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. Author manuscript; available in PMC: 2012 Dec 3.
Published in final edited form as: Vitam Horm. 2012;88:55–72. doi: 10.1016/B978-0-12-394622-5.00003-1

Noncanonical Hedgehog Signaling

Donna Brennan *,†, Xiaole Chen *, Lan Cheng *, My Mahoney †, Natalia A Riobo *
PMCID: PMC3513281  NIHMSID: NIHMS423224  PMID: 22391299

Abstract

The notion of noncanonical hedgehog (Hh) signaling in mammals has started to receive support from numerous observations. By noncanonical, we refer to all those cellular and tissue responses to any of the Hh isoforms that are independent of transcriptional changes mediated by the Gli family of transcription factors. In this chapter, we discuss the most recent findings that suggest that Patched1 can regulate cell proliferation and apoptosis independently of Smoothened (Smo) and Gli and the reports that Smo modulates actin cytoskeleton-dependent processes such as fibroblast migration, endothelial cell tubulogenesis, axonal extension, and neurite formation by diverse mechanisms that exclude any involvement of Gli-dependent transcription. We also acknowledge the existence of less stronger evidence of noncanonical signaling in Drosophila.

I. Definition

The main players of the so-called “canonical” hedgehog (Hh) signaling pathway in mammals have been the objects of intense study in the last decade. We have learnt that in the absence of any Hh ligand (Sonic (Shh), Indian (Ihh), or Desert (Dhh)), the receptor Patched1 (Ptc1) prevents activation of the seven-transmembrane protein Smoothened (Smo) by inhibiting its translocation into the primary cilium. In the presence of Hh, Smo accumulates in the cilium in an active conformation that initiates a complex signaling network that results in activation of the Gli family of transcription factors (Riobo and Manning, 2007). However, a vast number of recent studies have demonstrated that not all Hh signaling proceeds through Gli activation, and we have named this subset of Gli-independent responses “noncanonical” Hh signaling (Jenkins, 2009).

In this chapter, we review the current evidence for the existence of at least two distinct classes of noncanonical Hh signaling (Fig. 3.1): a Type I, which works through functions of Ptch1 that are unrelated to its inhibitory activity on Smo, and a Type II, which operates through Smo functions (or activities) beyond Gli regulation.

Figure 3.1.

Figure 3.1

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Schematic representation of the two types of noncanonical Hh signaling. Type I requires only binding of an Hh isoform to Ptc1 and is mediated by novel functions of Ptc1 unrelated to Smo repression, and it is by definition insensitive to Smo modulators. Type II is dependent on Smo and in some cases it has been shown to rely on signaling through Gi proteins, and it is both mimicked by Smo agonists and inhibited by Smo antagonists.

II. Type I Noncanonical Signaling: Pathways Engaged Exclusively by Ptc1

A. Apoptosis

Apoptosis, or programmed cell death, is an essential process for development, tissue homeostasis, and tumor suppression. Hh opposes apoptosis by at least three mechanisms: increased expression of pro-survival genes (such as Bcl-2) through the modulation of Gli transcription (Cayuso et al., 2006; Katoh and Katoh, 2009), activation of the survival kinase Akt (Riobo et al., 2006a), and Type I noncanonical pathway.

Growing evidence suggests that Ptc1 can promote or induce apoptosis independently of the canonical pathway (Chang et al., 2010; Chinchilla et al., 2010; Mille et al., 2009; Thibert et al., 2003). In the absence of an Hh ligand, ectopic expression of Ptc1 induces apoptosis in 293T and neuroepithelial cells (Thibert et al., 2003). This process is independent of the downstream elements of the canonical Hh pathway, as overexpression of Smo cannot prevent cell death. Soft-agar assays also showed that Ptc1 function as a tumor suppressor might be related to its capacity to induce apoptosis. In line with those findings, we found that siRNA-mediated depletion of Ptc1 in endothelial cells extends cell survival in the absence of serum and to limit caspase-3 activation (Chinchilla et al., 2010). We proposed that the pro-apoptotic effect of Ptc1 is exerted through a Type I noncanonical pathway for the following reasons: (1) lack of detectable canonical Hh signaling in endothelial cells both in vitro and in vivo (Chinchilla et al., 2010; Lavine et al., 2008; Pola et al., 2001); (2) lack of effect of the Smo antagonists SANT-1, cyclopamine, and KAAD-cyclopamine to prevent the Shh-dependent reduction in caspase-3 activity (Fig. 3.2); and (3) inability of the Smo agonist (SAG) to mimic the antiapoptotic effect of Shh (Fig. 3.2).

Figure 3.2.

Figure 3.2

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Apoptosis was assessed by caspase-3 activity measurement of serum starved HUVECs in the absence (control) or in the presence of 2.5μg/ml Shh (Shh), 0.5μM Smo agonist (SAG), or Shh plus the Smo inhibitors SANT-1, cyclopamine (CP), or KAAD-cyclopamine (KAAD) (Chinchilla et al., 2010).

Ptc1 has been proposed to function as a dependence receptor, meaning that cell survival is dependent on the presence of the ligand when the receptor is expressed (Bredesen et al., 2004; Mehlen and Thibert, 2004). There are more than 10 dependence receptors, most of which are involved in neural development and tumor suppression. Ptc1 shares the common features for the dependent receptors: (i) in the presence of ligand, receptors transduce signal, leading to the activation of the canonical signaling transcription pathway; (ii) in the absence of ligand, dependence receptors activate a different (noncanonical) pathway and induce apoptosis, which can be reverted by reintroducing the respective ligand; (iii) all dependence receptors contain the DART (dubbed dependence-associated receptor transmembrane) motif (del Rio et al., 2007); and (iv) in order to induce cell death, the receptor needs to be cleaved by a caspase to expose a pro-apoptotic domain. Thibert et al. showed that the C-terminal cytoplasmic domain of Ptc1 is a substrate for caspase-3, -7, and -8 (Thibert et al., 2003). Cleavage of Ptc1 is essential for induction of apoptosis because mutation of the caspase site (D1392N) abolishes Ptc1-induced cell death. Moreover, a truncated mutant constitutively exposing the caspase site Ptc1 (1–1392) induces cell death regardless of the presence of Shh, while full-length Ptc1 pro-apoptotic activity is exquisitely sensitive to Shh, demonstrating the functional importance of caspase cleavage. Further studies shed light on this mechanism, revealing that Ptc1 induces apoptosis by recruitment of a pro-apoptotic complex that includes caspase-9, DRAL, and TUCAN-1 to the C-terminal tail (Mille et al., 2009). However, it is still unknown whether caspase-9 is involved in the generation of the pro-apoptotic Ptc1 (1–1392) and whether recruitment of the complex is a requisite for or a consequence of the cleavage at D1392.

It is worth noting the importance of the C-terminal domain (CTD) of Ptc1, containing 273 aa, in the regulation of cell death. The CTD alone (soluble) or a fragment truncated at the caspase cleavage site, Ptc1 (1165–1392), induces cell death in 293T and neuroepithelial cells at similar extend as the full-length Ptc1 (Thibert et al., 2003). Mutation of the cleavage site (D1392N) acts as a dominant negative inhibitor for Ptc1-induced apoptosis. The CTD was suggested to also be a critical regulator of degradation and localization of Ptc1 (Chang et al., 2010; Kawamura et al., 2008; Lu et al., 2006). In addition, it was recently reported that Ptc1 undergoes proteolytic processing at the C-terminus and the soluble CTD translocates to the nucleus and mediates a new form of signal transduction (Kagawa et al., 2011). In Drosophila, the CTD is indispensible for repression of the canonical pathway when Hh is absent (Johnson et al., 2000). However, studies using mammalian cells and mouse models suggest that in mammals, the CTD is not required for the canonical signal transduction but most likely has a distinct function in apoptosis and/or regulation of proliferation (Makino et al., 2001; Mille et al., 2009; Nieuwenhuis et al., 2007; Sweet et al., 1996; Thibert et al., 2003). A spontaneous Ptc1 mouse mutant has a frameshift in the CTD that gives rise to retention of only the first 53 aa after the last transmembrane domain and addition of 63 nonsense residues. Named mesenchymal dysplasia (Ptc1mes), the mice exhibit epidermal hyperplasia, increased musculature and preaxial polydactyly (Makino et al., 2001; Nieuwenhuis et al., 2007; Sweet et al., 1996). Compared with Ptc1−/− homozygotes, which die between embryonic days 9 and 10.5 with severe neural tube defects as a result of hyperactivation of the canonical Hh signaling (Goodrich et al., 1997), the Ptc1mes/mes embryos exhibit normal spinal cord development, suggesting that most of the CTD is absolutely dispensable for canonical signaling in mammalian embryogenesis. Studies using Ptc1−/mes mice (Makino et al., 2001) also demonstrated that Ptc1mes can substitute a wild-type allele with regard to the canonical Hh pathway (Nieuwenhuis et al., 2007). However, the same report also indicates that the CTD plays an important role in body size control and skin development since the Ptc1mes/mess animals show a marked increase in proliferation of the basal cell layer of the skin (Nieuwenhuis et al., 2007). While they did not observe changes in cell death, the result is inconclusive due to the technical limitations on measuring the apoptosis in that system.

A different mouse model to study the function of Ptc-1 CTD in tumor suppression is a natural polymorphism (T1267N) responsible for a 600-fold change in susceptibility to develop Ras-induced skin squamous cell carcinomas in FVB versus C57BL/6 mice. The polymorphism was shown to reduce the binding of Ptc1 to Tid1, a tumor suppressor protein (Wakabayashi et al., 2007). Direct interaction of Ptc1 with other potential apoptosis-related proteins has also been reported, as mentioned above (Mille et al., 2009). Shh also activates the ERK pathway independently of Smo, probably through modulating the interaction of the SH3 interacting motif of Ptc1 CTD with SH3-containing proteins like Grb2 (Chang et al., 2010). Taken together, these findings strongly support the notion that the mammalian Ptc1 CTD functions in the induction of cell death independently of the canonical Hh signaling pathway.

B. Cyclin B1 and cell cycle regulation

A large number of studies have demonstrated that the canonical Hh signaling pathway plays an essential role in proliferation of some cell types, the best-characterized example being postnatal proliferation of cerebellar granule precursor cells (Dahmane and Ruiz i Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999). Induction of n-myc and cyclin D1 by Gli-dependent transcription mediates the mitogenic effect of Shh in those cells (Kenney and Rowitch, 2000; Kenney et al., 2003). Cyclins are a family of proteins and act as regulatory subunits for cyclin-dependent kinases, and these complexes are responsible for regulating cell cycle progression through the cell cycle by directly affecting a cell’s transcriptional regime (Murray, 2004). Dynamic modulation of the expression, phosphorylation, interactions, and subcellular localization of these complexes and their individual subunits all influence the activity of these complexes, and thus cell cycle progression. The Cyclin B1–Cdk1 complex (also known as M-phase promoting factor (MPF)) is specifically involved in the G2/M checkpoint, and its activation is essential for mitotic progression (Takizawa and Morgan, 2000). The activated form of this complex contains a series of phosphorylated serine residues on Cyclin B1. Interestingly, noncanonical Hh signaling also regulates the cell cycle at the level of Cyclin B1. An early report detected Ptc1 in a yeast two-hybrid assay designed to identify binding partners of phosphorylated Cyclin B1 (Barnes et al., 2001). Biochemical analysis revealed that Ptc1 interacts with phospho-Cyclin B1, but not with the unphosphorylated protein, through the large intracellular loop between transmembrane domains 6 and 7. Overexpression of Ptc1 in 293T cells resulted in a redistribution of Cyclin B1 from the nucleus to the cytoplasm and reduced cell proliferation, which was restored by addition of a Cyclin B1 mutant that cannot be phosphorylated. In a later study, the same group demonstrated that the Ptc1Q688X, a truncation mutant found in some basal cell carcinomas (BCCs), is unable to interact with Cyclin B1 and, unlike wild-type Ptc1, did not reduce the mitotic index (Barnes et al., 2005).

The identification of Ptc1 as a binding partner of Cyclin B1 suggested a new mode of cell cycle regulation by Hh signaling. Indeed, the presence of Shh disrupts Ptc1 interaction with Cyclin B1, allowing its nuclear translocation and promoting completion of mitosis. The regulation of Cyclin B1 by Shh/Ptc1 was not limited to overexpression experiments but was also evident with the endogenous proteins. These results support the notion that Shh regulates proliferation by modulating the interaction between Ptc1 and phosphorylated Cyclin B1 via a noncanonical signaling pathway.

While those studies were conducted in vitro, several histological observations are in line with those findings. Work characterizing Shh expression pattern in human urinary tract development revealed a correlation between Shh expression and Cyclin B1 localization: higher levels of Shh correlated to cytoplasmic and nuclear Cyclin B1, while when Shh levels go down Cyclin B1 relocalizes to the apical membranes together with Ptc1 (Jenkins et al., 2007). Another study showed that epidermal ablation of Ptc1 in mice coincided with an increase in nuclear Cyclin B1, when compared to normal epidermis (Adolphe et al., 2006). Wang et al. demonstrated that epidermal tumors that arise in Ptc1+/− mice are derived from hair follicle progenitor cells and are characterized by increased nuclear Cyclin B1. This is different from tumors developed in the SmoM2 mice model, which express a constitutively active from of Smo (Wang et al., 2011; Youssef et al., 2010). They suggested that nuclear Cyclin B1 and not increased Smo activity results in hair follicle-derived tumors. These data allude to a possible mechanism by which loss of Ptc1 can lead to tumorigenesis independently of the canonical Hh pathway, though more work will need to be done to determine if this alternative pathway is sufficient to induce tumor formation or synergizes with enhanced Gli1 activity.

Finally, studies in zebrafish provided some mechanistic insight into the regulation of Ptc1–Cyclin B1 interaction (Jiang et al., 2009). The authors reported that dissociation of the Ptc1–Cyclin B1 complex after Shh activation involves the formation of another complex between Ptc1 and G-protein receptor kinase-2 (GRK2). GRK2 silencing by siRNA abrogated the ability of Shh to redistribute Cyclin B1 to the nucleus, and exogenous GRK2 expression was able to rescue the antiproliferative phenotype associated with Ptc1 overexpression. Taken together, these results suggest that Shh stimulation may induce a conformational change in Ptc1 that increases its affinity for GRK2 at the expense of its interaction with Cyclin B1.

In summary, there is significant evidence for a noncanonical role for Ptc1 in cell cycle regulation through Cyclin B1 in an apparently Smo- and Gli-independent manner (Type I noncanonical signaling). The relative contribution of this noncanonical pathway on overall cell growth and survival mediated by Hh signaling remains to be established.

III. Type II Noncanonical Signaling: Pathways Engaged by Smoothened

A. Small GTPases

The discovery that Smo is a functional G-protein-coupled receptor (GPCR) with selectivity toward heterotrimeric Gi proteins had profound implications on Hh signaling (Riobo et al., 2006b). Several GPCRs have been shown to activate small GTPases, which play key roles in cytoskeletal reorganization (Ridley, 2006). Small GTPases are monomeric G proteins that act as molecular switches and, thus, rapidly regulate cellular processes (Vetter and Wittinghofer, 2001). Activated by guanine exchange factors (GEFs), they bound GTP in their “on” state, when they are able to interact with and activate their molecular targets. GTPases are then inactivated by nucleotide hydrolysis to GDP, an intrinsically slow process, enhanced by GTPase-activating proteins (GAPs). Small GTPases can be divided into four families: Ras, Rho, Arf, and Rab. The Rho family, which has been shown to mediate cytoskeletal reorganization, is further divided into three subfamilies: Rho, Rac, and Cdc42. These subfamilies differentially modulate distinct cytoskeletal rearrangements through polymerization of actin filaments, and these unique rearrangements can then regulate coordinated movement of the cell.

Recent work demonstrated that Hh proteins promote actin stress fiber formation and endothelial cell tubulogenesis in a Smo-dependent manner in endothelial cells (Chinchilla et al., 2010). Interestingly, while endothelial cells express all key proteins of the Hh signaling pathway, they are unable to activate the canonical pathway in response to Hh ligands, as measured by activation of the marker Gli-target genes gli1 or ptch1. However, the human umbilical vein endothelial cells (HUVECs) and human cardiac microvascular endothelial cells (HMVECs) utilized in this study exhibit strong morphological changes in response to Shh, Ihh, or Dhh stimulation. In this system, stimulation by Hh ligands results in the formation of actin stress fibers within minutes of treatment, thus suggesting a noncanonical pathway based on the time course and the lack of detectable Gli-dependent transcription. The acute cytoskeletal changes were shown to depend on Smo- and Gi-protein-mediated activation of the small GTPase RhoA (Fig. 3.3). In addition to short-term changes, stimulation of endothelial cells by the three Hh ligands resulted in a significantly increase in tubulogenesis after several hours by a process that was also the result of Smo-mediated, Gi-dependent activation of RhoA. Interestingly, this study also demonstrated that, unlike the canonical paradigm whereby the different Hh ligands exhibit distinct potencies for Gli activation (Shh≫Ihh>Dhh) (Pathi et al., 2001), endothelial cells responded equally to all three ligands in terms of stress fiber formation, RhoA activation, and tubulogenesis.

Figure 3.3.

Figure 3.3

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The three Hh isoforms promote tubulogenesis in endothelial cells by a Gi protein and RhoA-dependent mechanism. (A) HUVECs were cultured in a 3D collagen Type I matrix for 24h in the absence (control) or presence of Shh, Ihh, or Dhh (all at 2.5μg/ml). Photographs are representative tube densities. (B) Quantification of tube density in the absence (control) or presence of Shh, Ihh, and Dhh, and in the presence of Shh after preincubation with 0.5μM KAAD-cyclopamine (KAAD, Smo inhibitor) or 100ng/ml Pertussis toxin (PTX; inhibitor of Gi protein activation). (C) HUVECs were serum staved for 24h and stimulated with Shh, Ihh, or Dhh (all at 2.5μg/ml) during 15 min. Active RhoA (RhoA-GTP) was pulled down from whole cell lysates with a Rhotekin-binding domain–GST fusion protein coupled to GSH-sepharose beads. Active and total RhoA were evaluated by Western blot, and the densitometric values are shown in the bar graph (n=3). (D) RhoA pull-down assay as in (C) in cells preincubated with 0.5μM KAAD-cyclopamine (KAAD) or 100ng/ml PTX (n=3) (Chinchilla et al., 2010).

Another study demonstrated that Shh stimulates fibroblast migration via activation of small Rho GTPases (Polizio et al., 2011). In this system, both Shh and purmorphamine (PUR, a potent SAG) enhance migration of NIH-3T3 fibroblasts but have no effect on Smo−/− MEFs. Inhibition experiments demonstrated that activation of both Gi and PI3K by Smo was necessary for cell migration and activation of RhoA and Rac1. To confirm that these effects were indeed noncanonical, the canonical pathway was inhibited by the expression of Gli3R, the repressor form of the Gli3 transcription factor. Effective inhibition of the canonical pathway was confirmed, but expression of Gli3R did not perturb Shh- or PUR-mediated migration, indicating that fibroblast migration is affected by a noncanonical Hh signaling pathway.

While these studies provide solid evidence that Smo-dependent, noncanonical Hh signaling elicits specific cellular responses via the activation of small GTPases, the mechanism by which Smo or Gi activates RhoA and Rac1 has not been yet established. A recent report looking at Hh signaling during dendritic spine formation sheds some light on this issue (Sasaki et al., 2010). Tiam1 (T-lymphoma invasion and metastasis) is a GEF specific for Rac1 that localizes to dendritic spines via interactions with spine-specific proteins. The Tiam1–Rac1 cascade is activated in response to Shh stimulation, without parallel activation of the canonical Hh pathway. Results indicated that both stimulation by Hh ligand and depletion of Smo via siRNA resulted in an increase in spine formation. Subsequent binding studies demonstrated that Smo interacts directly with Tiam1 and sequesters it, thus preventing Rac1 activation. This complex dissociates upon Smo activation, which in turn allows Tiam1 to activate Rac1. While these studies do not ascertain the requirement of Gi activity, they do provide a direct mechanism by which Smo activation leads to Rac1 activation. It will be interesting to see if these types of interactions are common to Smo-mediated activation of small GTPases and whether or not these events are Gi-dependent.

In summary, there is considerable evidence for Smo-dependent, noncanonical Hh signaling that is mediated through the activation of small GTPases. Future studies designed to elucidate the exact mechanisms by which Smo activates these molecular switches have the potential to identify specific molecular targets for modulating noncanonical Hh signaling responses as well as provide insights into the diverse phenotypes observed when Hh signaling is dysregulated.

B. Src activation in the axonal growth cone

The identification of a noncanonical Hh signaling pathway playing a role in cytoskeleton remodeling is reminiscent of the identification, over the past few years, of noncanonical Wnt and BMP signaling pathways. Before the identification of these noncanonical signaling pathways, all of the functions of Hh isoforms were thought to be executed by alteration of gene expression by the Gli family of transcription factors. Recently, it has been demonstrated that Shh can signal through stimulating the activity of Src family kinase (SFK) members in a Smo-dependent manner to control axon guidance (Yam et al., 2009). In this study, axons of dissociated commissural neurons placed in a Shh gradient turned rapidly toward increasing concentrations of Shh in a transcriptional-independent manner. Instead, Shh stimulates the activity of SFK members (Src and Fyn) in a Smo-dependent fashion. This study established the existence of a transcription-independent Shh signaling pathway, which acts locally at the axonal growth cone. Through this pathway, Shh gradients can elicit a rapid and spatially polarized response within the growth cone, without retrograde signaling to elicit transcription in the nucleus to induce global gene expression changes in the cell. It has been proposed that a graded activation of SFKs in response to a Shh gradient mediates changes in the actin cytoskeleton (Liu et al., 2007; Robles et al., 2005; Suter and Forscher, 2001). However, the regulation of Src by a noncanonical Hh pathway appears to be even more complex. In a screening for Ptc1-interacting proteins containing SH3 domains, it was found that the C-terminal tail of Ptc1 (1163–1435) binds to Src (Chang et al., 2010). These finding define a novel signaling cascade operating through SH3 domain-containing proteins that could be directly stimulated by the Hh ligands. Together, both studies suggest that Shh is able to stimulate Src activity via both Type I and Type II noncanonical signaling pathways, and that the latter is preferred in axonal guidance.

Contributions of the canonical Hh pathway to Src activation are unlikely since (1) Hh-responsive transcriptional reporters have failed to identify Src or Fyn as components of the canonical Hh signaling pathway (Lum et al., 2003; Nybakken et al., 2005; Varjosalo et al., 2008) and (2) SFK mutant mice lack phenotypes reminiscent of dysregulated canonical Shh signaling (Kuo et al., 2005).

C. Arachidonic acid metabolites

Arachidonic acid (ARA) is an important signaling molecule that mediates many cellular functions including inflammation and actin remodeling (Glenn and Jacobson, 2002; Peppelenbosch et al., 1993). ARA is synthesized in the cell upon receptor stimulation and can act directly as a second messenger or can be further metabolized into leukotrienes and prostaglandins by the action of lipoxygenase or cyclooxygenase, respectively (Funk, 2001).

Recent studies have found that ARA metabolites, particularly leukotrienes synthesized by the lipoxygenase pathway, are involved in Gli-independent Hh signal transduction. In fibroblasts, Shh induces the formation of lamelli-podia and promotes migration (Bijlsma et al., 2007; Polizio et al., 2011). A role of leukotrienes was proposed since these responses were partially inhibited by treatment with 5-lipoxygenase inhibitors, could be mimicked by addition of ARA to cell culture medium, and the ARA metabolite leukotriene was upregulated in the cells following Shh stimulation (Bijlsma et al., 2007). These results strongly support the speculation that ARA, together with trimeric G-protein-dependent activation of RhoA and Rac1, is one of the signals that mediate Shh-induced cytoskeletal rearrangements. This study also demonstrated that Shh-induced cell migration is Smo dependent but Gli independent (Type II noncanonical pathway). The authors claimed the following reasons for their conclusion: (1) acute response (only minutes after addition of Shh) and (2) insensitivity to overexpression of Sufu, a negative regulator of the Gli transcription factors.

The same group also reported that leukotrienes mediate the formation of neurite projections in motor neurons differentiated from embryonic stem cells (ESCs) treated with Shh (Bijlsma et al., 2008). Neurite formation is also Gli independent and mediated by Smo, since this response occurs in wild type and ESC deficient in various combinations of Gli transcription factors, but not in Smo-deficient ESCs. In conclusion, these studies point to ARA and leukotrienes as novel players in Type II noncanonical Hh signaling in fibroblast migration and neuronal development.

D. Calcium transients

Calcium ions regulate a vast number of neuronal cell functions, including proliferation, differentiation, apoptosis, and migration (Berridge, 1998; Komuro and Rakic, 1996; LoTurco et al., 1995). Stimulation of transient elevations of intracellular calcium (Ca2+i) activates protein kinases, regulates transcription, and influences motility and morphology (Catterall, 2010). It is not surprising that Ca2+has also been shown both to influence expression of some Hh pathway genes and to mediate some cellular responses to Hh signaling.

Calcium has repeatedly reported to induce expression of Hh ligands. It was reported that Ca2+ chelation inhibits ihh gene expression in chick chondrocytes (Zuscik et al., 2002). Also, classical protein kinase C (PKC) isoforms, which are activated by Ca2+ release, sustain shh gene expression in the chick wing bud (Lu et al., 2001). In mouse primary gastric cultures, gadolinium-, thapsigargin-, and carbachol-mediated release of intracellular Ca2+ induces Shh expression as well (El-Zaatari et al., 2010). In contrast, Ca2+-chelation with BAPTA–EGTA reduced Shh expression. Thus, during gastric acid secretion, intracellular calcium release and PKC activation stimulate shh gene expression.

On the other hand, since Smo was proved to act as a GPCR, and stimulation of GPCRs often engages second messengers such as Ca2+, the interplay between Ca2+ transient and Shh signaling was recently investigated. Belgacem and Borodinsky reported that recombinant N-terminal Shh peptide acutely increases Ca2+ spike activity in a dose-dependent manner in the developing spinal cord (Belgacem and Borodinsky, 2011). This effect is mimicked by a SAG and is prevented by cyclopamine, a Smo antagonist. They further demonstrated that this effect depends on both extracellular Ca2+ and intracellular Ca2+ stores and on Pertussis toxin (PTX)-sensitive heterotrimeric Gi proteins. While the exact mechanisms are not yet clear, they proposed that activation Smo, resulting in the activation of a PTX-sensitive Gi protein, leads to activation of Phospholipase C-γ (PLC) and increases IP3 generation. Opening of IP3R-operated stores will deplete intracellular Ca2+ stores, which leads to the subsequent activation of transient receptor potential channel 1 (TRPC1) and voltage-gated channels (Cav), resulting in increased Ca2+ spike activity. This hypothesis is supported by the observed inhibition of Ca2+ spike activity by inhibitors of PLC, IP3R, and TRCP1 channels. Also in support, the authors found that acute stimulation of Smo with SAG results in sequential IP3 and Ca 2+ transients in the primary cilia of neurons, and that this effect is abolished by the Smo inhibitor cyclopamine.

This provoking finding suggests that Hh signaling might regulate a cohort of physiological processes involving fluctuations of Ca2+ that acutely affect membrane potential and Ca2+-dependent signaling pathways.

IV. Noncanonical Hh Signaling in Drosophila

The concept of noncanonical Hh signaling has not been formally extended to Drosophila, despite the existence of an old controversy about a subset of Hh target genes that seem to be independent of the Gli ortholog Cubitus interruptus (Ci). A large number of studies on Hh signaling in Drosophila focus on the effect of mutations that alter the body segment pattern at the larva stage. Each segment has a denticle-cover anterior part and a naked cuticle posterior part. Drosophila Hh is expressed and secreted from two rows of cells in the posterior compartment, from where it patterns the expression of wingless (wg), the Wnt ortholog, and of rhomboid (rho), ortholog of the mammalian EGFR protein. Absence of Hh results in lack of expression of wg and rho resulting in abnormal segmentation and lack of specification of the naked cuticle, leading to an “Hh-like” larva. If all Hh signaling in Drosophila is channeled through Ci activation, thus absence of Ci should result in a phenotype identical to that of the hh mutant. However, Gallet et al. showed that a null allele of Ci (ci94) has a much weaker phenotype than the Hh null (hhAC) (Gallet et al., 2000). Moreover, overexpression of Hh in a Ci-deficient background induces early stage expression of wg and a stronger expression of rho than in the presence of Ci, indicating that these are noncanonical effects. This group further demonstrated that expression of rho is mediated by the C2H2-zinc finger transcription factor Teashirt (tsh), which would act redundantly with Ci to transduce Hh signaling in the fly.

Another group presented a different point of view: instead of activating the pathway by Hh overexpression, they used two ptc-null alleles (ptcIIW and ptcS2) in the Ci-null background and analyzed the cuticle phenotype but not wg and rho expression (Méthot and Basler, 2001). While they agreed that the cuticle phenotype of ci94 is different from hhAC, they showed that an allele encoding just the repressor form of Ci (ciCell) resembles more closely the Hh-null phenotype. Notwithstanding, some stripes of naked cuticle are still present, suggesting that Ci-independent signaling may occur. In addition, this group analyzed the requirement for Ci in Hh-dependent imaginal wing disc patterning using somatic recombination techniques. In this model, clones that lack Ci cannot induce expression of engrailed (en) or ptc, when the Hh pathway was activated by loss of function of Ptc. The authors concluded that all Hh signaling in Drosophila requires Ci activity, either the activator or the repressor. However, an alternative explanation is that, as demonstrated in vertebrates, some Ci-independent signaling could be independent of Smo, for example, mediated by other Hh-interacting proteins (iHog, Boi, Dsp, etc.) and thus cannot be induced by removal of Ptc but is evidenced by Hh overexpression.

Another aspect of fly development that appears to utilize a noncanonical Hh pathway is the formation of the Bolwig’s organ, a light-sensing organ at the larval stage. Hh induces expression of atonal (ato) in the precursors of the Bolwig’s organ and that ato expression can be induced by loss of Ptc and blocked in a smo mutant (Susuki and Saigo, 2000). Elegant genetic analysis revealed that (1) loss of Ci does not affect Bolwig’s organ formation or ato expression, (2) an activator form of Ci (ciZnC) cannot rescue ato expression in an Hh mutant, and (3) the Ci repressor (ciCell) also fails to rescue the Hh mutants. These observations strongly argue in favor of the existence of at least some form of Ci-independent Hh signaling in Drosophila.

V. Concluding Remarks

We have presented ample evidence that noncanonical Hh signaling regulates key physiological functions in mammals during development and after birth. The recognition of those Gli-independent functions has been delayed due to the earlier consensual notion in the research community that true Hh-dependent effects are those requiring Smo and Gli activity, which was initially assessed using genetic models and later with pharmacological and molecular biology tools. The availability of recombinant active Hh isoforms and more specific inhibitors leads to discredit such belief and opened up a new area of research that teaches us that Hh signaling is more complex and regulates more physiological processes than previously thought.

Abbreviations

ARA

arachidonic acid

Ci

Cubitus interruptus

CTD

C-terminal domain

Hh

hedgehog

Ptc1

Patched1

Shh

sonic hedgehog

Smo

Smoothened

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