Attenuation of EPO-dependent erythroblast formation by death-associated protein kinase-2

Abstract

The adult erythron is maintained via dynamic modulation of erythroblast survival potentials. Toward identifying novel regulators of this process, murine splenic erythroblasts at 3 developmental stages were prepared, purified and profiled. Stage-to-stage modulated genes were then functionally categorized, with a focus on apoptotic factors. In parallel with BCL-X and NIX, death-associated protein kinase-2 (DAPK2) was substantially upmodulated during late erythropoiesis. Among hematopoietic lineages, DAPK2 was expressed predominantly in erythroid cells. In a Gata1-IE3.9int-DAPK2 transgenic mouse model, effects on steady-state reticulocyte and red blood cell (RBC) levels were limited. During hemolytic anemia, however, erythropoiesis was markedly deficient. Ex vivo analyses revealed heightened apoptosis due to DAPK2 at a Kit ؊ CD71 high Ter119 ؊ stage, together with a subsequent multifold defect in late-stage Kit ؊ CD71 high Ter119 ؉ cell formation. In UT7epo cells, siRNA knockdown of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated. DAPK2 therefore comprises a new candidate attenuator of stress erythropoiesis. (Blood. 2008;112:886-890) IntroductionErythroid progenitor cell survival is regulated by unique networked mechanisms. As cell-extrinsic factors, FASL and TRAIL can induce apoptosis, while erythropoietin (EPO) provides essential survival signals via JAK2/STAT5, PI3K/AKT, and RAS/MEK/ ERK1,2 routes. 1 Cell-intrinsic regulators include BCL-X and NIX, plus GATA1 as a caspase-3 target. [2][3][4] To define new potential key survival-regulating factors, we presently profiled differentially staged primary murine splenic erythroblasts. One discovered erythroid-predominant factor was death-associated protein kinase-2 (DAPK2). Among 3 DAPK serine/threonine kinases, 5,6 DAPK1 first was identified as an IFN-␥-induced factor which facilitates cell death initiated by IFN-␥, TNF-␣, FAS, or oncogene expression. 7 DAPK1 possesses a DAP kinase upper-lobe signature, CaM regulatory domain, ankyrin repeats, and a C-terminal death domain. 5,6 ZIPK (zipper-interacting protein kinase/DAPK3/DLK) lacks a CaM domain, but possesses leucine zipper and nuclear localization motifs. 5,6,8 DAPK2 retains a related CaM domain, but possesses a unique C-terminal region. 5,6,9 Such structural differences further suggest that DAPK1, ZIP-K and DAPK2 likely play unique biologic roles.DAPK1 can exert proapoptotic effects, potentially via caspaseindependent type-II mechanisms. [5][6][7] Gene disruption experiments further place DAPK1 upstream of p53, 9 and decreased DAPK1 expression is linked to multiple myeloma, ALL, and colon, breast and lung cancers. 10 For DAPK2, overexpression studies in cell lines also point to potential proapoptotic effects, 11,12 but to date this has not been investigated in primary cells which normally express, and regulate endogenous DAPK2. Via transcriptome analyses, transgenic mouse experiments, and analyses in EPO-dependent erythroid...

Diminished Hematopoietic Activity Associated with Alterations in Innate and Adaptive Immunity in a Mouse Model of Human Monocytic Ehrlichiosis

“…To determine whether the increase in the level of Ter119 ϩ cells reflected an increase in the number of reticulocytes belonging to a particular subset, we next examined the splenocytes for expression of Ter119, CD71 (the transferrin receptor), and c-Kit (CD117). The level of expression of CD71 on immature reticulocytes is typically high, but it decreases as reticulocytes mature; early-stage reticulocytes also exhibit high surface expression of c-Kit (10,11,13). On the basis of the expression of these surface markers, the Ter119 ϩ cells in the E. muris-infected spleens were largely immature erythroid cells as a majority of the Ter119 ϩ cells expressed high levels of CD71 and c-Kit (Fig.…”

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

“…Overexpression of DAPK2 results in morphological changes reminiscent of apoptosis in adherent cells, such as membrane blebbing and condensation of nuclei (5, 6), and in reduced viability and survival in suspension cells (12,13). In line with these findings, restoration of DAPK2 activity through fusion of a constitutively active DAPK2 to CD30 in Hodgkin lymphoma cells resulted in selective apoptosis in tumor cells in vitro and in prolonged survival in a Hodgkin lymphoma mouse model (14), and depletion of DAPK2 sensitizes resistant cells to TRAIL-induced killing (15).…”

DAPK2 (death-associated protein kinase 2)

“…DPAK2 is substantially up-modulated during late erythropoiesis (Fang J et al, 2008). In UT7epo cells, siRNA knock-down of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated.…”

“…In UT7epo cells, siRNA knock-down of DAPK2 enhanced survival due to cytokine withdrawal, and DAPK2's phosphorylation and kinase activity also were erythropoietin (EPO)-modulated. DAPK2 therefore comprises a new candidate attenuator of stress erythropoiesis (Fang J et al, 2008). The physiological substrate of DAPK2 is unknown although it is known to phosphorylate the myosin light chain in vitro (Inbal B et al, 2000).…”