CELLULAR PHYSIOLOGY OF STAT3

细胞因子在多细胞生物体的细胞之间的通讯中起重要作用，如它们在介导哺乳动物（包括人）对感染和损伤的“急性期”反应中的作用所例示的。 许多细胞因子，如干扰素、白细胞介素（IL）和造血生长因子，目前广泛用于治疗各种形式的人类癌症。 许多细胞因子，如白介素-6型细胞因子（其是肝急性期反应的主要介质）的共同特征是它们与通过激活JAK-STAT信号传导途径向细胞核发出信号的“非酪氨酸激酶”细胞表面受体类结合。 在IL-6的情况下，肝脏中STAT 3的活化是主要的生理学靶点。 在精氨酸诱导的STAT活化的“标准”模型中，假设STAT 3从胞质单体库中募集到受体复合物中。 文献中没有数据支持这一假设。 我们与该领域所有先前工作的出发点是我们发现在肝细胞的胞质溶胶中很少或没有单体STAT 3（91 kDa）。 我们已经发现，大量的细胞质STAT 3（以及STAT 1和STAT 5）以大小范围为200-400 kDa（“Statosome I”）和1-2 MDa（“Statosome II”）的两种宽分布的高分子量复合物存在，如通过Superose-6凝胶过滤色谱表征的。 我们已经开发了一种新的“抗体消减差异蛋白展示”技术，以确定从肝癌Hep 3B细胞中纯化的Statosome I含有估计8个多肽，其中至少3个以IL-6依赖的方式缔合。 本申请的焦点是来自人肝癌细胞系（Hep 3B）和来自大鼠肝细胞的含有细胞溶质STAT 3的平衡体的多肽组分的纯化、鉴定和分子克隆和表征。 这对于理解哺乳动物细胞中STAT 3的细胞生理学是重要的。 在不含IL-6和经IL-6处理的细胞中建立含有细胞溶质200-400 kDa和1-2 MDa STAT 3的复合物的基本亚基结构是该领域进一步进展的必要条件。

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.