This is a revised application for renewal of a highly productive program to research Epo-activated mechanisms of erythroid progenitor cell survival, and differentiation. The Epo receptor system has emerged as a paradigm for studies of mitogenic signalling via type 1 receptors. However, little is understood concerning receptor subdomains and effectors that mediate Epo's critical roles in inhibiting cell death, and stimulating late erythroid gene expression. Based on experiments in which Epo receptor proximal subdomains are shown to mediate inhibition of apoptosis, studies are proposed to test roles for box-1 & -2 motifs and STAT binding sites in this pathway. Roles for Jak2, Pim1 and STAT5 also will be assessed (using dominant-negative mutants), and whether Epo engages Bcl2-related factors via induced expression, phosphorylation or regulated dimerization will be investigated (aim #2).
Aims #1 & 3 are designed to advance an understanding of mechanisms of Epo-induced erythroid differentiation events. The PI has established erythroleukemic SKT6 cells as an important model for studies of Epo-induced globin expression; has demonstrated the activity of EpoR/EGFR chimeras in this model; and has provided primary evidence for a role for STAT5 in this response pathway.
In aim #1, Epo receptor domains that mediate globin induction in SKT6 cells will be defined, and roles for STAT5 proves to be a critical effector, suppressor PCR-based subtractive hybridization may be applied to clone targets for STAT5 in this unique context. In new studies Epo receptor-, GATA1 - dependent expression of endogenous GATA1, EKLF & BETA- globin genes also has been reconstituted in myeloid FDC2 cells. Here, Epo-dependent mechanisms of GATA1 transcript accumulation will be studied; possible involvements of NFE2, Rbtn2, GATA2, Evil and/or FOG in erythroid gene activation will be investigated; and the ability of GATA1 subdomains to affect Epo-dependent differentiation vs. Growth will be tested (aim #3). Proposed studies will advance an understanding of Epo action in red cell development, and are relevant to the development of novel therapies for red cell disorders, hemoglobinopathies, and leukemias via induced differentiation.
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