First published online 28 August 2008
doi: 10.1242/dev.022871
Development 135, 3291-3300 (2008)
Published by The Company of Biologists 2008
Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors
Susana R. Parathath1,*,
Lori Anne Mainwaring1,2,*,
Africa Fernandez-L1,
Dane Ohlosson Campbell1 and
Anna Marie Kenney1,2,3,
1 Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer
Center, New York, NY 10021, USA.
2 Program in Biochemistry, Cell, and Molecular Biology, Weill Cornell Graduate
School, New York, NY 10021, USA.
3 Program in Neurobiology, Weill Cornell Graduate School, New York, NY 10021,
USA.
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Fig. 1. IRS1 protein is upregulated in proliferating CGNPs. (A) CGNP
cultures were prepared from different litters on different dates. Preparations
of the cells were treated with SHH or left untreated for 24 hours prior to
lysis. The autoradiograph depicts a western blot for several downstream
components of IGF signaling pathway and the cell cycle progression marker
cyclin D2. Only IRS1 levels are upregulated in SHH-treated samples, which
correlates with increases in cyclin D2. β-Tubulin demonstrates equal
protein loading. (B) The autoradiographs show western blots for CGNPs
treated with two SHH signaling pathway inhibitors, forskolin (10 µM) or
cyclopamine (1 µg/ml), for increasing time points. In the absence of
continuous SHH signaling, levels of IRS1 decrease, which also correlates with
decreased cyclin D2 expression. (C) SHH-treated CGNPs were fixed and
immunostained for IRS1 (red) and p27 (green). IRS1 and p27 mark different
populations of cells. Confocal imaging (right panel) confirms IRS1 presence in
the nucleus (arrowhead) and cytoplasm (arrow). (D) Left: cerebellar
section from post-natal day 7 mouse immunostained for IRS1 (red) and GFAP
(green). Middle (low power) and right (high power): SHH-treated primary CGNP
cultures immunostained for IRS1 (red) and GFAP (green). IRS1 is excluded from
glia both in vivo and in vitro. (E) In the left (low power) and middle
(high power) panels, PN 7 mice were pulsed with BrdU and stained for BrdU
(green) and IRS1 (red). IRS1 colocalizes to proliferating CGNPs in the EGL. In
the far right panel, IRS1 (red) is expressed in the cytoplasm of BrdU-positive
cells (green) in CGNP cultures.
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Fig. 2. SHH signaling stabilizes IRS1 protein. (A) RNA was collected
from SHH-treated CGNPs and RT-PCR analysis was conducted for IRS1, cyclin D2
and β-actin to verify equal RNA input. (B) qPCR was used to
quantify levels of IRS1 expression. Expression levels of IRS1, depicted as
arbitrary units, do not change in response to SHH treatment. (C) CGNPs
were treated with SHH, cyclopamine or lactacystin (10 µM) for indicated
times. Levels of IRS1 protein are stabilized in the presence of the
lactacystin and cyclopamine. (D) Western blot analysis of IRS1 protein
levels in CGNPs infected with GLI1 retroviruses and subsequently cultured
without SHH for 36 hours. GLI1 infection can sustain proliferation as
indicated by cyclin D2 levels and as previously reported
(Oliver et al., 2003 ). IRS1
was present but at lower levels than in CGNPs infected with GFP-expressing
retroviruses and treated with exogenous SHH, suggesting the existence of
GLI-mediated and non-GLI-mediated mechanisms that promote IRS1 protein
accumulation.
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Fig. 3. SHH signaling inhibits S6 kinase activation and hence IRS1
degradation. (A) Treatment of CGNPs with 1 µg/ml cyclopamine
after increasing periods of time reduced IRS1 protein levels and increased
detectable phosphorylated IRS1. The value under each lane represents the ratio
of phosphorylated IRS1 to IRS1 as measured by densitometry. (B) CGNPs
were treated with SHH and/or 10 nm rapamycin (Rapa) as indicated. The final
lane represents CGNPs from which SHH was removed at the time of rapamycin
addition. Rapamycin in combination with SHH causes an accumulation of IRS1 and
rapamycin prevents reduction in IRS1 when SHH is removed. The value under each
lane represents densitometric measurement of the IRS1 signal using the
vehicle:tubulin value set to 1. (C) Treatment of CGNPs with rapamycin
in the presence of SHH caused an accumulation of IRS1 over time. The value
under each lane represents densitometric measurement of the IRS1 signal using
the vehicle:tubulin value set to 1. (D) SHH, cyclopamine and rapamycin
were given to CGNPs, as indicated above the lanes. Cyclopamine caused
reduction in IRS1 levels, whereas rapamycin caused additional IRS1
accumulation in the presence of SHH. Rapamycin partially rescued the
cyclopamine-mediated reduction of IRS1 in SHH-treated cells. (E)
Treatment with SHH reduces the phosphorylation of S6K and hence its
activation. Inhibition of PP2A with okadaic acid (OA, 100 nM) restores S6K
activation and reduces N-myc as well as cyclin D2 levels in CGNPs. (F)
Treatment with OA restores S6 phosphorylation and reduces IRS1 levels even in
the presence of SHH.
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Fig. 4. IRS1 is necessary to maintain CGNP proliferation. (A) CGNPs
were treated with SHH for 24 hours prior to infection with IRS1 shRNAs for 3
hours at which time the medium was replaced with either SHH treated or
untreated medium. Treatment with shRNAs knocked down levels of IRS1 (red box)
as well as levels of cyclin D2 without affecting the activation of AKT or
levels of IRS2. β-Tubulin confirms equal loading. (B) Infection of
cerebellar slices with lentiviruses targeting IRS1 causes reduced thickness of
the EGL (broken white lines) and reduced BrdU incorporation (red) in the EGL.
Blue represents DAPI counterstaining. (C) Levels of proliferation in
primary CGNP cultures in response to IRS1 shRNA lentivirus infection were
determined by measuring BrdU incorporation after a 2-hour BrdU pulse. Reduced
BrdU incorporation is evident in shRNA plus SHH compared with SHH-treated
CGNPs. BrdU-positive cells were counted and values expressed as percent of
total cells per field. Values are represented as fold BrdU-positive cells over
untreated CGNPs. *P<0.05, n=4. (D)
Levels of BrdU incorporation were assessed as above in Percoll
gradient-purified CGNP cultures. Trends of BrdU incorporation remain the same
as mixed cultures. **P<0.01. (E) Cell survival
was assessed by immunostaining for cleaved caspase 3. Based on quantification
of cleaved caspase 3-positive cells, there was no change in cell survival
regardless of treatment. (F) Cell survival in response to shRNA
treatment remained the same in purified cultures. Cleaved caspase 3-positive
cells were counted and values are expressed as percent of total cells per
field. Error bars represent s.e.m., n=5.
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Fig. 5. IRS1 overexpression maintains CGNP proliferation in the absence of
SHH. CGNPs were treated with SHH for 24 hours prior to infection with
retrovirus expressing IRS1. (A) Western blotting shows that IRS1
overexpression maintains increased levels of cyclin D2 after SHH withdrawal.
(B) RT-PCR analysis shows no effect of IRS1 overexpression on GLI1 or
GLI2 expression, but did show an increase in N-myc expression. (C)
Levels of CGNP proliferation in response to IRS1-expressing retrovirus
infection were assessed with BrdU incorporation. Numbers of BrdU-positive
cells are increased with treatment of SHH, as well as treatment with IRS1
retrovirus. Quantification of BrdU incorporation was performed as described in
Fig. 4D. (D) To assess
effects of virus treatment on cell death CGNPs were stained with antibodies to
activated caspase 3. Levels of cleaved caspase 3 remain the same for all
treatment groups. Cleaved caspase 3-positive cells were counted and values are
expressed as percent of total cells per field. Error bars represent s.e.m.,
n=5. Asterisks indicate statistically significant difference to
untreated CGNPs (P<0.05, n=4).
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Fig. 6. IRS1 is present in SHH-mediated mouse medulloblastoma. (A)
Hematoxylin and Eosin staining of a Neuro-D2-SmoA1 medulloblastoma depicts a
desmoplastic medulloblastoma. (B) Adjacent normal cerebellar areas in
the SmoA1 tumor model show very little IRS1 (red) immunostaining.
(C,D) Increased IRS1 protein can be seen within the tumor at
both 10x (C) and 63x (D) magnification (blue=DAPI). (E)
Western blot analysis for both Ptch1+/- and SmoA1
medulloblastoma models show increased IRS1 expression coinciding with
increased cyclin D2 expression. IGL, internal granule layer; ML, molecular
layer.
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Fig. 7. Model suggesting how SHH mediates IRS1 expression during CGNP
proliferation. In the absence of SHH, IGF signaling sends survival cues
through IRS2 and/or GAB1 and PI-3K signaling (left panel). IRS1 mRNA is
present. In the presence of SHH, IRS1 is upregulated by a mechanism that may
involve both enhanced translation and protein stabilization. SHH stabilizes
IRS1 protein by inhibiting mTOR-mediated activation of S6K, which is known to
phosphorylate IRS1 leading to its degradation.
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© The Company of Biologists Ltd 2008
