CELLULAR PHYSIOLOGY OF STAT3

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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.