Erythropoietin Pathway
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Erythropoietin Pathway
Erythropoiesis is a major pathway for Erythrocyte production, by which pluripotent Hematopoietic Stem Cells give rise to mature end stage cells via a series of differentiations. Epo (Erythropoietin), a glycoprotein hormone and a multifunctional Hematopoietic Cytokine ligand, is the master regulator of Erythropoiesis. As a major function, it monitors the safe passage of the committed Erythroid progenitor cells through several physiological and developmental stages by stimulating Growth, preventing Apoptosis, and promoting terminal differentiation. In addition to its immense survival strategies, Epo initiates Hemoglobin synthesis and is an essential viability and Growth factor in the maintenance of a steady physiological level of circulating Erythrocyte mass which ensure adequate tissue Oxygen levels and maintain an appropriate Oxygen supply throughout the body (Ref.1 & 2). It is produced in the Kidney, Liver and Brain in response to low Oxygen levels and is bound by circulating Red Blood Cells. Epo elicits a variety of biologic and cell-specific responses on interacting with its receptor, EpoR (Erythropoietin Receptor), a Type-I Cytokine Receptor expressed on the surface of cells. Activation of EpoR by ligand binding results in receptor homodimerization and induces a cascade of signaling events including Tyrosine phosphorylation of the EpoR correlated with mitogenesis, gene transcription and subsequent activation of intracellular Antiapoptotic Proteins, Kinases and Transcription Factors; generation, activation and transduction of several downstream pathways from the cell surface to the Nucleus, such as the JAK (Janus Kinase)-STAT (Signal Transducers and Activators of Transcription Factor) Pathway, PI3K (Phosphatidylinositde-3 Kinase) Pathway and the Ras (Ras Oncogene Homolog)-MAPK (Mitogen-Activated Protein Kinase) Pathway (Ref.3 and 4).

Following Epo stimulation, the Cytokine Receptor signal transduction pathway is initiated, as EpoR associates with JAK2 (Janus Kinase-2) via the membrane-proximal Cytoplasmic tail. This association results in the autophosphorylation and subsequent activation of JAK2, which induces rapid phosphorylation of several Tyrosine residues on the EpoR-Cytosolic domain and the downstream effector STAT5 (Signal Transducers and Activators of Transcription factor-5) (Ref.3). STAT5, a latent cryptic Cytoplasmic transcription factor, is phosphorylated on a conserved Tyrosine residue, which induces its homodimerization, Nuclear translocation, and DNA binding at PIE (Prolactin-Inducible Element), and leads to the induction of Epo-responsive genes. STAT5 activates transcription of the BclXL (Bcl2 Related Protein Long Isoform) gene. BclXL has an essential role in preventing Apoptosis of primitive and definitive Erythrocytes at the end of maturation, and the STAT5-BclXL signaling pathway promotes cell proliferation. However, Epo signal-promoting cascade encounters stringent regulation by terminating events such as; activation of the Tyrosine Phosphatase SHP1 (Tyrosine Phosphatase Shp1) and SHP2 (Tyrosine Phosphatase Shp2) that dephosphorylate JAK2, and induction of the negative regulatory proteins CIS (cytokine-inducible SH2 domain-containing protein), SOCS1 (Suppressor of Cytokine Signaling-1), SOCS3 (Suppressor of Cytokine Signaling-3), PIAS (Protein Inhibitor of Activated STAT), and PTPs (Protein Tyrosine Phosphatases) (Ref.5 and 6).

Epo is a major upstream activator of the Ras/MAPK pathway. This multi-step signaling is achieved by the association of several adapter molecules that have been identified as components of the tyrosyl phosphorylation sites of activated EpoR complex. These include the SH2 (Src Homology-2) domain-containing adapter proteins GRB2 (Growth Factor Receptor Bound Protein-2), SHC (Src Homology-2 Domain Containing Transforming Protein) and GAB2 (GRB2-Associated Binding Protein-2) and the PTB (Phosphotyrosine Binding) domain containing proteins. GRB2 constitutively associates with the Guanine Nucleotide-Releasing Factor SOS (Son of Sevenless), and binds to the Tyrosine-phosphorylated EpoR either via an interaction with GAB2 (which is activated on phosphorylation at Tyr577 by SHC) or by binding to EpoR-associated Tyrosine-phosphorylated SHC. SHC recruits the GRB2-SOS complex to the plasma membrane thereby promoting Ras activation as it accounts for the SOS-dependent increased conversion of Ras from the inactive GDP (Guanosine Diphosphate)-bound state to the active GTP (Guanosine Triphosphate)-bound form. The activity of Ras is however limited by the hydrolysis of GTP back to GDP by GAP (GTPase Activating Proteins) and alternatively by another Cytoplasmic Phosphatase SHIP (SH2-Containing Inositol Phosphatase), which competes with GRB2 to offer a second check to the downstream Ras signaling. EpoR activates the Tyrosine phosphorylation of SHIP and that SHIP binds to the EpoR in an SH2-dependent fashion through multiple phosphotyrosine residues, including EpoR Tyr401, Tyr429, and Tyr431. Once in the GTP-bound state, Ras associates with and activates members of the Raf family of Serine/Threonine Kinases, specifically Raf1 (v-Raf1 Murine Leukemia Viral Oncogene Homolog-1) (Ref.7). Stimulation of Erythroid cells with Epo induces a RhoA (Ras Homolog Gene Family Member-A) mediated phosphorylation and activation of calcium-dependent isoforms of the PKC (Protein Kinase-C) family of Serine/Threonine Kinases. PKC, a potent activator of Ras and Raf1, plays an important role in the Epo-induced activation of MAPKRaf1 inhibits the activity of VDAC (Voltage Dependent Anion Channel Protein) and functions as an important Anti-apoptotic factor. Raf blocks VDAC-dependent PTPC (Permeability Transition Pore Complex) formation, which is responsible for the release of mitochondrial products that triggers Apoptosis. Activated Raf1 phosphorylates and activates the dual functional protein kinases MEKs (MAPK/ERK Kinases) which then phosphorylate the MAPKs on both threonine and Tyrosine residues. These phosphorylation events activate ERK (Extracellular Signal-Regulated Kinase) (Ref.8). ERK activation is required for the phosphorylation-dependent regulation of various Cytosolic proteins and a number of Nuclear DNA binding transcription factors, including some specific transcription factors such as  Elk1 (ETS-domain protein Elk1) and CREB (cAMP Response Element-Binding Protein), which regulate the early gene expression in the Erythroid cells (Ref.9).

In Hematopoietic cells, Epo activates MAPK passing through a novel pathway dependent on Gi association to EpoR. A PT (Pertussis Toxin)-sensitive heterotrimeric Gi protein constitutively associates through the C-terminal end of the intracellular Cytoplasmic domain of EpoR. Following Epo stimulation, Gi is released from the receptor with its concomitant dissociation into GN-AlphaI (Guanine Nucleotide-Binding Protein G (i) Subunit-Alpha) and GN-Beta (Guanine Nucleotide-Binding Protein-Beta)-GN-Gamma (Guanine Nucleotide-Binding Protein-Gamma) subunits followed by their corresponding activations. MAPK activation is initiated by the GNBeta-Gamma subunit-mediated Tyrosine phosphorylation of SHC and proceeds through the Ras-dependent pathway stimulating MEK phosphorylation. In addition to this, Gi also independently regulates MAPK activation through the GN-AlphaI subunit. The Tyrosine Kinase JAK2 contributes to this new pathway by acting downstream of GNBeta-Gamma and upstream of Ras and PI3K (Ref.10). Epo signaling also activates the Ras/MAPK/ERK pathway by the use of a third group of interacting partners other than the SHC/GRB2/SOS complex or the Gi proteins. The adaptor protein CrkL (v-Crk Avian Sarcoma Virus Ct10 Oncogene Homolog-Like), interacts with and recruits C3G (Guanine Nucleotide Releasing Protein C3G), another Guanine Nucleotide Exchange Factor like SOS, to the activated EpoRC3G independently initiates Epo-mediated MAPK signaling pathway via the activation of Ras. Activated Ras then stimulates the Raf/MEK/ERK signaling cascade leading to the activation of Elk1, which enhances transcription of Growth-related proteins, such as the members of the AP1 (Activating Protein-1) family of transcription factors, through the serum response element. CrkL and C3G are also implicated in Raf1 activation via a Ras/PI3K dependent mechanism involving the activation of the Serine-Threonine Kinase Rac (Related to A and C Serine/Threonine Protein Kinase) (Ref.11).

Phosphorylated transcription factors turn on transcription and subsequent gene expression of certain specific sets of target genes. The activation and post-translational processing of the AP1 complex, a dimer composed of c-Fos (Cellular Oncogene Fos) and c-Jun family members, is one of the earliest Nuclear events induced by ERKs. The AP1 complex binds to a palindromic DNA element referred to as the TRE (TPA responsive element). The transcriptional activation and stability of c-Jun is accomplished by JNK1 (c-Jun Kinase-1), CKII (Casein Kinase-2) and PPtase (Protein Phosphatase), whereas in case of c-Fos, ERK phosphorylates multiple residues within the carboxylterminal transactivation domain of c-Fos, resulting in its increased transcriptional activity. PIN1 (Peptidyl-Prolyl Cis/Trans Isomerase-NIMA-Interacting-1) regulates AP1-mediated transcription. As a consequence, the mitogenic actions of Epo is directly linked to transcriptional regulatory events utilizing Ras as a molecular switch converting upstream Tyrosine Kinase signals into a Serine/Threonine Kinase cascade. Constitutively active ERK, in a c-Jun dependent approach, also activates CcnD1 (Cyclin-D1), the critical governor of the cell-cycle clock apparatus directing the G1 to S-phase progression during the cell cycle (Ref.12, 13 and 14).

In early human Erythroid progenitor cells, a low concentration of Epo efficiently mediates a moderate rate of proliferation of Erythroid progenitor cells following the activation of PI3K, and PI3K-Gamma (PI3-Kinase-p110 subunit-Gamma) in particular, using its regulatory p85 subunit. Activated EpoR ensures its control over this trail via Tyrosine Kinase Src (v-Src Avian Sacroma (Schmidt-Ruppin A-2) Viral Oncogene). JAK2 activated FAK1 (Focal Adhesion Kinase-1) plays a role in PI3K activation in a Syk (Spleen Tyrosine Kinase)-dependent fashion. PI3K then phosphorylates membrane bound PIP2 (Phosphatidylinositol-4, 5-Bisphosphate) to generate PIP3 (Phosphatidylinositol-3, 4, 5-Trisphosphate) and PTEN (Phosphatase and Tensin Homolog) acts as a negative watchdog of this process. PI3K then triggers the phosphorylation of Akt (v-Akt Murine Thymoma Viral Oncogene Homolog) with the help of PDKs (Phosphoinositide-Dependent Kinases), such as PDK-1 (Phosphoinositide-Dependent Kinase-1) (Ref.2). The activation of the Akt/PKB (Protein Kinase-B) pathway, which protects cells from Apoptosis, is an example of the indispensable Cyto-protective effect of Epo. Akt phosphorylates various signaling molecules, including GSK3 (Glycogen Synthase Kinase-3), BAD (Bcl2-Antagonist of Cell Death) and FkhR (Forkhead In Rhabdomyosarcoma), to promote cell survival. Akt prevents the Nuclear translocation FoxO3A (Forkhead Box-O3A), facilitating the Akt-dependent binding of the protein 14-3-3, which inhibits Apoptosis. BAD phosphorylation also stimulates certain Anti-apoptotic signals that facilitate the inhibition of mitochondrial CytoC (Cytochrome-C) release and the inactivation of several Pro-apoptotic proteins such as Caspase1, Caspase3Caspase8 and Caspase9 (Ref.15 and 16). Besides EpoR downstream signaling pathways, an optimum function of GATA1 (GATA Binding Protein-1) Erythroid differentiation factor is essential for the coordination of Epo through Erythropoiesis. GATA1 exerts different activities at various steps of the molecular program of Erythroid differentiation. Akt directly phosphorylates GATA1 at Ser310 and this site-specific phosphorylation is required for the transcriptional activation of the TIMP1 (Tissue Inhibitor of Metalloproteinase-1), which plays an important role in stimulating Erythroid progenitors via their Erythroid-Potentiating Activity (Ref.17).

In addition to its essential role in baseline Erythropoiesis, Epo drives a GR (Glucocorticoid Receptor) mediated Erythropoietic response to hypoxic stress which is coupled with Erythrolysis and EpoR targets a broader spectrum of progenitors during stress. The death receptors Fas and FasL (Fas Ligand), fundamentally expressed by the early Spleen Erythroblasts act as negative regulators of Erythropoiesis and their suppression by Epo-EpoR represents a novel stress response pathway that facilitates Erythroblast expansion by increasing the Erythropoietic rate (Ref.18). The Epo gene expression during stress is induced by HIF (Hypoxia-Inducible Transcription Factors) and the stress kinase p38-Alpha (Ref.2 and 4). In Hematopoietic cells, Epo promotes the formation of a membrane complex between PLC-Gamma (Phospholipase-C-Gamma) and the EpoR. This association stimulates the Ca2(+) (Calcium ion)-activated Ca2(+)-permeable Channels and results in an enhanced Ca2(+) internalization via a voltage-independent Ca2(+) conductance. Epo also regulates GPI (Glycosylphosphatidylinositol) Hydrolysis via Tyrosine phosphorylation of its receptor and by PLC-Gamma activation. This provides a mechanism for the Vasoconstriction and Hypertension observed during the clinical use of Epo for treatment of Chronic Anemias. Deficits in Epo production result in Anemia in humans and in such cases, treatment with Recombinant Human Epo is efficient and safe in improving the management of the Anemia associated with Chronic Renal Failure (Ref.19).

For several years Epo has been believed to act exclusively on Erythroid cells; however evidences suggest extensive continuum of potential roles of Epo other than Erythropoiesis. Epo exerts Neuronal, Vascular and Cardiac protection through multiple signaling pathways operating during tissue and cell injury. Recently, Epo and its receptor have been localized to several non-Hematopoietic cell types, including the Central and Peripheral Nervous Systems, Endothelial Cells and Heart, where it acts as an effective defensive molecule against several kinds of degenerative conditions and toxic insults. Epo functions as a Neuroprotective agent and is employed in the treatment of Neurological and Cardiac disorders (Ref.3). Activation of the EpoR in Neurons triggers a cross-talk between the JAK2 and NF-KappaB (Nuclear Factor-KappaB) signal transduction system through which Epo protects Cortical Neurons from Excitotoxic- and Nitric Oxide induced Apoptosis. JAK2 phosphorylates and degrades I-KappaB (Inhibitor of Kappa Light Chain Gene Enhancer in B-Cells), resulting in the Nuclear translocation of NF-KappaBNF-KappaB has important mitogenic and Anti-apoptotic effects in Hematopoietic cells and facilitates Neurogenesis and differentiation of Neuronal Stem Cells into Astrocytes by transcriptional activation Neuroprotective genes (Ref.20). Multiple Anti-apoptotic and Pro-survival strategies, potentially organized by the EpoR, therefore interact to produce a remarkable dynamic range of Erythropoiesis. A better understanding of interplay between all the cellular pathways and molecules modulated by Epo signaling is crucial in determining the potential therapeutic application of recombinant human Epo and may provide further insights in the development of better synergistic therapies to the treatment of certain types of Cancer and in the battle against AIDS (Ref.2, 3 and 4).

 

References

  1. Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation
  2. Erythropoietin regulations in humans under different environmental and experimental conditions
  3. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors
  4. Erythropoietin: a novel neuroprotective cytokine
  5. Molecular biology of erythropoietin
  6. The cytokine-inducible Scr homology domain-containing protein negatively regulates signaling by promoting apoptosis in erythroid progenitor cells
  7. SLIM trims STATs: ubiquitin E3 ligases provide insights for specificity in the regulation of cytokine signaling
  8. Human RAS superfamily proteins and related GTPases
  9. A double cysteine trkA mutant exhibiting reduced NGF binding and delayed Erk signaling
  10. Activation of MAPKs by 1alpha,25(OH)2-Vitamin D3 and 17beta-estradiol in skeletal muscle cells leads to phosphorylation of Elk-1 and CREB transcription factors
  11. Activation of the mitogen-activated protein kinases Erk1/2 by erythropoietin receptor via a G(i )protein beta gamma-subunit-initiated pathway
  12. Regulation of the transcriptional activity of c-Fos by ERK. A novel role for the prolyl isomerase PIN1
  13. CKS1B, overexpressed in aggressive disease, regulates multiple myeloma growth and survival through SKP2- and p27Kip1-dependent and -independent mechanisms
  14. Rewired ERK-JNK signaling pathways in melanoma
  15. New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway
  16. EPO tecting the endothelium
  17. Erythropoietin stimulates phosphorylation and activation of GATA-1 via the PI3-kinase/AKT signaling pathway
  18. Molecular insights into stress erythropoiesis
  19. Erythropoietin-modulated calcium influx through TRPC2 is mediated by phospholipase Cgamma and IP3R
  20. Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways