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PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells.

ABSTRACT: Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate the effects of PDCD2 loss on HPC clonogenic potential and on 12-O-tetradecanoyl-phorbol-13-acetate-and arabinofuranosylcytosine-induced terminal differentiation. In CD34(+) clonogenic progenitors, PDCD2 knockdown decreased the total number of colony-forming units, increased the number of colony-forming units-granulocyte-erythroid-macrophage-megakaryocyte and burst-forming unit-erythroid primitive colonies, and decreased the number of burst-forming unit-erythroid mature colonies. Similar results were observed in K562 cells, suggesting that PDCD2 is important for HPC differentiation and/or survival, and for erythroid lineage commitment. Furthermore, 12-O-tetradecanoyl-phorbol-13-acetate-induced megakaryocytic differentiation and proliferation of K562 cells was not affected by PDCD2 knockdown. In contrast, arabinofuranosylcytosine-induced erythroid differentiation of K562 cells was significantly reduced with PDCD2 knockdown, with no effect on cell proliferation. The effects of PDCD2 knockdown were attributed to a cell cycle arrest at G(0)/G(1), along with increased messenger RNA expression of early progenitor factors c-MYB and GATA-2, and decreased expression of erythroid factors GATA-1, EpoR, and ?-globin. We conclude that PDCD2 loss of function(s) impedes erythroid differentiation by inducing cell cycle arrest and increasing expression of early hematopoietic progenitor factors. These findings suggest that PDCD2 has a novel regulatory role in human hematopoiesis and is essential for erythroid development.

SUBMITTER: Kokorina NA

PROVIDER: S-EPMC5218995 | biostudies-other | 2012 Dec

REPOSITORIES: biostudies-other

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PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells.

Kokorina Natalia A NA Granier Celine J CJ Zakharkin Stanislav O SO Davis Stephani S Rabson Arnold B AB Sabaawy Hatem E HE

Experimental hematology 20120822 12

Programmed cell death-2 (PDCD2) protein is enriched in embryonic, hematopoietic, and neural stem cells, however, its function in stem/progenitor cell differentiation is unclear. We investigated the effects of PDCD2 knockdown on the development and differentiation of hematopoietic progenitor cells (HPC). CD34(+) cells derived from normal human bone marrow and K562 leukemic cells were effectively transduced with short-hairpin RNA to knockdown PDCD2. Colony-forming assays were used to investigate t ...[more]

PMID: 22922207

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Project description:BACKGROUND:Lymphotoxin (LT) is a lymphokine mainly expressed in lymphocytes. LT? binds one or two membrane-associated LT? to form LT?2?1 or LT?1?2 heterotrimers. The predominant LT?1?2 binds to LT? receptor (LT?R) primarily expressed in epithelial and stromal cells. Most studies on LT?R signaling have focused on the organization, development, and maintenance of lymphoid tissues. However, the roles of LT?R signaling in the nervous system, particularly in neurogenesis, remain unknown. Here, we investigated the role of LT?R-mediated NF?B signaling in regulating neural lineage differentiation. METHODS:The C57BL/6J wild-type and GFAP-dnI?B? transgenic mice were used. Serum-free embryoid bodies were cultured from mouse embryonic stem cells and further induced into neural stem/progenitor cells (NSCs/NPCs). Primary neurospheres were cultured from embryonic and adult mouse brains followed by monolayer culture for amplification/passage. NF?B activation was determined by adenovirus-mediated NF?B-firefly-luciferase reporter assay and p65/RelB/p52 nuclear translocation assay. LT?R mRNA expression was evaluated by quantitative RT-PCR and LT?R protein expression was determined by immunohistochemistry and Western blot analysis. Multilabeled immunocytochemistry or immunohistochemistry followed by fluorescent confocal microscopy and quantitative analysis of neural lineage differentiation were performed. Graphing and statistical analysis were performed with GraphPad Prism software. RESULTS:In cultured NSCs/NPCs, LT?1?2 stimulation induced an activation of classical and non-classical NF?B signaling. The expression of LT?R-like immunoreactivity in GFAP+/Sox2+ NSCs was identified in well-established neurogenic zones of adult mouse brain. Quantitative RT-PCR and Western blot analysis validated the expression of LT?R in cultured NSCs/NPCs and brain neurogenic regions. LT?R expression was significantly increased during neural induction. LT?1?2 stimulation in cultured NSCs/NPCs promoted astroglial and oligodendrocytic lineage differentiation, but inhibited neuronal lineage differentiation. Astroglial NF?B inactivation in GFAP-dnI?B? transgenic mice rescued LT?R-mediated abnormal phenotypes of cultured NSCs/NPCs. CONCLUSION:This study provides the first evidence for the expression and function of LT?R signaling in NSCs/NPCs. Activation of LT?R signaling promotes glial lineage differentiation. Our results suggest that neurogenesis is regulated by the adaptive immunity and inflammatory responses.

Dataset Information

PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells.

Publications

PDCD2 knockdown inhibits erythroid but not megakaryocytic lineage differentiation of human hematopoietic stem/progenitor cells.

Similar Datasets

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