For many years, nuclear growth hormone receptor (GHR) has been demonstrated both in vivo
and in vitro.
It is now becoming apparent that many growth factors, cytokines, and their receptors are nuclear localised, and in many cases nuclear localisation is necessary for full function. The functional role of the nuclear GHR is currently unknown. However from the published studies, a common feature of tissues and cells demonstrating nuclear GHR is the potential for a high proliferative status. Examples include the chondrocytes of the epiphysial growth plate, gastrointestinal tract, placenta and preimplanation embryo, as well as a number of cancers. Despite this, correlation of nuclear GHR with proliferative status has not been reported. In this thesis, I report that in regenerating liver nuclear localised GHR is strongly correlated with proliferative status at the cellular level. This finding is the first evidence for a biologically functional role of the nuclear GHR.
Further, I provide evidence to support a functional role for the nuclear GHR in proliferation, using a cell model where the GHR is constitutively targeted to the nucleus by addition of the nuclear localisation sequence (NLS) of the SV40 Large T antigen. Using this model, I have been able to dissect the relative contributions of the plasma membrane bound GHR versus the nuclear localised GHR, to proliferative signaling. A profound change was found in the proliferative status associated with increased nuclear targeting of GHR in the NLS lines. While the WT lines were GH dependent for both nuclear translocation of the GHR and cell cycle progression, the NLS lines exhibited constitutive nuclear localised GHR, and GH independent proliferation. Using microarray analysis of these model lines, I have identified the genes for Cytokine Inducible SH2 domain protein CIS, Myb binding protein (MybBP1a), and Survivin (identified in independent experiments) as being important in this process.
Finally, I report a novel putative "moonlighting" function of the nuclear GHR as a transcriptional activator directly involved in the regulation of a subset of GH induced genes. In support of this role, I show that GHR contains a potent transactivation domain when fused to a heterologous DNA binding domain. Using this transactivation domain of the GHR as the "bait" in an affinity chromatography approach, several nuclear proteins have been identified as binding to the GHR to form a large multiprotein complex. The GHR interacting proteins include two important coactivator proteins, Coactivator activator (CoAA) and Thyroid receptor uncoupling protein (TRUP). Several components of the nuclear import complex, including Importin α, Importin β, the GTPase Ran and Nucleoporin 145 have also been identified as associating with the GHR. This finding is concordant with results from a collaboration, indicating that GHR utilises the classical nuclear import pathway for its nuclear uptake. The final piece of evidence to support a transactivation function for the nuclear GHR is its ability to associate with DNA. Accordingly, a modified chromatin immunoprecipitation (ChIP) technique was used to isolate and identify DNA sequence to which the GHR is associated. Blast analysis using the Ensembl mouse genomic database, revealed the sequence to be localised upstream of a cluster of genes including the gene for Survivin. The association of the GHR WT with this sequence was tested by independent ChIP assay using the Baf-GHR WT line, and was found to be specific and GH dependent, occurring concomitant with the GHR translocation into the nucleus.
Thus, the direct regulation by the nuclear GHR of a subset of GH target genes appears be a mechanism for determining the specificity of GH's diverse actions. The induction of the STAT5 inhibitor CIS, may act to downregulate STAT5 dependent genes, many of which mediate GH's metabolic and differentiation processes. At the same time, the direct induction by the nuclear GHR of Survivin and MybBP1a may act to initiate cell cycle progression in quiescent cells, as occurs in the hepatocytes during the proliferative phase of liver regeneration. This mechanism may further apply to other regenerating tissues, and is worth investigating in the future. Conversely, the dysregulation of subcellular localisation of GHR to result in increased levels of nuclear GHR, as has been reported in carcinoma as discussed above, may contribute to uncontrolled cell cycle progression associated with proliferative disorders. This study may provide an important piece of the GH signaling scheme, which has been previously overlooked in the current paradigm of GHR signal transduction. Potentially this work may supply an insight into a new level of regulation by GHR, and provide novel targets in the treatment of proliferative disorders, as well as in the initiation of tissue regeneration.