User:Kinkreet/MCBII/Hedgehog

Many pathways are involved in animal patterning, they include hedgehog, Notch, Wnt, epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF) and bone morphogenetic protein/transforming growth factor β (BMP/TGFβ) pathways as well as other peptide growth factor–tyrosine kinase receptor intracellular signaling cascades.

The Wnt and hedgehog signalling pathways are integrated and cooperates together, most notably because they share a common regulator - GSK3. Hedgehog interacts with other transcription factors as well as other developmental genes, e.g. Nkx2.2 and Pax6.

Genetics
There are many similarities between Hedgehog and Wnt signalling; for one, they are involved in development of organs - of motor neurons, of digits and limbs, of the closing of the neural tube, of cell maintenance. Hedgehog is viewed to be similar to Wnt. In vertebrates, there are known to be 3 or more hedgehog proteins (compared to 19 Wnt proteins in humans); they are sonic hedgehog (SHH), desert hedgehog and Indian hedgehog. The latter two are less studied and are thought to be involved in eye development. All the hedgehog proteins are conserved (like Wnt), but have different roles in different species.

There are 2 Ptc genes (Ptc 1 and Ptc 2) as well as three Ci homologues known as Gli1, Gli2 and Gli3. All Gli proteins are transcription factors with 5 zinc-binding domains (zinc fingers).

Function
Hedgehog was a gene first identified in Drosophila which was responsible for segmentation during development. Drosophila embryos which lacks this gene did not develop normally and gave a phenotype which resembles a hedgehog, hence its name. Therefore, hedgehog proteins can be thought of as morphogens.

Therefore, hedgehog signalling is very important in embryogenesis, especially in pattern formation and differentiation. It works by forming a concentration gradient from specific sites which expresses hedgehog, and based on a specific concentration threshold of a specific hedgehog protein, in concert with other signals, a cell will differentiate in a certain way. The translation of a concentration gradient to actvity of the cell is hypothesised to occur according to the Gli code. It suggests that the signal from all three Gli proteins (Gli1, Gli2 and Gli3), both activating and inhibiting, are integrated at the same time, to specifically control the expression of genes.

Therefore, the production, release and trafficking must be precisely regulated; dysregulation often lead to birth defects and cancer.

It is known to be involved in determining ventral fate in nervous system, anterior-Posterior axis of limb, vasculogenesis, bone and cartilage formation, lung branching and maintenance of stem cells. Apart from inducing/repressing transcription, hedgehog signalling can also affect cell motility, and regulates cytoskeleton by acting as a chemoattractant. Hedgehog signalling remains important in maintaining of the pool of self-renewing stem cells population during adulthood.

Synthesis
Hedgehog signals are synthesized as a precursor which undergoes auto-proteolytic cleavage of its signalling sequence, to give a 19kDa N-terminal fragment (N-Hh). The N-Hh is processed further; N-Hh has a slightly larger C-terminus which gets cholesterol moieties added to it, and palmitate added to the N-terminus by the acyltransferase Skinny Hedghog (Skn) - also known as Central missing (Cmn), Rasp and Sightless.

Dispatched (Disp) is a 12-pass transmembrane protein that helps membrane-tethered Hh to be released, and Tout-velu (Ttv) regulates proteoglycan synthesis, which allows Hh to signal over a long distance.

These lipid modifications are important, but their specific role is not well understood.

Receptor-specific cells
The hedgehog signal are transported out of the cell as multimers, which makes them soluble and able to diffuse. Cells which display glypicans (heparan sulfate proteoglycans bound to plasma membrane via GPI anchor), megalin (a LRP) or Patched/iHog on the plasma membrane can bind to a hedgehog protein. When bound by megalin, some of the Hh signals are degraded while some are trafficked to other areas of the cell membrane, and gets passed along cell-by-cell.

Diffusion
Hedgehog can also move through a tissue by simple diffusion, the mechanism which is required for its role as morphogen.

Indirect Signalling Cascade
A cell which has a received the hedgehog signal can produce a signal of its own which gets passed on to other cells.

Cytonemes
Cytonemes are long and thin tubes formed from the plasma membrane that connect different animal cells over long distances. Two types of nanotubes have been observed. The first type are less than 0.7 micrometres in diameter, contain actin and carry portions of plasma membrane between cells in both directions. The second type are larger (>0.7 μm), contain both actin and microtubules and can carry components of the cytoplasm between cells, such as vesicles and organelles.

Signal Absent
In Drosophila, Patched (Ptc) is a 12-pass hedgehog receptor     which is located on the plasma membrane; Smoothened (Smo) is a 7-pass transmembrane protein which transduces hedgehog signalling, its structure is similar to Fz (might be evolutionary linked). In the absence of a hedgehog signal, Ptc, via Vitamin D3, keeps Smo inactivated in an intracellular endosome and promotes Smo's degradation by destabilizing it using lipids.

Cubitus interruptus (Ci, a transcription factor), Fused (Fu, a serine/threonine kinase), Costal 2 (Cos2, a kinesin-like molecule) and Supressor of fused (Sufu) forms the Hedgehog signaling complex (HSC). In the absence of hedgehog signals, the HSC remains bound to microtubules and membranes using Cos2. The HSC forms a complex with Smo using Cos2. Cos2 also binds to protein kinase A (PKA), protein kinase CK1 (formerly casein kinase 1) and glycogen synthase kinase 3 (GSK3), which are other kinases that are implicated in the Hedgehog signaling pathway.

Ci in the HSC is analogous to beta-catenin in Wnt signalling, in that it is the effector molecule. When Ci is cleaved, it acts as a repressor for transcription; when it is whole, it acts as a transcriptional activator.

In the absence of a signal, Sufu and Cos2 is associated to Ci, and prevents its translocation to the nucleus. PKA, CK1 and GSK3 associated with Cos2, phosphorylates and ubiquitinylate Ci, directing it for degradation in a proteasome, which cleaves it into a small protein (repressor form), and this enters into the nucleus and acts as a transcriptional inhibitor of the hedgehog genes.

Signal Present
When there is a hedgehog signal, the protein binds to Ptc and its co-receptor iHog. iHog is a single-pass protein containing multiple immunoglobulin and fibronectin type III (FNIII) repeats. The first FNIII repeat binds to the HhN in a heparin-dependent manner; iHog is called CDO or CDON in mammals. The binding of HhN to Ptc and iHog induces the activation of Smo via oxysterols. It also induces the endocytosis of Patched; this stops the inhibition on Smo. The HSC is then freed from being bound to microtubules and membranes. Smo is then phosphorylated by PKA and CK1, the two kinases associated with Cos2, and causes Smo to move to the plasma membrane by vesicle fusion. Plasma membrane-bound Smo recruits Ci, Fused and Cos2, causing PKA, CK1 and GSK3 to dissociate from Cos2 and the rest of the HSC, thus preventing the phosphorylation and degradation of Ci. Full-length Ci can then enter the nucleus and act as a transcriptional activator for hedgehog genes, such as engrailed and decapentaplegic (encodes Bone Morphogenetic Proteins in vertebrates); it will also include genes encoding proteins in the hedgehog pathways, such as Ptc, creating a feedback loop.

Hh can also bind to Smo and changes its conformation, regulating Smo recruitment and degradation.

Controlling levels of hedgehog signals
Similar to Wnt, there are proteins which are secreted that binds to hedgehog signals to prevent them from binding to receptors, thus lowering the effective signal. Hip1 is a membrane-bound gylcoprotein found in vertebrates (but not in Drosophila) which binds to and inhibits sonic hedgehog signalling; growth arrest specific1 (Gas1) is a 45 kDa glycophosphatydlinosotol (GPI)-linked protein expressed during development and when cells arrest their growth; it functions by binding to sonic hedgehog, like Hip1, reducing the signal's range. This permits Ptc to continue its inhibiting effects on Smo. In this study, it was shown that even in the absence of Shh, Gas1 was able to inhibit cell growth and promote apoptosis. Because of this property, Gas1 has been proposed as an anti-cancer drug. However, in this study conducted 2 years earlier, it was found that Gas1 promoted hedgehog signalling, espcially when hedgehog was acting at low concentrations. They found that Gas1 binds cooperatively with an unrelated protein, Cdo, to Ptc1 and promotes hedgehog signalling, especially for neural tube patterning, craniofacial, and vertebral development.

Therefore, it is proposed that Gas1 is a pleiotropic protein that is able to induce or suppress apoptosis, arrest cell growth, suppress tumour cells, with its precise function dependent on its cellular context.

Glypicans
Glypicans are cell-surface receptors which recognizes hedgehog signals. Glypican-1 is found to be over-expressed in human breast cancer cells compared to normal breast cells.

Cyclopamine
In cases where hedgehog signal is over-expressed, the stem cell population will continue to divide, giving rise to cancer stem cells. In this case, cyclopamine can be used. Cyclopamine binds to Smo and stops the signal transduction of hedgehog, and thus limit the rate at which the cancer stem cells divide.

However, because it blocks hedgehog signalling, it interferes with the development of the embryo, specifically by preventing the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). Because of these effects, it is too toxic to be used in humans.

Hedgehog in other vertebrates
In humans, there are three Ci equivalent - Gli1, Gli2 and Gli3; Gli1 is not cleaved and usually acts as transcriptional activator, Gli2 is also not cleaved but can act as an activator or inhibitor, whereas Gli 3 is processed and acts most like Ci, being able to function both as a transcriptional repressor and activator (though it is usually a repressor). In the absence of a hedgehog signal, Gli2 and Gli3 are expressed, whereas Gli1 is silenced; Gli2 and Gli3 both acts as repressors. In the presence of a hedgehog signal, the Gli code is changed, and Gli1 is expressed, Gli2 becomes an activator, and Gli3 is not cleaved anymore, and thus acts as an activator.

SIL functions downstream of Ptc. Missing in metastasis (MIM or BEG4) is an actin-binding protein that regulates Gli-dependent transcriptional activation in vertebrates, thereby modulating Hedgehog signaling.

Primary Cilia
In most vertebrate cells, the activated (phosphorylated) Smo is concentrated at the primary cilia. This suggests that the cilia acts as a sort of attenna, where it senses the position of itself in relation to the whole tissue; this hypothesis fits in with the proposed role of Hh - in development, where segmentation of cells are important.