CDNAs Expressed During During Hematopoietic Commitment

造血承诺期间表达的 cDNA

• 批准号: 6983694
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6983694
• 关键词: cadherins; cell adhesion molecules; cell differentiation; cell population study; colony stimulating factor; complementary DNA; differential display technique; erythropoietin; fluorescent in situ hybridization; gene expression; genetic mapping; glycosylation; hematopoiesis; hematopoietic stem cells; human tissue; in situ hybridization; messenger RNA; molecular cloning; myeloid stem cell; thrombopoietic factor; tissue /cell culture; tumor suppressor genes; western blottings

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. Human GC-1 (hGC-1) is a novel olfactomedin-like glycoprotein that is expressed during granulocytic differentiation. To further characterize the function of GC-1, we have cloned the cDNA for the mouse homologue of human GC-1 and investigated the pattern of GC-1 gene expression in the mouse during embryonic development and in adult tissues. Genomic and cDNA clones corresponding to mouse GC-1 (mGC-1) were isolated. The mGC-1 protein shares 93% homology with hGC-1 at the amino acid level. The protein sequence of mGC-1 indicates that it belongs to the olfactomedin-related glycoprotein family, which includes olfactomedin, TIGR, NOELIN-2 and latrophilin-1. Fluorescence in situ hybridization (FISH) was used to map the mGC-1 gene locus to chromosome 14D3. Like other olfactomedin-like genes with tissue-restricted patterns of expression, mGC-1 is expressed at high levels in the small intestine and kidney, at moderate levels in the stomach, thymus, spleen, and no expression was detected in brain, heart, liver or lung. These results are consistent with the distribution of hGC-1 in human tissues. Analysis of mGC-1 expression by in situ hybridization in mouse embryos showed that mGC-1 is not expressed until day E15. mGC-1 expression was detected in the small intestine and stomach. Interestingly, a very clear demarcation of the mGC-1 signal was evident in the intestine, where mGC-1 was specifically expressed between the enterocytes lining the villi, but not in the lamina propria or in the muscularis layers. There was a similar, albeit weaker, pattern of mGC-1 expression in stomach. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abundantly expressed in tissues which has secretion capability. On further analysis, we find high level expression of mGC-1 in pro-B and pre-B cells and low-level expression in mature B and T-cells that co-localizes in this region of the small intestine. When the myeloid progenitor 32D cell line was exposed to G-CSF for 7 days, mGC-1 expression was induced. Taken together, these results suggest that mGC-1 play an important role in granulocytic differentiation, and quite likely in mucousal immunity. To gain further insight into the potential pathways involved in GC-1 effects we performed further analysis in the 293 cell line. In situ hybridization showed mGC-1 is strongly expressed in the crypts of small intestine. hGC-1 cDNA has been transfected into 293 cells transiently and stably. In transfected 293 cells, hGC-1 was detected in the culture medium after 48h of transfection. In the Western blot, hGC-1 showed a multimer form in non-reducing gel, which was reduced to monomer after adding 10mM DTT. hGC-1 was also detected in the perinuclear region and cell surface.These data suggest hGC-1 is a secreted glycoprotein. This is consistent with tissue expression result that hGC-1 is only abuuundantly expressed in tissues which has secretion capability. Based on the data that hGC-1 is a glycoprotein and associated with cell adhesion, experiments have been performed to screen some lectins and cell adhesion proteins which might interact with hGC-1. Agarose bound lectin pull down assay shows hGC-1 strongly bound RCAI, weakly with ConA and WGA and no binding was detected with DSA, PNA and SNA. The affinity of hGC-1 with RCAI provide a potential approch to purify hGC-1 with RCA I. Co-immunoprecipitation demonstrated a association of hGC-1 with cadherin in 293 cells transiently transfected with hGC-1 cDNA. The potential association of hGC-1 with cadherin might explain the involvement of hGC-1 with cell adhesion. E-cadherin in particular, serves as a widely acting suppressor of invasion and growth of epithelial cancers, and its functional elimination represents a key step in the acquisition of the invasive phenotype for many tumors. Further analysis of the interaction of hGC-1 and the cadherin fam

我们克隆了一个新的造血粒细胞集落刺激因子（G-CSF）诱导的嗅调节素相关糖蛋白，称为hGC-1（人G-CSF刺激克隆-1）。mRNA差异显示结合改良的两相液体培养系统使用。培养物富集红细胞系、髓细胞系和巨核细胞系的早期前体细胞，这些细胞系在分别用促红细胞生成素、G-CSF和促血小板生成素诱导后分离。对来自富集细胞的RNA进行差异显示分析以鉴定谱系特异性表达的基因。人GC-1（human GC-1，hGC-1）是一种新的嗅调节素样糖蛋白，在粒细胞分化过程中表达。为了进一步表征GC-1的功能，我们克隆了人GC-1的小鼠同源物的cDNA，并研究了GC-1基因在小鼠胚胎发育和成年组织中的表达模式。分离了对应于小鼠GC-1（mGC-1）的基因组和cDNA克隆。mGC-1蛋白与hGC-1蛋白在氨基酸水平上有93%的同源性。mGC-1的蛋白质序列表明其属于嗅觉调节蛋白相关糖蛋白家族，该家族包括嗅觉调节蛋白、TIGR、NOELIN-2和latrophilin-1。采用荧光原位杂交（FISH）将mGC-1基因定位于染色体14 D3。与其他具有组织限制性表达模式的嗅觉调节素样基因一样，mGC-1在小肠和肾脏中以高水平表达，在胃、胸腺、脾脏中以中等水平表达，在脑、心脏、肝脏或肺中未检测到表达。这些结果与hGC-1在人体组织中的分布一致。在小鼠胚胎中通过原位杂交分析mGC-1表达表明，mGC-1直到E15天才表达。在小肠和胃中检测到mGC-1表达。有趣的是，mGC-1信号的非常明确的分界在肠中是明显的，其中mGC-1特异性地表达在衬于绒毛的肠上皮细胞之间，但不在固有层或肌层中。在胃中有类似的mGC-1表达模式，尽管较弱。原位杂交结果显示，mGC-1在小肠隐窝中有强表达。将hGC-1cDNA瞬时稳定转染293细胞。在转染的293细胞中，在转染48 h后在培养基中检测到hGC-1。在Western印迹中，hGC-1在非还原性凝胶中显示多聚体形式，其在加入IOmM DTT后被还原成单体。hGC-1还在核周区和细胞表面检测到，这些数据表明hGC-1是一种分泌型糖蛋白。这与hGC-1仅在具有分泌能力的组织中大量表达的组织表达结果一致。在进一步分析中，我们发现mGC-1在前B和前B细胞中高水平表达，而在共定位于小肠的该区域的成熟B和T细胞中低水平表达。当骨髓祖细胞32 D细胞系暴露于G-CSF 7天时，诱导mGC-1表达。总之，这些结果表明，mGC-1在粒细胞分化中起重要作用，并且很可能在粘膜免疫中起重要作用。为了进一步深入了解GC-1效应中涉及的潜在途径，我们在293细胞系中进行了进一步的分析。原位杂交结果显示，mGC-1在小肠隐窝中有强表达。将hGC-1cDNA瞬时稳定转染293细胞。在转染的293细胞中，在转染48 h后在培养基中检测到hGC-1。在Western印迹中，hGC-1在非还原性凝胶中显示多聚体形式，其在加入IOmM DTT后被还原成单体。hGC-1在核周区和细胞表面也有表达，提示hGC-1是一种分泌型糖蛋白。这与组织表达结果一致，即hGC-1仅在具有分泌能力的组织中大量表达。基于hGC-1是一种与细胞粘附相关的糖蛋白的研究结果，本研究筛选了一些可能与hGC-1相互作用的凝集素和细胞粘附蛋白。琼脂糖结合凝集素下拉试验显示hGC-1与RCAI强结合，与ConA和WGA弱结合，与DSA、PNA和SNA未检测到结合。hGC-1与RCAI的亲和性为用RCA I纯化hGC-1提供了一种可能的方法。免疫共沉淀显示，在瞬时转染hGC-1 cDNA的293细胞中，hGC-1与钙粘蛋白相关联。hGC-1与cadherin的潜在关联可能解释hGC-1参与细胞粘附。特别是E-钙粘蛋白，作为一种广泛作用的上皮癌的侵袭和生长抑制剂，其功能消除代表了许多肿瘤获得侵袭性表型的关键步骤。进一步分析hGC-1与钙粘蛋白家族的相互作用，