Growth hormone, JAKs and STATs : A model cytokine signal transduction system

The haematopoietic cytokine family of signalling molecules comprises a variety of polypeptides which have pleiotropic effects on cell differentiation, tissue development and homeostasis. The family includes several interleukins, a variety of growth and colony stimulating factors, erythropoietin, leukaemia inhibitory factor, ciliary neurotrophic factor, the interferons, as well as leptin, prolactin and growth hormone. The receptors for a large number of cytokines have been cloned and shown to be membrane-spanning glycoproteins with their amino termini in the extracellular space. On the basis of their possession of certain structural similarities these have been grouped together into the haematopoietic cytokine / interferon (HCI) receptor family. This thesis concerns the mechanism of signal transduction of the HCI receptors. The regulation of reporter gene transcription by growth hormone in cultured cell lines has been established as a model system for the study of cytokine signal transduction. Using a combination of gel mobility shift assays and reporter gene constructs we have identified an element in the growth hormone-regulated serine protease inhibitor (SPI) 2.1 promoter which shares sequence similarity with STAT (signal transducer and activator of transcription) transcription factor response elements found in a variety of cytokine-regulated gene promoters. Growth hormone treatment of cells in culture and rat liver was found to result in the stimulation of STAT 5-binding to this element. This was the first demonstration of growth hormone-dependent activation of a clearly defined transcription factor. STAT 5, but not STATs 1 or 3, was found to be capable of mediating growth hormone's effect on transcription of a reporter gene containing the SPI 2.1 STAT response element. In addition to the growth hormone receptor, the ligand-bound receptors for prolactin and erythropoietin were also found to stimulate gene transcription via the SPI 2.1 STAT response element. This indicates that although the SPI 2.1 STAT response element is STAT 5 specific it is not cytokine specific. The transport of growth hormone receptor into the nucleus was also investigated. Full length growth hormone receptor was found in the nuclear membranes and nucleoplasm. This nuclear receptor is speculated to have a role in activating Janus kinase 2 (JAK 2) in the nucleus. Finally, we have investigated the activation of STAT 5 by the recently cloned leptin receptor. This receptor shares sequence similarity with members of the HCI receptor family. We present data indicating that the leptin receptor activates STATs, particularly STAT 5, via a mechanism involving receptor homodimerisation. Based upon the work presented in this thesis, together with experiments performed in other laboratories, it is now possible to hypothesise a working model for the cytokine-dependent stimulation of gene transcription via JAK 2 and STAT transcription factors. Ligand-binding leads to activation of JAK 2. This is followed by phosphorylation of the kinase, the associated receptor and STAT molecules. Following phosphorylation on tyrosine residues STATs form dimers as a result of phosphotyrosine - SH2 domain association. This dimerization is accompanied by translocation to the nucleus which results in DNA-binding to specific response elements and stimulation of gene transcription. Kewords: Growth hormone, cytokine, receptor, signal transduction, Janus kinase, STAT. Larserics Digital Print AB, Sundbyberg 1996 ISBN 91-628-2296-