Anita Hall - Hedgehog

8/7/2019 Anita Hall - Hedgehog

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Molecular Cell Biology II Signalling During Vertebrate Development II - Hedgehog

[Page 1]

Hedgehog (HH) signalling has strong clinical relevance in embryonic development and stem cell biology,

amongst other fields.Drosophila signalling is well known.

Hedgehog Ligands:

More than 3 found in vertebrates

Sonic hedgehog (SHH) ² well characterised

Desert hedgehog

Indian hedgehog

Essential for positional identity in development and adult tissue homeostasis across species

e.g. Control of digit formation

e.g. brain and spinal cord formation

Also involved in stem cells and cancer in adults

Main molecules involved:

Patched ² 12-pass transmembrane HH receptor

iHog ² Single-pass HH receptor, probably co-receptor of patched

Smoothened ² 7-pass, similar to Frizzled, transduces the HH signal

When there is no HH signal«

Patched keeps Smoothened inactive in intracellular endosomes

Smoothened is regulated by the conformation of patched, which changes when HH binds, which could

regulate the recruitment and degradation of smoothened.

A gene regulator called Cubitusinterruptus (Ci) is processed

in a complex which contains«

PKA (phosphorylates)

GSK3 (phosphorylates)

CK1 (phosphorylates)

Fused kinase

Costal2 (scaffold protein)

Ci is triple phosphorylated in the complex, then

ubiquitinylated and then cleaved in a proteasome, to leave a

small peptide. This then enters the nucleus and interacts

with a co-repressor to repress the HH target genes.

When there is a HH signal«




[Page 2]

HH binds to Patched and iHog, inhibiting Patched and inducing its endocytosis and degradation

Smoothened is then phosphorylated by PKA &CK1 and moves to the cell surface

Smoothened in the plasma membrane can recruit the Ci complex, Fused and Costal 2

Costal 2 can·t bind the 3 kinases (PKA, GSK3, CK1) so Ci is not phosphorylated or cleaved

Unprocessed full-length Ci enters the nucleus and wil l activated HH target genes

HH Signalling in

Vertebrates:

3 Ci-like gene regulators (Gli1/2/3)

Gli1/2 are not processed and probably act as transcriptional activators

Gli3 is processed and so can keep genes off (-HH) or switch on transcription (+HH)

The primary cilium is a HH signalling centre in vertebrate cells

+HH, activated smoothened and Gli proteins are concentrated in a primary cilium

All of the components of a signalling pathway in one of these extensions p a quick response

Defects in cilia can disrupt Shh signalling, can cause brain defects




[Page 3]

Processing of HH proteins:

Signal sequences are cleaved

HH precursor is autocatalytically cleaved

C-terminus is modified ² covalently linked to cholesterol

o Errors in this linkage are bad as they can effect the morphological gradient

N-terminus has a fatty acid added ² palmitate by skinny hedgehog

The HH is now fully active after having dual lipid modification

Potential use of lipid modifications«

Targeting lipid rafts

Regulating HH release and trafficking

Multimerisation of HH to increase solubility

Formation of diffusion gradientsetc«

HH release from cells:

Trafficked to cell surface

Can be released as lipoprotein oligomers by 12-pass transmembrane protein Dispatched

Mechanisms of release«

HH multimers are more soluble

Dispatched releases HH

LRP in target cell takes up HH and passes on signal by cell trafficking

Movement through tissue«

Diffusion

Indirect ² via signalling cascades

Via cytonemes (thin finger-like projections from plasma membrane)

HH can act as a morphogen, the effect on cell will vary depending on concentration

Seemingly small changes in [HH] can give hugely different cellular responses

Regulating the amount of HH signal a target receives:

ECM (extra cellular matrix) can bind HH and present it to a receptor

o Or isolate it from receptor

Ptc 2

Hip (Hedgehog Inhibitory Protein)

Gas1 (Growth Arrest Specific gene) binds Shh and promotes signalling

All of these factors will determine how much HH can bind to a receptor




[Page 4]

Gli Code:

How are the different concentrations of HH binding sensed and converted to effect on cells?

The three Glis in vertebrates can activate / repress gene transcription

Glis are transcription factors with 5 zinc-binding domains

3 Ci-like gene regulators (Gli1/2/3)

Gli1/2 are not processed and probably act as transcriptional activators

Gli3 is processed and so can keep genes off (-HH) or switch on transcription (+HH)

The balance between 1,2,3 will determine the cellular outcome

The cell integrates the activation / repression of all 3 to give the overall result of the HH signal

Levels of each Gli changes over time, so the result of the HH signal will change over time

Regulation of the Gli code«

Other signalling pathways can modify the activity of PKA, CK1 and GSK3 that regulate Gli cleavage to

a repressor form

Ligases can tag Gli with ubiquitin

Proteins can bind to Glis and prevent them from entering the nucleus e.g. SuFu

Wnt/F-catenin can oGli activity etc«

State of Gli activity will influence the binding of other transcription factors

e.g. Nkx2.2 and Pax6 (both involved in cell fate)

Feedback loops can occur with HH target genes, such as Patched

+HH will increase transcription of patched and inhibit further HH signalling

Transcription independent effects of HH:

Cell motility ² important in the spreading of cancer

Chemoattraction ² important for axon growth

Roles of HH signalling:

Ventral fate in nervous system (determining what is top / bottom)

Anterior ² Posterior axis of limb (thumb / little finger)

Vasculogenesis ² important for cancer

Bone / cartilage formation

Lung branching

Maintenance of stem cell populations post-development




[Page 5]

More detailed example ² Dorso-ventral patterning in the CNS

A floor plate makes HH, which builds up a morphological gradient

Works in combination with other morphological gradients to determine cell fate

HH also acts as a mitogen to ensure the embryo has enough of each cell type

e.g. In the developing cerebellum, HH is used to match the number of Purkinje cells with the number of

granule cells (number matching)

Also regulates stem cell division / number which can be affected by disease / ageing

Due to its role in cell cycle control HH is directly linked to cancer

e.g. Basal cell carcinoma

Gli codes are disrupted

e.g. Brain cancer

Very aggressive form of cancer

Gli code is deregulated

Widespead with severe side effects

Therapeutics:

e.g. cyclopamine binds to Smo and stops downstream HH signal transduction

The chemical is very toxic makes developing foetuses Cyclops

Has been shown to eliminate lung cancer in mice

Crosstalk between Shh and Wnt:

A lot of interaction between the two pathways particularly in limb development

Wnt and HH signalling have many similarities

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Anita Hall - Hedgehog