First published online February 21, 2008
Stem Cells
Vol. 26 No.
5
May 2008, pp.
1138
-1145
doi:10.1634/stemcells.2007-0841; www.StemCells.com
© 2008 AlphaMed Press
Leukotriene Synthesis Is Required for Hedgehog-Dependent Neurite Projection in Neuralized Embryoid Bodies but Not for Motor Neuron Differentiation
Maarten F. Bijlsmaa,b,
Maikel P. Peppelenboschc,
C. Arnold Speka,
Henk Roelinkb
aCenter for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands;
bDepartment of Biological Structure, University of Washington, Seattle, Washington, USA;
cDepartment of Cell Biology, University of Groningen, Groningen, The Netherlands
Key Words. Development • Stem cells • Leukotrienes • Neurons • Cytoskeleton
Correspondence:
Correspondence: M.F. Bijlsma, M.Sc., Center for Experimental and Molecular Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Telephone: 0031-0-20-5667062; Fax: 0031-0-20-6977192; e-mail: m.f.bijlsma{at}amc.uva.nl
Received on October 4, 2007;
accepted for publication on February 18, 2008.
First published online in STEM CELLS EXPRESS February 21, 2008.
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ABSTRACT
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The hedgehog (Hh) pathway is required for many developmental processes, as well as for adult homeostasis. Although all known effects of Hh signaling affecting patterning and differentiation are mediated by members of the Gli family of zinc finger transcription factors, we demonstrate that the Hh-dependent formation of neurites from motor neurons, like migration of fibroblasts, requires leukotriene synthesis and is different from the Gli-mediated Hh response. Smoothened activity is required for the use of the leukotriene metabolism, and inversely, the leukotriene metabolism is required for mediating the Hh effects on neurite projection. These data establish a function for the previously described arachidonic acid-dependent Hh pathway in a developmentally relevant model system.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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Hedgehog (Hh) signaling is involved in a large number of developmental processes, as well as in adult pathophysiology [1]; however, its role in inducing ventral neurons was one of the first and best characterized functions to be found for Sonic hedgehog (Shh) signaling [2]. Activation of the Hh pathway involves a particularly complicated sequence of events. The Hh ligands for the pathway are autocatalytically cleaved [3], sterolated [4], and palmitoylated [5], and despite its apparent hydrophobicity, Hh mediates long-range signaling. Hh binds to one of the two receptors of the pathway, patched-1 (Ptch1) [6], which results in the internalization of the Ptch1/Hh complex [7]. The 12-pass transmembrane protein Ptch1 probably inhibits the activity of the 7-pass transmembrane protein smoothened (Smo) via the redistribution of (pro-)vitamin D3 or similar molecules [8]. Smo accompanies the Ptc1/Hh complex into the cell, after which it segregates away from the complex, thereby removing itself from the inhibitory action of Ptch1 and becoming active [9]. This activity is relayed to the pathway's transcription factors (the family of zinc finger-containing glioma-associated oncogene [Gli] proteins) through an intracellular complex of pathway components, the exact composition and function of which is not yet clear and appears to be different in distinct phyla [10]. In the absence of a signal from Smo, the pathway's transcription factors are either kept inactive by sequestration or processed into repressor forms by this complex [11].
There were several reasons to postulate an Hh response independent of the Gli-mediated activation or suppression of transcription. For instance, Shh has been shown to be an important chemotactic factor with respect to axonal guidance during development of the central nervous system [12], an action that fits poorly with Shh acting through Gli-dependent transcription, as these actions take place outside the cell body and on a time scale seemingly incompatible with transcription/translation. We recently showed that besides the traditional Hh pathway, an arachidonic acid-dependent but Gli-independent pathway exists [13]. This pathway was shown to function in mediating fibroblast motility independently of transcriptional activity and to require a functional arachidonate metabolism. We set out to investigate the existence and function of the leukotriene-dependent Hh pathway in a neuronal model for Shh-mediated differentiation.
By virtue of their pluripotency and sensitivity to differentiation cues, embryonic stem (ES) cells provide a good in vitro model for neural differentiation [14]. In neuralized ES cells, the response to Shh results in expression of HB9, a marker of motor neuron differentiation [15]. By using ES cells derived from an embryo transgenic for an HB9 promoter-driven green fluorescent protein (GFP) [14], we were thus able to assess motor neuron differentiation in neuralized embryoid bodies (EBs) derived from these ES cells. By staining for class III β-tubulin (a neuron-specific cytoskeletal protein), we could simultaneously visualize neurites of neurons induced in neuralized EBs [16], allowing us to assess the effect of Hh signaling on neuronal differentiation and neurite formation independently.
To address the role of leukotrienes in neurite formation, we focused on a key enzyme involved in their synthesis, 5-lipoxygenase (5-LOX), which catalyzes the formation of the leukotriene precursor 5-hydroperoxyeicosatetraenoic acid (5-HPETE) from arachidonic acid. Because of its function in generating leukotrienes, the role of 5-LOX in immunology is clear and well established, and very specific inhibitors have been developed for pharmaceutical use. We showed that one of these inhibitors, MK-886, inhibited neurite outgrowth in of Shh-induced motor neurons, while not affecting the induction of motor neuron-specific gene expression. We further showed that Smo function is required for MK-886 to inhibit neurite growth. These results indicate the presence of two independent, Smo-dependent mechanisms to relay the Hh response, the classic pathway, resulting in the induction of motor neurons, as well as a 5-LOX-dependent response affecting neurite outgrowth.
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MATERIALS AND METHODS
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ES Cell Culture
HB9::GFP or Smo–/– mouse-derived ES cells were maintained in ES medium (Dulbecco's modified Eagle's medium [DMEM] with 4.5 g/l D-glucose, L-glutamine, 110 mg/l sodium pyruvate (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 3.7 g/l sodium bicarbonate (Mallinckrodt Baker, Phillipsburg, NJ, http://www.mallbaker.com) supplemented with 0.1 FM2-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 15% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA, http://www.gembio.com), 1% penicillin-streptomycin-glutamine, 1% nonessential amino acids (all from Invitrogen), 1% ES cell nucleosides, and 1,000 units/ml recombinant murine leukemia inhibitory factor (both from Chemicon, Temecula, CA, http://www.millipore.com). For EB differentiation, cells were trypsinized, washed, and diluted to a concentration of 50,000 cells per milliliter in 25% DMEM with 4.5 g/l D-glucose, L-glutamine, 110 mg/l sodium pyruvate (Invitrogen), 3.7 g/l sodium bicarbonate (Mallinckrodt Baker), 48% neurobasal medium, 25% Ham's F-12 medium (both from Invitrogen) supplemented with 80 µM FM2-mercaptoethanol (Sigma-Aldrich), 1% penicillin-streptomycin-glutamine, and 1% B-27 supplement (DFNB) (all from Invitrogen). To induce motor neurons, the cells were grown in nonadherent, bacterial-grade Petri dishes for 2 days to allow aggregation into EBs. On day 2, medium was changed to DFNB supplemented with appropriate combinations of 1 mM retinoic acid (Sigma-Aldrich) and Hh agonist. Produced EBs were grown for an additional 3 days in this supplemented DFNB with a medium change after 2 days. For the experiments described in Figure 3C, EBs were grown in the supplemented DFNB for 6 days.
Shh Response Quantification
After culture, EBs were fixed in 4% phosphate buffered paraformaldehyde for 30 minutes, blocked and permeabilized in phosphate buffered saline 0.1% Triton X-100 with 10% normal goat serum for 1 hour, and incubated in 1:500 
-Tuj1 primary antibody (Covance, Princeton, NJ, http://www.covance.com) overnight. After overnight incubation with Cy3-conjugated secondary antibody, EBs were mounted in ProLong Gold (Invitrogen) and imaged on a Nikon fluorescence microscope (Nikon, Tokyo, http://www.nikon.com). For classification, EBs were counted on a Carl Zeiss dissection microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). EBs were simultaneously quantified for proper neurite projection (details given in text body and Fig. 1) and GFP expression.
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Figure 1. HhAg treatment induces motor neurons and a dense network of Tuj1-positive neurite projections in neuralized EBs. (A–F): As indicated by HB9 promoter-driven GFP expression in neuralized EBs, HhAg induced differentiation to motor neurons (GFP expression) (D–F). This differentiation was not abrogated by treatment with 5 µM MK-886. In the presence of HhAg, a dense network of class III β-tubulin-positive neurite projections (stained by -Tuj1) (A–C) could be seen. This network was, however, disturbed by inhibition of 5-lipoxygenase with 5 µM MK-886, a concentration that did not affect GFP expression. EBs were stimulated with 500 nM HhAg and 1 µM retinoic acid for 3 days after 2 days of allowing aggregation in the absence of stimulus. MK-886 was added simultaneously with HhAg. (G): Frequency distribution analysis of neurite length (in pixels) in between crossing neurites as a measure of network reticularity. Two distinct classes of neurite network density could be seen. All conditions included retinoic acid treatment. n = 40. Quantification of percentage of class II EBs showed strong inhibition by MK-886 treatment (as in [C]) (H), whereas the population of GFP-positive (motor neuron) EBs was hardly disturbed (I). Approximately 50 EBs were quantified in at least three individual experiments. Shown is the mean ± SEM. NDGA treatment in above-mentioned experimental setup and quantification shows a similar response (J) but slightly more pronounced effect on motor neuron differentiation, as indicated by GFP expression (K). Statistics in (J, K) are the same as those in (H). Abbreviations: GFP, green fluorescent protein; HhAg, hedgehog agonist; NDGA, nordihydroguaiaretic acid.
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Neural Plate Explants
Ventral neural plate explants were obtained as previously described [17, 18] and maintained on growth factor-reduced Matrigel substrate (BD Biosciences) in the presence of Hh agonist for 3 days. Leukotriene synthesis was inhibited by adding 500 nM or 5 µM MK-886. Explants were processed for microscopy as described above, and neurite projection length was quantified using ImageJ (NIH, public domain software). Image colors shown were inverted for better visibility of projections.
Leukotriene Measurement
Cells from wild-type, Gli1–/–2–/–, and Gli2–/–3–/– double knockout mice [19] were grown in 145-mm culture dishes and stimulated with arachidonate or solvent control. Cells were lysed in 2 ml of methanol, after which the methanol was evaporated in a SpeedVac concentrator (Thermo Scientific, Waltham, MA, http://www.thermo.com). After reconstitution in 200 µl of enzyme immunoassay (EIA) buffer, cysteinyl leukotriene content was assayed according to the manufacturer's directions (Cysteinyl Leukotriene EIA Kit; Cayman Chemicals, Ann Arbor, MI, http://www.caymanchem.com) [13]. Measured values of all samples were midrange in the standard curve.
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RESULTS
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Motor Neuron Neurite Projection in Neuralized Embryoid Bodies Is Dependent on 5-Lipoxygenase Activity
To study the possible involvement of the arachidonic acid-dependent Hh pathway in a model for neuronal development, we investigated motor neuron differentiation in HB9::GFP EBs. Motor neuron differentiation is known to be critically dependent on Gli transcription factor activity [14, 20], and this differentiation thus represents the classic Hh signaling pathway in our assay. Following neuralization by retinoic acid treatment and ventralization by a pharmacological Hh agonist, all EBs contained motor neurons, as shown by GFP expression (Fig. 1E). The Hh agonist used is similar to the previously described HhAg1.3 [14, 21] and specifically binds and activates Smo.
In addition to assessing proper motor neuron differentiation, staining for Tuj1 enabled us to visualize the projection of class III β-tubulin-positive neurites in these EBs. In response to Smo activation, a dense network of neurite projections was formed. As we used conventional epifluorescence microscopy, we predominantly visualized motor neuron projections on the outside of the EBs (Fig. 1B). In the absence of Hh agonist, this fine neurite network was largely absent, and only a few projections could be seen per optical field (Fig. 1A). When the EBs were stimulated with Hh agonist as well as with the specific inhibitor of 5-LOX, MK-886, the number of neurites was significantly diminished (Fig. 1C), whereas motor neuron differentiation as measured by GFP expression was unaffected (Fig. 1F).
To quantify the density, or reticularity, of this network, we measured neurite length between crossing neurites (nodes). This quantification showed two distinct classes of neurite network density evident from by the frequency distribution analysis of the measured lengths (Fig. 1G). In the dense network that formed in the presence of Hh agonist, the distances between crossing neurites were small, whereas longer distances between these nodes characterized a less dense network. The latter EBs were referred to as class I, whereas EBs containing a dense network were referred to as class II. Activation of the Hh response by Hh agonist caused the formation of class II EBs, whereas in the presence of Hh agonist as well as MK-886, the neurite network became significantly less dense, and these EBs were classified class I. All subsequent analyses of proper neurite projection were performed using this classification.
Quantification of the percentage of class II EBs demonstrated that increasing concentrations of MK-886 decreased the induction of class II EBs by Hh agonist (Fig. 1H). Conversely, quantification of the percentage of GFP-positive EBs showed that motor neuron differentiation was not affected by 5-LOX inhibition (Fig. 1I). Thus, although MK-886 left the developmental program leading to motor neurons unaltered, it efficiently disturbed the formation of neurite projections from these motor neurons. This suggests that neurite extension, but not the differentiation of motor neurons, relies on leukotriene synthesis. Using a more general leukotriene inhibitor, nordihydroguaiaretic acid (NDGA), we found a similar response on class II EB induction (Fig. 1J), although NDGA also caused some inhibition of GFP expression (Fig. 1K), probably because of the relative low specificity of NDGA compared with MK-886 in inhibiting LOX enzymes.
Neurite Projection from Ventral Neural Explants Is Dependent on 5-Lipoxygenase Activity
To be able to quantitatively study single-neurite dynamics under leukotriene inhibition and confirm the observations from the EB model, we used a ventral quail neural explant model [18] in which we observed neurite projections, likely from motor neurons, exiting the explant and adhering to the substrate in the presence of Hh agonist (Fig. 2A). In this experimental setup, we observed that MK-866 caused shortening of the neurites outside the explant in a dose-dependent manner (Fig. 2B). Trypan blue exclusion revealed that this was not due to cytotoxicity (data not shown). Quantification of the effect of MK-866 on the length of neurite outgrowth is shown in Figure 2C. Although these explant data do not allow us to precisely separate the effects of Hh signaling on differentiation and neurite extension, they do emphasize that 5-lipoxygenase activity is required for neurite outgrowth from ventral neural explants.
Specificity of Leukotriene Inhibition for Class II EB Induction
Arachidonate is generated from membrane phospholipids by phospholipase A2 (PLA2; overview given in Fig. 3A). Leukotrienes (LTs) are subsequently synthesized from arachidonate by the lipoxygenases (LOX), through HPETE intermediates, whereas the cyclooxygenases mediate synthesis of prostaglandins. The group of cysteinyl leukotrienes includes the leukotrienes that are indirectly synthesized by 5-lipoxgenase and consists of leukotrienes C4, D4, and E4. These leukotrienes are the focus of this study.
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Figure 3. Specificity of various blockers of arachidonate metabolizing enzymes for induction of class II EB fraction. (A): Schematic of arachidonate metabolism leading to production of leukotrienes and prostaglandins. Gray boxes indicate enzymes. (B): Following differentiation in the presence of hedgehog agonist and various blockers of arachidonate metabolizing enzymes, class II- and GFP-positive EBs were quantified as shown in Figure 1H and 1I, and IC50 values were determined. Abbreviations: CDC, cinnamyl 3,4-dihydroxy--cyanocinnamate; COX, cyclooxygenase; CysLT, cysteinyl leukotrienes; GFP, green fluorescent protein; HPETE, hydroperoxyeicosatetraenoic acid; IC50, 50% inhibitory concentration; LOX, lipoxygenase; NDGA, nordihydroguaiaretic acid; PG, prostaglandin; PLA2, phospholipase A2.
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We determined the 50% inhibitory concentration (IC50) for inhibition of class II EB formation, as well as the IC50 for inhibition of GFP expression by compounds inhibiting lipoxygenases. The lipoxygenase inhibitors MK-886 (specific for 5-LOX) and NDGA, the 12-LOX inhibitors baicalein and cinnamyl 3,4-dihydroxy--cyanocinnamate, and the cyclooxygenase inhibitor indomethacin were tested. Inhibition of cyclooxygenases was approximately 25-fold less efficient in inhibiting class II EB formation, whereas its effect on GFP expression was comparable to that of MK-886 (Fig. 3B). These data suggest a specific role for leukotrienes as the primary arachidonate metabolites required for the projection of neurites from motor neurons.
To confirm that inhibition of leukotriene synthesis does not inhibit motor neuron differentiation in EB development but rather affects the projection of neurites after completed motor neuron differentiation, EBs were stimulated for 3 days with Hh agonist, and during the last 24 hours, MK-886 was added (Fig. 4A). After 2 days, we observed widespread motor neuron differentiation. Subsequent addition of MK-886 affected only processes in differentiated motor neurons and resulted in significant inhibition of class II EB induction (Fig. 4B) The magnitude of this inhibition was similar to that found in Figure 1H. To formally exclude a detrimental effect of the inhibitors used on cell viability in general, we assayed cell viability in EBs treated with various concentrations of MK-886 and NDGA by trypan blue exclusion. NDGA and MK-886 were only toxic to cells at concentrations 10-fold higher (50 µM) than those than those affecting neurite outgrowth (Fig. 4B). At lower concentrations, these compound appeared to slightly enhance cell viability.
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Figure 4. The inhibition of class II EB formation by MK-886 is not caused by abrogation of motor neuron differentiation or diminished cell viability. (A): EBs were stimulated with retinoic acid and HhAg for 2 days, and during the last 24 hours, MK-886 was added at the concentrations as indicated. EBs were fixed and stained with -Tuj1 and classified as for Figure 1H. (B): EBs were treated as for Figure 1, with increasing concentrations of MK-886 or NDGA, and subsequently incubated for 5' in 0.1% trypan blue. EBs were mounted in aqueous medium and photographed, and subsequently, intensity of the blue channel was quantified using ImageJ software. Statistics were as Figure 1H; *, p < .05; **, p < .01; Student's t test. Abbreviations: HhAg, hedgehog agonist; NDGA, nordihydroguaiaretic acid.
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The Effects of 5-Lipoxygenase Inhibition on Neurite Projection Require an Active Shh Response
To verify that the effects of MK-886 on the induction of class I EBs are dependent on Hh signaling, we assessed the effects of the leukotriene inhibitors on neuralized EBs in the absence of Hh signaling. We achieved this by either including the Shh-inhibiting antibody 5E1 in the culture medium or by using EBs derived from Smo–/– ES cells, which are unable to activate an Hh response. The presence of the Shh-blocking antibody 5E1 even resulted in a slight reduction of neurite outgrowth, indicating some low level of Shh expression in the EBs (Fig. 5A). The inhibition of basal class II EB induction by 5E1 also tells us that this basal level is not due to any non-Hh-related artifacts, such as the induction of PLA2 by retinoic acid [22]. Inclusion of MK-866 in the presence of 5E1 had little effect, which might indicate that those neurites that form in the absence of Hh agonist are not sensitive to this compound. Similarly, Smo–/– EBs, which are insensitive to Hh agonist, were not affected by MK-866, although at 5 µM MK-886, no EBs remained, probably because of nonspecific toxicity to Smo–/– ES cells (Fig. 5B). Since very few neurites formed under these conditions in Smo–/– EBs in the first place, we wanted to test whether the neurite extensions that appear in the neuralized EBs in the absence of Hh pathway activation are MK-886-sensitive. Note that the cells from which these neurites extended were probably Lim1/2 interneurons, representing the dorsal and intermediate population of neurons, as formed in neuralized tissue in the absence of Shh.
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Figure 5. The inhibition of motor neuron neurite projections by MK-886 is Hh pathway-specific. (A): EBs were treated with 1 µg/ml Shh-blocking antibody (5E1) simultaneously with retinoic acid, resulting in a low amount of class II EBs. In these EBs, MK-886 addition showed no effect. (B): Low baseline levels of class II among Smo–/– EBs after 3 days of HhAg treatment could not be lowered by MK-886 treatment. (C–E): Following incubation for 6 days, Smo–/– EBs formed proper (Hh-independent) neurite projections. These were insensitive to treatment with MK-886; quantification in (F). (G): To exclude Gli action on the leukotriene metabolism, fibroblasts from WT, Gli1–/–2–/–, and Gli2–/–3–/– double knockouts were stimulated with 30 µM arachidonate or solvent control for 5 minutes. Intracellular cysLT content was determined and baseline as well as stimulated conditions were not found to differ significantly between WT and the knockout cells. n = 6. Abbreviations: cysLT, cysteinyl leukotriene; HhAg, hedgehog agonist; Smo, smoothened; WT, wild-type.
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When Smo–/– EBs were cultured for an additional 3 days, we could observe the induction of class II EBs (Figs. 4F, 5C–5E). The appearance of these EBs was insensitive to MK-886 (Fig. 5D–5F), demonstrating that Smo-independent formation of neurite projections does not rely on leukotriene synthesis. This implies that the observed effect of 5-LOX inhibition on motor neuron projections is Smo-specific and -dependent.
Gli Transcription Factors Do Not Influence Leukotriene Synthesis
To consolidate the Gli independence of neurite projections from motor neurons, it is important to uncouple leukotriene synthesis from transcriptional activity of the Hh pathway through the Gli proteins. To formally exclude the family of Gli transcription factors to be responsible for modulation of leukotriene synthesis, we investigated whether fibroblasts from mice knock-out for these transcription factors had an inhibited or enhanced leukotriene synthesis. We used cells from wild-type, Gli1–/–2–/–, and Gli2–/–3–/– double knockout mice. Although none of the cells used were knocked out for all 3 Gli proteins, Gli3 is a potent inhibitor of the Hh response. Consequently, the Gli1–/–2–/– knockout fibroblasts lack all activating functions of the Gli transcription factors (also described in [19]).
Cells were treated with 30 µM arachidonate for 5 minutes, and leukotriene content was determined using a cysteinyl leukotriene EIA. The excess of arachidonate (not recognized by the EIA) served as a positive control for a functional leukotriene synthesis machinery [23]. Intracellular leukotriene content was not different between the different cell lines under both basal and stimulated (arachidonate) conditions, meaning that the presence or absence of Gli transcription factors does not influence leukotriene synthesis and suggesting that the requirement for leukotriene synthesis for motor neuron neurite projection is Gli-independent as well.
Exogenous Arachidonate Bypasses the Requirement for Hh Agonist for Neurite Projection
We reasoned that if Hh activates and requires the arachidonate metabolism for cytoskeletal rearrangement in motor neurons as it does in mesenchymal fibroblasts, enhancing the production of leukotriene synthesis via the addition of substrate (arachidonate) while Smo activity is low should mimic the Hh agonist effect on neurite outgrowth. Indeed, we observed that the addition of 5 µM arachidonate was sufficient to induce class II EBs in the absence of Hh agonist (Fig. 6A). Under these conditions, motor neuron differentiation did not occur, but the low Smo activity was adequate to use the exogenously added arachidonate to increase neurite outgrowth. In other words, by adding arachidonate in excess, we augmented the non-Gli Hh pathway, and this is shown by the presence of neurites but absence of GFP expression. In the presence of Hh agonist, arachidonate was not able to further increase the number of neurite-rich EBs, which had apparently reached the highest possible level (Fig. 6A).
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Figure 6. Arachidonate treatment is sufficient to recover proper neurite projection under low Smo activity conditions. (A): EBs in the absence of HhAg (0 nM HhAg) or EBs from a Smo knockout background (Smo–/–) were treated with arachidonate, and class II EBs were scored. Addition of arachidonate in the absence of HhAg was able to induce class II EBs, whereas addition of arachidonate to Smo–/– EBs did not significantly raise the number of class II EBs. Addition of arachidonate in the presence of HhAg did not further increase neurite-rich EB formation, possibly indicating the upper limit of the assay. Statistics were as Figure 1H. (B): Schematic explaining the mechanisms elucidated in Figures 1 –5; Smo stimulation by a synthetic agonist induces Gli transcription factor activity, as well as a response that is independent of transcription factors that requires leukotriene synthesis and functional Smo. (C): If Smo is functional but has low activity in the absence of agonist, as for the experiments shown in Figures 5 and 6, the addition of excess arachidonate is sufficient to drive the Gli-independent response. Low Smo activity due to the absence of agonist, however, precludes a transcriptional Hh response as determined by differentiation markers. (D): In the total absence of Smo, as shown in Figure 6A, excess arachidonate does not induce class II EBs, proving that the leukotriene-dependent Hh pathway requires Smo. Abbreviations: HhAg, hedgehog agonist; N.S., not significant; PLA2, phospholipase A2; Smo, smoothened.
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Although pathway activation by Hh agonist was not required for arachidonate-induced class II EB formation, basal Smo activity was required; in Smo–/– EBs, the increase in class II EBs after arachidonate stimulation was nonsignificant. This is important, as it confirms that Smo signals to the leukotriene-dependent Hh pathway. This is consistent with the notion that Smo mediates two distinct activities, one that signals to the Gli transcription factors and mediates patterning, and another that uses leukotrienes and is involved in cytoskeletal rearrangement [13].
Figure 6B summarizes the experiments as performed in Figures 1–5. Stimulation of EBs with Hh agonist activates Smo, and this induces a transcriptional response as well as a Gli-independent, nontranscriptional response. This latter pathway is sensitive to leukotriene inhibition and requires Smo. If Smo activity is low, the addition of excess substrate (arachidonate) is required and sufficient to induce the nontranscriptional response (Fig. 6C). Under these conditions, there is no transcriptional Hh response, as determined by the lack of HB9 promoter-driven GFP expression, separating the effects of the two pathways. If Smo is absent, additional arachidonate has no effect, and no class II EBs are formed (Fig. 6D). These experiments provide solid evidence for a leukotriene-dependent Hh pathway that is Gli-independent but mediated through Smo.
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DISCUSSION
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In this study, we describe the requirement of arachidonic acid for Hh action in an in vitro model for neurite projection. A major advantage of deriving neurons from embryonic stem cells in vitro is that this allows us to use small molecule activators and inhibitors in a controlled manner, in contrast to in vivo systems, where pharmacokinetics and side effects greatly complicate interpretation of results. Another advantage is that we can use stem cell lines with specific mutations that would be lethal in experimental animals. For example, these stem cells allow us to easily visualize neurons in cell lines mutant for Smo, which are thus completely insensitive to Hh; this method is inherently superior to the use of Smo inhibitors, such as cyclopamine.
We have shown earlier that Shh induces cytoskeletal rearrangement and migration regulated by arachidonate metabolites, independently of transcription. By analogy, neurite extension, as observed in the present study, also involves significant cytoskeletal rearrangements. Previous studies have shown that Shh signaling is involved in neural guidance [12, 24]. However, the Shh response, as mediated via the normal pathway, involving Ptch1 and Smo trafficking, followed by activation of the Gli transcription factors, occurs at a time scale incompatible with the fast responses that take place in the growth cone. In addition, this response would be incompatible with maintaining directional information. Our observation that Shh mediates neurite outgrowth via arachidonate metabolites is compatible with a fast-acting, directional response. It is easy to imagine that this signaling pathway is contained within the growth cone and is expected to be able to mediate a directional response on a short time scale.
Our results also demonstrate that Smo is required for leukotriene-dependent signaling, as is the Gli-dependent pathway. The molecular discrimination between the leukotriene-dependent and Gli-dependent response must occur at or downstream of Smo. Smo activity is know to be modulated, both positively and negatively, by several small, lipophilic molecules [8, 21, 25, 26], and it is not a major conceptual leap to hypothesize that Smo interacts with yet another class of membrane-associated molecules to influence the arachidonate metabolism.
Involvement of the leukotriene pathway in neurite projection of neurons is consistent with the observation that infants with impaired leukotriene synthesis show a syndrome of seemingly Hh-related phenotypes [27]. Most strikingly, the phenotype is characterized by impaired motor neuron functioning, consistent with our finding that leukotriene synthesis is necessary for proper formation of motor neuron neurites.
Another interesting implication of our findings is that motor neurons appear to go through multiple developmental phases where they are dependent on Hh signaling. Initially Hh dependence is via the classic pathway involving Gli activity, but the later phases, involving the establishment of the correct connectivity, rely on the leukotriene-dependent pathway focused on in this study. Since we have shown that neurite extension is independent of Gli-mediated Shh signaling, it is expected that Hh-mediated neurite extension can affect many aspects of Hh-mediated growth cone guidance. This might be particularly important for the many axons that cross the ventral midline. The morphogen Shh is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance [12]. This chemoattraction is known to require the Hh coreceptor brother of cdo, and this Shh-mediated pathfinding is a prime candidate to be mediated in a Smo-dependent, leukotriene-dependent pathway. Not only the axons of dorsal sensory relay neurons cross the midline. Other examples are the axons in the lateral corticospinal tract and the vast majority of retinal ganglion cells. In particular, in these cases, the growth cones are at a significant distance from the transcriptional machinery, making any involvement of Gli-mediated signaling unlikely, but instead it would be consistent with our observations that some of the required guidance occurs via arachidonic acid metabolites.
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CONCLUSION
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Given the analogy between neurite formation and membrane ruffling, it is possible that this Hh-dependent process also plays a role in tumor metastasis. Not only is the activation of the Shh pathway the cause of many tumors, increased Hedgehog signaling is also know to enhance metastasis of some tumors [28]. Inhibitors of leukotriene synthesis are commonly used as antiallergens, and such compounds could be anticipated to have a role in the inhibition of Hedgehog-induced metastases. It has been shown that inhibition of the arachidonic acid metabolism has an inhibitory effect on pancreatic tumors [29], which are often induced in response to Shh, and it could be argued that the novel Hh pathway is responsible for the observed beneficiary effects [30]. In addition, the use of leukotriene-inhibiting compounds might need more scrutiny in regard to their use during pregnancy. Conversely, the widespread use of these leukotriene synthesis inhibitors might help in determining the in vivo importance of this pathway in Hedgehog-dependent cellular migration, neuron guidance, and other biological phenomena.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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H.R. has acted as a consultant to Berlex Healthcare.
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ACKNOWLEDGMENTS
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We thank T.Q. Crawford for technical advice and R.J. Lipinski for the Gli knockout cell lines. M.F.B. is supported by the Stichting Technische Wetenschappen. H.R. is supported by National Institute of General Medicine grant 1R01-HD042307. H.R. is currently affiliated with the Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, CA.
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Abstract
Introduction
5; *, p < .05; ***, p < .005; Student's t test. Abbreviation: HhAg, hedgehog agonist.