The role of syndecan-4 in the regulation of JNK activation in human neutrophils

描述（由申请方提供）：单核细胞向急性炎症区域的募集是炎症过程的后期阶段。我们以前已经表明，无论是p38或c-Jun氨基末端激酶（JNK）的全身抑制限制中性粒细胞招募到肺中的LPS诱导的急性炎症模型。然而，正如我们将展示的，JNK而不是p38的抑制限制了单核细胞的肺募集，这通过单核细胞趋化蛋白1（MCP-1）和中性粒细胞依赖性途径发生。因此，了解导致中性粒细胞中JNK激活的机制对于了解单核细胞募集至关重要。虽然中性粒细胞中p38的活化发生在悬浮条件下，但JNK的活化需要中性粒细胞在粘附条件下培养，尽管调节细胞粘附的细胞表面蛋白以及由此调节JNK活化的细胞表面蛋白知之甚少。一个这样的家族，多配体蛋白聚糖，结合细胞外基质（ECM）诱导细胞粘附。此外，syndecan-4已被证明与CXCR 4共缔合，并且我们将在这里显示中性粒细胞中与IL-8受体CXCR 2。我们拟通过检测多配体蛋白聚糖-4是否通过上调蛋白激酶C α（PKC α）活性增强JNK活化，检测多配体蛋白聚糖-4与CXCR 2共结合的机制，以及共结合增强IL-8诱导的JNK活化的必要性，来检测多配体蛋白聚糖-4在调节中性粒细胞中IL-8诱导的JNK活化，从而调节MCP-1表达中的作用。我们将使用几种技术，包括化学抑制剂，封闭抗体，小干扰RNA，中性粒细胞和PLB-985细胞系的蛋白转导，它可以分化成嗜中性粒细胞样细胞。此外，为了证实我们从嗜中性粒细胞和PLB-985细胞中的发现，我们将利用来自多配体蛋白聚糖-4，CXCR 2和PKC（缺陷小鼠）的嗜中性粒细胞。利用上述技术，我们将检查我们假设调节多配体蛋白聚糖-4诱导的PKC β和JNK活化的组分，包括PKC β和PP 2A，或调节多配体蛋白聚糖-4与CXCR 2的共缔合的组分，包括衔接蛋白CASK和syntenin。这些研究将为中性粒细胞中JNK激活的调节机制提供重要的见解，并将提示新途径在肺部炎症调节中的作用，特别是肺单核细胞募集。

DESCRIPTION (provided by applicant): The recruitment of monocytes to areas of acute inflammation is a later stage in the inflammatory process. We have previously shown that systemic inhibition of either p38 or c-Jun NH2-terminal kinase (JNK) limits neutrophil recruitment to the lung in an LPS-induced model of acute inflammation. As we will show, however, inhibition of JNK, but not p38, limits the pulmonary recruitment of monocytes, which occurs through monocyte chemoattractant protein 1 (MCP-1) and neutrophil dependent pathways. Understanding mechanisms that lead to JNK activation in neutrophils will, therefore, be fundamental to the understanding of monocyte recruitment. While activation of p38 in neutrophils occurs in suspended conditions, activation of JNK requires neutrophils to be cultured in adherent conditions, although the cell surface proteins that regulate cell adhesion, and thereby JNK activation, are poorly understood. One such family, the syndecans, bind to the extracellular matrix (ECM) inducing cell adhesion. In addition, syndecan-4 has been shown to co-associate with CXCR4, and we will show here with the IL-8 receptor CXCR2 in neutrophils. We propose to examine the role of syndecan-4 in regulating IL-8-induced JNK activation in neutrophils, and thereby expression of MCP-1, by examining if syndecan-4 enhances JNK activation via an upregulation of protein kinase C alpha (PKC() activity, examine mechanisms for the co-association of syndecan-4 with CXCR2, and the necessity of the co-association for the enhancement of IL-8-induced JNK activation. We will use several techniques including chemical inhibitors, blocking antibodies, small interfering RNAs, and protein transduction of neutrophils and the PLB-985 cell line, which can be differentiated into neutrophil-like cells. In addition, to confirm our findings from neutrophils and PLB-985 cells, we will utilize neutrophils derived from syndecan-4, CXCR2, and PKC( deficient mice. Utilizing the above techniques we will examine components that we hypothesize to regulate syndecan-4-induced PKC( and JNK activation, including PKC-( and PP2A, or to regulate the co-association of syndecan-4 with CXCR2, including the adaptor proteins CASK and syntenin. These studies will provide vital insights into mechanisms that regulate JNK activation in neutrophils and will suggest roles for novel pathways in the regulation of lung inflammation, and in particular pulmonary monocyte recruitment.