Unrestrained erythroblast development in Nix-/- mice reveals a mechanism for apoptotic modulation of erythropoiesis - PubMed (original) (raw)

. 2007 Apr 17;104(16):6794-9.

doi: 10.1073/pnas.0610666104. Epub 2007 Apr 9.

Andrew G Koesters, Amy M Odley, Suvarnamala Pushkaran, Christopher P Baines, Benjamin T Spike, Diedre Daria, Anil G Jegga, Hartmut Geiger, Bruce J Aronow, Jeffery D Molkentin, Kay F Macleod, Theodosia A Kalfa, Gerald W Dorn 2nd

Affiliations

Unrestrained erythroblast development in Nix-/- mice reveals a mechanism for apoptotic modulation of erythropoiesis

Abhinav Diwan et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Normal production of RBCs requires that the antiapoptotic protein Bcl-xl be induced at end stages of differentiation in response to erythropoietin (Epo) signaling. The critical proapoptotic pathways inhibited by Bcl-xl in erythroblasts are unknown. We used gene targeting in the mouse to evaluate the BH3-only factor Nix, which is transcriptionally up-regulated during Epo-stimulated in vitro erythrocyte differentiation. Nix null mice are viable and fertile. Peripheral blood counts revealed a profound reticulocytosis and thrombocytosis despite normal serum Epo levels and blood oxygen tension. Nix null mice exhibited massive splenomegaly, with splenic and bone marrow erythroblastosis and reduced apoptosis in vivo during erythrocyte maturation. Hematopoietic progenitor populations were unaffected. Cultured Nix null erythroid cells were hypersensitive to Epo and resistant to apoptosis stimulated by cytokine deprivation and calcium ionophore. Transcriptional profiling of Nix null spleens revealed increased expression of cell cycle and erythroid genes, including Bcl-xl, and diminished expression of cell death and B cell-related genes. Thus, cell-autonomous Nix-mediated apoptosis in opposition to the Epo-induced erythroblast survival pathway appears indispensable for regulation of erythrocyte production and maintenance of hematological homeostasis. These results suggest that physiological codependence and coordinated regulation of pro- and antiapoptotic Bcl2 family members may represent a general regulatory paradigm in hematopoiesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Nix effects on isolated mitochondria and general phenotype of Nix gene ablation in mice. (A) Isolated WT mouse liver mitochondria were incubated with increasing concentrations of GST-Nix, GST-sNix, and GST-Bax. Resultant mitochondrial pellet and supernatant underwent Western blotting for cytochrome c and cytochrome oxidase IV (COX-IV) (n = 3). (B) Time-course studies (0–180 min) as in A. (C) Swelling of isolated WT mitochondria induced by GST-Nix, 250 μM Ca2+, and GST-Nix + 250 μM Ca2+ (means of n = 2). (D) Schematic of Nix deletion strategy. Exons 1–6b (filled rectangles) and restriction sites are depicted (

see SI Methods

). (E) Southern blot (Left) and PCR (Right) screening of _Nix_-targeted mice. (F) Multiple-tissue Northern blot hybridized to Nix probes. (G) Hypomorphism. (H) Splenomegaly of WT (Upper) and _Nix_−/− (Lower) mice.

Fig. 2.

Fig. 2.

Splenic erythroblastosis and erythrocyte abnormalities in _Nix_−/− mice. (A and B) H&E-stained splenic sections. (Magnification: A, ×4; B, ×20.) (C) Ter119-stained (brown) splenic sections. Blue is counterstained lymphoid tissue. (D) Representative flow-cytometric quantification of Ter119+ splenocytes. (E) Wright-Giemsa-stained peripheral blood smears (1, polychromatic cells; 2, immature erythrocytes with redundant membrane; 3, discocytes).

Fig. 3.

Fig. 3.

Erythroblast hyperplasia and diminished apoptosis in _Nix_−/− bone marrow and spleens. (A and B) Ter119 and CD71 expression in freshly isolated bone marrow (A) or splenic (B) cells. Cells: yellow, proerythroblasts (ProE); blue, basophilic erythroblasts (BasoE); pink, chromatophilic erythroblasts (ChromoE); green, orthochromatic erythroblasts (OrthoE). (C and D) Representative flow-cytometric analysis of Lin-, Sca-1/c-kit+ fraction (C), and Hoechst 33342-excluding “side population” (D) bone marrow cells. (E and F) Analysis of in vivo apoptosis in splenocytes. NonE, nonerythroblasts; ∗, P < 0.05.

Fig. 4.

Fig. 4.

Epo-hyperresponsiveness and apoptosis resistance of _Nix_−/− splenocytes. (A) CFU-E colony formation with and without increasing doses of Epo (n = 6–7 paired experiments; ∗, P ≤ 0.001 compared with WT). (B) Survival (Left, n = 5) and apoptosis (Right, n = 4) of splenocytes in monoculture. (C and D) Proportional change in Ter119+ splenocytes (C) and Ter119 and CD71 expression (D) after 48 h of suspension monoculture as in B (n = 4 paired experiments). (E) Splenocyte survival after apoptotic provocation with ionomycin 1 μg/ml (Left) or PMA 2 ng/ml (Right; n = 5 paired experiments; ∗, P < 0.05). (F) Survival of Ter119+ splenocytes in vitro (n = 4 paired experiments; ∗, P < 0.05).

Fig. 5.

Fig. 5.

Altered patterns of gene expression in _Nix_−/− spleens. (A) Enrichment (red) and disenrichment (blue) of selected functional gene groups in _Nix_−/− spleens. (B) Dendrogram and heat map depiction (Left) and abbreviated list of regulated genes (Right). Color intensity (red:highest to blue:lowest) displays relative expression.

Fig. 6.

Fig. 6.

Schematic depiction of Nix involvement in erythroid maturation pathway. Fas L, Fas ligand.

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