Spry1 Plays Selective Hematopoietic Roles as An Erythropoietin - Modulated Positive Regulator of Erythropoiesis.

Abstract 777

The four vertebrate Sprouty (Spry1-4) proteins are molecular adaptors, best known as negative regulators of MAP kinase activation mediated by FGFR, VEGFR and RET. Prior studies in human hematopietic stem cells and zebrafish implicated Spry proteins in stem cell development. Presently, we have ascertained the role of Spry1 in erythroid development using cellular models and in knockout mice. Treatment of UT7 erythropoietin-responsive cells led a strong increase in phosphorylation of Spry1 and Spry2 on critical N-terminal tyrosine sites of these proteins (Y53 and Y55, respectively). UT7 cells engineered to constitutively express Spry1 also demonstrated decreased ERK activation in response to erythropoietin treatment. Spry expression was measured in developing primary bone marrow (pro)erythroblasts by real time PCR. Spry1 was expressed most prominently in erythroblasts at a level 40 times higher than Spry 2-4. Furthermore, Spry1 expression rose markedly as erythroblasts matured. To determine the role of Spry1 in murine hematopoiesis, conditional, LoxP flanked allele of Spry1 was crossed with Mx1-Cre transgenic mice and Spry1 was deleted in murine marrow by injection of mice with poly pIpC. Efficient deletion of the Spry1 gene in murine marrow did not affect lymphocytes or granulocytes and selectively led to an increased reticulocyte count (8.9% +/- 0.2% in Spry1 deleted vs. 4.9 +/- 0.5% in control mice, p<0.002). Deletion of Spry1 led to activation of splenic erythropoiesis with a four fold enrichment of CD71^high, Ter119^pos precursors in Mx1-Cre; Spry1^flox/flox animals compared to Mx1-Cre;Spry1^flox/+ animals. In ex vivo expansion cultures, however, erythroid progenitors, were significantly compromised in their intrinsic capacity to form Kit^negCD71^highTer119^neg and Kit^negCD71^highTer119^pos erythroblasts. Collectively these data suggest that during hematopoiesis, SPRY1 acts selectively as a non-redundant novel positive effector of EPO- dependent red cell formation.