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PHGY 313 > Erythroid cells > Flashcards

Erythroid cells Flashcards

1
Q

Explain the RBC lineage steps

A

Mixed myeloid progenitor –> Burst forming unit erythroid –> colony forming unit erythroid –> mature RBC

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2
Q

What are the different waves of erythropoiesis?

A

Primitive and definitive

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3
Q

What is primitive erythropoiesis? When does it start in mice? in humans?

A

transient embryonic 1st wave (E7 – 11, mouse) of hematopoiesis in mammals that produces mostly red blood cells (EryP), some megakaryocytes and macrophages
 Starts at ~ day 18 in humans
o First primitive erythroblasts found in extra-embryonic yolk sac (mouse, human)
o Presumably providing maximal oxygen delivery to young embryo
o Production of mostly NUCLEATED RBCs. Denucleation occurs in the bloodstream

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4
Q

What is definitive erythropoiesis? When does it start in mice? in humans?

A

o additional independent waves of hematopoiesis in mammals that generate red blood cells (EryD) and all other blood cell lineages.
o originating from Yolk Sac (2nd wave), seeding fetal liver
o originating from AGM region, major blood vessels, placenta (3rd wave), HSCs produced in these tissues seed fetal liver, thymus (T cells), bone marrow
Starts E9 (mice) and E24 (humans)

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5
Q

Differentiate EryP from EryD

A

EryP
…arise from yolk sac only
…are larger (macrocytic)
…retain their nuclei until midgestation, and eventually enucleate after several days in the bloodstream
…Proerythroblasts go in the bloodstream directly (EryD terminally differentiate and enucleate before entering the bloodstream)
…express mostly embryonic globins (higher oxygen carrying capacity)
…differ in requirements for cytokines, transcription factors, signaling pathways
…EryP primitive erythroid progenitor not able to self-renew, when cultured (definitive progenitors can); not sustainable
EryD –> enucleation happening in fetal liver or bone marrow
Already enucleated and called a RBC when reaching bloodstream

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6
Q

What is an advantage of not having a nucleus in human RBCs?

A

Flexibility of the cell

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7
Q

Why has the RBC adopted a biconcave shape?

A
  1. Optimal for diffusion of O2 as it maximizes surface to volume ratio
  2. Maximizes laminar flow (vs. turbulent flow)
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8
Q

How much time does it take to make BFU-E in vitro? in mice?

A

14 days (mouse 7-9 days)

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9
Q

How much time does it take to make CFU-E from BFU-E in vitro? in mice?

A

7 days (mouse 2-3 days)

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10
Q

What is a blood island?

A

Cells line up to a macrophage (aka “nurse macrophage”) –> blood island; and this is where they develop (progenitor stages to end), eventually leave macrophage and go in bloodstream. Macrophage is the one that takes the nucleus of the RBCs leaving

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11
Q

What is the EPO-dependant process in RBC synthesis?

A

Between CFU-E and Pro-EB is the highest expression of EPO-R

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12
Q

What differentiates an erythroblast from a reticulocyte?

A

Enucleation

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13
Q

What differentiates a reticulocyte from a mature RBC?

A

Degradation of residual organelles, microvesicle exocytosis

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14
Q

Where are blood islands found?

A

fetal liver, bone marrow, spleen

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15
Q

What is the role of central macrophages?

A
  • supporting erythroblast proliferation
  • supplying iron for hemoglobin (recovers it at the end of the RBC’s lives)
  • promoting enucleation
  • clearing nuclear debris
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16
Q

Name the different stages of erythroblasts (erythropoiesis)

A
  1. Proerythroblast
  2. Basophilic erythroblast
  3. Polychromal erythroblast
  4. Orthochromatic erythroblast
  5. Reticulocyte
  6. Mature RBC
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17
Q

Explain the macrophage’s role in erythrophagocytosis/iron recycling

A

After life of ~ 120 days, RBCs are taken up by macrophages and different components of Hgb are reused
Heme is broken down in the endoplasmic reticulum to biliberdin and CO and Iron
Iron is either …
- Stored in ferritin and used later by lysosome degradation
- Taken our of the cell by ferroportin 1 with help of ceruloplasmin to Fe3+ and taken up by (apo)transferrin

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18
Q

Besides GFs and TFs, name something that participates to the regulation of erythroid differentiation and explain how it does it.

A

miRNA
Normal circumstances = low miRNA (so low Gcn5); and HDACs active –> chromatin condensation is allowed for enucleation
Overexpression of microRNA blocks of chromatic condensation (blocks enucleation) by a cascade of TFs.

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19
Q

Proerythroblasts:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? ++
  • rRNA/tRNA synthesis? ++
  • Globin mRNA synthesis? +/-
  • Globin protein synthesis? +/-
  • Heme synthesis? +
  • oxidative phosphorylation? ++
  • Anaerobic glycolysis? ++
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20
Q

Basophilic erythroblasts:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? +
  • rRNA/tRNA synthesis? +
  • Globin mRNA synthesis? ++
  • Globin protein synthesis? +
  • Heme synthesis? ++
  • oxidative phosphorylation? ++
  • Anaerobic glycolysis? ++
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21
Q

Polychromatophillic erythroblasts:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? +
  • rRNA/tRNA synthesis? +
  • Globin mRNA synthesis? +
  • Globin protein synthesis? ++
  • Heme synthesis? ++
  • oxidative phosphorylation? ++
  • Anaerobic glycolysis? ++
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22
Q

Orthochromatic erythroblasts:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? 0
  • rRNA/tRNA synthesis? +
  • Globin mRNA synthesis? +
  • Globin protein synthesis? ++
  • Heme synthesis? ++
  • oxidative phosphorylation? ++
  • Anaerobic glycolysis? ++
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23
Q

Reticulocytes:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? 0
  • rRNA/tRNA synthesis? 0
  • Globin mRNA synthesis? 0
  • Globin protein synthesis? +
  • Heme synthesis? +
  • oxidative phosphorylation? +
  • Anaerobic glycolysis? ++
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24
Q

Young RBCs:

  • DNA synthesis?
  • rRNA/tRNA synthesis?
  • Globin mRNA synthesis?
  • Globin protein synthesis?
  • Heme synthesis?
  • oxidative phosphorylation?
  • Anaerobic glycolysis?
A
  • DNA synthesis? 0
  • rRNA/tRNA synthesis? 0
  • Globin mRNA synthesis? 0
  • Globin protein synthesis? 0
  • Heme synthesis? 0
  • oxidative phosphorylation? 0
  • Anaerobic glycolysis? +
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25
Q

Erythroblasts:

  • Nuclear DNA?
  • RNA in cytoplasm?
  • Mitochondria?
  • In marrow?
  • In blood?
A
  • Nuclear DNA? Yes
  • RNA in cytoplasm? Yes
  • Mitochondria? Yes
  • In marrow? Yes
  • In blood? No
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26
Q

Reticulocytes:

  • Nuclear DNA?
  • RNA in cytoplasm?
  • Mitochondria?
  • In marrow?
  • In blood?
A
  • Nuclear DNA? No
  • RNA in cytoplasm? Yes
  • Mitochondria? Yes
  • In marrow? Yes
  • In blood? Yes
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27
Q

Mature RBCs:

  • Nuclear DNA?
  • RNA in cytoplasm?
  • Mitochondria?
  • In marrow?
  • In blood?
A
  • Nuclear DNA? No
  • RNA in cytoplasm? No
  • Mitochondria? No
  • In marrow? Yes
  • In blood? Yes
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28
Q

When was EPO first discovered, how and by who?

A

1906: Carnot & DeFandre
o Made rabbits anemic by bleeding and injected blood from these rabbits in other rabbits –> increased of RBC production
o Thought that something might help rabbits make more blood; called it “hemopoietine”

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29
Q

Explain the discovery of EPO’s history

A
  • 1906: Carnot & DeFandre
    o Made rabbits anemic by bleeding and injected blood from these rabbits in other rabbits  increased of RBC production
    o Thought that something might help rabbits make more blood; called it “hemopoietine”

1950’s: Reissman, Erslev
o Made experiments in other animal species
o Humoral factor in plasma –> Hematopoietin

1960’s: Fisher
o Kidney as the production site of EPO
o Development of EPO assays

1970’s: Goldwasser, Kung, Miyake
o Purification of EPO
o Had to use throusand of liters of urine of anemic patients

1980’s: Lin/Jacobs
o Recombinant EPO

1990’s: D’Andrea
o Recombinant EPO receptor

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30
Q

What is EPO?

A

A glycoprotein of 166 AAs (193 aa, 27 leader peptides)
(mature protein: removal of Arg residue at C-terminus)
34,000
o (11 % sialic acid, 11 % hexose, 8% n-acetylglucosamine)
o Heavily glycosylated – makes it more stable in the bloodstream

31
Q

Where is EPO produced?

A
  • inner cortex of kidney (peritubular interstitial cells)

- liver, brain, spleen, lung, bone marrow

32
Q

What is NESP?

A

Novel Erythropoiesis Stimulating Protein (NESP)
o “Artificial” EPO
o hyperglycosylated erythropoietin –> makes it even more stable in the blood
o increased metabolic stability & half-life

33
Q

Explain EPO dependant signalling

A

Only 1 EPO molecule engages 2 EPO receptors

Intracellular Kinase domains (JAK2) come together and autophosphorylate (activate each other)
Activation of signalling –>
1. STAT (which can activate DNA themselves or activate other TFs and engage signalling)
2. PI3K
3. RAS/RAF

Reversed by phosphatases –> SHIP removes phosphates from JAK2 and IC domain of EPO-R thus terminating signalling

34
Q

What does the RAS/SAF pathway lead to in EPO signalling?

A

Proliferation

35
Q

What does the PI3K pathway lead to in EPO signalling?

A

Survival differentiation

36
Q

What does the STAT pathway lead to in EPO signalling?

A

Survival proliferation

37
Q

What does EPO-R KO do?

A

similar to EPO KO
Yolk sac very pale; decreased number of cells in cross section
Embryos die day 13-14

38
Q

What is HIF?

A

Hypoxia inducing factors
HIF-2alpha in humans
Its is activated by hypoxic conditions and activates gene expression

39
Q

What happens to HIF-1alpha in hypoxic conditions?

A

o Stabilization
o Heterodimerization
o DNA-binding
o Transcriptional co-activation

allows HIF-1alpha to enter the cell and dimerize with HIF-1beta –> binds to 3’ enhancer of EPO gene with other proteins –> activation of expression of EPO

40
Q

What happens to HIF-1alpha in conditions of normoxia?

A

o Rapid degradation via ubiquitin-proteasome pathway (5-15 min)

O2 activates protyl hydroxylases (3) which hydroxylates proline residues on HIF-1alpha
Asparaginyl hydroxylase hydroxylates Asparagine on HIF-1alpha
Makes protein more prone to degradation by VHL E3 ligase and blocks interaction with co-activates which would normally make protein transcription active

41
Q

Which 2 proteins are activate by oxygen and participate to the breakdown of HIF-1alpha?

A

Asparaginyl hydroxylase

protyl hydroxylases

42
Q

What do protyl hydroxylases do?

A

Hydroxylation of HIF-1alpha activate VHL E3 ligase which inactivates HIF-1alpha by ubiquitin-mediated proteolysis

43
Q

What do asparaginyl hydroxylases do?

A

Hydroxylate Asparagine 803 which deactivates p300 co-activator which normally activates HIF-1alpha transcription cascade (erythropoiesis, angiogenesis, glucose metabolism, iron transport, ECM modelling, apoptosis)

44
Q

What does HIF-1 bind to?

A
  • 3’ enhancer (downstream)

- Kidney inducible element (upstream)

45
Q

Does an increase in Hgb change EPO levels?

A

Yes. Low Hgb = more EPO

46
Q

Sickle cell disease: Does EPO increase with low Hgb?

A

Yes

47
Q

Renal failure/anephric children: Does EPO increase with low Hgb?

A

No. Linear relationship, can’t make EPO

48
Q

Name surface markers / receptors important in erythropoiesis

A 
EpoR
CD117 (c-kit)
CD71 (TFR)
CD36
GPA
49
Q

Name transcription factors important in erythropoiesis

A

SCL
GATA 1
GATA 2

50
Q

Name an effector molecule important in erythropoiesis

A

Hgb

51
Q

When is EPO-R expressed in erythropoiesis?

A

Between BFU-E and erythroblasts

52
Q

When is c-kit expressed in erythropoiesis?

A

Between pluripotent SC and proerythroblasts

53
Q

When is TFR expressed in erythropoiesis?

A

between CFU-E and reticulocytes

54
Q

When are CD36 and GPA expressed in erythropoiesis?

A

between proerythroblasts and reticulocytes

55
Q

When is SCL expressed in erythropoiesis?

A

between pluripotent SC and erythroblasts

56
Q

When is GATA1 expressed in erythropoiesis?

A

Between BFU-E and erythroblasts

57
Q

When is GATA2 expressed in erythropoiesis?

A

Between pluripotent SC and CFU-E

58
Q

When is hemoglobin expressed in erythropoiesis?

A

Between proerythroblasts and RBCs

59
Q

What is the effect of EPO following interaction with EPO-R?

A 
Increased Intracellular Ca2+
Increased Hemoglobin synthesis
Increased Transferrin receptor
Increased Synthesis of integral membrane proteins
Increased DNA synthesis
60
Q

Explain the feedback loop of EPO synthesis

A

Hypoxia (which increases HIF and increases EPO mRNA) –> increased plasma EPO
EPO tells the marrow to make more RBCs
More RBCs –> normoxia –> decreased HIF and decreased EPO mRNA

61
Q

Name the tissues affected by EPO other than the hematopoietic system

A 
Adipocytes
Macrophages
Pancreatic beta cells
Muscle myoblasts
Myscle myotubules
Hypothalamus
Endothelial cells
Heart
62
Q

What is the effect of EPO on adipocytes?

A
  • Energy metabolism
  • mitochondrial function
  • Inhibiting differentiation
63
Q

What is the effect of EPO on macrophages?

A
  • Anti-inflammation

- Immuno-modulary

64
Q

What is the effect of EPO on pancreatic beta cells?

A
  • Growth
  • Survival
  • Antiapoptosis
65
Q

What is the effect of EPO on muscle myoblasts?

A
  • Proliferation

- Wound repair

66
Q

What is the effect of EPO on muscle myotubules?

A
  • Inhibiting differentiation

- Fiber type specification

67
Q

What is the effect of EPO on the hypothalamus?

A
  • Food intake control

- Neuroprotection

68
Q

What is the effect of EPO on endothelial cells?

A
  • Angiogenesis

- NO production

69
Q

What is the effect of EPO on the heart?

A
  • Cardioprotection

- Mitochondrial biogenesis

70
Q

What are the overall effects of EPO on the body?

A
  • Anti-OB
  • Anti-metabolic disorders and diabetes
  • Improving insulin resistance and glucose tolerance
  • Cytoprotection
  • Protective against brain ischemia
71
Q

Explain EPO’s role in tumor growth

A

it causes:
- Lymphangiogenesis
- Tumor growth
- Angiogenesis (which can promote tumor growth)
- erythropoiesis (which enhances O2 perfusion to tumors)
Tumors can metastasize to lymph nodes

However EPO can increase QOL…

72
Q

Explain how EPO detection is performed

A 
Radio immunoassay (RIA)
ELISA (enzyme-linked immunosorbent assay)

anti-EPO monoclonal capture antibody is linked to plastic of well plate and captures EPO; detection antibody also binds to EPO
streptavidin-HRP conjugate
tetramethylbenzidine substrate reaction (spectrophotometry)

73
Q

Who is Eero Mäntyranta

A

Athlete skiing
Had high Hct level naturally (55-60 range…)
Had constant EPO-R signal  benigh erythrocytosis

PHGY 313 (10 decks)

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