutively active form of HA-tagged MEK2 (CA-MEK2) or with the empty
vector (CMV) using antibodies that recognize phospho-active ERKs and
hemagglutinin (HA). (B) A luciferase assay for detection of activated JNKs
(see Materials and Methods); luciferase activity (units/plates) was assayed in
total lysates from RD cells co-transfected with a constitutively active form
of MEK2 (CA-MEK2) or with empty vector (CMV) and both with activator plasmid
GAL4-Jun and reporter plasmid GAL4-luc. The data shown are representative of
two independent experiments.
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Fig. 7. Effects of anisomycin on JNK and p38 phosphorylations. (A) Immunoblots of
total lysates of control (C) and TPA-treated cells both in the absence (TPA)
and in the presence of 10 ng/ml anisomycin (AN, AN + TPA) for 30 minutes and 5
days using antibodies specific for phospho-active JNKs and p38. For
normalization, filters were re-probed with antibodies that recognize total
protein. (B) Immunoblots, using anti-sarcomeric MHC antibody, of total cells
lysates from RD cells untreated (C) or treated with different doses of
anisomycin (5, 10, 50 ng/ml, AN) for 3 days and chased for a further 3 days in
the presence and in the absence of TPA. The data shown are representative of
three different experiments.
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Fig. 8. Effects of SB203580, U0126 and anisomycin on growth potential. Growth
curves of RD cells untreated (C) or pre-treated for 1 hour with SB203580 (SB)
and (A) treated with anisomycin (AN), (B) treated with TPA or (C) with U0126
for 2, 4 and 6 days. Each value represents the mean±s.e.m. of three
samples. (D) Immunoblots of nuclear extracts from RD cells, either untreated
(C) or treated with TPA, anisomycin and U0126 using the anti-PCNA antibody.
The data shown are representative of three independent experiments.
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Fig. 9. Effects of SB203580, U0126 and anisomycin on myosin expression. (A)
Immunoblot, using anti-MHC antibody, of total lysates from RD cells, untreated
(C) or treated with U0126 in the absence (U) or presence of SB203580 (SB+U),
with TPA in the absence (TPA) or in the presence of U0126 (U+TPA), SB203580
(SB+TPA) and anisomycin (AN+TPA) for 6 days. (B) Immunofluorescence microscopy
of cells treated as indicated above. The data shown in A and B are
representative, respectively, of four and two independent experiments.
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Fig. 10. Effect of anisomycin-mediated JNK re-activation on MHC accumulation.
Immunoblots of total lysates from RD cells untreated (C) and treated with
anisomycin (AN), with TPA and U0126 in the absence (TPA, U) and in the
presence of anisomycin (AN + TPA, AN + U) for 30 minutes, 3 and 5 days.
Filters were probed with antibodies recognizing phospho-active JNKs and MHC.
The data are representative of three independent experiments.
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Fig. 11. A model of PKC -mediated signal transduction pathways in
differentiating RD cells. A schematic presentation of PKC-mediated
MAPKs cascades induced by TPA and the proposed roles of ERKs, JNKs and p38 in
the regulation of growth arrest and myogenic differentiation in RD cells
(solid arrows). The MAPK pathways targeted by the agonist, as well as
inhibitors and a negative regulator of JNK's effect on p38, are indicated by
dashed and dotted lines. PKC-mediated MAPK activation induces growth
arrest and myogenic differentiation. When ERK and JNK are downregulated
(encircled), by U0126, growth arrest and p38-mediated myogenic differentiation
occur. Inhibition of the p38 pathway, by SB203580, prevents myogenic
differentiation. Activation of both p38 and JNKs, by anisomycin, induces
growth arrest but prevents myogenic process owing to a persistent and highly
activated JNK, which negatively regulates the p38 pathway.
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© The Company of Biologists Ltd 2002
. The filter was normalized with an antibody specific to p54/JNK2.
The data shown are representative of four independent experiments. The
increases (fold over the CMV-transfected cells) in phosphorylation levels are
indicated for each sample. (C) Luciferase assay for detection of activated
c-Jun and Elk1 (see Materials and Methods). RD cells cotransfected with
activator plasmid GAL4-jun or GAL4-Elk1 and reporter plasmid GAL4-luc together
with empty vector (CMV), WT PKC