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The G12 family of heterotrimeric G proteins and Rho GTPase mediate Sonic hedgehog signalling

Kenji Kasai1,2,*, Masanori Takahashi3, Noriko Osumi3, Srikumar Sinnarajah4, Tomohiro Takeo1, Hiroshi Ikeda1, John H. Kehrl4, Gen Itoh1 and Heinz Arnheiter2

1 Department of Pathology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
2 Mammalian Development Section, Laboratory of Developmental Neurogenetics, NINDS, NIH, Bethesda, MD 20892, USA
3 Department of Developmental Neurobiology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan
4 B cell Molecular Biology Section, Laboratory of Immunoregulation, NIAID, NIH, Bethesda, MD 20892, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Sonic hedgehog (Shh) is a secreted morphogen crucial for cell fate decision, cellular proliferation, and patterning during vertebrate development. The intracellular Shh signalling is transduced by Smoothened (Smo), a seven-transmembrane spanning protein that belongs to the G-protein coupled receptor family. Among four families of G{alpha} subunits, G{alpha}i has been thought to be responsible for transducing Shh signalling, while several lines of evidence indicated that other signalling pathways may be involved. We found that the G12 family of heterotrimeric G proteins and the small GTPase RhoA are involved in Shh/Smo-mediated cellular responses, including stimulation of target gene promoter and inhibition of neurite outgrowth of neuroblastoma cells. We also found that the G12/RhoA pathway is responsible for Smo-induced nuclear import of GLI3 which is thought to transduce Shh signals to nucleus. Furthermore, misexpression of a G12-specific GTPase-activating protein in rat neural tubes leads to pertubation of motor neurone and interneurone development, mimicking the effects of decreased Shh signalling. These results show that Shh signalling is mediated in part by activating G12 family coupled signalling pathways. The participation of RhoA, a pivotal molecular switch in many signal transduction pathways, may help explain how Shh can trigger a variety of cellular responses.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Sonic hedgehog (Shh) is a vertebrate homolog of the secreted glycoprotein Drosophila Hedgehog (Hh) and controls the morphogenesis of various organs (for review, Ingham & McMahon 2001). The first step in Shh action is the binding of Shh to Patched (Ptc), a 12-transmembrane protein. While Ptc serves as the Shh receptor, signal transduction requires a second protein, Smoothened (Smo), that shares the structural features of a seven-transmembrane G-protein coupled receptor (GPCR). In the absence of Shh, Ptc suppresses the activity of Smo at substoichiometrical levels (Taipale et al. 2002), but when Shh binds to Ptc, Smo is de-repressed and capable of inducing Shh signals. In fact, Smo seems to be responsible for all of the cellular responses to Shh since Smo null mutant mice are phenotypically identical with compound mutants for Shh and its close relative, Indian hedgehog (Zhang et al. 2001). Also, Smo ectopically expressed in the dorsal neural tube induces multiple ventral neural cell types that are normally induced by Shh secreted from the notochord and floorplate (Hynes et al. 2000). Recent findings with small non-peptidylic agonists and antagonists of Smo support the notion that Smo acts as a typical GPCR. It has been postulated therefore that Smo activation may depend on a hypothetical endogenous ligand whose access to Smo is controlled by Ptc in a Shh-dependent fashion (Frank-Kamenetsky et al. 2002). Given Smo's GPCR-like properties, it is of critical importance to determine which G proteins, if any, are stimulated following Smo activation.

In Drosophila, Smo transmits Hh signals into the cell nucleus by regulating the activity and intracellular distribution of the zinc finger transcription factor Cubitus interruptus (Ci) (Chen et al. 1999; Wang & Holmgren 2000). It is known that phosphorylation by protein kinase A (PKA) renders Ci susceptible to proteolytic cleavage. Hh signalling reduces the PKA-dependent cleavage of Ci and increases the accumulation of uncleaved Ci (Ci-155) in the nucleus, thereby leading to transcriptional activation of Hh target genes (Ingham & McMahon 2001). It has been speculated therefore that the activation of G{alpha}i, which decreases the intracellular cAMP levels and the activity of PKA, is part of the Shh signalling pathway. Indeed, observations in a frog melanophore system were consistent with the activation of G{alpha}i upon Shh exposure (DeCamp et al. 2000). However, Shh stimulation does not change the intracellular cAMP levels in cultured mammalian cells (Murone et al. 1999), and the blockage of G{alpha}i-coupled receptors by pertussis toxin during zebrafish development did not yield conclusive results (Hammerschmidt & McMahon 1998). Furthermore, pka null mutations in Drosophila do not fully mimic gain-of-function mutations of hh (Wang & Holmgren 2000), and proteasome inhibitors, though interfering with Ci degradation, do not by themselves lead to transactivation of Hh target genes (Chen et al. 1999). These observations suggest that even though a role for G{alpha}i cannot be excluded, additional signalling pathways are likely involved in mediating Shh action.

Here we present evidence indicating that among the members of four distinct families of {alpha} subunits of heterotrimeric G proteins (G{alpha}i, G{alpha}q, G{alpha}s and G{alpha}12), the G{alpha}12 (G12) family plays a significant role in Shh signalling. G12 proteins (which include G{alpha}12 and G{alpha}13) are expressed ubiquitously and activate Rho GTPase and its downstream effectors, Rho-associated kinase (Rho-kinase)/ROK/ROCK. In fact, we found that G12/RhoA/Rho-kinase pathway is involved in a variety of cellular responses to Shh. A link between Shh signalling and G12/RhoA/Rho-kinase pathway may thus help to explain many facets of Shh's action.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
G12 mediates Shh/Smo-induced activation of the Gli1 promoter

In order to examine which G proteins might participate in Shh action, we first tested the effects of constitutively active mutants (G{alpha}QLs, Fromm et al. 1997) of the four G{alpha} families (G{alpha}i, G{alpha}q, G{alpha}s, G12), as well as wild-type Gß1 and G{gamma}2, on the transcriptional stimulation of hgli-Luc, a luciferase-based reporter plasmid containing a fragment of the Gli1 promoter that responds to Shh/Smo-stimulated GLI3 (Dai et al. 1999). These experiments were performed in Hek 293 cells which are commonly used for the analysis of G protein signalling and express G{alpha} proteins of all four families. They also express Ptc1, Smo, three Gli genes (Gli1–3), and are capable of transducing Shh signals as suggested by the facts that hgli-Luc is stimulated upon over-expression of Shh or Smo (Fig. 1B,C,E) and that the expression of the endogenous Ptc1 gene, a known Shh target gene, is increased upon Shh exposure (data not shown).



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  		Figure 1  The G12 family of heterotrimeric G proteins and RhoA mediate Shh/Smo-induced transactivation of the Gli1 promoter. Hek 293 cells (A–C) or Neuro2A cells (E) were transiently transfected with the indicated expression plasmids, hgli-Luc (a Gli1 promoter/firefly luciferase reporter plasmid) and pRL-TK (an internal control Renilla luciferase expression plasmid used for normalization of reporter activity). Reporter activities are shown relative to those obtained in LacZ transfectants (means ± SD from three or more independent experiments. *P < 0.001). (A) Reporter activities after expression of GTPase-deficient, constitutively active mutants (G{alpha}QLs) of the indicated G{alpha} family members or wild-type Gß1 or G{gamma}2. (B) Dose-dependent suppression of Shh/Smo-induced hgli-Luc activation by RGSp115. (C) Dose-dependent suppression of the Shh/Smo-induced activation of SRE-Luc, a serum response element/luciferase reporter plasmid, by RGSp115. (D) Increased amounts of GTP-binding, activated RhoA in Hek 293 cells as determined in a Rhotekin pull-down/Western blotting assay after exposure to recombinant mouse Shh, Shh-N. This increase can be blocked by including the inhibitor Cyclopamine. Total RhoA shown for control. (E) Suppression of Shh/Smo-induced hgli-Luc activation by dominant-negative variants of PDZ-RhoGEF (RGSm/{Delta}DH), RhoA (RhoAN19) or Rho-kinase (Rho-kinaseRB/PH(TT)).

 
As shown in Fig. 1A, while constitutively active forms of G{alpha}i (G{alpha}iQL) and G{alpha}q (G{alpha}qQL) showed only weak activation of the reporter gene, constitutively active forms of G{alpha}12 (G{alpha}12QL) and G{alpha}13 (G{alpha}13QL) activated hgli-Luc 5–6-fold. This suggested that these latter two G proteins play a role in Gli1 activation. Shh- or Smo-stimulated hgli-Luc activity could be suppressed by co-transfection of RGSp115, an expression plasmid for the RGS (Regulator-of-G protein Signalling) domain representing residues 1–246 of p115RhoGEF (Kozasa et al. 1998). This RGSp115 has been shown to have GAP activity specifically toward G12 but not toward the other families (Kozasa et al. 1998). In addition, SRE-Luc, a reporter plasmid under control of the serum response element of the c-fos gene, which responds to G{alpha}12/13 signalling (Fromm et al. 1997), was also activated by Shh or Smo, and this activation could likewise be suppressed by RGSp115 (Fig. 1C). These results imply that G{alpha}12/13 are involved in the stimulation of Shh/Smo-responsive promoters.

RhoA mediates Shh/Smo-induced activation of the Gli1 promoter

Since the above results indicated that Shh signalling activates G{alpha}12/13, and since G{alpha}12/13 are known to activate RhoA (Fromm et al. 1997; Schmidt & Hall 2002), we anticipated that Shh treatment would increase the amounts of the active, GTP-bound form of RhoA. Indeed, as determined by a Rhotekin binding assay shown in Fig. 1D, activated RhoA was increased when cells were exposed to Shh, and this increase was inhibited by co-treatment with Cyclopamine, a steroidal alkaloid specifically blocking the function of Smo (Chen et al. 2002). Furthermore, in the mouse neuroblastoma cell line Neuro2A, over-expression of dominant-negative mutants of PDZ-RhoGEF (RGSm/{Delta}DH) (Driessens et al. 2002), RhoA (Rho AN19) or Rho-kinase (Rho-kinaseRB/PH(TT)) (Amano et al. 1997), all of which interfere with RhoA or Rho-kinase action, suppressed the Shh/Smo-induced activation of hgli-Luc (Fig. 1E) or SRE-Luc (data not shown). Thus, the transcriptional activation of Shh/Smo-responsive promoters is mediated by the G12/RhoA pathway.

The G12/RhoA pathway is responsible for Smo-induced inhibition of neurite outgrowth

One among the many cellular responses triggered by RhoA and Rho-kinase is the inhibition of neurite outgrowth of neuroblastoma cells after serum starvation (Hirose et al. 1998; Togashi et al. 2000). In fact, we found that consistent with the notion that Shh/Smo activate G{alpha}12/13 and RhoA/Rho-kinase, mouse neuroblastoma Neuro2A cells transfected with fluorescently tagged Smo (pDsRed-Smo) lacked neurites despite serum starvation, as did those transfected with haemaggulutinin (HA)-tagged G{alpha}12QL (or G{alpha}13QL, data not shown). In contrast, those transfected with the fluorescent tag alone (pDsRed) or with HA-tagged G{alpha}iQL extended neurites normally (Fig. 2A). Moreover, co-transfection with expression plasmids for RGSp115, or the dominant-negative proteins RGSm/{Delta}DH, RhoAN19 or Rho-kinaseRB/PH(TT), rescued neurite outgrowth in the presence of pDsRed-Smo (Fig. 2B). Thus, the activation of the G12/RhoA/Rho-kinase is responsible for the Smo-induced inhibition of neurite outgrowth in neuroblastoma cells and may explain the previous finding that Shh suppresses axonal growth of retinal ganglion cells (Trousse et al. 2001).



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  		Figure 2  The G12/RhoA pathway mediates Smo-induced inhibition of neurite outgrowth. Neuro2A cells were transiently transfected with the indicated expression plasmids and cultured in serum-starved culture medium. (A) Over-expression of pDsRed-Smo as well as G{alpha}12QL inhibit neurite outgrowth while control pDsRed and G{alpha}iQL do not. (B) The inhibition of the neurite outgrowth by pDsRed-Smo is blocked by either RGSp115, RGSm/{Delta}DH, RhoAN19 or Rho-kinaseRB/PH(TT) Transfectants were immunostained with anti-HA antibody (for G{alpha}iQL, G{alpha}12QL, RhoAN19 transfectants) or anti-myc antibody (for RGSp115, RGSm/{Delta}DH, Rho-kinaseRB/PH(TT) transfectants).

 
The G12/RhoA pathway is involved in Smo-induced nuclear accumulation of GLI3

In Drosophila, Shh regulates Ci in two ways, by stabilizing full length Ci-155 and by inducing the translocation of this full-length protein into the nucleus. While stabilization of Ci depends on pka-mediated phosphorylation, nuclear translocation is pka-independent, suggesting that a signalling pathway distinct from G{alpha}i/PKA might be involved in this latter process (Wang & Holmgren 2000). Among the three vertebrate Ci-homologs, only GLI3 shares a multitude of structural, biochemical, and genetic characteristics with Ci, including the presence of PKA phosphorylation sites (Dai et al. 1999; Wang et al. 2000), proteasome-mediated, phosphorylation-dependent proteolysis (Wang et al. 2000), the capacity to bind the transcriptional coactivator CBP/p300 (Dai et al. 1999), and the ability to function both as a transcriptional activator and repressor in vivo (Litingtung & Chiang 2000). Hence, GLI3 is the closest vertebrate counterpart of Ci, and so we sought to test whether the G12/RhoA pathway of Shh signalling might regulate the nuclear accumulation of GLI3. To visualize GLI3, we used GLI3-EGFP, an expression plasmid encoding human GLI3 protein fused at its carboxyl terminus with the fluorescent EGFP. Consistent with previous reports (Dai et al. 1999; Wang et al. 2000), immunoblot analyses showed that over-expressed GLI3-EGFP was cleaved following treatment of cells with forskolin, which increases intracellular cAMP levels and PKA activity, but not following co-transfection with Shh or Smo (data not shown). Thus, the fluorescent signal of GLI3-EGFP in pDsRed-Smo transfectants likely represents full length GLI3 protein. As reported previously (Shin et al. 1999) and shown in Fig. 3A, the majority of Hek 293 cells coexpressing the control plasmid pDsRed displayed GLI3-EGFP in the cytoplasm (cytoplasmic fluorescence in 79% of co-expressing cells, n = 43). In contrast, co-expression of pDsRed-Smo increased the number of cells with nuclear GLI3-EGFP (nuclear fluorescence in 69% of co-expressing cells, n = 59). In the absence of pDsRed-Smo, GLI3-EGFP did not efficiently accumulate in the nucleus even when the cells were treated with leptomycin B which blocks CRM 1-mediated nuclear export (Kogerman et al. 1999) (data not shown), suggesting that the Smo-induced nuclear accumulation of GLI3 was due to an enhanced nuclear import and not a suppression of nuclear export. This observation further supports the notion that GLI3 is the vertebrate counterpart of Ci whose Hh-induced nuclear accumulation also results from enhanced nuclear import (Chen et al. 1999; Wang & Holmgren 2000).



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  		Figure 3  The G12/RhoA pathway mediates Smo-induced nuclear accumulation of GLI3. Hek 293 cells were transiently transfected with GLI3-EGFP along with the indicated expression plasmids. Transfectants were immunostained with the appropriate anti-tag antibodies. Arrows indicate nuclei. (A) pDsRed-Smo induces nuclear accumulation of GLI3-EGFP while control pDsRed, G{alpha}iQL, G{alpha}12Q do not. (B) Nuclear accumulation of GLI3-EGFP following pDsRed-Smo expression is inhibited by either RGSp115, RhoAN19or Rho-kinaseRB/PH(TT). The constitutively active mutant Rho AV14 alone does not induce nuclear accumulation of GLI3-EGFP. A quantification of the number of cells co-expressing nuclear GLI3-EGFP and co-transfected plasmids/total number of cells expressing GLI3-EGFP and co-transfected plasmids is given in the text.

 
With this co-transfection system in hand, we then tested whether Smo-induced nuclear import of GLI3 depended on the activation of the G12/RhoA/Rho-kinase pathway. In fact, we found a substantial reduction of cells expressing nuclear GLI3 after co-transfection of pDsRed-Smo with RGSp115 (nuclear fluorescence in 17% of coexpressing cells, n = 48), dominant-negative RhoAN19 (15%, n = 60), or dominant-negative Rho-kinaseRB/PH(TT) (11%, n = 36) (Fig. 3B). These results suggested that the G12/RhoA pathway is indeed involved in mediating Smo-induced nuclear import of GLI3. Neither G{alpha}12QL nor a constitutively active mutant of RhoA (RhoAV14) (Amano et al. 1997) increased the number of cells expressing nuclear GLI3-EGFP (8%, n = 49; 7%, n = 42, respectively), suggesting that the activation of the G12/RhoA pathway, though necessary, is not sufficient for nuclear import of GLI3. This interpretation was consistent with the fact that neither RGSp115 nor the dominant-negative proteins RhoAN19 or Rho-kinaseRB/PH(TT) showed complete suppression of the Shh/Smo-induced activation of the Gli1 promoter (Fig. 1B,E). As suggested by the G{alpha}i/PKA independence of Ci nuclear import in Drosophila, co-transfection of G{alpha}iQL did also not affect the number of cells exhibiting nuclear EGFP-GLI3 (15%, n = 54).

Blockage of G12 activation perturbs the development of motor and interneurones

It is well-established that Shh secreted from the notochord and the floorplate of the neural tube governs the development of motor neurones and interneurones (Ingham & McMahon 2001). Specific ventral motor neurones and interneurones are absent in Shh null mutants (Chiang et al. 1996). In explanted chicken neural tube, recombinant Shh induces MNR2- and Isl1/2-positive motor neurones (Ericson et al. 1996; Tanabe et al. 1998) while the addition of Shh-neutralizing antibodies leads to the generation of Lim1/2-positive interneurones at the expense of motor neurones (Ericson et al. 1996). We anticipated, therefore, that the misexpression of RGSp115 in neural tube might perturb the development of these neuronal subpopulations. Using an established system of neural tube electroporation (Takahashi & Osumi 2002), we over-expressed either FLAG-tagged RGSp115 or control EGFP in rat neural tube. Rat embryos were electroporated at E11.5, cultured for 42 h, and then fixed, sectioned, and examined for the expression of RGSp115, EGFP and specific neuronal markers. As shown in Fig. 4A,C, the numbers of MNR2- and Isl1/2-positive somatic motor neurones were reduced and those of Lim1/2-positive interneurones were increased in the RGSp115-expressing, left side of the neural tube compared to the non-expressing, right side of the tube. In contrast, control EGFP revealed no significant differences between the two sides in any of the markers tested (Fig. 4C). Since we observed both increases and decreases of specific neuronal subpopulations, it is unlikely that the RGSp115 effects were due to adverse effects of the electroporation procedure itself. Moreover, the expression of Shh in the floorplate (Fig. 4A) and of the floorplate marker HNF3ß (data not shown) was not altered in RGSp115 or control EGFP-expressing neural tubes, suggesting that the changes seen in motor neurone and interneurone subpopulations were not due to quantitative changes in the synthesis of Shh.



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  		Figure 4  Expression of RGSp115 in electroporated rat neural tube perturbs the development of motor neurones and interneurones. The left sides of the rat neural tubes were electroporated with the expression vectors for either FLAG-tagged RGSp115 (RGSp115) or control EGFP (EGFP). The embryos were then cultured, fixed, cryostat-sectioned and the sections were stained with anti-FLAG antibody for RGSp115 together with either anti-Lim1/2, anti-MNR2/HB9, anti-Isl1/2, or anti-Shh antibodies. Anti-FLAG antibody was revealed with FITC-labelled second antibody while antibodies for the other markers were revealed with appropriate Cy3-labelled second antibody. (A) MNR2/HB9- and Isl1/2-positive somatic motor neurones are reduced, and Lim1/2-positive interneurones are increased in number in the RGSp115-expressing side of neural tube. Overlayed images of green (RGSp115) and red (indicated markers) are shown in ‘Merged’. Note that Shh expression in the floorplate is not altered. (B) Higher magnification of the region highlighted by arrowhead in (A). Many of the Lim1/2-positive cells co-express electroporated RGSp115. (C) Quantification of the changes in the numbers of Lim1/2-, MNR2/HB9- and Isl1/2-positive cells following RGSp115 or EGFP expression. n = 5 sections; mean + SD. (D) Higher magnification of the region marked by white square in Fig. 4A and its corresponding region of EGFP-electroporated neural tube. Many cells showing pyknotic nuclei (labelled blue with Hoechst33342 staining, and colabelled with anti-active Caspase3 antibody in red in insert), were seen in RGSp115-expressing (RGSp115-Exp) but not in EGFP-expressing (EGFP-Exp) neural tube. Overlayed images of green (RGSp115 or EGFP), red (MNR2 staining) and blue (Hoechst) are shown in ‘Merged’. Note that many cells with pyknotic nuclei were positive for RGSp115 and that the number of MNR2/HB9-positive cells was reduced. (E) Quantification of the numbers of apoptotic cells with pyknotic nuclei in RGSp115- or EGFP-electroporated neural tubes (RGSp115-Exp, EGFP-Exp, respectively). n = 5 sections; mean ± SD.

 
The increase in the number of Lim1/2-positive cells resulted from an increase of such cells in the ventral margin of the Lim1/2-positive domain (Fig. 4A, highlighted by arrowhead). Since many of these ventral margin cells co-expressed Lim1/2 and RGSp115 (Fig. 4B), their increased number was likely a cell-autonomous response to RGSp115. Conversely, RGSp115-expressing neural tube showed an increased number of cells with pyknotic nuclei selectively in the somatic motor neurone progenitor (SM) domain where MNR2-positive cells normally develop (white square of Fig. 4A, higher magnification in Fig. 4D, quantified in Fig. 4E). The cells with pyknotic nuclei were stained with an antibody that selectively recognizes the active form of Caspase3, suggesting cell death by apoptosis (insert in Fig. 4D). An increase in the number of apoptotic cells in the SM domain following RGSp115-misexpression is consistent with the fact that Shh null mutant mouse embryos also show apoptotic cells mainly in this domain (Litingtung & Chiang 2000). We previously found that cells in the SM domain strongly express Shh target genes, including Gli1 (Takahashi & Osumi 2002). It is possible therefore that this domain might be particularly sensitive to a decrease in the level of Shh signalling. We conclude that the misexpression of RGSp115 in the neural tube had effects on the development of motor neurones and interneurones that were similar to those resulting from a reduction of Shh signalling (Ericson et al. 1996; Tanabe et al. 1998).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Here we present evidence that the G12/RhoA/Rho-kinase pathway participates in Shh signalling. In a first set of experiments we show that constitutively activated forms of G{alpha}12 and G{alpha}13 stimulate the Shh-responsive Gli1 promoter and also that Shh/Smo-induced activation of this promoter is inhibited by RGSp115, a GAP specific toward G{alpha}12 and G{alpha}13. These results are consistent with the observation that Shh/Smo stimulate the SRE of c-fos gene, a known target of G{alpha}12/13. In a second set of experiments we show that the activated GTP-binding form of RhoA is increased in Shh-stimulated Hek293 cells which was suppressed by co-treatment with Cyclopamine, a steroidal alkaloid that is known to block the function of Smo. We also show that dominant-negative forms of PDZ-RhoGEF, RhoA and Rho-kinase inhibit Shh/Smo-induced activation of Gli1 promoter. The involvement of G12/RhoA/Rho-kinase in Shh signalling is confirmed by experiments showing Smo-induced inhibition of neurite outgrowth in Neuro2A cells and Smo-dependent nuclear import of GLI3 in Hek293 cells. Furthermore, using whole embryo culture system, we demonstrate that misexpression of RGSp115 in developing neural tubes leads to changes in the ratio of interneurones to motor neurones and in regional apoptosis, mimicking the in vivo effects of reduced Shh signalling. The notion of the involvement of RhoA/Rho-kinase in Shh signalling is supported by a recent independent study showing that in transgenic embryos, over-expressiion of dominant-negative RhoAN19 or dominant-negative Rho-kinaseRB/PH(TT) causes a reduction in the numbers of motor neurones by triggering apoptosis (K. Kobayashi, personal communication). Taken together, our studies indicate that the G12/RhoA/Rho-kinase pathway mediates, at least in part, Shh signalling.

Smo inhibits neurite outgrowth in G12/RhoA-dependent manner

Previous studies have shown that the inhibition of neurite outgrowth of neuroblastoma cell lines is one among many cellular reponses that involves the activation of G12 and Rho/Rho-kinase (Hirose et al. 1998; Togashi et al. 2000). Therefore, we reasoned that if Shh signalling activated G12/RhoA, it should affect neurite outgrowth. Indeed, we found that the expression of Smo inhibited neurite outgrowth in neuroblastoma cells and that the co-transfection of either RGSp115 or dominant-negative forms of PDZ-RhoGEF, RhoA and Rho-kinase relieved this inhibition. These findings supported the notion that Shh signalling activates the G12/RhoA/Rho-kinase pathway and are consistent with the previous observation that Shh stimulation suppressed the axonal growth of retinal ganglion cells (Trousse et al. 2001).

The activation of either G12 or Rho also enhances cell proliferation and can lead to cellular transformation (Xu et al. 1993; Sahai et al. 1999; Schmidt & Hall 2002). Acutually, Shh has been shown to promote proliferation of, for instance, neural tube (Britto et al. 2002), cerebellar granular cells (Dahmane & Ruiz i Altaba 1999; Wallace 1999; Wechsler-Reya & Scott 1999), retinal precursor cells (Jensen & Wallace 1997), myoblasts (Duprez et al. 1998), and haematopoietic stem cells (Bhardwaj et al. 2001). Mutations of Ptc1 in human and mouse, which lead to activation of Shh signalling, cause a high incidence of a variety of tumours (for review, Hahn et al. 1999). Furthermore, the human squamous cell carcinoma cell line A431, in which both Ptc1 alleles are mutated, shows PTC1-dependent oncogenic potential including anchorage-independent growth (Koike et al. 2002), and another human cancer cell line, LK-2, secrets Shh and proliferates in a Shh-dependent manner (Fujita et al. 1997). It has not been examined, however, whether Shh-induced proliferation described in above studies is G12- or Rho-dependent, but we observed that Y-27632, a selective blocker for Rho-kinase, inhibits Shh-induced proliferation of other Shh-secreting human carcinoma cells (unpublished data). This observation supports the notion that Rho-kinase is involved in Shh-induced cellular proliferation.

Smo induces G12/Rho-dependent nuclear accumulation of GLI3

In Drosophila all aspects of the intracellular Hh signals are mediated by Ci (Chen et al. 1999; Wang & Holmgren 2000). Full length Ci (Ci-155) primarily localizes in cytoplasm. In the absence of Hh signalling, Ci-155 is proteolytically processed to a truncated form (Ci-75) that accumulates in the nucleus and represses the transcription of Hh target genes. This proteolysis is proteasome-dependent and requires PKA-induced multisite phosphorylation of Ci-155 (Chen et al. 1999). In contrast, Hh signalling stabilizes Ci-155, likely by reducing Ci-155 proteolysis, and leads to nuclear import of Ci-155 and thereby to activation of target genes (Chen et al. 1999; Wang & Holmgren 2000).

Despite these facts, however, blocking Ci-155 processing with proteasome inhibitors is not sufficient to lead to the transactivation of Hh target gene (Chen et al. 1999). Furthermore, in loss-of-function mutants of pka, the intracellular distribution of Ci-155 is not altered (Wang & Holmgren 2000). These observations suggest that the stabilization and the nuclear import of Ci are differently regulated and that pka is dispensable for the nuclear import of Ci-155. In contrast, loss-of-function mutants of smo lack nuclear Ci-155 and ptc mutant cells in which intracellular Hh signalling is constitutively activated accumulate Ci-155 in their nuclei, leading to strong activation of Hh target genes (Wang & Holmgren 2000).

Here we show that Shh signalling induces nuclear accumulation of GLI3, the closest mammalian homolog of Ci-155, in a G12/RhoA-dependent manner. Ci-155 has a leucine-rich nuclear export signal (NES), and an analogous sequence exists in GLI3 and also in GLI1 (Kogerman et al. 1999). However, we and others observed that in the absence of Shh stimulation, GLI1 accumulates in the nucleus following treatment with leptomycin B, a drug that specifically inhibits CRM1-mediated nuclear export of NES-containing proteins (Kogerman et al. 1999; our unpublished data), while we found that the GLI3 distribution was not altered. This evidence suggests that even though both GLI1 and GLI3 have NES, their intracellular distribution is differently regulated. Indeed, we observed that as reported previously (Kogerman et al. 1999), the intracellular distribution of GLI1 was not altered in Smo-transfected cells, in contrast to GLI3 as shown in this paper.

There are indeed precedents for Rho-dependent nuclear accumulation of transcription factors. For example, Rho signalling triggers the phosphorylation and proteolysis of I{kappa}B, and the translocation of p50 homodimers and p50/p65 heterodimers to the nucleus (Perona et al. 1997). It is therefore conceivable that RhoA/Rho-kinase post-translationally modulate the protein complex that tethers the GLI3 protein to microtubules and so allow its nuclear translocation. In Drosophila cells, cytoplasmic Ci-115 associates with multimolecular protein complexes, including the kinesin-like protein Costal2 (Cos2), the serine/threonine kinase Fused (Fu) and its putative suppressor Suppressor-of-Fused (Su(fu)) (Ingham & McMahon 2001). In the absence of Hh stimulation Cos2 binds microtubules to anchor the protein complex in the cytoplasm. Hh signalling is thought to modulate the biochemical status of the complex and lead to nuclear accumulation of some of the members in the complex, including Ci-155. Indeed, Hh signalling was shown to phosphorylate Fu and Cos2 by unknown mechanism (Therond et al. 1996; Fukumoto et al. 2001; Nybakken et al. 2002).

Until today, many substrates of Rho-kinase have been successfully identified (for review, Fukata et al. 2001), including myosin-binding subunit of myosin phosphatase (Matsui et al. 1996; Kawano et al. 1999), vimentin (Goto et al. 1998), and collapsin response mediator protein (CRMP)-2 (Arimura et al. 2000). These studies suggest the consensus sequence of Rho-kinase phosphorylation site is RXXS/T or RXS/T, sequences also found in Fu protein. It is conceivable, therefore, that the increased activity of Rho-kinase after Hh stimulation leads to Fu phosphorylation and in turn regulates the intracellular distribution of Ci. It has not been examined, however, whether phosphorylation of these proteins by itself is sufficient to influence the intracellular distribution of Ci. In any event, it is conceivable that the nuclear import of Ci is mediated by Rho-kinase after G12 activation, while the stabilization of Ci is modulated by PKA after G{alpha}i activation.

The use of positive and negative regulators of the G12/RhoA/Rho-kinase pathway in transfection and electroporation assays strongly suggests the involvement of this pathway in Shh signalling. In fact, a link between Shh signalling and the G12/RhoA/Rho-kinase pathway, which is known to participate in many distinct cellular responses, provides a novel view on how Shh can exert its multiple cellular functions. Based on this evidence, it will be important to examine the relation between Shh/Hh signalling and G12/RhoA/Rho-kinase pathway genetically. In Drosophila, genetic studies place Concertina(cta), encoding a G{alpha} subunit belonging to the G12 family, between the secreted protein Folded gastrulation (Fog) and DRhoGEF2 (Hacker & Perrimon 1998; Morize et al. 1998). Fog binds to a yet unidentified receptor, which triggers cell shape changes during Drosophila gastrulation. However, cta is a maternal effect gene whose mutations also lead to deficient gastrulation at a time point before hh or smo mutations become phenotypically manifest (Parks & Wieschaus 1991). Furthermore, in mice, G{alpha}12/13 double null mutant embryos die at E8.5, that is, at an earlier developmental stage than Smo null mutant embryos die (Gu et al. 2002). We anticipate that future tests with conditional mutations will help overcome these complications.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmids

Expression and reporter plasmids were obtained from multiple sources: G{alpha}QL mutants, wild-type Gß1, G{gamma}2 and the reporter plasmid SRE-Luc from J.S. Gutkind; mouse Shh from P.A. Beachy; mouse Smo from H. Akiyama; myc-tagged RGSp115 from T. Kozasa; HA-tagged dominant-negative RhoA (RhoAN19), constitutive active RhoA (RhoAV14), and myc-tagged dominant-negative Rho-kinase (Rho-kinaseRB/PH(TT)) from K. Kaibuchi; myc-tagged dominant-negative PDZ-RhoGEF (RGSm/{Delta}DH) from K-i. Nagata. The reporter plasmid hgli-Luc was constructed by introducing the human genomic sequence of the Gli1 promoter region (–397 to +216), provided by H. Hahn, into pGL2-basic luciferase reporter plasmid (Promega). RGSp115 and EGFP expression plasmids for the electroporation experiments were constructed by introducing coding sequences of FLAG epitope tagged-RGSp115 or EGFP into the CMV-IE enhancer and chicken ß-actin promoter-based expression vector pCAX (Takahashi & Osumi 2002). GLI3-EGFP was constructed by introducing human GLI3 cDNA (L. G. Biesecker) into pEGFP-N2 (Clontech). pDsRed-Smo was constructed by introducing mouse Smo cDNA into pDsRed-N1 (Clontech), leading to a carboxyl-terminally fused Smo protein. This pDsRed-Smo was found to activate hgli-Luc and SRE-Luc as well as Smo plasmid as shown in Fig. 1.

Luciferase assay

Hek293 cells and Neuro2A cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS). Cells were transferred to 24-well plates and transiently transfected with mixtures of the indicated expression plasmids (150 ng), the firefly luciferase reporter plasmids (100 ng) and the internal control Renilla luciferase expression plasmid pRL-TK (Promega) (20 ng) by using Lipofectamine (Invitrogen). The total amount of DNA was kept constant by adding appropriate amounts of control plasmids (LacZ expression plasmid or appropriate empty plasmid). After a 4 h incubation with DNA-Lipofectamine complex, the cells were washed and cultured in DMEM containing 0.5% FBS. Luciferase activity (Dual-luciferase system, Promega) was measured after 24 h. Luciferase activity was normalized to a Renilla control and the results are shown as mean ± SD from at least three independent experiments, normalized to the results of LacZ transfectants.

Rhotekin pull-down assay

Hek 293 cells were stimulated with recombinant mouse Shh protein (R & D Systems) along with Cyclopamine (Toronto Research Chemicals) or corresponding vehicle. GTP-binding, activated RhoA protein in crude cell lysates was precipitated with the GST-tagged Rho binding domain of Rhotekin (Upstate Biotechnology) and subsequently revealed by anti-RhoA antibody in Western blots (Santa Cruz Biotechnology). The amount of precipitated GTP-RhoA protein was compared based on the amount of total RhoA proteins revealed in crude cell lysates.

Neurite retraction assay and GLI3 nuclear accumulation assay

Cells were plated on poly L-lysine-coated glass chamber slide (IYAKI) and transiently transfected with the indicated combination of expression plasmids as described above. Transfectants were stained with anti-tag antibodies: anti-HA11 monoclonal antibody (Babco) for HA tagged-G{alpha}iQL, G{alpha}12QL, G{alpha}13QL, RhoAN19, RhoAV14; anti-myc monoclonal antibody (Invitrogen) for myc tagged-RGSp115, RGSm/{Delta}DH, Rho-kinaseRB/PH(TT); anti-FLAG monoclonal or polyclonal antibodies (SIGMA) for FLAG tagged-RGSp115.

Neural tube electroporation assay and whole embryo culture

Electroporation and subsequent whole embryo culture have been described (Takahashi & Osumi 2002). After cryostat sectioning, protein expression in electroporated neural tubes was detected by staining with anti-FLAG monoclonal and polyclonal antibodies (SIGMA) for FLAG tagged-RGSp115 plasmid or by visualizing fluorescent signal of EGFP. Motor neurones and interneurones were labelled with the appropriate antibodies generated by T. M. Jessell and collaborators and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA. Anti-active Caspase3 antibody was from Promega.


   Acknowledgements
 
We thank Drs L. Hudson, M. Dubois-Dalcq, J. Battey, L. G. Biesecker, R. D. McKay, and members of LDN/NINDS/NIH for numerous discussions and critical reading the manuscript; Drs J. S. Gutkind, P. A. Beachy, H. Akiyama, T. Kozasa, L. G. Biesecker, K. Kaibuchi, K-i. Nagata for plasmids; Dr A. Nakayama for Neuro2A cells; and Dr H. Hahn for a genomic fragment of the Gli1 promoter. We also thank Dr K. Kobayashi for sharing unpublished information. This work was supported in part by a grant to K. Kasai from Daiko Foundation and to N.Osumi from Uehara Memorial Foundation.


   Footnotes
 
Communicated by: Kozo Kaibuchi

* Correspondence: E-mail: kkasai{at}amugw.aichi-med-u.ac.jp


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 Discussion
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Received: 6 October 2003
Accepted: 29 October 2003




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