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Keywords:

  • astrocyte;
  • G-protein receptor kinase;
  • p38;
  • smoothened;
  • sonic hedgehog

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The molecular determinants of Sonic Hedgehog (Shh) signaling in mammalian cells and, in particular, those of the CNS are unclear. Here we report that primary cortical astrocyte cultures are highly responsive to both Shh protein and Hh Agonist 1.6, a selective, small molecule Smoothened agonist. Both agonists produced increases in mRNA expression of Shh-regulated gene targets, Gli-1 and Patched in a cyclopamine- and forskolin-sensitive manner. Using this model we show for the first time that Shh pathway activation mediates rapid increases in p38 MAPK phosphorylation, without altering phosphorylation of either extracellular-signal-regulated kinases or c-jun N-terminal kinases. Selective inhibition of p38 MAPK significantly attenuated Shh-dependent up-regulation of Gli-1, inter-alpha trypsin inhibitor and thrombomodulin mRNA, however did not affect expression of insulin-like growth factor 2 or a novel Shh target, membrane-associated guanylate kinase p55 subfamily member 6. Using RNAi and a constitutively-active mutant we show that Shh signaling to p38 MAPK and subsequent Gli-1 transcription requires G-protein receptor kinase 2. Taken together, these findings provide evidence for a central role of G-protein receptor kinase 2-dependent p38 MAPK activity in regulating Shh-mediated gene transcription in astrocytes.

Abbreviations used
BSA

bovine serum albumin

ERK

extracellular signal-regulated kinase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GFAP

glial fibrillary acidic protein

GFP

green fluorescent protein

GRK

G-protein regulated kinase

GSK

glycogen synthase kinase

HBSS

Hank’s balanced salt-solution

IGF2

insulin-like growth factor 2

ITIH3

inter-alpha trypsin inhibitor heavy chain 3

JNK

c-jun N-terminal kinase

MAGUK MPP6

membrane-associated guanylate kinase p55 subfamily member 6

PBS

phosphate-buffered saline

PKA

protein kinase A

Ptc

Patched

Shh

Sonic Hedgehog

Smo

Smoothened

Sonic Hedgehog (Shh) is a secreted morphogen that is critically important for the development of many tissues and organs (Hooper and Scott 2005; Ingham and Placzek 2006) including the mammalian central nervous system (Bertrand and Dahmane 2006; Dessaud et al. 2008). Shh signaling also contributes to tissue homeostasis in the adult central nervous system by regulating neural progenitor cell proliferation (Lai et al. 2003; Machold et al. 2003). However, mutations that constitutively activate the pathway result in endodermally derived human cancers including basal cell carcinomas (Hahn et al. 1996) and medulloblastomas (Taylor et al. 2002). Despite the recognized importance of this pathway, the details of signal transmission in mammalian systems have only recently been addressed, predominantly using recombinant expression systems, with a paucity of data from central nervous system derived cells (Lum and Beachy 2004; Osterlund and Kogerman 2006; Riobo and Manning 2007).

In both invertebrates and vertebrates, Hh/Shh signal activation occurs following binding of the Hh/Shh protein to a twelve-transmembrane domain protein, Patched (Ptc), relieving the inhibition of Ptc on the seven-transmembrane protein, Smoothened (Smo) resulting in activation of the Ci/Gli family of zinc finger transcription factors (Lum and Beachy 2004; Hooper and Scott 2005). In Drosophila, activated Ptc induces extensive phosphorylation, by protein kinase A (PKA) and casein kinase I, of the Smo C-terminal tail leading to cell surface accumulation and activation by a conformational shift (Jia et al. 2004; Zhang et al. 2004; Apionishev et al. 2005; Zhao et al. 2007). However, most of these phosphorylation sites are not conserved in mammalian Smo (Lum et al. 2003; Osterlund and Kogerman 2006) suggesting that alternative mechanisms for the activation of Smo have evolved in vertebrates.

The molecular determinants of Smo signaling to Gli proteins remain a topic of debate (Osterlund and Kogerman 2006; Riobo and Manning 2007). Based on the structural similarity of Smo to seven-transmembrane G-protein coupled receptors, recent studies, using recombinantly expressed vertebrate Smo, have demonstrated that heterotrimeric G-proteins, specifically Gi, can be activated by Smo (DeCamp et al. 2000; Kasai et al. 2004; Riobo et al. 2006c; Low et al. 2008; Ogden et al. 2008). Additional studies have identified G protein-coupled receptor kinase (GRK) 2 and β-arrestin as novel signaling components in the Shh pathway, although the precise role of these proteins in Shh signaling have not been defined (Chen et al. 2004; Wilbanks et al. 2004; Meloni et al. 2006). In the current study we have investigated Shh signal regulation in rat primary cortical astrocytes that are highly responsive to both Shh protein and a potent, selective, small molecule Smo agonist. We have identified p38 MAPK, but not extracellular-signal-regulated kinases (ERKs) or c-jun N-terminal kinases (JNKs), as a novel determinant of Shh signaling and gene transcription. In addition, we provide evidence to show that GRK2 functions to transduce Shh signaling from Smo to p38 MAPK and Gli-1.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Chemicals

SB-203580, SB-202190, U0126, anisomycin, cyclopamine, deoxyribonuclease I from bovine pancreas (DNase I), bovine serum albumin, Fraction V (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). CompleteTM protease inhibitor cocktail was purchased from Roche (Basal, Switzerland). HaltTM phosphatase inhibitor was purchased from Pierce (Rockford, IL, USA). The Hh protein described here (denoted as octyl-Hh) consists of the amino-terminal human Shh modified at its amino-terminal cysteine with an octyl maleimide moiety (Frank-Kamenetsky et al. 2002). The small molecule agonist, Hh Agonist 1.6, 3-chloro-N-{[4-(2,6-dimethylpyridin-4-yl)thien-2-yl]methyl}-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-1-benzothiophene-2-carboxamide, structure shown in Fig. 1(a), was derived from a compound class previously described (Frank-Kamenetsky et al. 2002). Pharmacological characterization indicates that these compounds bind specifically, and with high affinity, to recombinant Smo (Frank-Kamenetsky et al. 2002).

image

Figure 1.  Cortical astrocytes respond to octyl-Hh and a Smoothened agonist (Hh Agonist 1.6). Cortical astrocytes were treated with octyl-Hh or Hh Agonist 1.6 at the indicated concentrations and total RNA was isolated 24 h later (unless otherwise indicated). Levels of Gli-1 transcript were determined using real-time quantitative PCR, as described in the Experimental Procedures and expression values were normalized against GAPDH. (a) The structure of Hh Agonist 1.6 is shown. (b) Astrocytes were treated with 0.1–100 nM octyl-Hh or Hh Agonist 1.6 (up to 10 μM) for 24 h prior to isolation of total RNA. Data show mean fold change ± SEM Gli-1 following normalization to GAPDH from three separate experiments. (c and d) Astrocytes were treated with 5 nM octyl-Hh or 2.5 nM Hh Agonist 1.6 and total RNA was collected at the time-points (2–96 h) indicated. Representative time-courses of Gli-1 or Ptc up-regulation from three experiments are shown. (e and f) Octyl-Hh or Hh Agonist 1.6-activated Gli-1 expression is attenuated by forskolin (10 μM) or cyclopamine (5 μM) pre-treatment (2 h). Control samples (ctrl) contained equivalent concentrations of DMSO vehicle used to solubilize forskolin and cyclopamine. Representative data from three separate experiments are shown.

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Primary astrocyte cultures

Mixed glial cultures were prepared using a protocol adapted from Marriott et al. (1995). Neonatal rats (postnatal days, 1–3) were decapitated and cortices were dissected and placed into 4°C Hank’s balanced salt-solution (HBSS). Cortical tissue was cut coarsely and incubated in HBSS containing 0.025% trypsin, 0.3% BSA and DNase (40 μg/mL) for 20 min at 37°C. The solution was replaced by HBSS containing BSA and DNase and triturated 20 times. After allowing for debris to settle the supernatant was passed through a 40-μm cell strainer (BD Biosciences, Bedford, MA, USA) and the process was repeated twice more. The supernatant was centrifuged at 300 g for 5 min and the pellet was resuspended in 10 mL of Dulbecco’s modified Eagle’s medium containing 50 units of penicillin, 50 mg/mL streptomycin and 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Suspended cells were divided into T175 flasks, grown for 10 days in vitro and purified by overnight shaking (120 rpm). Remaining adherent cells, containing ∼95% glial fibrillary acidic protein (GFAP)-positive astrocytes, were plated 48 h prior to experiments.

Transfection of astrocytes

All siRNA duplexes were purchased from Ambion (Austin, TX, USA). Pre-designed GRK2 siRNA duplexes siRNA#200413 and siRNA#200414 are denoted as GRK2 siRNA#1 and GRK2 siRNA#2 respectively. Scrambled siRNA duplex (validated by Ambion) was used as negative control and is denoted as control siRNA. Plasmid encoding constitutively active GRK2 (denoted as GRK2*) was obtained from Molecular Devices (Sunnyvale, CA, USA). Where indicated, astrocytes were transfected using the Nucleofection system (Amaxa Biosystems, Gaithersburg, MD, USA), according to the manufacturer’s optimized protocol. In brief, 4 × 106 cells were transfected with 0.5–5 μg siRNA using Program T-20 on the Nucleofector. Transfected cells were seeded into six-well plates for experimentation at the time-points indicated. Data represent the mean ± SEM for at least three separate transfections.

Quantitative real-time PCR

Total RNA was isolated from astrocytes using the RNAeasy kit (Qiagen, Valencia, CA, USA) and diluted to 50 ng/μL rat gli-1, Ptc-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts were amplified and measured using Dual-labeled 5′-FAM/3′BHQ-1 TaqMan probes (Biosearch Technologies, Novato, CA, USA) and QuantiTect Probe RT-PCR Kit (Qiagen). Primer and probe sequences were: rat gli-1 forward, 5′-CGTCACTACCTGGCCTCACA-3′, reverse, 5′-CCCCCTGGCTGAAGCATAT-3′ and probe, 5′-FAM-CCAGCACTACATGCTCCGGGCAA-BHQ-1-3′; patched-1 forward, 5′-TCACAGAGACAGGGTACATGG-3′, reverse, 5′-CCCGGACTGTAGCTTTGC-3′ and probe, 5′-FAM-CCTTCCCAGAAGCAGTCCAAAGGTG-BHQ-1-3′; GAPDH forward, 5′-ACCTGCCAAGTATGATGACATCA-3′, reverse, 5′-TGTTGAAGTCACAGGAGACAACCT-3′ and probe, 5′-FAM-CCCTCGGCCGCCTGCTTCA-BHQ-1-3′. Additional genes were investigated using Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) designed for specific amplification of thrombomodulin (assay ID: Rn00582226_s1), insulin-like growth factor 2 (IGF2; assay ID: Rn00580426_m1), inter-alpha trypsin inhibitor, heavy chain 3 (ITIH3; assay ID: Rn00569293_m1), membrane-associated guanylate kinase p55 subfamily member 6 (MAGUK MPP6; assay ID: Rn01410223_m1) transcripts. PCR parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Data were captured using a 7500 Fast Real-Time PCR System (Applied Biosystems) to obtain CT values for the gene of interest. RNA expression levels were extrapolated from CT values corresponding to known quantities of rat brain total RNA and normalized against a housekeeping gene, GAPDH. All measurements were performed in triplicate.

Immunoblot analysis

Prior to experiments astrocytes were incubated in Krebs-Henseleit buffer (in mM: NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.3, KH2PO4 1.2, NaHCO3 4.2, HEPES 10, glucose 11.7, pH 7.4) at 37°C for 1 h. Astrocytes were treated with compounds as indicated, prior to lysis in 300 μL of extraction buffer (Willets et al. 2001) containing CompleteTM protease and HALTTM phosphatase inhibitors (according to the manufacturer’s instructions). Lysates were sonicated and diluted using 4× LDS sample buffer prior to loading into NuPAGE 10% Bis-Tris gels (Invitrogen). Blots were transferred to nitrocellulose, blocked for 1 h at 21°C using Odyssey® Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) and incubated with primary antibody overnight at 4°C. The antibodies used were: anti-GRK2 (Santa Cruz, 1 : 200); anti-p38 MAPK phospho-site [pTpY180/182] specific (Invitrogen, 1 : 1000) or anti-(pan)-p38 MAPK antibodies (Invitrogen, 1 : 1000), anti-ERK or JNK phospho-site specific (Cell Signaling Technologies, Danvers, MA, USA, 1 : 1000); anti-GAPDH (Sigma, 1 : 10 000) and anti-α-tubulin (Upstate, 1 : 10 000). Immunoreactivity was captured and quantified using Odyssey® Infrared imaging (LI-COR Biosciences). IRDye infrared secondary antibodies (LI-COR Biosciences) were used to allow multiplex detection of p38 MAPK and α-tubulin immunoreactivity from the same nitrocellulose membrane.

Immunocytochemistry

Primary cultures of astrocytes or microglia were seeded into 8-well BD-Biocoat poly-d-lysine chamber slides. After 2 days cells were fixed using 4% paraformaldehyde for 20 min at 21°C and washed three times with phosphate-buffered saline (PBS). Blocking and permeabilization was carried out for 1 h at 21°C using blocking solution (5% goat serum in 0.3% Triton-X100 PBS). Cells were subsequently incubated with primary antibodies diluted 1 : 500 in blocking solution for 2 h at 37°C. Primary antibodies used were rabbit anti-GFAP (DAKO, Carpinteria, CA, USA) and mouse anti-OX-42 (Millipore, Billerica, MA, USA). Following an additional 3× PBS wash step, cells were exposed to secondary antibodies diluted 1 : 500 with blocking solution for 1 h at 21°C. Secondary antibodies were anti-rabbit Alexa Fluor 488 conjugate and anti-mouse Alexa Fluor 595 conjugate (Molecular Probes, Eugene, OR, USA). Cells were washed 3× PBS prior to mounting using ProLong Antifade (containing DAPI) reagent (Invitrogen). Cells were viewed using a Leica SP5 confocal microscope (Bannockburn, IL, USA).

Data analysis

Data was analyzed and plotted using Prism 4.0 software. (GraphPad Software, Inc., San Diego, CA, USA). Statistical differences between data sets were determined by one-way analysis of variance for multiple comparisons, followed by Bonferroni’s multiple-range test at < 0.05 (using Prism 4.0 software), or Student’s t test (unpaired; < 0.05 being considered significant).

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Cortical astrocytes respond to Sonic Hedgehog protein or a Smoothened agonist by increasing Gli-1 and Patched mRNA levels

In the current study we utilized a novel primary astrocyte model to identify candidate regulators of Shh signaling in the brain. Activation of the Shh pathway was achieved using Hh Agonist 1.6 (see Fig. 1a for structure) or octyl-Hh. Shh-mediated Gli-1 up-regulation, measured at 24 h was concentration-dependent following treatment with 0.1–100 nM octyl-Hh or 0.1–10 μM Hh Agonist 1.6 (Fig. 1b). Gli-1 and ptc-1 mRNA increases in response to either agonist were significant (< 0.05) at 24, 48, 72 and 96 h, and persisted up to 4 days in vitro (Fig. 1c and d). Based on these findings the agonist treatment time for subsequent experiments was 24 h. Consistent with these observations, optimal Shh-dependent Gli-1 up-regulation occurred following 2–4 days of pathway activation studies using C3H10T1/2 and NIH3T3 cells (Taipale et al. 2000; Ingram et al. 2002). Considering the relatively delayed onset of response it was of significant interest to note that a just brief exposure (∼5 min) to either agonist was sufficient to initiate pathway activation, determined 24 h later (data not shown).

Subsequent experiments demonstrated that octyl-Hh or Hh Agonist 1.6-mediated Gli-1 mRNA increases in astrocytes are attenuated by pre-treatment with the Hh pathway-specific antagonist, cyclopamine (5 μM) (Fig. 1e and f). Furthermore, the inhibitory action of 10 μM forskolin (Fig. 1e and f) confirmed the potential for this pathway to be regulated by PKA which, along with glycogen synthase kinase (GSK) 3β phosphorylates Gli transcription factors as a prerequisite to Gli protein degradation and/or processing (Taipale et al. 2000; Riobo et al. 2006d).

Immunocytochemical characterization of the astrocyte cultures confirmed that 95% of cells were GFAP-positive astrocytes, suggesting that a low level of other cell types (determined to be microglia) may be present at low levels (Fig. S1a and b). It was therefore important to determine whether any of the mRNA changes that were measured were because of other contaminating cell types such as microglial cells. We compared Gli-1 mRNA levels in primary cultures enriched with microglial cells (Fig. S1b) to the astrocyte cultures (Fig. S1a) following 24 h challenge with 2.5 nM Hh Ag 1.6. The Gli-1 mRNA induction in the astrocytes was 14.5 ± 3.2 fold over basal, consistent with previous observations, whereas the induction in the microglial culture was 1.1 ± 0.1 fold over basal (Fig. S2). This provides evidence that the primary Shh-responsive cells in these cultures are GFAP-expressing astrocytes.

Sonic Hedgehog pathway activation mediates p38 but not JNK or ERK1/2 MAPK phosphorylation

Previous studies have proposed that ERK1/2 MAPK is required for Shh signaling (Riobo and Manning 2007). In the current study we examined the effects of octyl-Hh and Hh Agonist 1.6 on phosphorylation of p38, JNK and ERK1/2 MAPK. Phosphorylation of p38 MAPK was increased 4.0 ± 1.3 fold (< 0.05 compared to control) and 3.7 ± 1.3 fold (< 0.05 compared to control) in response to octyl-Hh (5 nM) and Hh Agonist 1.6 (2.5 nM), respectively (Fig. 2a–d). This experiment also highlighted an agonist-specific difference in the temporal profile of p38 phosphorylation. Thus, the earliest increase in p38 phosphorylation reaching significance (< 0.05) occurred at 10 min for the Hh Agonist 1.6 compared to 30 min for octyl-Shh. It is likely that this delay may reflect the fact that Ptc (providing the binding site for octyl-Hh) is positioned upstream of Smo (providing the binding site for Hh Agonist 1.6) in the Shh signaling paradigm (Frank-Kamenetsky et al. 2002), and that p38 MAPK phosphorylation is proximal to Smo. In contrast with p38 MAPK, phosphorylation of both JNK and ERK1/2 MAPK were unchanged (Fig. 2e and f).

image

Figure 2.  Sonic Hedgehog pathway activation mediates p38 but not JNK or ERK1/2 MAPK phosphorylation in cortical astrocytes. (a–d) Astrocytes cultures were treated with 2.5 nM octyl-Shh (a, c) or 5.0 nM Hh Agonist 1.6 (b, d) and lysates were collected at the time points indicated. Phosphorylation of p38 MAPK was determined using immunoblot analysis as described in the Experimental Procedures. Representative immunoblots for phospho-p38 MAPK (pho-p38), α-tubulin and total p38 MAPK in response to octyl-Hh (a) or Hh Agonist 1.6 (b). (c and d) Data show mean ± SEM (*< 0.05 compared to basal) changes in phospho-p38 MAPK expressed as fold-change relative to basal (after normalization to α-tubulin) in response to octyl-Hh (c) or Hh Agonist 1.6 (d). (e and f) Representative immunoblots demonstrating that ERK1/2 MAPK phosphorylation (pERK, upper panels) and JNK MAPK phosphorylation (pJNK, lower panels) are unaffected by octyl-Hh (e) or Hh Agonist 1.6 (f) treatment. Where indicated astrocytes were also treated with anisomycin (ans; 10 μM, 10 min) as a positive control. Equal protein loading was confirmed by measuring α-tubulin immunoreactivity. Data were obtained from at least four separate experiments.

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Selective modulation of Sonic Hedgehog activated gene expression by p38 MAPK

The potential role of p38 MAPK in mediating Shh signaling events was investigated using the selective inhibitors, SB-203580 and SB-202190 (Bain et al. 2007). In the first set of experiments we examined the inclusion of 10 μM SB-203580 that significantly attenuated Gli-1 mRNA induction measured at 24 h in response to multiple concentrations (0.1–3 nM) of Hh Agonist 1.6 (Fig. 3a). Subsequent experiments showed that the effect of SB-203580 and an additional inhibitor, SB-202190 was concentration-dependent (Fig. 3b). 10 μM SB-203580 and SB-202190 significantly inhibited Hh Agonist 1.6 (2.5 nM)-induced Gli-1 expression by 62% (1.3 ± 0.3 to 0.5 ± 0.12, < 0.05) (Fig. 3b). Additional experiments showed that structurally similar, inactive analogue, SB-202274 (Lee et al. 1994) did not significantly inhibit Hh Agonist 1.6-mediated Shh signaling (10 μM, > 0.05) suggesting that these effects are p38 MAPK-specific (Fig. 3c). These observations were recapitulated using octyl-Hh, confirming that Ptc activation is comparable to small molecule activation of Smo (data not shown).

image

Figure 3.  Sonic Hedgehog signaling is attenuated by inhibitors of p38 MAPK. Astrocytes were treated with Hh Agonist 1.6 for 24 h. The cells were co-treated with vehicle or SB-203580, SB-202190 and SB-202474 (a SB-203580 analogue lacking p38 inhibitory activity) for the first 2 h as indicated. Control cells (basal) contained the equivalent amount of vehicle used to solubilize Hh Agonist 1.6 and, where indicated, astrocytes that were stimulated in the absence of inhibitor instead received the maximal vehicle concentration used to solubilize the inhibitor (denoted as veh). (a) Effect of SB-203580 (10 μM) on Hh Agonist 1.6 (0.1–3 nM)-activated Gli-1 signaling. (b) Data show the concentration-dependent effects of SB-203580 and SB-202190 on Hh Agonist 1.6 (EC90)-mediated Gli-1 up-regulation. (c) Comparison of the inhibitory action of 10 μM SB-202190 with 10 μM SB-202474 on Shh pathway-activated Gli-1 increases. Data show mean fold change ± SEM Gli-1/GADPH from at least three independent experiments (*< 0.05, significant inhibition compared to vehicle control).

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To further determine the role of p38 MAPK in Shh signaling we examined the regulation of additional genes that have been identified previously as downstream targets of the Shh pathway in a neuroepithelial cell line, MNS-70, and a fibroblast cell line, C3H10T1/2 (Kato et al. 2001; Ingram et al. 2002). Exposure to either octyl-Hh or Hh Agonist 1.6 mediated robust and equivocal increases in the mRNA expression level of thrombomodulin (2-fold relative to basal) ITIH3 (7-fold relative to basal), IGF2 (2-fold relative to basal). In addition to known targets of this pathway, we also identified MAGUK MPP6 as a novel Shh-gene target that was significantly up-regulated (2-fold) in response to octyl-Hh or Hh Agonist 1.6 (Fig. 4a–d). Consistent with our experiments measuring Gli-1, the inclusion of SB-202190 (10 μM) to inhibit p38 MAPK, resulted in a significant attenuation of Shh-activated ITIH3 gene transcription (Fig. 4a), suggesting that this gene is also regulated downstream of a common p38-dependent pathway. Whilst SB-202190 attenuated the Shh-activated thrombomodulin gene expression, the p38 MAPK inhibitor also decreased the basal gene expression (Fig. 4b) whereas the induction of IGF2 and MAGUK MPP6 expression by either agonist was completely unaffected by SB-202190 (Fig. 4c and d). These data suggest that some degree of divergence in the Shh pathway and gene regulation occurs at the level of p38 MAPK. Importantly, this observation also precludes the possibility that p38 inhibitors instill a non-specific inhibitory effect on astrocyte function/gene transcription. The gene changes observed appear to be specific to Shh-pathway activation as these effects were attenuated by pre-treatment with the Hh-pathway antagonist, cyclopamine (5 μM).

image

Figure 4.  Selective modulation of Sonic Hedgehog activated gene expression by p38 MAPK. Astrocytes were treated with 5 nM octyl-Hh or 2.5 nM Hh Agonist 1.6 for 24 h in the presence of absence of 10 μM SB-202190 and RNA was extracted as described under Experimental Procedures. In these experiments veh indicates an equivalent concentration of DMSO to Hh Agonist 1.6. Control samples contained the equivalent concentration of DMSO that was used to solubilize SB-202190. Data show mean fold change ± SEM (*< 0.05, significant inhibition compared to control) thrombomodulin (a), inter-alpha trypsin inhibitor (b), MAGUK MPP6 (c) and insulin-like growth factor 2 (d) following normalization to GAPDH. (e) Data show the effect of the Shh-specific antagonist, cyclopamine (5 μM), on Hh Agonist 1.6 activated gene expression from three separate experiments.

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Sonic Hedgehog signaling to Gli-1 and p38 MAPK is GRK2-dependent

In addition to an established role in homologous desensitization of G-protein coupled receptors (GPCRs), recent studies have revealed a role of GRK proteins in mediating additional pathways including MAPK signaling (Violin and Lefkowitz 2007). In relation to the current work, GRK2 has been implicated in Shh signal regulation in transfected HEK293 cells and a mouse fibroblast cell line (Chen et al. 2004; Meloni et al. 2006). In view of these findings we hypothesized that GRK2 may function to transduce Shh signals to p38 MAPK and Gli-1 in astrocytes. We therefore used RNAi-induced knockdown of GRK2 protein expression in the primary astrocyte cultures and examined the effects on Shh signaling by measure agonist-induced Gli-1 mRNA up-regulation and p38 MAPK phosphorylation. Transfection of two separate siRNA duplexes significantly reduced GRK2 protein expression levels (up to 90% compared to control) measured at 72 h (Fig. 5a), while levels of GAPDH expression remained unchanged. Samples treated with GRK2 siRNA also reduced GRK2 mRNA by approximately 75% whereas GAPDH mRNA expression was unchanged compared to control (data not shown). Transfection with a green fluorescent protein (GFP)-expressing plasmid confirmed that at least 50% of astrocytes were successfully transfected (Fig. S3). Previous experiments have indicated that the transfection efficiency of siRNA duplexes is typically higher than GFP, which provides an explanation for the robust (up to 90%) silencing of protein expression observed. Knockdown of GRK2 protein significantly reduced Hh Agonist 1.6 (2.5 nM) activated Gli-1 up-regulation by 91% (GRK2 siRNA #1) and 90% (GRK2 siRNA #2) (Fig. 5b). In contrast, Gli-1 mRNA up-regulation was enhanced following transfection with increasing quantities of constitutively active GRK2 (GRK2*). In control cells transfected with 3.0 μg of GFP, Gli-1 mRNA was increased 8.9 ± 3.4 and 7.6 ± 0.4 fold above basal levels in response to EC50 concentrations of Hh agonist 1.6. In cells transfected with an equivalent amount of GRK2*, these responses were increased to 15.9 ± 2.1 fold (< 0.05, compared to control GFP) (Fig. 5c). The relatively small magnitude of this effect might suggest that the quantity of endogenous GRK2 appropriately localized for Shh signal transduction may already be optimal in these cells. Furthermore, it was noted that responses were reduced following plasmid transfection, perhaps indicating that strong cytomegalovirus promoter activity may compromise the transcriptional machinery required for Gli-1 up-regulation.

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Figure 5.  Sonic Hedgehog signaling to p38 MAPK and Gli-1 is GRK2 dependent. Astrocytes were transfected with control siRNA, GRK2 siRNA#1, GRK2 siRNA#2 or plasmids expressing GFP or GRK2*, as described in the Experimental Procedures. (a) Representative immunoblot showing the expression of endogenous GRK2 protein in astrocytes treated with control or GRK2 siRNAs; GAPDH expression levels in the same samples are also shown to indicate equal protein loading. (b) Data show the mean ± SEM fold-increases in Gli-1 mRNA expression in response to 2.5 nM Hh Agonist 1.6 (after 24 h) following treatment with ctrl or GRK2 siRNA for 72 h. (c) Data show the mean ± SEM (#< 0.05, compared to control) increases in Gli-1 mRNA expression in response to EC50 concentrations of Hh Agonist 1.6 following transfection of GFP (3.0 μg) or GRK2* (1.5–5.0 μg) for 72 h. Data were collected from at least three experiments using separate cultures and transfections. (d and e) After 72 h transfection with control or GRK2 siRNA, astrocytes were treated with 2.5 nM Hh Agonist 1.6 and lysates were collected at 10 min. (d) Representative immunoblot demonstrating the effect of GRK2 knockdown on Shh pathway-mediated phospho-p38 MAPK immunoreactivity in astrocyte lysates. (e) Data show the mean ± SEM phospho-p38 MAPK responses (expressed as fold-change relative to basal, after normalization to α-tubulin) in GRK2 siRNA knockdown astrocytes compared to control (*< 0.05, compared to control).

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Next, we examined the effect of RNAi-mediated GRK2 knockdown on Shh-activated p38 MAPK. In astrocytes transfected with control siRNA, Hh Agonist 1.6 (2.5 nM)-mediated p38 phosphorylation (measured at 10 min) was 2.1 ± 0.3 fold over basal (Fig. 5d and e). This response was reduced by ∼50% (to 1.1 ± 0.3 fold over basal) in astrocytes transfected with GRK2 siRNA (Fig. 5d and e). These data provide new evidence to suggest that GRK2 transduces Shh signals from Smo to p38 MAPK which is required to activate specific gene transcription in astrocytes.

Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

This study utilized primary cortical astrocytes to help elucidate potential signaling mechanisms that may be important in Shh pathways in the mature CNS. In these cells, which are highly responsive to both Shh protein and a small molecule Smo agonist, we demonstrate that p38 MAPK is a novel regulator of Shh signaling and downstream gene expression. In addition we show that Shh-mediated p38 MAPK signaling and gene transcription in primary astrocytes requires GRK2, a kinase previously linked to Shh signaling in immortalized mouse fibroblasts (Meloni et al. 2006).

Although Shh pathway activation can be determined readily by measuring Gli-1 mRNA expression, the molecular determinants of pathway activation are unclear. Recent data suggest that the Shh activation is subject to complex, context-dependent regulation with parallel intracellular signaling cascades (Riobo et al. 2006b). Canonical GPCR signaling stimulation promotes the activation of heterotrimeric G-proteins and initiates receptor phosphorylation at serine/threonine residues by GRKs. Although evidence for a partial involvement of G-proteins in Smo signal transduction exists, the Smo C-terminal domain, containing consensus sites for GRK phosphorylation, is essential for signal transduction (Riobo et al. 2006c; Varjosalo et al. 2006). Accordingly, activated Smo is subject to GRK2-mediated phosphorylation and internalization when expressed in human embryonic kidney cells (Chen et al. 2004) and endogenous GRK2 is required for Shh signaling in a mouse fibroblast cell line (Meloni et al. 2006). Experiments examining downstream modulators of vertebrate Shh signaling, particularly in the CNS, are limited and inconclusive (Riobo and Manning 2007).

In the current study it was of significant interest to discover that activation of either Smo or Ptc resulted in an increase in p38 MAPK phosphorylation. To our knowledge, this is the first study to report direct activation of p38 MAPK in the Shh signal transduction pathway. Basal p38 MAPK activity has been implicated in the regulation of the level of Shh transcript (Androutsellis-Theotokis et al. 2006) although this effect was not observed in astrocytes (data not shown). Other studies suggest that basal ERK1/2 MAPK activity is indirectly required for Shh signaling in oligodendrocyte progenitors and NIH3T3 cells (Kessaris et al. 2004; Riobo et al. 2006a) and that Shh protein mediates ERK1/2 phosphorylation in adult muscle cells (Osawa et al. 2006; Elia et al. 2007). In contrast, we have shown that, in astrocytes, Shh pathway activation does not lead to ERK or JNK phosphorylation demonstrating that MAPK phosphorylation in this model is p38-selective. Furthermore pre-incubation of the astrocyte cultures with the MAPK/ERK kinase inhibitor, U0126 (up to 25 μM) had no significant effect on octyl-Hh or Hh Agonist 1.6-mediated Gli-1 up-regulation (data not shown). This is in contrast with a study showing that Shh signaling in NIH3T3 cells could be abolished in the presence of U0126 (Riobo et al. 2006a). It is therefore likely that Shh-dependent recruitment of MAPK is context-dependent, perhaps unsurprisingly given the diverse functional role of this protein in vivo. Interestingly, the onset of p38 phosphorylation was delayed in response to octyl-Hh compared to the Smo agonist. It is possible that this represents the position of the octyl-Hh binding site, Ptc, which is located upstream in the pathway relative to Smo. The delayed onset may be reconciled when considering the proposed mechanism by which Ptc controls Smo. A significant body of evidence from Drosophila and mammalian cell types indicate that pathway activation requires Ptc-dependent protein trafficking events that target Smo to specific subcellular signaling domains (Rohatgi and Scott 2007). Our study suggests that p38 MAPK activation is proximal to Smo, and that a subset of receptors is appropriately compartmentalized for signal activation via direct agonist activation.

Our observation that the p38 MAPK inhibitors, SB-203580 and SB-202190 attenuate Gli-1 up-regulation in a concentration-dependent manner implicates this kinase as a key player in regulating Shh signaling. In general, p38 MAPK activation is associated with mediating cellular stress signals and inflammatory events, however more recent work implicate this protein in many other aspects of cell physiology including: survival, myogenesis, migration, proliferation, cardiac hypertrophy and synaptic plasticity (Cuenda and Rousseau 2007). To further elucidate the role of Shh-dependent p38 MAPK activity in astrocytes we examined the expression of several additional gene transcripts that can be regulated by this pathway (Kato et al. 2001; Ingram et al. 2002). Thrombomodulin, IGF2 and ITIH3 transcripts were significantly up-regulated in response to Shh or Hh Agonist 1.6. In addition, as part of a distinct transcriptional profiling study (unpublished observations), we have identified the scaffolding protein, MAGUK MPP6 as a novel Shh-dependent gene target in astrocytes. Of specific interest in these experiments was the observation that Shh-dependent increases in IGF2 and MAGUK MPP6 expression were completely insensitive to p38 MAPK inhibition with SB-209120. In the same samples, thrombomodulin, ITIH3 and Gli-1 transcripts were all inhibited, suggesting that distinct subsets of genes do not require p38 MAPK activity. Similarly, Kasper et al. (Kasper et al. 2006) showed that Shh-dependent gene targets in human keratinocytes were modulated selectively by epidermal growth factor and ERK signaling and inhibition.

In determining how Smo may transduce Shh signals to p38 MAPK and subsequent gene transcription we next examined the role of GRK2. This follows recent evidence that suggest this kinase may be important for Shh signaling (Meloni et al. 2006), and that in general GRKs and associated arrestin proteins are known to recruit signaling pathways such as MAPK, Akt and Src (Violin and Lefkowitz 2007). We show that a near-complete knockdown of endogenous GRK2 protein expression mediated by two distinct siRNA duplexes results in an equivocal reduction in the activation of Gli-1 up-regulation by maximal concentrations of a Smo agonist. Conversely, expression of a GRK2 constitutively active mutant (GRK2*) enhanced Shh signaling in a concentration-dependent manner. Furthermore, knockdown of GRK2 expression also inhibited Shh-activated p38 MAPK phosphorylation suggesting GRK2 facilitates Smo interaction/activation of p38 MAPK. This is an attractive hypothesis in view of recent studies showing that another GPCR, the kappa opioid receptor, mediates GRK3- and arrestin-dependent p38 MAPK activation in striatal (Bruchas et al. 2006) and spinal cord astrocytes (Xu et al. 2007).

In speculating how p38 MAPK may regulate Shh signaling it is possible that this kinase affects the stability of Gli transcription factors, which are subject to phosphorylation by PKA and GSK3β prior to degradation. Interestingly a recent study showed that p38 MAPK can specifically phosphorylate and inactivate GSK3β in the brain leading to accumulation of the transcription factor β-catenin in the canonical Wnt pathway (Thornton et al. 2008). It will be of significant interest to determine whether analogous regulation of Gli proteins occurs in the Shh signaling paradigm.

The sensitivity of astrocytes to Shh is intriguing given the crucial role of these cell types in various aspects of central nervous system physiology and pathophysiology (Seifert et al. 2006). Although the role of Shh in directing fate and specification of neuronal precursors in the developing central nervous system is well-documented (Traiffort et al. 1998; Bertrand and Dahmane 2006; Dessaud et al. 2008), studies describing Shh-responsive astrocytes have been limited (Wallace and Raff 1999; Dellovade et al. 2006). Importantly, a recent transcriptome study has demonstrated that components of the canonical Shh signaling pathway, in particular Smo, were enriched in acutely isolated immature and mature astrocytes relative to other central nervous system cell-types (Cahoy et al. 2008), consistent with the functional observations of the present study. Furthermore, Gli-1-lacZ knock-in mice have been characterized, where GFAP expressing astrocytes respond to sonic hedgehog signaling in the adult CNS (Garcia et al. 2008). In this model, BrdU labeling indicates that the majority of Shh-responding cells in the adult CNS are not proliferating, suggesting that in contrast to development, Shh signals primarily to a terminally differentiated cell population (Garcia et al. 2008).

Taken together these data suggest that the response of astrocytes to Shh in the current study may indeed reflect the capacity of these cells to orchestrate some of the Shh-mediated events in the mature brain, including neuroprotection (Dass et al. 2002; Rafuse et al. 2005; Dellovade et al. 2006) and neural progenitor cell proliferation (Lai et al. 2003; Machold et al. 2003; Han et al. 2008). In summary, the present study demonstrated that GRK2 and p38 MAPK are regulators of Shh-dependent signaling and gene-regulation in astrocytes.

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  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Figure S1. (a and b) Immunocytochemical characterization of astrocyte cultures. Immunocytochemistry on cultures enriched with astrocytes (a) or microglia (b) was carried out as described in the Experimental Procedures. Images show cells stained with the astrocyte marker, GFAP (green), the microglial marker, OX-42 (red) and the nuclear marker, DAPI (blue). Also shown is an overlay of all three markers to illustrate the relative density of each cell type in the cultures.

Figure S2. Primary cultures enriched with astrocytes and not microglia are responsive to Hh Agonist 1.6. Cortical astrocytes or microglial cultures (see Fig.  S1a and b) were treated with 2.5 nM Hh Agonist 1.6 total RNA was isolated 24 h later (unless otherwise indicated). Levels of Gli-1 transcript were determined using real-time quantitative PCR, as described in the Experimental Procedures and expression values were normalized against GAPDH. Data show mean fold change ± SEM Gli-1 following normalization to GAPDH from three separate experiments.

Figure S3. Transfection of cortical astrocytes with GFP. Astrocytes were transfected using the Nucleofection system (Amaxa Biosystems, Gaithersburg, MD, USA) as described in the Experimental Procedures. The image shows a representative field of view following transfection of astrocytes with 1.5 μg GFP (green) and co-staining with the nuclear marker, DAPI (blue).

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