Regulation of IL-3 and GM-CSF genes and their receptors

1.T细胞中细胞因子基因的调控。利用具有TH1、TH2和TH0表型的人和小鼠T细胞克隆，我们一直在研究IL-3、IL-4、IL-5和GM-CSF基因的协调和差异表达。小鼠GM-CSF启动子的CLE2/GC-box和CLE0对于PMA和钙离子载体的转录激活是必不可少的。CLE2定义了一个与核因子-kappaB结合的位点。纯化了主要的GC-box结合活性A1，并鉴定为Sp1。我们从激活的Jurkat提取液中纯化了一个人的核因子-AT蛋白，结果表明它与GM-CSF启动子中的CLE0强烈结合，并与AP1相关。我们分离到了一个新的蛋白，它带有锌指基序，与IL-3启动子的CT/GC富集区结合。我们还鉴定了IL-5、IL-4和IL-2启动子中的顺式调控元件。这些工作是与荒井直子博士和De Vries Jan博士在DNAX.2中合作完成的。我们一直在研究GM-CSF/IL-3受体的结构和功能。在GM-CSF受体下游有几条信号通路。Betac膜近端区域对于细胞增殖、c-myc和Pim-1的诱导以及JAK2的结合是必不可少的。Betac的C末端区域在抑制细胞凋亡、RAS、Raf、MAPK的激活和c-fos、c-jun的诱导中起重要作用。我们还表明，从结构性表达hGM-CSF受体的转基因小鼠获得的骨髓细胞具有增殖能力，可以诱导所有髓系细胞对hGM-CSF做出反应。此外，我们还产生了携带Betac和BetaIL-3零突变的小鼠。Betac突变小鼠也表现出肺病理，包括淋巴细胞渗透和类似肺泡蛋白沉积症的区域。这些工作是与DNAX的宫岛敦博士和汉堡大学的奥斯特塔格·沃尔夫勒姆博士合作完成的。

1.Regulation of cytokine genes in T cells. Using human and mouse T cell clones with TH1, TH2, and TH0 phenotype, we have been working on the coordinate and differential expression of the IL-3, IL-4, IL-5 and GM-CSF genes. The CLE2/GC-box and CLE0 of the mouse GM-CSF promoter are essential for transcriptional activation in response to PMA and Ca ionophore. CLE2 defines a binding site for NF-kappaB.The major GC-box binding activity A1 was purified and was identified as Sp1. We purified a human NF-AT protein from activated Jurkat extract and showed that it strongly bound to the CLE0 within the GM-CSF promoter in association with AP1. We isolated a novel protein with zinc finger motifs which binds to the CT/GC-rich region of the IL-3 promoter. We also identified cis-regulatory elements within IL-5, IL-4 and IL-2 promoters. These works were done in collaboration with Drs.Naoko Arai and de Vries Jan in DNAX.2.Receptor and Signal transduction. We have been working on the structure and function of GM-CSF/IL-3 receptors. There are several signal pathways downstream of the GM-CSF receptor. The membrane proximal region of betac is essential for proliferation, c-myc and pim-1 induction and Jak2 association. The C-terminal region of betac is important for the suppression of apoptosis, Ras, Raf, MAPK activation and c-fos, c-jun induction. We have also shown that bone marrow cells derived from transgenic mice which constitutively express hGM-CSF receptor have the proliferative capacity to induce all the myeloid cell lineages in response to hGM-CSF.Furthermore, we have generated mice carrying a null mutation of betac and betaIL-3. betac mutant mice also showed lung pathology consisting of lymphocytic infiltration and areas resembling alveolar proteinosis. These works were done in collaboration with Dr.Atsushi Miyajima in DNAX and Dr.Ostertag Wolfram in Hamburg University.