An Overview of Hedgehog Signaling Pathway

Proteins of the Hedgehog (Hh) family act as morphogens during development in vertebrate and invertebrate species. These powerful signaling molecules were initially discovered in a genetic screen conducted on a cuticle embryo to better comprehend the body segmentation of Drosophila melanogaster (Nusslein-Volhard and Wieschaus, 1980). In this genetic screen, mutant embryos for Hh developed as tiny prickly balls which were akin to a hedgehog (hence the name of the protein).

First identified in Drosophila melanogaster, the main components of the Hh pathway are preserved in vertebrates, where the pathway has retained the same mechanisms of action via species, albeit with some exceptions. Interestingly, developmental defects and cancer are caused by deregulation of the Hh pathway.

Hh signaling cascade in Drosophila

Hh maturation, release, and movement

Initially synthesized as a precursor, Hh goes through autoproteolytic cleavage where a palmitic acid molecule (Ingham and McMahon, 2001) and a cholesterol molecule (Porter et al., 1996) are added to the end product.

These modifications play an important role by directing the mature signal to communicate with a series of cellular components responsible for secretion, movement, and reception of Hh. Cholesterol, in particular, is involved in the trafficking and movement of Hh (Gallet et al., 2003), while palmitoylation plays a role in Hh signaling (Chamoun et al., 2001; Liu et al., 2007).

Once modified, Hh is ready to be released from the cells (Burke et al., 1999). Following its secretion, Hh easily interacts with the extracellular matrix and navigates through it in order to reach the receiving cells, where it eventually forms a concentration gradient.

A number of models have been suggested to demonstrate the way Hh moves away from its source;  its movement within lipoprotein particles which are unique structures (Bolanos-Garcia and Miguel, 2003; Olofsson et al., 1999), and via its interaction with heparan sulphate proteoglycans (HSPGs) (Jia et al., 2003; Nakato et al., 1995), for example.

At the plasma membrane

At the plasma membrane, Hh signal transduction is initiated where Hh communicates with its 12 transmembrane protein receptor Patched (Ptc) (Ingham and McMahon, 2001). The Ihog/Cdo family of coreceptors facilitates Hh-Ptc interaction (Zhang et al., 2010). The Hh-Ptc binding has two major roles:

1. Limiting the spreading of Hh: Internalization of Ptc and Hh occurs as result of their binding, targeting Hh to lysosomes for the purpose of degradation (Gallet and Therond, 2005).
2. Increase of Smoothened (Smo) expression and activation: (Chen and Struhl, 1996; Denef et al., 2000; Lum et al., 2003; Taipale et al., 2002) this leads to a cascade of signal transmission that plays a role in the regulation of transcription factor Cubitus interruputs (Ci) (Alexandre et al., 1996; Méthot and Basler, 1999).

Following the binding between Ptc and Hh, the seven-pass transmembrane protein Smo goes through a number of phosphorylation events (Hh dose-dependent) (Fan et al., 2012). Phosphorylation of Smo takes place at its cytoplasmic tail (C-tail) that contains a number of phosphorylation sites of GSK3, PKA, and CK1(Zhang et al., 2004). Following are the key results of Smo phosphorylation:

1. Inhibiting ubiquitation-mediated endocytosis and degradation (Fan et al., 2012) to promote the expression of Smo cell surface.
2. Regulating Smo conformation, which takes place on the Smo dimer’s C-tail that results in an ACTIVE (C-tails opening and approach in the presence of Hh) and INACTIVE (C-tails distant from each other in the absence of Hh). Phosphorylation events contribute to this conformation change (Zhao et al., 2007).

Within the cytoplasm

A multi-protein complex (Hh signaling complex, HSC) downstream of Smo regulates the inhibition or activation of the Hh pathway. The components of the HSC complex are as follows:

• The serine/threonine kinase Fused (Fu)
• The transcription factor Ci
• Suppressor of fused (Sufu)
• Costal 2 (Cos2) is a kinesin-like molecule which also adheres to GSK3, PKA, and CK1, and is also implicated in the Hh signaling pathway (Aza-Blanc et al., 1997).

In the absence of Hh (Robbins et al., 1997; Sisson et al., 1997; Stegman et al., 2000), the HSC complex is associated with microtubules but in the presence of Hh it dissociates from the microtubule. The Cos-Fu-Ci complex then interacts with the Smo’s C-tail (Hooper, 2003; Ingham et al., 1991; Lum et al., 2003; Ogden et al., 2003; Ruel et al., 2003), while the Sufu-Ci complex stays cytoplasmic.

The status of the transcription factor Ci is regulated by both Sufu-Ci and Cos-Fu-Ci complexes. Being a 155 kDa protein (Ci-FL, full length), Ci contains a zinc finger domain which accounts for its DNA binding (Slusarski et al., 1995). Ci is transformed to an ACTIVE FORM (Ci-A, 155 kDa) which is responsible for activating a target gene in the presence of Hh, or it is converted to a REPRESSOR FORM (Ci-R, 75 kDa) that stills bind DNA but, in the absence of Hh, inhibits the pathway.

Phosphorylation events, which are mostly under the control of Cos2, mediate the control of the active and inactive form of Ci. Further, Sufu-Ci and Cos2-Fu-Ci complexes, in the absence of Hh, promote the formation of Ci-R and prevent its activation (Robbins et al., 1997; Sisson et al., 1997; Wang et al., 2000; Wang and Holmgren, 2000; Wang and Jiang, 2004; Zhang et al., 2004).

The Cos2-Fu-Ci complex communicates with the C-tail of Smo domains in the presence of Hh. Cos2 phosphorylation regulates this C-tail of Smo domains (Liu et al., 2007; Nybakken et al., 2002; Ranieri et al., 2012; Ranieri et al., 2014; Ruel et al., 2007) and promotes the formation of Ci-A and the resultant activation of the pathway.

Figure 1. Drosophila Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs, such as Dally and Dally-like (Dlp). In the absense of Hh, Ptc inhibits Smo allowing Ci to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Ci to a partial proteolytic cleavage (mediated by Slimb/β TRCP) to generate the repressor form (Ci-R). Binding of Hh to its receptor Ptc and co-receptor Ihog releases Ptc inhibition on Smo, which undergoes phosphorylation mainly by PKA and CK1. Consequently, Smo accumulates at the cell surface recruiting the Cos2-Fu-Ci complex. Here, according to the amount of Hh received by the cell, phosphorylation events on Cos2 and Fu regulate the activation of Ci and therefore of the pathway itself.

Hh signaling orthologues in vertebrates

Three paralogous Hh genes are present in mammals. They include:

• Indian hedgehog (Ihh, mainly involved in bone differentiation)
• Sonic hedgehog (Shh, the best studied and the most widely expressed Hh molecule)
• Desert hedgehog (Dhh, involved in gonad differentiation)

The key difference between Hh signaling in vertebrates and Drosophila is the need for the vertebrate intraflaggular transport (IFT) consisting of large multisubunits complexes that play a role in the bidirectional transport of proteins between the tip cilia and the base (Huangfu et al., 2003).

Both Smo and Ptc are capable of localizing to primary cilia in a mutually beneficial manner, where the binding between Ptc and Shh enables Smo to shift into the cilium and promotes the activation of the pathway via the Gli transcription factors (Rohatgi et al., 2007).

Following are the key differences and similarities between vertebrate and Drosophila Hh signaling:

• The Smo structure is highly preserved between vertebrates and Drosophila. The most interesting fact is that the phospho-sites on the Smo C-tail, as well as their dimerization mechanism, are also preserved, yet the kinases involved are somewhat different (Chen et al., 2011).
• Three Ci homologues called Gli1, Gli2 and Gli3. While Gli3 acts as a transcriptional repressor, the Gli1 and Gli2 functions as transcriptional activators (Ding et al., 1998; Matise et al., 1998; Park et al., 2000; Tempé et al., 2006).
• Vertebrate Sufu is different from Drosophila Sufu and plays an extremely critical role in the Shh pathway (Svärd et al., 2006), but both proteins share high sequence homology (Merchant et al., 2004; Stone et al., 1999).
• kif7 and kif27 – the Cos2 homologues – have preserved their negative role within the pathway by regulating the function and abundance of Gli (Cheung et al., 2009; Tay et al., 2005; Wilson et al., 2009).
• Mammalian Fu is capable of associating with kif27 and plays a role in ciliogenesis, whilst a compensatory Fu kinase, associated with kif7, is required for Hh signaling (Wilson et al., 2009).

The above factors indicate an evolutionary preservation in the Shh intracellular cascade, although more studies are required to gain a better understanding about the molecular mechanisms of the protein involved.

Figure 2. Mammal Hh signal transduction pathway (Chen and Jiang, 2013). The mature Hh molecule reaches Hh receiving cells by binding with HSPGs (such as GPC3, GPC4 and GPC6). In the absence of Hh, Ptc inhibits Smo allowing Gli to be phosphorylated by PKA, CK1 and GSK3. These phosphorylation events target Gli to a partial proteolytic cleavage (mediated by β-TRCP) to generate the repressor form (Gli-R). In the presence of Hh, binding of Hh to its receptor Ptc and co-receptor Cdo releases Ptc inhibition on Smo, which undergoes phosphorylation by mainly CK1 and GRK2. Consequently, Smo accumulates at the cell surface (within the cilia). Sufu is the major negative regulator of the pathway (kif7 is a minor one). In the presence of Hh, Sufu destabilization and degradation allows the release of its repression on Gli, with consequent pathway activation.

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