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. 2008 Oct;135(19):3291-300.
doi: 10.1242/dev.022871. Epub 2008 Aug 28.

Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors

Susana R Parathath  1 , Lori Anne Mainwaring, Africa Fernandez-L, Dane Ohlosson Campbell, Anna Marie Kenney
Affiliations

Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors

Susana R Parathath et al. Development. 2008 Oct.

Abstract

Sonic hedgehog (SHH) and insulin-like growth factor (IGF) signaling are essential for development of many tissues and are implicated in medulloblastoma, the most common solid pediatric malignancy. Cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for specific classes of medulloblastomas, require SHH and IGF signaling for proliferation and survival during development of the cerebellum. We asked whether SHH regulates IGF pathway components in proliferating CGNPs. We report that SHH-treated CGNPs showed increased levels of insulin receptor substrate 1 (IRS1) protein, which was also present in the germinal layer of the developing mouse cerebellum and in mouse SHH-induced medulloblastomas. Previous roles for IRS1, an oncogenic protein that is essential for IGF-mediated proliferation in other cell types, have not been described in SHH-mediated CGNP proliferation. We found that IRS1 overexpression can maintain CGNP proliferation in the absence of SHH. Furthermore, lentivirus-mediated knock down experiments have shown that IRS1 activity is required for CGNP proliferation in slice explants and dissociated cultures. Contrary to traditional models for SHH signaling that focus on gene transcription, SHH stimulation does not regulate Irs1 transcription but rather stabilizes IRS1 protein by interfering with mTOR-dependent IRS1 turnover and possibly affects Irs1 mRNA translation. Thus, we have identified IRS1 as a novel effector of SHH mitogenic signaling that may serve as a future target for medulloblastoma therapies. Our findings also indicate a previously unreported interaction between the SHH and mTOR pathways, and provide an example of a non-classical means for SHH-mediated protein regulation during development.

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Figures

Figure 1
Figure 1
IRS1 protein is upregulated in proliferating CGNPs. (A) CGNP cultures were prepared from different litters on different dates. Each preparation 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 a 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 (carat) 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 co-localizes 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.
Figure 2
Figure 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 with out 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 promoting IRS1 protein accumulation.
Figure 3
Figure 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, while 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.
Figure 4
Figure 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 redcued thickness of the EGL (white dotted 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 to 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. * statistically significant difference (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. (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 SEM, n=5.
Figure 5
Figure 5
IRS1 over-expression 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 over-expression 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 Figure 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 SEM, n=5. * statistically significant difference to untreated CGNPs (p<0.05 n=4).
Figure 6
Figure 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 and 63X magnification (blue=DAPI). (E) Western blot analysis for both Patched +/- and SmoA1 medulloblastoma models show increased IRS1 expression coinciding with increased cyclin D2 expression.
Figure 7
Figure 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 (squiggle) is present. In the presence of Shh, IRS1 is up-regulated 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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