First published online 27 May 2003
doi: 10.1242/jcs.00525
Inhibition of p38 MAPK signaling promotes late stages of myogenesis
Andrea D. Weston1,*,
Arthur V. Sampaio1,
Alan G. Ridgeway2,3 and
T. Michael Underhill1,
1 Department of Physiology, Faculty of Medicine and Dentistry, The University of
Western Ontario, London, Ontario, N6A 5C1, Canada
2 Department of Biochemistry, Faculty of Medicine and Dentistry, The University
of Western Ontario, London, Ontario, N6A 5C1, Canada
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
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Fig. 1. Effects of p38 inhibition on chondrogenesis and myogenesis in PLM cultures.
Primary limb mesenchymal cells were plated at high density, and cultures were
fixed for detection of cartilage and muscle. Cartilage was detected by alcian
blue staining, and muscle cells were identified by immunofluorescence with a
mouse MyHC antibody. In untreated cultures, cartilage nodule formation
increases over time, whereas the number of MyHC-positive cells declines to
only a few by day 10 (A). Exposure of limb mesenchymal cultures to SB202190
(10 µm) (A) inhibits cartilage nodule formation, whereas there is a
substantial increase in the presence of MyHC-positive cells in comparison to
control cultures. At later stages the SB202190-treated cultures exhibit
extensive myocyte fusion accompanied by the organization of the myocytes into
parallel arrays (B). Bars, (A) bright field, 1.0 mm; fluorescence images, 500
µm; (B) 65 µm.
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Fig. 2. Myogenic effects of p38 inhibition rely on factors present in primary limb
mesenchymal cultures. G8 myoblasts (A-D) and C2C12 myocytes (E-H) were tagged
using the pMSV-tk-ßgeo retrovirus and were monitored by staining with
X-gal or magentagal. When SB202190 is added for 4 days, there is no noticeable
change in cell appearance, number or organization of either the G8 cells
(compare A with B) or the C2C12 cells (compare E with F). To analyze the
effects of SB202190 on the muscle cells in the primary limb mesenchymal
cultures, cells were mixed at a ratio of 19:1, primary cells:G8 cells (C,D) or
primary cells:C2C12 cells (G,H), and the tagged muscle cells were identified
by magenta-gal staining after 12 days. When mixed into the primary cultures,
both tagged cell populations become highly organized, resembling MyHC-positive
cells from primary cultures that were treated with SB202190 (compare to
Fig. 1). In contrast, in the
untreated cultures, G8 and C2C12 cells appear to lose their muscle phenotype,
becoming less bipolar and more round in appearance. Bars, (A,B) 125 µm;
(C-D) 500 µm; (E-H) 250 µm.
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Fig. 3. Attenuation of p38 MAPK promotes expression of the myogenic phenotype. The
normal increase in Col2a1 expression over time in primary cultures is
blocked by SB202190, whereas myogenin, present at low levels early on,
decreases in expression in the untreated cultures while continuing to increase
in response to SB202190 (A). Mef2c is also more abundant in
SB202190-treated cultures by day 3 compared with the untreated control (A).
SB202190 causes a concentration-dependent decrease in the activity of a
pGL3(4X48) reporter gene (B), whereas the activity of a transfected
E-box-luciferase (pGL3-E4-Luc) reporter was increased in response to
increasing concentrations of SB202190 (C). A modest increase was also observed
in the activities of reporters containing the myogenin promoter
(pGL3-myogenin-Luc) and the cardiac actin promoter (pGL3-c-actin-Luc) (C).
When co-transfected with GAL4-MEF2A or GAL4-MEF2C, the activity of a GAL4
reporter (pG5-Luc) was enhanced by SB202190 (D). [ANOVAs (C-E)
P<0.0001; Bonferroni post-tests, (C-E): #P<0.05,
*P<.01, **P<0.001)].
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Fig. 5. Inhibition of p38 promotes myogenesis of pre-existing myoblasts. Cultures
were established from distal limb mesenchyme (A-F) or proximal limb mesenchyme
(G-L). Proximal cultures contain more MyHC-positive cells compared to distal
cultures (compare G with A). After 6 days, there are fewer detectable muscle
cells in untreated distal cultures (B) compared with proximal cultures (H),
but many more cartilage nodules form from distal mesenchyme (E) than from
proximal mesenchyme (K). Following 6 days of treatment with SB202190, in both
distal and proximal cultures, there is a decrease in nodule formation and an
increase in the formation of foci of MyHC-positive cells. The muscle cells in
treated proximal cultures (I), however, are much more prevalent, stain more
intensely and are more highly organized into parallel arrays compared with
muscle cells of distal cultures (C), and there are more cartilage nodules in
the distal cultures (F) compared with the proximal cultures (L). Bars,
fluorescence images (A-C, G-I) 750 µm; bright-field images (D-F, J-L) 1.5
mm.
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Fig. 6. Inhibition of p38 signaling rapidly alters morphology of myoblasts.
SB202190-treated and untreated PLM cultures were fixed and analyzed for MyHc
expression at various times over 24 hours. In control cultures, at all times
examined, MyHc-positive cells are only slightly bipolar, with small cellular
extensions, are randomly oriented and are distributed throughout the culture.
Within only a few hours of SB202190 addition, these cells acquire an enhanced
bipolarity, with the bipolar cellular extensions oriented in the same
direction. These bipolar cells are also in very close proximity to each other.
As early as 6 hours after treatment, this polarization and aggregation is
pronounced, as indicated by the presence of foci of myoblasts that become much
more discernible by 24 hours. Bar, top eight panels, 125 µm; bottom panel,
500 µm.
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© The Company of Biologists Ltd 2003
, as determined by western analysis using a pan-p38
and p38
42 kDa, consistent with the reported size of p38 isoforms, was
observed with both antibodies. The expression of p38 in limb mesenchymal
cultures does not noticeably change upon treatment with 10 µM SB202190. To
control for loading all blots were subsequently incubated with an antibody
against ß-actin. SB202190 effectively inhibits p38 activity in limb
mesenchymal cultures (B) and G8 and C2C12 cells (C). Activity of a GAL4-CHOP
fusion protein, measured by pG5-luc activity, is induced by co-transfection
with MKK6E and p38