REGULATION OF GLUCOSE TRANSPORT BY CTAP-III ISOFORMS

越来越多的证据表明，许多天然存在的肽 在体外激活结缔组织细胞， 促进细胞外基质分泌增加， 风湿性疾病中结缔组织细胞的复制。 一个这样 CTAP-III（结缔组织激活肽-III）是一种 同源蛋白家族（PF-4，NAP-1，MGSA，GRO，γ IP-10， 9 E3）被认为在炎症、伤口愈合和瘤形成中重要。 我们建议研究一个重要的，最早的事件之一， 将CTAP-III添加到培养的结缔组织细胞中-即 刺激葡萄糖转运。 用CTAP-III孵育30 min 增加葡萄糖转运30-110%（“即时”效应）; 显著的刺激（400-800%）（“延迟效应”）在17- 18天后是明显的。 41 h，在人成纤维细胞类型中， 胰岛素 只有延迟效应似乎需要mRNA的合成， 表明这些影响涉及不同的途径。 因为最近 研究表明，CTAP-III的活性形式可能是 蛋白水解加工，命名为CTAP-III（des 1 -15）/NAP-2， CTAP-III和des 1-15对结缔组织葡萄糖转运的影响 将比较细胞。 这两种形式将被测试他们的能力， 刺激中性粒细胞葡萄糖转运， CTAP-III（des 1 -15）/NAP-2的趋化性。 对运输的延迟影响是 伴随着小鼠GLUT 1葡萄糖转运蛋白数量的增加， 3 T3-F442 A成纤维细胞。 葡萄糖特异性多克隆抗体 转运蛋白结合免疫印迹和免疫沉淀 [35 S]甲硫氨酰标记的转运蛋白将用于测试CTAP-III是否 增加合成速率或降低降解速率 葡萄糖转运蛋白 由于初步数据显示CTAP-III增加 GLUT 1 mRNA水平，CTAP-III增加GLUT 1表达率的能力， 将测试转录和/或信息稳定性。 如果CTAP-III 增加转录速率，CAT构建体将用于启动 以确定GLUT 1基因赋予CTAP-III敏感性的部分。 CTAP-III亚型对葡萄糖转运的直接影响是否 通过增加转运体数量介导，增加内源性 将检测葡萄糖转运蛋白的活性或再分布， 假设CTAP-III对葡萄糖转运的影响是 通过PGE 2介导和随后的cAMP产生。 CTAP-III是否使用 对CTAP-III同种型特异的受体或与结构蛋白共有的受体 还将测定NAP-1和PF-4等同系物。 提供 深入了解第三阶段工作组成员发挥职能和 结构家族调节葡萄糖转运，分子机制是 这些局部释放的生长因子可以使特定的，局部的相对 胰岛素不敏感的身体区域，以满足增加的葡萄糖需求 将得到澄清。 了解所使用的信号传导机制 通过生长因子而不是胰岛素来调节葡萄糖转运， 为风湿性关节炎的最早期事件提供了有价值的见解， 疾病，炎症和伤口愈合，并可能提出策略， 治疗干预

Accumulating evidence suggests that many naturally occurring peptides activate connective tissue cells in vitro and may play important roles in promoting increased secretion of extracellular matrix and increased replication of connective tissue cells in rheumatic diseases. One such peptide, CTAP-III (connective tissue activating peptide-III), is a member of a family of homologous proteins (PF-4, NAP-1, MGSA, GRO, gamma IP-10, 9E3) thought to be important in inflammation, wound healing and neoplasia. We propose to study a vital, and one of the earliest events following addition of CTAP-III to cultured connective tissue cells - namely stimulation of glucose transport. Incubation with CTAP-III for 30 min increases glucose transport by 30-110% ("immediate" effect); a more substantial stimulation (400-800%) ("delayed effect") is evident after 17- 41 h in human fibroblast cell types that are relatively insensitive to insulin. Only the delayed effect appears to require mRNA synthesis, indicating that the effects involve different pathways. Since recent studies indicate that the active form of CTAP-III may be a product of proteolytic processing, designated CTAP-III (des1-15)/NAP-2, the effects of CTAP-III and the des 1-15 form on glucose transport in connective tissue cells will be compared. Both forms will be tested for their ability to stimulate glucose transport in neutrophils, a cell type stimulated to chemotaxis by CTAP-III (des1-15)/NAP-2. The delayed effect on transport is accompanied by increased numbers of GLUT 1 glucose transporters in murine 3T3-F442A fibroblasts. Specific polyclonal antibodies to the glucose transporter combined with immunoblotting and immunoprecipitation of [35S]methionyl-labeled transporters will be used to test whether CTAP-III increases the rate of synthesis or decreases the degradation rate of glucose transporters. Since preliminary data indicate CTAP-III increases GLUT 1 mRNA levels, the ability of CTAP-III to increase the rate of transcription and/or message stability will be tested. If CTAP-III increases the rate of transcription, CAT constructs will be used to start to determine the part of the GLUT 1 gene conferring CTAP-III sensitivity. Whether the immediate effects of CTAP-III isoforms on glucose transport are mediated via an increase in transporter number, increased intrinsic activity, or redistribution of glucose transporters will be tested as will the hypothesis that the effects of CTAP-III on glucose transport are mediated via PGE2 and subsequent production of cAMP. Whether CTAP-III uses a receptor specific for CTAP-III isoforms or ones shared by structural homologues such as NAP-1 and PF-4 will also be determined. In providing insight into the mechanism by which members of the CTAP-III functional and structural families regulate glucose transport, the molecular mechanisms by which locally released growth factors enable specific, localized relatively insulin-insensitive areas of the body to meet increased demands for glucose will be clarified. Understanding the signal transduction mechanisms used by growth factors other than insulin to regulate glucose transport will provide valuable insight into the earliest events involved in rheumatic disease, inflammation and wound healing, and possibly suggest tactics for therapeutic intervention.