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Erythropoietin Pathway

Erythropoiesis is one major pathway by which a pluripotent hematopoietic stem cell gives rise to mature end stage cells. Erythropoietin (Epo) is a lineage-specific hematopoietic cell required for survival, proliferation and differentiation of committed erythroid progenitor cells. Its major effects are to promote erythroid differentiation and to initiate hemoglobin synthesis. Therefore, Epo has been identified as the major hormone required for erythropoiesis. Epo exerts its function through the EpoR (Epo Receptor), a member of the classI Cytokine receptor family (Ref.1). Following binding of Epo to its receptor, the receptor forms homodimers and undergoes phosphorylation by physically associating and interacting with the tyrosine kinase JAK2 (Janus Kinase-2). Once phosphorylated, these tyrosine residues allow the recruitment and activation of a number of downstream adaptors and effectors including STAT5 (Signal Transducers and Activators of Transcription factor-5), PI3K (Phosphoinositide-3 Kinase), SHIP (SH2-containing Inositol Phosphatase), the tyrosine phosphatase SHP1 and SHP2 and the ERKs (Extracellular Signal Regulated Kinases), JNK (Jun N-terminal Kinases) and p38 MAPK (Mitogen Activated Protein Kinase) (Ref.2). STAT5 act as a transcription factor, binds to nuclear DNA carrying the signal from the membrane to the nucleus (Ref.3).

Adapter molecules that have been identified as components of the activated EpoR complex include the SH2 domain-containing adapter proteins GRB2 (Growth Factor Receptor Bound Protein-2) and SHC. GRB2, which is constitutively associated with the guanine nucleotide-releasing factor SOS (Son of Sevenless), binds the tyrosine-phosphorylated EpoR either directly through its SH2 domain or indirectly through binding to EpoR-associated tyrosine-phosphorylated SHC. Binding of GRB2 to the receptor is thought to translocate SOS to the membrane, where it activates the exchange of GDP for GTP on Ras guanine nucleotide-binding proteins. Ras-GTP has been shown to activate the Raf1/MAPK cascade by binding Raf1 and anchoring it at the cell membrane where it is phosphorylated and activated by other kinases. Activated Raf1 then phosphorylates and activates the dually specific kinase MEKs, which phosphorylates and activates MAPK. Although the specific kinases involved in Raf1 activation have not been completely identified, members of the PKC (protein kinase-C) family of serine kinases have been implicated as potential activators of Raf1. It has been demonstrated that PKC-mediated serine phosphorylation directly activates Raf1 in hematopoietic cells stimulated with IL-3 (Interleukin-3) and in other cell types stimulated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Stimulation of erythroid cells with Epo induces phosphorylation and activation of calcium-dependent isoforms of the PKC family of serine/threonine kinases. In addition, PKC is required for Epo-induced activation of MAPK in normal erythroid progenitor cells. However, studies using a variety of mitogen- and growth factor-activated cells have demonstrated the existence of both PKC-dependent and PKC-independent modes of Raf1 activation, and the requirement for PKC in Raf1 activation varies with the specific growth factor receptor being stimulated (Ref.4). Epo also induces tyrosine phosphorylation of Akt. Activation of Akt by Epo is dependent on PI3K (Phosphatidylinositol 3-Kinase) activity. The PI3K signaling pathway is important for the regulation of a number of cellular responses.

Overexpression of SOCS1 (Suppressor of Cytokine Signaling-1), SOCS3 (Suppressor of Cytokine Signaling-3), or CIS (Cytokine Inducible SH2-containing Protein) negatively regulates Epo-mediated cell proliferation (Ref.5). JAK2 seems to activate a new downstream target, the transcription factor NF-κB (Nuclear Factor-κ B). Epo treatment of neurons caused the nuclear translocation of NF-κB and a sustained increase in NF-κB association with DNA. The neuroprotective effects of Epo were abolished when JAK2 or NF-κB action was inhibited. JAK2 may phosphorylate IKKs (I-κB Kinase), a modification that would degrade I-κB and free NF-κB from inhibition. As NF-κB regulates the expression of antiapoptotic proteins, the proposed EpoR-JAK2-NF-κB signaling pathway may be a critical component of preventing neuronal cell death.

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