L2. Development of Blood Cells.pdf

Physiology Dr. Farhan Cyprian Development of Blood Cells 9th March 2020 Basant Moustafa Selim Yazan Al-Dali 02 1 This document resorted to: 1. “Development of Blood Cells” Lecture Slides. 2. “Hoffbrand’s Essential Haematology 7th edition” Chapters 1 & 2. Overview: This sheet will cover the process o...

Physiology Dr. Farhan Cyprian Development of Blood Cells 9th March 2020 Basant Moustafa Selim Yazan Al-Dali 02 1 This document resorted to: 1. “Development of Blood Cells” Lecture Slides. 2. “Hoffbrand’s Essential Haematology 7th edition” Chapters 1 & 2. Overview: This sheet will cover the process of erythropoiesis and few related concepts. Blood overview Blood is a connective tissue that is consisting of two compartments: 1) Liquid compartment or Plasma (55% of blood volume): ▸ Water → 90% of plasma volume ▸ Plasma proteins → 7% of plasma volume - Albumin, the most abundant plasma protein (60% of plasma protein). - Globulin. Homeostasis is - Fibrinogen, which is produced from the liver and, along with platelets, helps maintenance of to maintain the hemostasis (clotting). the internal ▸ Nutrients → glucose, amino acids, vitamins and lipids in forms of cholesterol, environment (HDL, LDL or fatty acids) ▸ Salts (electrolytes / ions) → sodium, potassium, calcium, chloride and bicarbonate. ▸ Waste products of metabolism → urea, uric acid and amine waste products ▸ Respiratory gases → CO2 and O2 ▸ Drugs and hormones. 2) Cellular compartment (45% of blood volume): ▸ Erythrocytes (RBC) → the most dominant cell type that forms 99.1% of the cellular compartment. ▸ Leukocytes (WBC) ▸ Platelets both make 0.9% of the cellular compartment. Introduction Erythro (red blood cell) poiesis (development) is the formation of red blood cells which form a great portion of the blood. This process takes place in the bone marrow, which is a very conserved environment due to the availability of ① proliferation and ② differentiation factors. Factors can be cytokines, hormones or any substance that is produced in the bone marrow. These factors can bind to the cell receptors that are present on the membrane or on the nucleus. We have two types of factors: 1. Proliferation factors: a factor that act on the cell and induce multiplication and simple cell division producing a clone that is typical to the original cell. 2. Differentiation factor: a factor that act on the cell and change its behavior to produce a clone with different properties compared to the original cell. 2 The bone marrow has a lot of factors that sustain and maintain one population of cells, which are the Pluripotent Stem Cells (PSC). The PSC that give rise to blood cells are known as Pluripotent Hematopoietic Stem Cells (PHSC). Therefore, PHSCs are the group of cells that will give rise to blood cells. Similarly, the group of cells that give rise to chondrocytes, adipocytes, and osteoblasts among others are known as Mesenchymal Stem Cells. In conclusion, PSCs have the ability to give rise to different cell types, hepatocyte or a neuron, depending on how you differentiate them. Pluripotent hematopoietic stem cells (PHSC) have the capacity to renew themselves by using the proliferation factors present in the bone marrow. However, differentiation factors along with different cell signaling pathways will cause the PHSC to produce different types of blood cells instead of remaining in a cycle of self-division. That is done by the PHSC gaining different attributes and losing others. Overview of hematopoiesis The PHSCs will become common myeloid progenitor (CMP) or common lymphoid progenitor (CLP). A. CLP → Can only give rise to lymphoid cells (B and T lymphocytes) & Natural Killer Cells. B. CMP → Gives rise to: ① MEP which is responsible for giving rise to RBCs and platelets. ② GMP which is responsible for giving rise to granulocytes such as neutrophils, monocytes, eosinophils and mast cells. 3 The road map: HSC → CMP → MEP → Mature RBC (Erythrocyte) Stem cell (HSC) 1. DNA in the nucleus. 2. Receptors on the cell membrane, nucleus or cytoplasm. In order to respond to the external stimuli (Factors). 3. Mitochondria is the source of energy. 4. Protein synthesis machinery (Endoplasmic Reticulum and Ribosomes): an external stimuli will bind to and activate the receptors. Then, the receptors will downstream some sort of transcription factors that will tell the DNA to make particular mRNA. The mRNA will be transcribed to produce the protein of interest. Mitochondria is the source of energy through Electron transport chain that is oxygen dependent. Mature RBC (Erythrocyte) 1. Hemoglobin molecules that transport gases. 2. Bilipid layer with sodium potassium pumps. 3. Cytoskeletal proteins: Erythrocytes contain some structural proteins that help the blood cells maintain their unique structure and enable them to change their shape to squeeze through capillaries. This includes the protein spectrin. 1. Glycolysis (80-90% of energy) which is cytoplasmic in nature and needs enzymes inside the cell as well. 2. Hexose monophosphate shunt (10-20% of energy required by the RBC). “The energy is required to maintain the structure and function of RBC including the sodium potassium ATPase pumping” Why RBCs lack Mitochondria as the factory of energy? Within the mitochondria, electron transport chain takes place which consume high amount of oxygen to provide the energy needed in the form of ATP. Because the function of the RBC is to transport oxygen, it does not have any mitochondria. In other words, RBCs rely on anaerobic respiration. This means that they do not utilize any of the oxygen they are transporting, so they can deliver it all to the tissues. Why RBCs lack nucleus and other cell organelles (Golgi apparatus, endoplasmic reticulum)? An RBC does not have a DNA nor a nucleus. Therefore, no mRNA will be produced, and no protein will be synthesized. Hence, the protein synthesis machinery will be absent, so no ribosomes nor endoplasmic reticulum are present in the mature red blood cell. Will the RBC survive very long? No, it only survives for 120 days and after that they will be broken down in the spleen and the liver, which are the main sites for RBCs destruction “Majority takes place in the spleen, minority in the liver”. ATP outside the cell will activate the immune system and cause inflammation, similar to DNA. 4 Sites of Erythropoiesis Embryonic erythropoiesis: 1. Mesoblastic Stage (3rd week): Nucleated RBCs are developed from yolk sac and mesothelial layers of placenta. 2. Hepatic Stage (6th week): RBCs develop mainly in Liver. Spleen and lymphoid tissue also make RBCs. 3. Myeloid Stage (3rd month onwards): The bone marrow starts to carry on hematopoiesis and by the last month of gestation, the bone marrow is exclusively the site of RBC synthesis. Erythropoiesis after birth: During adulthood, almost exclusively all of our RBCs are made by the bone marrow. By 5 years old, all bones are sites for erythropoiesis. As time progress, a significant number of fat cells will appear in the diaphysis of the human long bones. After 20 years, hematopoietic marrow is found only in the membranous bones (vertebrae, sternum, ribs, skull, Ilium) & the proximal epiphyses of humeri, tibiae and femora. FYI The preference of hematopoietic tissue for centrally located bones has been ascribed to higher central tissue temperature with greater vascularity. Adipocytes are negative regulators in the hematopoietic microenvironment that decrease or suppress the hematopoietic regeneration. Extramedullary hematopoiesis: (blood formation outside the medullary canal of the bone): During adulthood, what happens if the bone marrow fails to function? The erythropoiesis machinery that existed in the primitive state during embryonic stages of development will take over. As adults, we do not have the yolk sac, nor the placenta, hence, the liver and the spleen start making RBCs. 5 Hemoglobin Alpha Chain → 141 AAs Beta Chain → 146 AAs Structure of hemoglobin: Hb is a large molecule made up of proteins and iron. It consists of: 1) Globin: which is four folded chains of a protein, designated alpha 1 and 2, and beta 1 and 2. Each of these globin molecules is bound to a red pigment molecule called heme. 2) Heme group: it looks like a protoporphyrin ring and it contains an ion of iron in the ferrous state (Fe2+). As long as iron is in its ferrous state, it can bind to oxygen molecules. However, when it gets oxidized, becoming Ferric (Fe+3), it is not able to bind oxygen anymore. Methemoglobin is a hemoglobin in the form of metalloprotein, in which the iron in the heme group is in the Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal hemoglobin and it cannot bind oxygen. Hence, agents which strongly oxidize the Hb, converts it methemoglobin and hinders its oxygen carrying capacity. Function of Hb: Each iron ion in the heme can bind to one oxygen molecule; therefore, each hemoglobin molecule can transport four oxygen molecules. RBC is a bag or sac of hemoglobin; hence an individual erythrocyte may contain about 280 million hemoglobin molecules, and therefore can bind to and transport up to 1.12 billion oxygen molecules. Significance of Hb: One important function of blood, which is 55% liquid and 45% cells, is to transport O2 to the tissues. Oxygen is found within the RBCs bound to heme groups, also minor proportion of oxygen can be dissolved in the plasma. So, why we don’t depend on the plasma solely to transport oxygen? The plasma can carry oxygen; however, the capacity is very low. If we need to satisfy the body needs of oxygen depending ONLY on plasma, the volume of blood required need to be 70 times more than normal (350 L instead of 5 L). Because this is impossible (350 L of blood in your body !!), hemoglobin is needed. So, hemoglobin at normal concentration increases that O2 carrying capacity by 70 folds. Therefore, if your blood was only liquid, its oxygen carrying capacity would be very low. 6 Possible outcomes of stem cells division 1. Asymmetric cell division: When: stem cells in healthy individuals at any given time are going through asymmetric cell division. How: the stem cells that are in your body, are actually giving rise to more stem cells and giving rise to progenitors. In other words, both differentiation and proliferation (growth) factors are almost equally present. Therefore, growth inducers will give rise to more stem cells, and differentiation inducers will give rise to more progenitors. This is what is happening at any given time point. 2a. Symmetric cell division (stem cells → progenitors only): When: After blood loss (menstrual cycles in females, trauma after an accident or donation). How: The body needs to have a mechanism to replace the blood very fast. Therefore, stem cells will undergo symmetric cell division → progenitors. In other words, the differentiation inducers will be highly expressed compared to the growth inducers, hence stem cells will differentiate and give rise to more progenitors. For example, after blood loss, erythropoietin pushes the stem cells into directly becoming progenitors. 2b. Symmetric cell division (stem cells → stem cells only): When: after exposure to radiation or certain drugs that cause stem cells loss. How: to replace the damaged stem cells, the remaining stem cells can undergo symmetric cell division where they can give rise to more stem cells to repopulate the environment. In this case, the growth (proliferation) inducers are highly expressed compared to the differentiation inducers. 7 Stages of erythropoiesis “overview” Early stage: PHSC → Common Myeloid Progenitor → Burst Forming Unit-Erythroid (BFU-E) → Colony Forming Unit-Erythroid (CFU-E) → Late Stage Location: This stage takes place in the bone marrow and it is primarily focused on giving rise to committed cells (BFU-E & CFU-E) that cannot change or push to any other linage. Characteristics: In the early stages, you are not able to differentiate between cells morphologically (what it looks like). However, they behave in different ways. Meaning that if you take PHSC and grow them in-vitro culture (cell cultures outside the body where we put the cells in incubators to grow them), and put with them different kind of growth factors or differentiating factors, they show different kind of behavior. For example: Burst Forming Unit-Erythroid (BFU-E) Colony Forming Unit-Erythroid (CFU-E) Morphologically unidentifiable “They have the same morphology (shape) and look exactly the same under the microscope” - They will give rise to blast cells that clump together. - Single or multiple cell clusters. - >200 erythroblast per colony. - They do not proliferate very fast, but aggregate and clump together to form small colonies. - 1-2 cell clusters. - 8 - 200 erythroblast per colony. Late stage: Proerythroblast → Polychromatophil Erythroblast → Orthochromatophil Erythroblast → Reticulocyte → Erythrocyte. Location: Most of this stage takes place in the bone marrow, however the reticulocytes will come out of the bone marrow to the blood until they mature and become erythrocytes. Characteristics: In this stage, all cells are morphologically identifiable. Under a microscope, proerythroblast will look different in comparison to polychromatophil, orthochromatophil, reticulocyte or erythrocyte. That difference is due to the different content of the cells. The development of Orthochromatophil Erythroblast from PHSC takes place in the bone marrow (although they are early and late stages) and this takes 4-5 days. However, outside the bone marrow, the reticulocytes will come out and they will remain in the blood for around 1 to 2-3 days till they mature to erythrocyte. The majority of blood, normally, consists of mature erythrocytes and a few reticulocytes. However, when the RBCs requirements increase like in blood loss or high altitude, more EPO will be produced and that will push in the direction of RBCs formation. Hence, more and more reticulocytes are formed (reticulocytosis = increase in the number of reticulocytes because of increase demand). 8 The PHSC have some level of self-renovative capacity, as described above, which is mediated by 3 things: ① Stem cell factor, ② IL-6, ③ IL-11. All these three factors give rise to more of PHSC, that by default are 0.01% of total bone marrow cells. Note that they are considered as a minority, yet they have the capacity of self-renewal. After several stages in the bone marrow, they end up coming out to the blood as reticulocytes and then RBCs. For this process to happen, growth factors must be available. We have two types of growth factors: 1. Common or general growth factor: - They are impacting all the precursor cell types: - Examples: Stem cell factor, IL-6, Granulocyte monocyte Colony stimulating factor. 2. Lineage specific growth factors: - They only promote the direction of formation of one cell type or the other, and not any. - Examples: ① EPO (erythropoietin), a lineage specific growth factor that gives rise to RBCs only. ② TPO (Thrombopoietin), a lineage specific growth factor that gives rise to megakaryocytes that will give rise to Platelets. ③ G-CSF (Granulocyte colony stimulating factor), a lineage specific growth factor that gives rise to neutrophil development. Stages of Erythropoiesis “Detailed” Myeloid The growth and differentiation factors will play an important role in the formation of BFU-E Progenitor from CMP. For example, ① IL-3 pushes the CMP towards differentiation, ② Stem cell factor support its proliferation and ③EPO causes maturation and blocks apoptosis. BFU-E CFU-E The burst forming unit- erythroid (BFU-E) will give rise to blast cells in clusters. They are several 1000s cells with satellite clusters around core of erythroid cells “BURST”. They also have the ability to replicate and proliferate very fast. On their surfaces, we can see ① CD34+ as all stem cells have it, ② CD45+, ③ absence or down regulation of IL-3 receptors (IL 3R−) because they have undergone differentiation, they do not require the signal anymore from IL-3, therefore, the way they shut off the signaling is just decreasing the receptor expression on the surface as they do not have the control of stopping IL-3 production by the environment, ④ low expression of CD71 that still has a debatable role, which is a transmembrane protein, called Transferrin receptor protein 1 (TfR1), that is responsible for iron import from transferrin into cells by endocytosis. Then, under the influence of EPO, Colony forming unit Erythroid CFU-E will be produced. They are still CD45+ but they lose CD34, remain IL3R- and start expressing more CD71. Colony forming unit erythroid is the last cell where you can say that it has the stem cell like properties, because it is losing them by downregulating CD34 from its surface. EPO pushes the CMP to become BFU-E then CFU-E “lineage specific signal”. 9 “Let’s pause a little bit to understand H&E stain and its basis, so we will easily figure out the function and structure of the upcoming group of cells” Rule1 “North pole attracts South pole” If something is acidic, it would like to go towards the opposite. Acids likes to get entangled with bases and vice versa. The reason is that basic dyes are cationic (+ charge) so they form salts with tissue anions. For example, phosphate group of nucleic acids. However, acid dyes are anionic (- charge), so they form salts with tissue cations. For example, ionized amino groups of proteins Rule 2 “Cell Structures” Now it should be clear in our minds that nucleic acids are acids “very logic” and anions (-). So, they are basophilic “likes to bind basic dye”. DNA is mainly confined to the nucleus with a small quantity in the mitochondria (absent in the cytoplasm). However, RNA is found mainly in the cytoplasm with a small quantity in the nucleus. On the other hand, cytoplasmic proteins are bases and cations (+). So, they are acidophilic or eosinophilic “likes to bind the acidic eosin dye”. Summary: ▪ Basic dye “hematoxylin binds acidic structure “Nucleic acids” and stain them deep blue-purple. ▪ Acidic dye “eosin” binds basic structures “cytoplasmic proteins” and stain them pink. ▪ The proportion of nucleic acids and cytoplasmic proteins will determine the color of different compartments of the. In other words, the fact that cytoplasm takes the pinkish stain explains that the majority of cytoplasm is cytoskeletal proteins, but still small amount of nucleic acid are present in it. ▪ The nucleus of any cell type will always be basophilic, hence purple, because they have nucleic acid. ▪ Cytoplasmic granules are acidic in Basophils that is why they take the purple basic dye, and basic in Eosinophils that is why they take the pinkish acidic dye. Neutrophil does not take any dye (the deep purple dye nor the pink dye). 10 Proerythroblast Proerythroblasts are the immature cells with proliferation capacity that will give rise to the erythroblast. They can multiply very fast and undergo 4-5 divisions. There is no real function for this cell except that it is in an intermediate stage. Proerythroblasts are characterized by high nucleic to cytoplasmic ratio (large nucleus and scanty cytoplasm), large central nucleus, very prominent nucleoli and small projections in the cytoplasm which are ear-like membrane projections. Proerythrocytes are the largest precursors and don’t produce hemoglobin. Basophilic Erythroblast The proerythroblast stage progresses very fast into the basophil erythroblast. As the name implies, basophilic erythroblast has a cytoplasm that likes to take a deep purple stain. Why? you know that what takes the basic dye, a basophilic structure, should be acidic and anion like nucleic acids. And because we know that DNA cannot be found within the cytoplasm and granules are absent in those cells, we can clearly understand that high proportion of RNA molecules are found within the cytoplasm, that is why it is called basophilic erythroblast. It is characterized by lost/not apparent nucleoli, deep blue cytoplasm (basophilic staining) due to excessive ribosomes and increase in RNA and Little Hb “Hb starts to be produced”. It also show mitosis. Polychromatic Erythroblast Now, the RNA that used to give the purple color in basophilic erythroblast will be decreased because protein synthesis is started, so the production of cytoplasmic proteins increases (mainly hemoglobin). When the cell is losing the RNA to synthesize proteins, it will have both colors and we call it polychromatic erythroblast (cell with many colors). In other words, as the color is changing from a basophilic going towards eosinophilic, in between, we have a stage where the proportion of basophilic as well as eosinophilic structures in the cell is equal (RNA in cytoplasm = cytoplasmic proteins). Polychromatic erythroblasts are characterized by dark staining smaller nucleus with coarse chromatin (checkerboard pattern) and polychromatic appearance in which the cytoplasm is pinkish due to Hb and purplish due to RNA giving greyish look to the cell rather than pink or blue. Orthochromatic Erythroblast Moving on to the orthochromatic erythroblast, also known as normoblast, in which the nucleus shrinks and more Hb is being produced while RNA content is gone away giving rise to the pinkish staining of the cytoplasm. It is characterized by smaller and darker nucleus, pinkish cytoplasm due to high Hb content. At the end, the nucleus is pushed to one side of the cell and enucleation takes place. What is not clear is whether the cell is pushed out of the cell or whether a macrophage eats it. But generally, the nucleus moves out and the macrophage phagocytose it. 11 Reticulocyte After enucleation, we have the reticulocytes that will leave the bone marrow to the circulation by a process of diapedesis similar to neutrophils when they move out of the blood vessel towards the site of inflammation. The reticulocyte contains cytoplasm, cytoplasmic organelles, many ribosomes, few mitochondria and remnants of endoplasmic reticulum. Over the next two days, while the reticulocyte is circulating, it loses all of those structures by the process of proteasomal degradation and it undergoes certain cytoskeletal changes. Mature As a result, it develops into a mature erythrocyte after Erythrocyte spending 1 to 2 days in the peripheral blood. (RBC) Mature erythrocytes are red, biconcave disks packed with hemoglobin, an oxygen-carrying compound. RBC has more cell membrane than the cytoplasmic content, therefore it has a high surface area to the volume ratio, that’s why it has a biconcave shape and it can be squeezed without being stretched or rupture. In other words, it has high flexibility. Substrates required for erythropoiesis: 1) Iron: ▸ The importance: hemoglobin synthesis. ▸ The source: the Iron that is stored in the macrophages after RBC breakdown is the main source, even though the dietary iron also goes to the bone marrow through the blood itself (bound to transferrin), it is not enough for rapid proliferative cycle that happens in erythropoiesis. How it is formed? 1. Majority of RBC breakdown takes place in the spleen by the macrophages. In the spleen (follow the next picture), the macrophages break down the RBC and its content, which is mainly Hb. The Hb will be broken down into ① Globin, which will be hydrolyzed to free amino acids and recycled, ② Heme group, which in turn will be broken down into ① Porphyrin ring that will end up being bilirubin and ② Iron. However, not all of the iron will be exported from the macrophages by ferroportin, rather it remains sequestered in the macrophage itself. Macrophages 12 2. Those macrophages go to the bone marrow, and give rise to erythroblastic islands, which are small islands formed by macrophages surrounded by human erythroid cells in different erythroblastic stages. 3. Then, the iron in the macrophages is slowly being released into the cells to make hemoglobin. The erythroid cells that need iron for hemoglobin formation are basophilic erythroblasts, polychromatophilic erythroblast and orthochromatophilic erythroblasts. 2) Folate and B12: The importance: nucleic acids synthesis, so they are important for the early than the late stages. For example, basophilic erythroblast and polychromatophilic erythroblast needs both substrates for the cell division that takes place in them. While folate and B12 are less required in orthochromatophilic and reticulocytes. Erythroblast Enucleation “detailed but good for your understanding” The nuclei of orthochromatic erythroblasts are polarized to one side of the cell. Based on the cytokinetic model, nuclear extrusion of orthochromatic erythroblasts is a form of cell division, in which nucleus is separated from the cytoplasm by an active process, the Asymmetric cell division. Eventually these cells enucleate to form: - Reticulocyte → Enucleated cells. - Pyrenocyte → The extruded nucleus with a thin rim of cytoplasm surrounded by a plasma membrane. Steps: 1. First, one or more cellular signals initiate the process of enucleation. At least one study 2. 3. 4. 5. 6. suggested that MAPK signaling “described later” is involved in late erythroid differentiation and enucleation. The actin cytoskeleton polarizes the condensed nucleus, free it from intermediate filament attachments, to one side of the cell. Then, the region of plasma membrane in close proximity to the nucleus yields to form a small extrusion that includes a portion of the nucleus. The subsequent formation and coalescence of U-shaped channels and vesicles that have accumulated in the region between the nucleus and incipient reticulocyte allows for separation of the reticulocyte from the pyrenocyte. Finally, pyrenocyte will be attached to a nearby macrophage coupled with actin-mediated movement of the reticulocyte away from the pyrenocyte. Pyrenocytes gradually start expressing phosphatidyl serine on their surface, providing an “eat me” signal for macrophages, which engulf them. The engulfed nucleus is then digested in lysosome where DNase II digests the DNA within the engulfed nuclei. 13 Arrow coding: Direction of force applied over the nucleus by actin cytoskeleton. Protein trafficking, which directs the proteins that are destined to reach the pyrenocyte Direction of the force exerted on the pyrenocyte by a bound macrophage The movement of the reticulocyte away from the center.. The figure shows the erythroblast in a marrow film in which an orthochromatic erythroblast with the nucleus in the final phases of extrusion Note the characteristic rim of cytoplasm encircling the extruded nucleus (Pyrenocytes). The erythroblast (moments away from being an erythrocyte) still contains mitochondria. 14 Erythropoietin: Structure: Erythropoietin is a heavily glycosylated polypeptide hormone Source: 90% of EPO is produced by renal peritubular interstitial cells, while 10% is produced by liver and elsewhere. Some cells make a basal level of EPO, in hypoxia other cells join in and make more. How it is released? The interstitial peritubular cells act as oxygen sensors, but they do not immediately kick in erythropoietin production. In other words, you don’t have performed EPO stores like the case in insulin, which is a preformed hormone that is released the moment you eat food, rather a sustained hypoxia, ↓O2 tension, for at least 15-18 hours is required for the EPO to be synthesized, and then released. The EPO levels maintain the RBC levels, just like IL7 levels maintain the T lymphocytes levels. Therefore: ↓ EPO → ↓erythropoiesis and EPO →  erythropoiesis When EPO level increases? When EPO level is decreased? 1. Anemia 1. Increased O2 supply to the tissues (because of an increased red 2. Hemoglobin inability to give up O2 cell mass or because hemoglobin is able to release its O2 more normally due to metabolic or readily than normal). structural reasons 2. Severe renal disease 3. Low atmospheric O2 3. Polycythemia vera. In this case, there is a cancer in the precursor 4. Defective cardiac or pulmonary cells of the RBCs (PHSC) that give rise to more reticulocytes and function RBCs. As a result of having increased RBC mass in the circulation, 5. Damage to the renal circulation that the production of EPO will be suppressed, and its level will affects O2 delivery to the kidney. decrease. Thus, the erythropoiesis is no longer under the control of EPO. 15 FYI EPO and Erythropoiesis: Erythropoietin stimulates erythropoiesis by increasing the number of progenitor cells committed to erythropoiesis. The transcription factor GATA 2 is involved in initiating erythroid differentiation from pluripotential stem cells. The erythropoietin receptor stimulation activates the transcription factors GATA1 and FOG1. Both are important in enhancing expression of erythroid-specific genes (e.g. globin, heme biosynthetic and red cell membrane proteins) and also enhancing expression of antiapoptotic genes and of the transferrin receptor (CD71). FYI Hypoxia: It induces synthesis of hypoxia-inducible factors (HIF -1αandβ), which stimulate: ▸ Erythropoietin production. ▸ New vessel formation. ▸ Iron absorption by increasing transferrin receptor synthesis and reducing hepcidin synthesis. Erythropoietin signaling EPO stimulates erythropoiesis by binding and activating a high affinity receptor (EpoR) that is expressed predominantly on the surface of immature erythroid cells. The EpoR is a member of the type I cytokine receptor superfamily and it has two extracellular immunoglobulin-like domains. The predominant pathway activated by EpoR is the Jak/STAT signaling cascade: 1. EpoR depends on the JAKs to initiate signaling in the nucleus for gene expression. JAK 2, belongs to Janus family, is a tyrosine kinase that is constitutively associated with the membrane-proximal regions of the EpoR intracellular domains and it activates phosphorylation. 2. When the erythropoietin binds to the EpoR, the receptor will undergo conformational changes that brings the two JAK 2 proteins close enough to phosphorylate each other “trans phosphorylation” or “autophosphorylation”. In other words, the two JAKs are almost mirror images and when one is phosphorylated, it phosphorylates the other. 3. The phosphorylated Jak2 will phosphorylate tyrosine residues in the intracellular region of the EpoR, providing docking sites for signaling molecules with phosphotyrosine binding motifs, including the signal transducer and activator of transcription protein STAT5. 4. STAT5 proteins are principal signaling molecules for EpoR, undergoing homodimerization after recruitment and phosphorylation by the receptorJak2 complex. STAT5 mediates erythroid survival, proliferation and differentiation signals. 16 FYI Activation of intracellular multiple signaling pathways by erythropoietin (EPO): EPO binds to its receptor (EPOR) and triggers activation of Janus Kinase 2 (JAK2). JAK2, in turn, phosphorylates tyrosine residues on the EPOR as well as signal transducer and activator of transcription factors (STATs). After EPOR phosphorylation, phosphatidylinositol-3 kinase (PI3-K), phospholipase C (PLC), and growth factor receptorbound protein 2 (Grb2) bind to the receptor and become activated resulting in the stimulation of multiple intracellular signaling processes. PI3-K activates its downstream effector protein kinase B (Akt). PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2) into two second-messenger proteins; inositol trisphosphate (IP3) and diacylglycerol (DAG). Finally, Grb2 binds the Ras-guanine exchange factor son of sevenless (SOS) which, in turn activates Ras, which activates the classic mitogen activated protein kinase (MAPK) pathway involving Raf, MAPK/extracellular signaling related kinase (ERK) kinase, and ERK. All the pathways result in proliferation, growth and survival of RBCs’ precursors and their differentiation until reticulocytes are formed. Take-home points: 1. Function of macrophages is crucial for RBC synthesis. People who have got defective macrophages, are actually not able to make RBCs. 2. These macrophages are important in iron supply in erythropoiesis and in erythrocytes enucleation. 3. EPO is the key factor of driving erythropoiesis. Don’t hesitate to contact Basant/ Yazan regarding any clarification, concern or suggestion! 17