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Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Shibayama H, Takai E, Matsumura I, Kouno M, Morii E, Kitamura Y, Takeda J, Kanakura Y - J. Exp. Med. (2004)

Bottom Line: Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules.Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation.Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Yamada-oka, Suita, 565-0871, Japan.

ABSTRACT
Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin-/- mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin-/- mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

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Phenotypes of Anamorsin (AM)−/− embryos, spleens, and FLs. (a) E16.5 embryos of Anamorsin−/− and the littermate control are shown. (b) The spleens (arrows) with stomach of Anamorsin+/+ and Anamorsin−/− mice at birth are shown. (c) Transverse sections of FL at E14.5 obtained from Anamorsin+/+ (top) or Anamorsin−/− (bottom) embryos were stained with hematoxylin and eosin for histological examination (left) and with TUNEL to detect apoptotic cells (right). Apoptotic cells are stained with brown in this TUNEL assay. ×400. (d) Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–Ter-119 and annexin V, and subjected to flow cytometric analysis. (e) The proportions of CD34+ c-kit+, CD34low CD44high, and Ter-119+ c-kit+ cells in Anamorsin+/+ and Anamorsin−/− FL cells at E14.5. Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–c-kit, anti-CD34, anti-CD44, or anti–Ter-119, and subjected to flow cytometric analysis.
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fig4: Phenotypes of Anamorsin (AM)−/− embryos, spleens, and FLs. (a) E16.5 embryos of Anamorsin−/− and the littermate control are shown. (b) The spleens (arrows) with stomach of Anamorsin+/+ and Anamorsin−/− mice at birth are shown. (c) Transverse sections of FL at E14.5 obtained from Anamorsin+/+ (top) or Anamorsin−/− (bottom) embryos were stained with hematoxylin and eosin for histological examination (left) and with TUNEL to detect apoptotic cells (right). Apoptotic cells are stained with brown in this TUNEL assay. ×400. (d) Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–Ter-119 and annexin V, and subjected to flow cytometric analysis. (e) The proportions of CD34+ c-kit+, CD34low CD44high, and Ter-119+ c-kit+ cells in Anamorsin+/+ and Anamorsin−/− FL cells at E14.5. Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–c-kit, anti-CD34, anti-CD44, or anti–Ter-119, and subjected to flow cytometric analysis.

Mentions: Genotypic analysis of embryos from Anamorsin+/− mice intercrosses revealed that Anamorsin−/− embryos began to die between E12.5 and E14.5. The rate of dead Anamorsin−/− embryos increased after E14.5 (0% until E12.5, 18.8% at E14.5, 36.4% at E16.5, and 44.4% at E18.5) and all Anamorsin−/− mice died at birth, suggesting that Anamorsin−/− mice expire in late gestation (Table I). Anamorsin−/− embryos were smaller in body size than Anamorsin+/+ embryos, whereas Anamorsin+/− embryos displayed phenotypes similar to Anamorsin+/+ embryos (Fig. 4 a). Despite no significant difference in the formation of blood islands in yolk sacs, the FLs and spleens of Anamorsin−/− embryos were remarkably smaller than those of Anamorsin+/+ embryos. The size of FL of Anamorsin−/− embryos was about one third of that of Anamorsin+/+ embryos, and the spleen of Anamorsin−/− embryos appeared scarce (Fig. 4 b). Furthermore, Anamorsin−/− embryos later than E14.5 looked anemic (Fig. 4 a). Anamorsin−/− embryos at E18.5 showed nearly half of RBCs, hemoglobin, and hematocrit in the peripheral blood (RBC: 365 ± 50.8, 335.5 ± 50.6, and 210 ± 67.0×104/mm3; hemoglobin: 11.7 ± 1.5, 10.6 ± 1.3, and 6.7 ± 2.3 g/dl; hematocrit: 39.7 ± 5.8, 37.8 ± 5.3, and 26.3 ± 7.6% in Anamorsin+/+ [n = 10], Anamorsin+/− [n = 22], and Anamorsin−/− [n = 8] embryos, respectively). Moreover, RBCs of Anamorsin−/− embryos were macrocytic (mean corpuscular volume in Anamorsin+/+, Anamorsin+/−, and Anamorsin−/− embryos were 108.8 ± 4.3, 113.1 ± 9.5, and 128.2 ± 16.6, respectively). In addition to defects in hematopoietic organs, the heart walls of Anamorsin−/− embryos were thinner than those in Anamorsin+/+ or Anamorsin+/− embryos, whereas Anamorsin−/− embryos displayed no apparent macroscopic or histological abnormalities in other organs (not depicted). It was unlikely that the abnormality in the heart walls caused death in Anamorsin+/− embryos.


Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Shibayama H, Takai E, Matsumura I, Kouno M, Morii E, Kitamura Y, Takeda J, Kanakura Y - J. Exp. Med. (2004)

Phenotypes of Anamorsin (AM)−/− embryos, spleens, and FLs. (a) E16.5 embryos of Anamorsin−/− and the littermate control are shown. (b) The spleens (arrows) with stomach of Anamorsin+/+ and Anamorsin−/− mice at birth are shown. (c) Transverse sections of FL at E14.5 obtained from Anamorsin+/+ (top) or Anamorsin−/− (bottom) embryos were stained with hematoxylin and eosin for histological examination (left) and with TUNEL to detect apoptotic cells (right). Apoptotic cells are stained with brown in this TUNEL assay. ×400. (d) Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–Ter-119 and annexin V, and subjected to flow cytometric analysis. (e) The proportions of CD34+ c-kit+, CD34low CD44high, and Ter-119+ c-kit+ cells in Anamorsin+/+ and Anamorsin−/− FL cells at E14.5. Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–c-kit, anti-CD34, anti-CD44, or anti–Ter-119, and subjected to flow cytometric analysis.
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fig4: Phenotypes of Anamorsin (AM)−/− embryos, spleens, and FLs. (a) E16.5 embryos of Anamorsin−/− and the littermate control are shown. (b) The spleens (arrows) with stomach of Anamorsin+/+ and Anamorsin−/− mice at birth are shown. (c) Transverse sections of FL at E14.5 obtained from Anamorsin+/+ (top) or Anamorsin−/− (bottom) embryos were stained with hematoxylin and eosin for histological examination (left) and with TUNEL to detect apoptotic cells (right). Apoptotic cells are stained with brown in this TUNEL assay. ×400. (d) Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–Ter-119 and annexin V, and subjected to flow cytometric analysis. (e) The proportions of CD34+ c-kit+, CD34low CD44high, and Ter-119+ c-kit+ cells in Anamorsin+/+ and Anamorsin−/− FL cells at E14.5. Cell suspensions isolated from Anamorsin+/+ (top) and Anamorsin−/− (bottom) FL cells at E14.5 were stained with anti–c-kit, anti-CD34, anti-CD44, or anti–Ter-119, and subjected to flow cytometric analysis.
Mentions: Genotypic analysis of embryos from Anamorsin+/− mice intercrosses revealed that Anamorsin−/− embryos began to die between E12.5 and E14.5. The rate of dead Anamorsin−/− embryos increased after E14.5 (0% until E12.5, 18.8% at E14.5, 36.4% at E16.5, and 44.4% at E18.5) and all Anamorsin−/− mice died at birth, suggesting that Anamorsin−/− mice expire in late gestation (Table I). Anamorsin−/− embryos were smaller in body size than Anamorsin+/+ embryos, whereas Anamorsin+/− embryos displayed phenotypes similar to Anamorsin+/+ embryos (Fig. 4 a). Despite no significant difference in the formation of blood islands in yolk sacs, the FLs and spleens of Anamorsin−/− embryos were remarkably smaller than those of Anamorsin+/+ embryos. The size of FL of Anamorsin−/− embryos was about one third of that of Anamorsin+/+ embryos, and the spleen of Anamorsin−/− embryos appeared scarce (Fig. 4 b). Furthermore, Anamorsin−/− embryos later than E14.5 looked anemic (Fig. 4 a). Anamorsin−/− embryos at E18.5 showed nearly half of RBCs, hemoglobin, and hematocrit in the peripheral blood (RBC: 365 ± 50.8, 335.5 ± 50.6, and 210 ± 67.0×104/mm3; hemoglobin: 11.7 ± 1.5, 10.6 ± 1.3, and 6.7 ± 2.3 g/dl; hematocrit: 39.7 ± 5.8, 37.8 ± 5.3, and 26.3 ± 7.6% in Anamorsin+/+ [n = 10], Anamorsin+/− [n = 22], and Anamorsin−/− [n = 8] embryos, respectively). Moreover, RBCs of Anamorsin−/− embryos were macrocytic (mean corpuscular volume in Anamorsin+/+, Anamorsin+/−, and Anamorsin−/− embryos were 108.8 ± 4.3, 113.1 ± 9.5, and 128.2 ± 16.6, respectively). In addition to defects in hematopoietic organs, the heart walls of Anamorsin−/− embryos were thinner than those in Anamorsin+/+ or Anamorsin+/− embryos, whereas Anamorsin−/− embryos displayed no apparent macroscopic or histological abnormalities in other organs (not depicted). It was unlikely that the abnormality in the heart walls caused death in Anamorsin+/− embryos.

Bottom Line: Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules.Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation.Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Yamada-oka, Suita, 565-0871, Japan.

ABSTRACT
Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin-/- mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin-/- mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.

Show MeSH