Cytokines and Growth Factors

Question 1

what is EPO produced by ?

Answer 1

Peritubular Interstitial Fibroblasts

Question 2

what are cytokines?

Answer 2

• small proteins 5-20 kDa
• released by cells
• affect other cells
• especially important for cells of the immune system

Question 3

how many cytokines do we know of?

Answer 3

> 50

Question 4

what are the four distinct structural families of cytokines?

Answer 4

• TNF tumor necrosis factor
• interferon
• chemokines
• hematopoeitin

Question 5

what are some principles for tumour necrosis factor type interactions?

Answer 5

• a trimeric receptor and a trimeric ligand
• ligands are beta strand type ptoteins

Question 6

what are some priniciples of inteferon (IFNy)?

Answer 6

• alpha helical ligand and binds to beta type receptor in similar way
• dimeric interferon binds to dimeric receptor

Question 7

what are some principles of chemokines?

Answer 7

• structure includes alpha helices and beta strands
• can be monomeric or dimeric
• found in cosmetics and facial creams and wound healing

Question 8

what are some priniciples of EPO hematopoetin?

Answer 8

• Erythropoietin
• erythrocytes are red blood cells
• two receptors one EPO
• EPO make RBCs

Question 9

what does expression of a cognate receptor mean?

Answer 9

Its how a cell asks to be able to recieve signal from the blood

Question 10

what does cytokine response (specifically EPO) depend on ?

Answer 10

• expression of a cognate receptor

Cell type and development:
- chromatin structure
- constellation of transcription factors
- developmental history

Question 11

If we have less oxygen we have…

Answer 11

more red blood cells and this decrease in oxygen is what EPO reacts to

Question 12

what tells the body EPO is needed ?

Answer 12

Hif1a (Hypoxia Inducible Factor)

Question 13

what do we need to raise the EPO response?

Answer 13

• Hif
• Transcription factor (to start transcription of EPO)

Question 14

what is the mechanism by which hif1a is taken out of circulation and destroyed?

Answer 14

so in the presence of normal oxygen levels…
- prolyl hydroxylase hydroxylates Hif1a and marks it for ubiquitatylation/ destruction

Question 15

what is the mechanism in hypoxic conditions using Hif1a?

Answer 15

asparagin hydroxylase hydroxylates Hif1a and its activation results in tagaet gene expression

Question 16

what does EPO activate?

Answer 16

erythroid progenitor cells

Question 17

what happens when EPO stimulates a Erythroid progenitor cell (CFU-E)?

Answer 17

it divides to two cells then 4, 8 , 16 etc
3-5 divisions and they slowly differentiate into red blood cells that slowly lose EPO receptors
leading to 30-100 erythroids

Question 18

how many ligands need to be occupied to get 80% of the physiological response?

Answer 18

50%

Question 19

what can be said about EPO ligand concentrations?

Answer 19

below the KdS

Question 20

how many EPO receptors does an EPC have?

Answer 20

1000

Question 21

what is a simple experiment to look at whether the patient has healthy blood ?

Answer 21

hematocrit experiment
centrifuge the blood and it will disperse into
1) RBCs
2) WBCs and platelets
3) Plasma

With men showing 41-50% RBCs
Women showing 36-44% RBCs

Question 22

what cancers can cause low hematocrit levels?

Answer 22

• lymphoma (increased B/ T Lymphocyte WBC)
• Hodgkin’s Lymphoma (increased lymphocyte WBC)
• Leukemia (increased immature WBC)
• Multiple Myeloma (increased myeloma cellls - malignant plasma cells in the bone marrow - reduces production of RBCs and other blood cells)

Question 23

why do cancers cause low hematocrit levels?

Answer 23

cancers use the blood producing stem cells on the expense of RBC production

Question 24

what diseases require the use of EPO to treat anemia?

Answer 24

• chronic kidney disease
• inflammatory bowel disease (Crohn’s disease and ulcer colitis)
• Myelodysplasia (blood disorder) resulting from:
  > exposure to chemicals (benzene)
  > treatment of cancer (chemotherapy and radiation)

Question 25

If JAK is deleted from the gene pool what happens?

Answer 25

failure of development in fetal mammals

Question 26

describe a simple structure of JAK

Answer 26

- a kinase - multiple domains (incl. linker, domain able to bind to the tail of the receptor and a lip domain) - two lobes

Question 27

Describe the JAK/ STAT pathway

Answer 27

1) cytokine binding and cytokine receptor dimerisation occurs 2) phosphorylation of associated JAK kinase - leads to activation (ATP to ADP) 3) phosphorylation of additional residues 4) recognition by inactive monomeric transcription factor STAT 5) phosphorylation and release of STAT 6) STAT dimerisation allows entry into the nucleus

Question 28

how does EPO act in three simple steps

Answer 28

- EPO binds to the EPO-receptor - monomeric EPOr dimerises on EPO binding - this leads to activation of an associated kinase resulting in a conversion of extracellular signal to intracellular change

Question 29

describe structure of STAT

Answer 29

C-terminal phosphorylated by JAK, Phosphate tail of one monomer binds to SH2 domain of the other monomer to create a dimer formation

Question 30

Explain the cascade of protein-protein interactions in the JAK/ STAT pathway

Answer 30

- EPO binds EPOr - dimerisation of EPOr occurs - activates cytosolic JAK-Kinase - Receptor phosphorylation - Receptor (P) recognised by SH2 - Activates STAT pathway - Exposes NLS, pathway continues in the nucleus

Question 31

What is the short term adaptation to deactivate the EPO system?

Answer 31

removes phosphate from lip domain to inactivate the kinase - has an SH2 domains - activated upon binding to the c terminus phosphotases = SHP1

Question 32

How does the cell achieve long term adapation (deactiviation) in regards to EPOr ?

Answer 32

using SOCS protein recognises SH2 domain and binds on the SH2 domain SOCs protein has a SOCs box which attracts the E3 Ubiquitin ligase which then marks the receptor for destruction

Question 33

Name the two ways to switch off EPO's activation through JAK/ STAT

Answer 33

1) Short term adaptation - phosphatase SHP1 dephosphorylates JAK --> OFF 2) Long term adaptation - SOCS --> block of SH2 binding sites - SOCS --> targets JAK for degredation via E3

Question 34

name the four processes that JAK2 and the EPO pathway can activate

Answer 34

a) STATS (Transcriptional activation) b) GRB2 or Shc (Ras - MAP kinase - transcriptional activation or repression) c) Phospholipase Cy (Elevation of calcium - Transcriptional activation or repression; modification of other cellular proteins) d)PI-3 kinase (Protein kinase B - Transcriptional activation or repression; modifications of other cellular proteins)

Question 35

what do STAT 1- 5 interact with

Answer 35

STAT-1: IFNy STAT-1 and 2: IFNa/B STAT-3,-5: growth hormones STAT-4 STAT-5: EPO

Question 36

what type of interferons do virus infected cells produce?

Answer 36

Dendritic cells: IFNa Fibroblasts: IFNb

Question 37

what two directions can type 1 interferons act after release by virus infected cells?

Answer 37

autocrine: towards the infected cell paracrine: towards uninfected neighbours

Question 38

what do INFy do in terms of viral defence via the Jak-STAT pathway?

Answer 38

signal through the Jak-STAT pathway to produce >300 gene products, including cytokines

Question 39

what type of interferond fo T cells and NK cell produce as part of the viral defence?

Answer 39

type II INFy

Question 40

How can INFy kill virus infected cells or cancerous cells ?

Answer 40

autocrine: inhibition of virus replication, via apoptosis paracrine: up-regulation of MHC-I and NK-cell activation