22 - RTKs and Wound Healing

Question 1

How can you study wound healing in a lab?

Answer 1

Work in the labs are done on culture models
o you can culture a surface monolayer of fibroblasts which are a cell line involved in wound healing as they produce an extracellular matrix to seal wounds
* These can be cultures in a monolayer in the lab on a petri dish

Question 2

How can you wound a monolayer on a Petri dish?

Answer 2

When you grow a monolayer on a petri dish you can wound it by dragging a pipette tip down the dish to make a gap
o The cells will migrate naturally an proliferate to fill the gap an that’s brough about through stimulation of growth pathways

Question 3

What is a classic example of RTKs and wound healing

Answer 3

A classic example of this is the PDGF receptor (platelet derived growth factor), it is produced by platelets and its produced at wounding sites which stimulate cells to close the wound

Question 4

How is the PDGF receptor arranged?

Answer 4

It is a dimer linked by PDGF, two molecules of PDGF linking the receptor together
o So one PDGF molecule is linked to one receptor, and the other to the other, and then two ligands linked together to draw the two monomers together to produce and active dimer
o The ligands crosslink two monomeric receptors

Question 5

What happens when you get a tissue injury?

Answer 5

If you have tissue injury, for example we cut our skin, this will cut into blood vessels so we start to bleed, in our blood we have platelets and when they start to accumulate at the wound site we get the clotting response

Question 6

What are the jobs of the fragments of ells that produce PDGF and TBFbeta?

Answer 6

o They activate the fibroblasts which are packing cells
o They trigger inflammation (recruitment of macrophages and neutrophils)

Question 7

What causes bleeding to stop in a wound?

Answer 7

• Formation of a clot stops bleeding
• Within the dermis, where the fibroblasts start to proliferate to block the wound, and they secrete extracellular matrix to form a mesh to block the plug, keratinocytes will proliferate too

Question 8

How is PDGF involved in fibroblast proliferation in wound healing?

Answer 8

• PDGF stimulates the fibroblast cells causing migration of the fibroblasts through the dermis to the wound
• Proliferation of the cells to make more fibroblasts which will secrete more collagen, elastin and exracellular matrix
• This all leads to wound healing

Question 9

What is one of the main routes by which PDGF stimulates proliferation and migration?

Answer 9

One of the main routes by which PDGF stimulates proliferation and migration is by triggering the ErkMAP kinase pathway
* As we know when the PDGF receptor is activated by a dimeric ligand we can transautophosphorylation of the receptor on many sites so we get recruitment of many signalling proteins
- of these Grb2 is important
* SOS is a guanine nucleotide exchange factor which converts Ras to the GTP form – not a phosphorylation, it is an exchange to GTP from GDP

Question 10

What happens when RAS is activated?

Answer 10

When RAS is activated it leads to the recruitment of Raf, the MAPKKK, to the cell membrane
* This triggers Raf activity
* This phosphorylates MEK which phosphorylates Erk
* Erk can then enter the nucleus where it can phosphorylate transcription factors like Elk

Question 11

Why is negative feedback control important in the RasMAP kinase pathway?

Answer 11

We need the RasMAP kinase pathway to be under strict negative control because it needs to stimulate proliferation to a limited degree for wound healing and then it needs to stop
* The types of negative feedback we have is recruitment ot tyrosine phosphatases, control of the MAPK pathway by a scaffold

Question 12

Negative feedback mechanism of RasMAP kinase pathway

Answer 12

Once the ERK is activated it also phosphorylates SOS, and this blocks interaction with Grb2, preventing the recruitment of SOS and any further nucleotide exchange on Ras
* We have influence of phosphatases- in particular a fast induced phosphatase gene called MAP kinase phosphatase
o Erk switches on this gene and MKP is produced, translated rapidly which dephosphorylates Erk
o MKP is a dual specificity phosphatase so it dephosphorylates Ser/Thr but it phosphorylates tyrosine
* Action of other phosphatases too

Question 13

What are the three forms of negative regulation involving the activated PDGF receptor?

Answer 13

1. Ras inactivation
2. Receptor dephosphorylation
3. receptor internalisation