CELLULAR PHYSIOLOGY OF STAT3

细胞因子在多细胞生物体细胞间的交流中发挥着重要作用，其在包括人类在内的哺乳动物对感染和损伤的“急性期”反应中的作用就是例证。许多细胞因子，如干扰素、白介素类和造血生长因子，目前被广泛用于治疗各种形式的人类癌症。许多细胞因子，如白细胞介素6型细胞因子(它们是肝脏急性期反应的主要或中介)的一个共同特征是，它们与一类“非酪氨酸激酶”细胞表面受体结合，该受体通过激活JAK-STAT信号通路向细胞核发出信号。在IL-6的情况下，肝脏中STAT3的激活是一个主要的生理靶点。在细胞因子诱导STAT激活的“标准”模型中，假设STAT3是从胞浆单体池中招募到受体复合体的。文献中没有数据支持这一假设。我们从这一领域所有先前工作的出发点是我们发现在肝细胞胞浆中很少或根本没有单体STAT3(91 KDa)。在Superose-6凝胶过滤层析中，我们发现细胞质中的STAT3(以及STAT1和STAT5)主要以两种大小分布的高分子复合体形式存在，大小分别为200-400 kDa(“Statosome I”)和1-2“MDA”(“Statosome II”)。我们发展了一种新的“抗体消减差异蛋白显示”技术来确定从肝癌细胞Hep3B中纯化的他汀体I含有大约8个多肽，其中至少有3个与IL-6依赖的方式相关。本申请的重点是从人肝癌细胞系(Hep3B)和大鼠肝细胞中分离纯化、鉴定、克隆和鉴定含有STAT3的胞浆状态体多肽成分。这对于理解哺乳动物细胞中STAT3的细胞生理学很重要。在无IL-6和IL-6处理的细胞中建立胞浆200-400 kDa和1-2MDA-STAT3复合体的基本亚基结构是这一领域进一步发展的必要条件。

Cytokines play an important role in the communication between cells of multicellular organisms as exemplified by their role in mediating the "acute phase" response of mammals, including humans, to infection and injury. Many cytokines, such as the interferons, interleukins (IL) and hematopoietic growth factors, are currently in widespread therapeutic use against various forms of human cancer. A common feature of many cytokines, such as the interleukin-6-type cytokines (which are the major or mediators of the hepatic acute phase response) is that they engage the class of "non-tyrosine kinase" cell surface receptors that signal to the cell nucleus by activation of the JAK-STAT signalling pathway. In the case of IL-6, the activation of STAT3 in the liver is a major phyisological target. In the "standard" model of cytokine-induced STAT activation, it is assumed that STAT3 is recruited to the receptor complex from within a cytosolic monomer pool. No data have been presented in the literature to support this assumption. Our point of departure from all prior work in this area is our discovery that there is little or no monomeric STAT3 (91 kDa) in the cytosol of liver cells. We have found that the bulk of cytoplasmic STAT3 (and STAT1 and STAT5) is present as high-molecular mass complexes in two broad distributions in the size range 200-400 kDa ("Statosome I") and 1-2 MDa ("Statosome II") as characterized by Superose-6 gel filtration chromatography. We have developed a new technique of "antibody subtractive differential protein display" to determine that statosome I purified from hepatoma Hep3B cells contains an estimated 8 polypeptides, at least 3 of which associate in an IL-6-dependent manner. The focus of this Application is the purification, identification and molecular cloning and characterization of the polypeptide components of cytosolic STAT3-containing statosomes from an human hepatoma cell line (Hep3B) and from the rat liver hepatocyte. This is important to an understanding of the cellular physiology of STAT3 in the mammalian cell. Establishing the basic subunit structure of the cytosolic 200-400 kDa and 1-2 MDa STAT3-containing complexes in IL-6-free and IL-6-treated cells is a sine qua non for further progress in this field.