 |
INTRODUCTION |
The capacity of skeletal muscle for self-repair is due to a rare
population of myogenic progenitor cells (previously termed muscle
satellite cells) that reside at the periphery of adult muscle fibers
(1, 2). Previous work has demonstrated the plasticity or multipotential
capacity of these adult progenitor cells (3), but the molecular events
that regulate their quiescence, proliferation, and differentiation
during cellular growth and regeneration remain poorly defined.
The forkhead/winged helix (Fox) transcription factor family
is characterized by a 100-amino acid winged helix DNA binding domain
and has been shown to play essential roles during embryogenesis and in
the control of cellular proliferation/determination in adult progenitor
cell populations (4-13). We have identified a novel member of the
forkhead/winged helix transcription factor family, termed
Foxk1 (previously referred to as myocyte nuclear factor or MNF) (14,
15). Foxk1 is expressed early, selectively, and transiently in the
developing neural tube, heart, and somites of the mouse embryo.
However, during the latter stages of embryogenesis and in the adult,
Foxk1 is restricted to a subpopulation of mononuclear cells within the
skeletal muscle, which were identified as myogenic satellite
(progenitor) cells (14). Importantly, Foxk1 was the first selective
molecular marker of the quiescent myogenic progenitor cell population.
Furthermore, we have demonstrated that mice lacking Foxk1 (Foxk1
/
)
exhibit a growth deficiency and a severe impairment in skeletal muscle
regeneration following injury.
The mechanistic defect associated with the impaired skeletal muscle
regeneration in Foxk1 mutant mice is the focus of the current work.
Overall, it was hypothesized that dysregulation, or loss, of Foxk1
attenuates skeletal muscle growth and regeneration due to an impairment
of the cell cycle regulatory genes within the myogenic progenitor cell
population. The data presented here establish Foxk1 as an important
regulator of the myogenic progenitor cell population and provide
evidence supporting the hypothesis that the
cyclin-dependent kinase inhibitor, p21CIP, is a
downstream target of Foxk1.
 |
EXPERIMENTAL PROCEDURES |
Mice--
Foxk1
/
(15), C57BL/6 wild type
(WT),1 and
p21CIP
/
mice (Jackson Laboratory, Bar Harbor, ME) were
used in these studies. Combinatorial mating of Foxk1
/
and
p21CIP
/
mice was undertaken to generate mice lacking
both Foxk1 and p21CIP. Genotyping was performed by PCR
analysis of genomic DNA using the following primer pairs: Foxk1 wild
type allele, For 5'-CAAAGTCCCTCGGTCCCAGGAGG-3' and Rev
5'-GCGGAAGCAGGAGACACCTCTCTG-3'; Foxk1 mutant allele, For 5'-CCTCCGAGCAGCAAGCAGATGCG-3' and Rev 5'-GCTTCTGAGGAGAGAACTGGCTGAG-3'; p21CIP wild type allele, For 5'-GGCTGAACTCAACACCCACCT-3'
and Rev 5'-GAGACAACGGCACACTTTGCTC-3'; p21CIP mutant allele,
For 5'-GGCTGAACTCAACACCCACCT-3' and Rev
5'-GCTATCAGGACATAGCGTTGGC-3'.
Thermocycler conditions for p21CIP are: step 1, 94 °C
for 4 min; step 2, 94 °C for 1 min; step 3, 62 °C for 1 min; step
4, 72 °C for 1.5 min; 5) 72 °C for 3 min. Steps 2-4 are repeated
for 40 cycles. Thermocycler conditions for Foxk1 are: step 1) 94 °C for 2 min; step 2, 94 °C for 15 s; step 3, 62 °C for 15 s; step 4, 72 °C for 15 s; step 5, 72 °C for 3 min. Steps
2-4 are repeated for 30 cycles.
Primary Myogenic Progenitor Cell Culture--
Asynchronously
dividing primary myogenic progenitor cell cultures were harvested from
hindlimb skeletal muscle of neonatal (2-day-old) mice (16). Cells were
preplated and grown on collagen coated plates in F-10 growth
medium (20% fetal bovine serum, 0.5% penicillin/streptomyocin, 25 ng/ml basic fibroblast growth
factor). In selected experiments, IGF-I (50 ng/ml) or WT
conditioned medium was added to the growth medium of WT
and Foxk1
/
cells and further cultured for 4 days.
Single Skeletal Muscle Fiber Isolation--
Single skeletal
muscle fibers were harvested from the fast twitch extensor digitorum
longus muscles of 2-4-month-old mice (17, 18). After 72 h in
culture, the number of myogenic progenitor cells that had migrated
from the fiber were quantified.
Electron Microscopy for Myogenic Progenitor Cell Identification
and Quantitation--
Fast twitch tibialis anterior muscles from
2-4-month-old WT, Foxk1
/
and Foxk1
/
:p21CIP
/
mice were harvested following perfusion fixation with 3%
gluteraldehyde. Samples were post-fixed with buffered 1% osmium
tetroxide, dehydrated with ethanol, embedded in Spurr resin, and
polymerized overnight at 60 °C. Sections (80 nm) were suspended on
200-mesh copper grids, stained with uranyl acetate and lead citrate,
and examined using a JEOL 1200EXII transmission electron microscope.
Myogenic progenitor cells and myonuclei were identified and quantified
according to criteria described previously (2).
Real-time RT-PCR, Semiquantitative RT-PCR, and Western
Analyses--
Real-time PCR was performed using 2 µl of cDNA and
the Quantitect SYBR Green PCR Master Mix (Qiagen, Valencia, CA) on a
Bio-Rad iCycler. Semiquantitative RT-PCR (using 1:10 and 1:50 dilutions of cDNA) was also performed under conditions in which the abundance of each amplified cDNA varied linearly with input RNA to confirm the results of Real-time PCR. Semiquantitative RT-PCR was performed using cDNA (1 µl) as a template for the PCR reaction in a 25-µl reaction volume including 40 ng of each primer, 1.5 mM
MgCl2, 0.2 mM dNTPs, 2.5 µl, 10×
Taq buffer, and 2.5 units of Taq polymerase (Invitrogen). Thermocycler conditions were: step 1) 96 °C for 2 min;
step 2, 96 °C for 15 s; step 3, 62 °C for 30 s; step 4, 72 °C for 30 s; step 5, 72 °C for 10 min. Steps 2-4 were
repeated for 30 cycles. Primers for real-time and semiquantitative
RT-PCR were: p27, For 5'-TTGGGTCTCAGGCAAACTCT-3'; p27, Rev
5'-TCTGTTCTGTTGGCCCTTTT-3'; p53, For 5'-AAGTCCTTTGCCCTGAACTG-3'; p53,
Rev 5'-CTGTAGCATGGGCATCCTTT-3'; p21, For 5'-TTGCACTCTGGTGTCTGAGC-3';
p21, Rev 5'-CTGCGCTTGGAGTGATAGAA-3'; cyclin B1, For
5'-AACCTGAGCCTGAACCTGAA-3', cyclin B1, Rev 5'-GCGTCTACGTCACTCACTGC-3'; cyclin D1, For 5'-CGGATGAGAACAAGCAGACC-3'; cyclin D1,
Rev5'-GCAGGAGAGGAAGTTGTTGG-3'; p57, For 5'-AGGAGCAGGACGAGAATCAAG-3';
p57, Rev 5'-ACATGAACGAAAGGTCCCAG-3'; cdk1, For
5'-GGTCCGTCGTAACCTGTTGA-3'; cdk1, Rev 5'-CTCCTTCTTCCTCGCTTTCA-3'; glyceraldehyde-3-phosphate dehydrogenase, For
5'-GTGGCAAAGTGGAGATTGTTGCC-3'; glyceraldehyde-3-phosphate
dehydrogenase, Rev 5'-GATGATGACCCGTTTGGCTCC-3'. Western analysis
of protein extracts isolated from wild type or Foxk1
/
progenitor
cell cultures was performed as described previously using rabbit
anti-p21 or rabbit anti-p53 sera (1:1000 dilution, Santa Cruz
Biotechnologies) (15).
Fluorescence-activated Cell Sorting (FACS) and BrdUrd
Incorporation--
Primary myogenic progenitor cell cultures
from WT, Foxk1
/
, p21CIP
/
, and
Foxk1
/
:p21CIP
/
skeletal muscle were fixed in 4%
paraformaldehyde, permeabilized with 0.3% Triton X-100, and incubated
with a propidium iodide staining solution (1.8 mg/ml RNase A, 50 µg/ml propidium iodide) for ~3 h to label DNA. Using a FACScan
(Becton Dickinson, Franklin Lakes, NJ) flow cytometer, 10,000+ cells
were sorted based on DNA content. The data were processed and the
percentage of cells in each phase of the cell cycle quantified using
Cellquest Software (BD Biosciences).
To assess cellular proliferation, primary myogenic progenitor cells
grown on collagen coated coverslips were incubated for 7 h with 10 µM BrdUrd. Following incubation, cells were fixed for 5 min with 4% paraformaldehyde and stained with anti-BrdUrd monoclonal
serum (as per the supplier's instructions, Roche Molecular Biochemicals). Cells were co-stained with propidium iodide (50 ng/ml)
to label all nuclei. Cellular proliferation was quantified as a
percentage of BrdUrd-positive (proliferating cells) to propidium iodide-positive nuclei (total cells).
Cardiotoxin Injury of Mouse Skeletal Muscle--
Gastrocnemius
muscles from WT, Foxk1
/
, and Foxk1
/
:p21CIP
/
mice (2-month-old) were injected with cardiotoxin (150 µl/muscle of
10 µM cardiotoxin (Naja nigricollis;
Calbiochem)) (15). Muscles were harvested 10 days post-injection and
fixed overnight with 4% paraformaldehyde. Paraffin-embedded sections
were stained with hemotoxylin/eosin to assess the skeletal muscle fiber architecture.
Data Analysis--
Student's t tests were performed
to test for significant differences in data obtained from WT
and Foxk1
/
or Foxk1
/
:p21CIP
/
mice. Data are
presented as mean ± S.E. unless otherwise noted.
 |
RESULTS |
Reduced Myogenic Progenitor Cell Population in the Absence of
Foxk1--
The impaired skeletal muscle regeneration observed in
Foxk1
/
skeletal muscle directed the initial analysis to examine the resident myogenic progenitor cell population in WT and Foxk1 mutant mice. Ultrastructural analysis revealed that, although Foxk1
/
skeletal muscle possesses a resident myogenic progenitor cell (satellite cell) population (Fig.
1a), the quantity of myogenic progenitor cells is significantly decreased by 71% compared with age/sex-matched WT mice (2.86 ± 0.05% in WT versus
0.84 ± 0.05% in Foxk1
/
; n = 3 for each
group; p < 0.05; Fig. 1c). Single fiber
myogenic progenitor cell quantitation also demonstrated a 73%
reduction in the number of myogenic progenitor cells migrating from
Foxk1
/
fibers compared with gender and size-matched WT fibers
following 72 h in culture (104 ± 10 in WT (n = 19) versus 29 ± 6 in Foxk1
/
(n = 29); p < 0.05; Fig. 1, b and c).
These results support the hypothesis that Foxk1 is an important
regulatory factor of the myogenic progenitor cell population.

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Fig. 1.
Myogenic progenitor cells isolated from
Foxk1 / mice are reduced
in number and have a cell autonomous G0/G1
arrest. a, electron microscopic images of representative WT
and Foxk1 / myogenic progenitor cells (muscle satellite cells) with
overlaying basal lamina (arrowheads) and separation between
the myofiber and the myogenic progenitor cell plasmalemma
(arrows). S, satellite cells or myogenic
progenitor cells. Bar = 500 nm. b, light
microscopic images of isolated skeletal muscle fibers following 72 h in culture. Arrowheads denote myogenic progenitor cells
that have migrated from the myofiber. Significantly more myogenic
progenitor cells have migrated from the WT myofiber than from the
Foxk1 / myofiber. The inset of fibers at a higher
magnification illustrates the reduced myogenic progenitor cell number
present on the Foxk1 / compared with WT myofiber. Arrows
denote myogenic progenitor cells (satellite cells) on the adult
myofiber. c, quantitative analysis demonstrating the reduced
myogenic progenitor cell number in the absence of Foxk1 using electron
microscopy and isolated single fiber methods. The asterisk
denotes significance (p < 0.05) compared with WT using
the same technique for quantitation. Electron microscopy (WT:
2.86 ± 0.05% versus Foxk1 / : 0.84 ± 0.05%;
n = 6 muscles with >500 myonuclei counted for WT
and Foxk1 / ) and single fiber (WT: n =
4 mice, total 29 fibers; Foxk1 / : n = 3 mice,
total 19 fibers) quantitation reveals significant differences between
WT and Foxk1 / . d, FACS analysis of asynchronously
dividing WT (n = 9) and Foxk1 / (n
= 3) myogenic progenitor cells demonstrates that myogenic
progenitor cells lacking Foxk1 / have significantly more cells in
the G0/G1 phase than WT myogenic progenitor
cells. Exposure to WT conditioned medium (CM;
n = 3) or supplementation with IGF-I (50 ng/ml;
n = 5) does not effect the
G0/G1 arrest in the FoxK1 / myogenic
progenitor cells, supporting the conclusion that the perturbed cell
cycle progression is a cell autonomous defect. The asterisk
denotes significant changes (p < 0.05) compared with
the WT controls.
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|
Cell Cycle Progression in the Foxk1
/
Myogenic Progenitor Cell
Is Impaired--
To examine the mechanism associated with the reduced
myogenic progenitor cell number in the absence of Foxk1, we compared the cell cycle profile of WT and Foxk1
/
myogenic progenitor cells
using FACS analysis. Loss of Foxk1 disrupts cell cycle progression, causing an increase in G0/G1 cells and a
concomitant decrease in S and G2/M cells as indicated by
the DNA content of the cells (Fig. 1d). Furthermore, the
proliferative capacity of the Foxk1
/
myogenic progenitor cell
population, as determined by BrdUrd incorporation, was decreased by
~50% compared with the WT myogenic progenitor cell cultures (43 ± 0.5% in the WT, n = 6 versus 22 ± 0.5% in the Foxk1
/
, n = 8; p < 0.05). These results establish that Foxk1 is involved in the cell cycle
process, since myogenic progenitor cells lacking Foxk1 show perturbed
cell cycle progression and decreased proliferative capacity.
The G0/G1 Arrest Observed in Foxk1
/
Myogenic Progenitor Cells Is a Cell Autonomous Defect--
To
ascertain whether the impaired cell cycle progression of the Foxk1
/
myogenic progenitor cells is the result of intrinsic or extrinsic
factors, Foxk1
/
myogenic progenitor cells were exposed to WT
conditioned medium or IGF-I. IGF-I was chosen as it has been
shown to modulate the transcriptional activity and cell cycle control
of other forkhead/winged helix transcription factors and
influence the G1/S cell cycle progression (19). We
hypothesized that if the cell cycle defect in the absence of Foxk1 was
the result of impaired extrinsic cues, then exposure of Foxk1
/
myogenic progenitor cells to WT conditioned medium or IGF-I
should rescue the cell cycle perturbation (i.e.
G0/G1 arrest). Neither WT conditioned
medium (n = 3) nor IGF-I (n = 5)
was capable of rescuing the G0/G1 arrest
observed in the Foxk1
/
myogenic progenitor cells (Fig.
1d), supporting the hypothesis that the cell cycle
perturbation in Foxk1
/
myogenic progenitor cells is the result
of intrinsic rather than extrinsic defects or cues.
Dysregulation of Cyclin-dependent Kinase Inhibitor,
p21CIP, Is Observed in the Foxk1
/
Myogenic Progenitor
Cell--
As these data supported a cell autonomous perturbation of
cell cycle progression in the Foxk1
/
myogenic progenitor cell population, we examined the transcriptional profile of cell cycle regulatory genes to identify putative candidates responsible for the
G0/G1 arrest. Real-time RT-PCR,
semiquantitative RT-PCR, and Western analyses revealed a significant
up-regulation of the cyclin-dependent kinase inhibitor,
p21CIP, independent of changes in other
cyclin-dependent kinase inhibitors (p53, p27, or p57 (Fig.
2, a-c)). A significant
decrease in cdk1 in the absence of Foxk1 was also observed. The
decrease in cdk1 expression is consistent with the hypothesis that
Foxk1
/
myogenic progenitor cells are arrested at the
G0/G1 phase. Collectively, these data support
the hypothesis that Foxk1 regulates cell cycle progression and the
cyclin-dependent kinase inhibitor, p21CIP, in
the myogenic progenitor cells.

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Fig. 2.
Foxk1 / myogenic
progenitor cells have increased expression of the
cyclin-dependent kinase inhibitor, p21CIP.
Real-time (a) and semiquantitative (b) PCR
analyses reveal a significant increase in p21CIP gene
expression in Foxk1 / myogenic progenitor cells that is independent
of changes in other cyclin-dependent kinase inhibitors,
including p53. Expression of cdk1 is down-regulated in the Foxk1 /
myogenic progenitor cells consistent with the
G0/G1 arrest in Foxk1 / myogenic progenitor
cells. The asterisk denotes significance (p < 0.05) compared with WT. c, Western analysis reveals
increased p21CIP protein expression in the Foxk1 mutant
myogenic progenitor cells compared with the wild type control. Note
that no changes in p53 protein expression are observed in the Foxk1 and
WT myogenic progenitor cells.
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|
Abolition of p21CIP Rescues the Impaired Myogenic
Progenitor Cell Number and Cell Cycle Perturbation of the Foxk1
/
Myogenic Progenitor Cells--
We hypothesized that the up-regulation
of p21CIP expression was responsible for the observed
G0/G1 arrest in the Foxk1
/
myogenic progenitor cells. The G0/G1 arrest would
ultimately result in the growth retardation of Foxk1
/
mice, since
myogenic progenitor cells are required to maintain muscle mass, even in
the absence of overt muscle injury. We therefore undertook a
combinatorial mating of the Foxk1
/
with
p21CIP-deficient mice to generate progeny lacking both
Foxk1 and p21CIP (Foxk1
/
:p21CIP
/
) and
determine whether we could rescue the defects observed in the Foxk1
mutant myogenic progenitor cells.
Quantitation of myogenic progenitor cell number using electron
microscopy revealed that mice lacking both Foxk1 and p21CIP
had a normal compliment of myogenic progenitor cells, restoring the
decreased number of myogenic progenitor cells in the Foxk1
/
skeletal muscle (2.76 ± 0.9% in
Foxk1
/
:p21CIP
/
versus 0.84 + 0.05% in
Foxk1
/
). The rescued myogenic progenitor cell number in the
Foxk1
/
:p21CIP
/
skeletal muscle was further
corroborated using single fiber myogenic progenitor cell quantitation
(Fig. 3a).

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Fig. 3.
Myogenic progenitor cell number and cell
cycle progression are rescued in
Foxk1 / :p21CIP /
mice. a, single fiber myogenic progenitor
cell quantitation shows a 73% reduction in myogenic progenitor cell
number in the absence of Foxk1 compared with WT values. Fibers lacking
both Foxk1 and p21CIP show restoration of myogenic
progenitor cell number (WT: 104 ± 10, n = 19;
Foxk1 / : 29 ± 6, n = 29;
Foxk1 / :p21CIP / : 109 ± 16, n
= 14 myogenic progenitor cells per fiber). The
asterisk denotes significant changes (p < 0.05) compared with the wild type controls. Quantitation of the
myogenic progenitor cells utilizing electron microscopy further
demonstrates a restoration of myogenic progenitor cells in skeletal
muscle lacking both Foxk1 and p21CIP (WT: 2.86 ± 0.05%,
n = 3; Foxk1 / : 0.84 ± 0.05%, n
= 3; Foxk1 / :p21CIP / : 2.76 ± 0.99%,
n = 4). b, FACS analysis of
asynchronously dividing WT, Foxk1 / , p21CIP / , and
Foxk1 / :p21CIP / myogenic progenitor cells
demonstrates that the G0/G1 arrest observed in
Foxk1 / myogenic progenitor cells is restored to WT levels in
myogenic progenitor cells lacking Foxk1 and p21CIP. Loss of
p21CIP alone has no significant effect on cell cycle
progression, consistent with previous studies (20). These results
support the hypothesis that p21CIP is a downstream target
for Foxk1. The asterisk denotes significant changes
(p < 0.05) compared with the wild type controls.
c, BrdUrd incorporation assays to assess proliferative
capacity confirms that the impaired cellular proliferation observed in
the absence of Foxk1 is rescued in the double mutant
(Foxk1 / :p21CIP / ) myogenic progenitor cells. Note
BrdUrd-positive nuclei are green, and all nuclei are stained
with propidium iodide (PI). d, quantitative
analysis of the BrdUrd incorporation assays comparing the number of
BrdUrd-positive nuclei (proliferating myogenic progenitor cells) as a
percent of propidium iodide-positive nuclei (total myogenic progenitor
cells). The percent of BrdUrd-positive myogenic progenitor cells in the
Foxk1 / :p21CIP / (40.6 ± 3.9%, n
= 3) is not significantly different from WT (43.4 ± 0.5%,
n = 6), while both have a significantly greater
percentage of BrdUrd positive nuclei than the Foxk1 / myogenic
progenitor cells (22.4 ± 0.5%, n = 8). The
asterisk denotes significant changes (p < 0.05) compared with the wild type controls.
|
|
FACS analysis of the Foxk1
/
:p21CIP
/
myogenic
progenitor cells demonstrated that the lack of p21CIP
expression in Foxk1
/
myogenic progenitor cells prevents the G0/G1 arrest observed in the Foxk1
/
myogenic progenitor cell alone (Fig. 3b). Cell cycle
analysis of the p21CIP
/
myogenic progenitor cells did
not reveal a significantly enhanced cell cycle progression, which is in
agreement with other published reports (20). An increase in cellular
proliferation of the double mutant myogenic progenitor cell was also
observed using BrdUrd incorporation (Fig. 3, c and
d). The percentage of BrdUrd-positive nuclei in the
Foxk1
/
:p21CIP
/
was significantly greater than that
observed in the Foxk1
/
myogenic progenitor cells (Fig.
3d), reaching values comparable with that observed in WT
myogenic progenitor cells. We conclude that myogenic progenitor cells
lacking Foxk1 have an increase in p21CIP resulting in an
impaired cell cycle progression.
The Growth Retardation and the Impaired Skeletal Muscle
Regeneration Observed in the Foxk1 Null Mice Are Rescued in Mice
Lacking Both Foxk1 and p21CIP--
Foxk1
/
mice are
grossly smaller than gender-matched WT littermates at all ages (15).
The absence of p21CIP in the Foxk1
/
mice resulted in a
rescue of the growth deficit in gender and age-matched mice (Fig.
4a). No significant
differences were noted between the gross size of the adult age and
gender-matched WT, Foxk1
/
:p21CIP
/
and
p21CIP
/
mice.

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Fig. 4.
Growth and regenerative Foxk1 defects are
rescued in
Foxk1 / :p21CIP /
mice. a,
Foxk1 / :p21CIP / mice are comparable in size and
indistinguishable from WT mice, while Foxk1 / mice remain
significantly growth retarded. b, ten days post-cardiotoxin
injection, WT skeletal muscle architecture is completely restored with
many newly regenerated myofibers visible (denoted by
arrows). Mice lacking Foxk1 still exhibit a hypercellular
response with little evidence of regenerated myofibers. In contrast,
Foxk1 / :p21CIP / skeletal muscle has a complete
restoration of the skeletal muscle architecture, which is
indistinguishable when compared with WT regenerating muscle. Note the
presence of numerous regenerated myofibers (arrows) in WT
and double mutant skeletal muscle.
|
|
The rescue of both the cell cycle defect in the myogenic progenitor
cell population and the growth deficit observed in the Foxk1
/
:p21CIP
/
mice prompted us to examine the
regenerative capacity of skeletal muscle deficient for both Foxk1 and
p21CIP. Ten days following cardiotoxin-induced skeletal
muscle injury in WT mice, skeletal muscle architecture is largely
restored. In contrast, Foxk1
/
mice manifest a myonecrotic response
that persists more than 3 weeks following the injury (15). The
impairment in Foxk1
/
skeletal muscle regeneration is completely
rescued in mice lacking both Foxk1 and p21CIP with
restoration of the skeletal muscle architecture within 10 days of
injury comparable with that observed in WT mice (Fig. 4b).
 |
DISCUSSION |
Elegant studies have challenged previously established paradigms
by demonstrating that adult stem cells have a broad capacity or
plasticity and are capable of contributing to multiple lineages when
placed in a permissive environment (3, 21, 22). In recent years,
cellular augmentation therapy has been pursued using alternative adult
stem cell populations, including myogenic stem (progenitor) cells, to
repopulate the failing heart and myopathic skeletal muscle (23, 24).
The studies utilizing the transfer of myogenic progenitor cells for the
treatment of debilitating myopathies and congestive heart failure have
unfortunately yielded disappointing results. Successful utilization of
these adult stem cell/progenitor cell populations for therapeutic
applications requires an understanding of the molecular regulation and
cell cycle control of these rare and unique cell populations.
Foxk1 Is Expressed Selectively in Myogenic Progenitor
Cells--
Myogenic progenitor cells are arrested at an early stage of
the myogenic program, such that they do not express any of the myogenic
basic helix-loop-helix proteins of the MyoD family (25-28). Although a
number of studies have examined the physiological responses of these
adult muscle progenitor cells to various stimuli, the molecular signals
governing their proliferative and differentiation capacity remain
poorly defined (4, 25, 28-30).
We have previously established that Foxk1 is restricted to the myogenic
progenitor cell population in adult skeletal muscle and is the first
molecular marker for this quiescent cell population (14). In the
present study, we establish that Foxk1 is necessary for the normal
complement of quiescent myogenic progenitor cells that reside in adult
skeletal muscle. In the absence of Foxk1 only a small subpopulation of
myogenic progenitor cells are established, and they have perturbed cell
cycle progression. This defect in myogenic progenitor cell number and
cell cycle progression results in the muscle regeneration deficit
observed in Foxk1-deficient mice. These results demonstrate that Foxk1
plays a critical role during muscle development and during muscle
regeneration in the adult mouse.
Foxk1 Is Important in the Cell Cycle Progression of the Myogenic
Progenitor Cell--
The exquisitely ordered, functional regulation of
the G1-S phase transition of the cell cycle is determined
by a balance between positive and negative regulatory pathways. The
principle control of cell cycle progression is mediated through the
regulation of the activity of cyclin-dependent kinases by
both cyclins and the cyclin-dependent kinase inhibitory
proteins. p21CIP is an important
cyclin-dependent kinase inhibitor that functions, in part,
to regulate the G1-S phase transition of the cell cycle. Expression of the p21CIP gene is induced by a wide range of
cell growth regulatory signals, including p53-dependent
(e.g. DNA damage) and by p53-independent mechanisms during
normal tissue development or cellular differentiation (31, 32).
Transgenic overexpression of p21CIP in the adult murine
hepatocyte was shown to result in a perturbed cell cycle progression
during hepatocyte regeneration, a runted liver, and a significant
growth deficit of the animal (33). Notably, this is a similar phenotype
to that observed in the Foxk1
/
mouse model. Here we present
evidence supporting the hypothesis that in the absence of Foxk1,
p21CIP expression is up-regulated, resulting in a
G0/G1 arrest in the myogenic progenitor cell
population under growth promoting conditions. The conclusion that Foxk1
regulates the myogenic progenitor cell population and modulates cell
cycle progression is further based on the rescue of the growth deficit,
decreased myogenic progenitor cell number, and cell cycle perturbation
in mice that are deficient in both Foxk1 and p21CIP.
Mice that are genetically deficient for p21CIP are viable
but are radiation-sensitive, and their cells display impaired
p53-dependent cell cycle arrest in response to DNA damage
(34). Furthermore, cultures of p21CIP null embryonic
fibroblasts reveal a mild decrease in G1 length with less
than a 5% decrease in G1 cells (20). While no skeletal muscle defects have been previously reported in the absence of a single
cyclin dependent kinase inhibitor, mice lacking both p21CIP
and p57 have severe defects in skeletal myogenesis characterized by the
failure to form differentiated myotubes (32). These results support the
essential role of coordinated cell cycle regulation for myoblast
proliferation and differentiation during myogenesis (32). In the
present study, we hypothesized that the growth retardation in
Foxk1
/
mice reflected a defect in the function of the myogenic
progenitor cell population, since myogenic progenitor cells are
required to maintain muscle mass, even in the absence of overt muscle
injury. In the present study, we provide data to support this
hypothesis as mice lacking both Foxk1 and p21CIP resulted
in a rescue of the growth deficit observed in Foxk1 mutant mice. In
addition, doubly mutant mice have normalized both the myogenic
progenitor cell number and cell cycle progression. The conclusion that
Foxk1 is an important regulator of the myogenic progenitor cell
population is further based on the rescue of chemical induced muscle
injury in Foxk1
/
:p21CIP
/
mice. Cardiotoxin-induced
injury results in an extensive myonecrotic response (>80% of the
muscle is destroyed) followed by a predictable, reproducible, and rapid
repair process within 10 days of the injury (15). The regenerative
response of both WT and Foxk1
/
:p21CIP
/
muscle is
indistinguishable, as normal skeletal muscle architecture was observed
within 10 days of injury. In contrast, the Foxk1 mutant mice have a
severe impairment in muscle regeneration with ineffectual repair even 3 weeks following injury due to the defect in the myogenic progenitor
cell population. These results establish Foxk1 as an important cell
cycle regulator of the myogenic progenitor cell and supports the
hypothesis that p21CIP is a downstream target of Foxk1.
Furthermore, preliminary studies undertaken in our laboratory have
identified a Foxk1 binding motif within one of three evolutionarily
conserved regions of the p21CIP promoter (the other two
regions are p53 binding sites). Additional studies are under way that
will further examine the transcriptional regulation of the
p21CIP gene by Foxk1.
forkhead/winged helix Factors and Stem Cell Biology--
Members
of the forkhead/winged helix family have a signature motif
that encodes a DNA binding domain and function as transcription factors
to exert important regulatory functions during development (i.e. cell specification and lineage segregation) and with
respect to stem cells and/or tissue repair (4-13). For example,
Genesis (Foxd3) is expressed selectively in embryonic stem cells (5), TWH (Foxb1) is a regulator of neural progenitor cells (35), and a
protein related to HNF3 (FOXM1) has been identified in regenerating hepatocytes (36). Other forkhead/winged helix proteins have also been implicated in the regulation of cell cycle progression through interactions with cyclin-dependent kinase
inhibitors. For example, overexpression of AFX (FOXO4) in multiple cell
lines results in a G1 arrest of the cell cycle, which is
dependent on the cyclin-dependent kinase inhibtor p27 (37).
It has also been proposed that the tumor-suppressing role of
PTEN is mediated through antagonization of the action of
phosphatidylinositol 3-kinase/protein kinase B on FKHR
(FOXO1a) and FKHR-L1 (FOXO3a). Both PTEN and activated
FKHR/FKHR-L1 were demonstrated to induce expression of p27 while having
no effect of p21CIP (8). Our current data concerning Foxk1,
however, provide direct evidence for a specific role in the regulation
of the cell cycle for members of this extended gene family in the
regulation of progenitor (or stem) cell function.
In summary, these studies establish a functional role for Foxk1 in the
regulation of the myogenic progenitor cell population and support the
hypothesis that p21CIP is a downsteam target gene. These
studies further provide a mechanistic understanding of the regulation
of the myogenic progenitor cells and will advance the use of cell
transfer technologies for therapeutic applications in the treatment of myopathies.