固有の分化経路と抗原提示能を有する新規CD135⁺単球サブセットの同定

京都大学新制・課程博士博士(医学)甲第24515号医博第4957号新制||医||1064(附属図書館)京都大学大学院医学研究科医学専攻(主査)教授 森信 暁雄, 教授 竹内 理, 教授 濵﨑 洋子学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDFA

Cyclic AMP Responsive Element Binding Proteins Are Involved in ‘Emergency’ Granulopoiesis through the Upregulation of CCAAT/Enhancer Binding Protein β

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPβ. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPβ-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPβ were involved in the positive regulation of C/EBPβ transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPβ mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPβ transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPβ upregulation

Lentivirus-based reporter system.

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without GM-CSF stimulation <i>in vivo</i>. *:<i>P</i><0.05, n = 3. Data are representative of two independent experiments. B. Schematic illustration of the lentivirus vector for reporter assays. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. C. Elongation factor-1 (EF-1) promoter activity in bone marrow cells revealed by the lentivirus-based reporter system. Bone marrow cells were analyzed by flow cytometry after two days incubation with GM-CSF following viral infection. Shaded histogram, promoter-less control; open histogram, EF-1 promoter.</p

Flow cytometric analysis of the C/EBPβ proximal promoter region using the lentivirus-based reporter system.

<p>A. Schematic illustration of the lentivirus vector. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. B and C. Bone marrow cells were transduced with the lentivirus vector containing the indicated fragment and were analyzed for Thy1.1 and d2EGFP expression after 48 hours stimulation with GM-CSF. D and E. Effects of the mutated CREs at −110 and −65 bp on the activity of the promoter fragment (−243 to +16 bp). Shaded histogram, promoter-less control; open histogram, promoter fragment. Wt, wild type; mut, mutated. *:<i>P</i><0.05, **:<i>P<0.01</i> (n = 3). Data are representative of three independent experiments.</p

Involvement of CREB-C/EBPβ pathway in candidemia-induced “emergency granulopoiesis.”

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without candidemia. *:<i>P</i><0.05, n = 3. B. Western blotting analysis of c-kit+ bone marrow cells with or without candidemia. C. Chromatin immunoprecipitation of CREB using c-kit+ bone marrow cells during candidemia induced emergency granulopoiesis. Data are representative of two independent experiments.</p

Use of bicistronic vectors in combination with flow cytometry to screen for effective small interfering RNA target sequences

10.1016/j.bbrc.2010.02.033Biochemical and Biophysical Research Communications3933498-50