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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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Anamorsin (AM) was expressed ubiquitously in various tissues and its expression was induced by various cytokines through the Ras signaling pathway. (a) The expression of the human Anamorsin homologue in various human organs (MTC panels, CLONTECH Laboratories, Inc.) was examined by Northern hybridization. (b) Ba/F3 cells were IL-3 depleted for 15–18 h and then cultured with 0.1 ng/ml mIL-3 for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. Ba/F3-Ad, Ba/F3-Ad in the absence of IL-3. (c) Ba/F3 cells each transfected with EPO receptor, c-kit, and c-mpl were deprived of IL-3 for 15–18 h and cultured with 5 u/ml mEPO, 100 ng/ml mSCF, and 30 ng/ml mTPO, respectively, for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. (d) Ba/F3 cells transfected with an empty vector (Mock) or oncogenic H-Ras (H-RasG12V) were deprived of IL-3 for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (e) Ba/F3 cells, in which dominant negative H-Ras (H-RasS17N) was inducibly expressed by a LacSwitch-II–inducible expression system, were cultured with IL-3 in the presence or absence of IPTG for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (f) The localization of Anamorsin was examined by an immunofluorescence staining with an anti-Anamorsin mAb. Nucleus was visualized with staining with DAPI. Green, Anamorsin; blue, nucleus.
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fig2: Anamorsin (AM) was expressed ubiquitously in various tissues and its expression was induced by various cytokines through the Ras signaling pathway. (a) The expression of the human Anamorsin homologue in various human organs (MTC panels, CLONTECH Laboratories, Inc.) was examined by Northern hybridization. (b) Ba/F3 cells were IL-3 depleted for 15–18 h and then cultured with 0.1 ng/ml mIL-3 for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. Ba/F3-Ad, Ba/F3-Ad in the absence of IL-3. (c) Ba/F3 cells each transfected with EPO receptor, c-kit, and c-mpl were deprived of IL-3 for 15–18 h and cultured with 5 u/ml mEPO, 100 ng/ml mSCF, and 30 ng/ml mTPO, respectively, for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. (d) Ba/F3 cells transfected with an empty vector (Mock) or oncogenic H-Ras (H-RasG12V) were deprived of IL-3 for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (e) Ba/F3 cells, in which dominant negative H-Ras (H-RasS17N) was inducibly expressed by a LacSwitch-II–inducible expression system, were cultured with IL-3 in the presence or absence of IPTG for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (f) The localization of Anamorsin was examined by an immunofluorescence staining with an anti-Anamorsin mAb. Nucleus was visualized with staining with DAPI. Green, Anamorsin; blue, nucleus.

Mentions: Next, we examined the expression profile of the human Anamorsin homologue in various organs using MTA panels (CLONTECH Laboratories, Inc.). As shown in Fig. 2 a, left, the homologue was expressed ubiquitously in various tissues, with especially high expression levels detected in heart, liver, and pancreas. As for hematopoietic tissues, the homologue was abundantly expressed in FL and spleen (Fig. 2 a, right). Also, we found that expression of Anamorsin was detectable in early stages (7 d) of embryogenesis by the PCR method (not depicted).


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)

Anamorsin (AM) was expressed ubiquitously in various tissues and its expression was induced by various cytokines through the Ras signaling pathway. (a) The expression of the human Anamorsin homologue in various human organs (MTC panels, CLONTECH Laboratories, Inc.) was examined by Northern hybridization. (b) Ba/F3 cells were IL-3 depleted for 15–18 h and then cultured with 0.1 ng/ml mIL-3 for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. Ba/F3-Ad, Ba/F3-Ad in the absence of IL-3. (c) Ba/F3 cells each transfected with EPO receptor, c-kit, and c-mpl were deprived of IL-3 for 15–18 h and cultured with 5 u/ml mEPO, 100 ng/ml mSCF, and 30 ng/ml mTPO, respectively, for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. (d) Ba/F3 cells transfected with an empty vector (Mock) or oncogenic H-Ras (H-RasG12V) were deprived of IL-3 for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (e) Ba/F3 cells, in which dominant negative H-Ras (H-RasS17N) was inducibly expressed by a LacSwitch-II–inducible expression system, were cultured with IL-3 in the presence or absence of IPTG for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (f) The localization of Anamorsin was examined by an immunofluorescence staining with an anti-Anamorsin mAb. Nucleus was visualized with staining with DAPI. Green, Anamorsin; blue, nucleus.
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fig2: Anamorsin (AM) was expressed ubiquitously in various tissues and its expression was induced by various cytokines through the Ras signaling pathway. (a) The expression of the human Anamorsin homologue in various human organs (MTC panels, CLONTECH Laboratories, Inc.) was examined by Northern hybridization. (b) Ba/F3 cells were IL-3 depleted for 15–18 h and then cultured with 0.1 ng/ml mIL-3 for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. Ba/F3-Ad, Ba/F3-Ad in the absence of IL-3. (c) Ba/F3 cells each transfected with EPO receptor, c-kit, and c-mpl were deprived of IL-3 for 15–18 h and cultured with 5 u/ml mEPO, 100 ng/ml mSCF, and 30 ng/ml mTPO, respectively, for the time indicated. Induction of Anamorsin gene was examined by Northern blot analysis. (d) Ba/F3 cells transfected with an empty vector (Mock) or oncogenic H-Ras (H-RasG12V) were deprived of IL-3 for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (e) Ba/F3 cells, in which dominant negative H-Ras (H-RasS17N) was inducibly expressed by a LacSwitch-II–inducible expression system, were cultured with IL-3 in the presence or absence of IPTG for the time indicated. The expression levels of Anamorsin were examined by Northern blot analysis. (f) The localization of Anamorsin was examined by an immunofluorescence staining with an anti-Anamorsin mAb. Nucleus was visualized with staining with DAPI. Green, Anamorsin; blue, nucleus.
Mentions: Next, we examined the expression profile of the human Anamorsin homologue in various organs using MTA panels (CLONTECH Laboratories, Inc.). As shown in Fig. 2 a, left, the homologue was expressed ubiquitously in various tissues, with especially high expression levels detected in heart, liver, and pancreas. As for hematopoietic tissues, the homologue was abundantly expressed in FL and spleen (Fig. 2 a, right). Also, we found that expression of Anamorsin was detectable in early stages (7 d) of embryogenesis by the PCR method (not depicted).

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
Related in: MedlinePlus