Role of the nuclear growth hormone receptor in cell proliferation and tumorigenesis

Miss Jong Wei Wooh (2008). Role of the nuclear growth hormone receptor in cell proliferation and tumorigenesis PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Miss Jong Wei Wooh
Thesis Title Role of the nuclear growth hormone receptor in cell proliferation and tumorigenesis
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2008-06
Thesis type PhD Thesis
Supervisor Professor Michael Waters
Professor George Muscat
Total pages 300
Total colour pages 53
Total black and white pages 247
Subjects 270000 Biological Sciences
Formatted abstract
The presence of growth hormone receptors (GHR) in the cell nucleus has been reported both
in vivo and in vitro in a range of proliferating cells, including cancers. The role and
mechanism of action of the nuclear GHR had not been established before this thesis work,
although recent studies have defined functional roles for some growth factor receptors and
their ligands in the nucleus.
In this thesis, I report that the proliferative function of the nuclear localized GHR is mediated
by autocrine GH. In the absence of exogenous GH, rabbit GHR (rbGHR) targeted to the
nucleus with the nuclear localization sequence (NLS) of the SV40 large T-antigen (NLSrbGHR)
can respond to murine autocrine GH secreted by pro-B cells (BaF cells) with
constitutive cell proliferation and constitutive Stat5 activation. In contrast, the NLS-tagged
human GHR (NLS-hGHR) which cannot respond to murine GH failed to proliferate in the
absence of exogenous hGH. Further, inhibition of murine GH action with the G120R GH
antagonist or siRNA to murine GH strongly inhibited the constitutive proliferation of stably
expressed NLS-rbGHR BaF cells, demonstrating that autocrine GH plays a significant role
in activation of the nuclear GHR. However, while the proliferative action of the nuclear
rbGHR requires autocrine GH, it is not known if there are novel functions regulated by the
nuclear GHR in the absence of GH binding.
Using microarray analysis of the transcriptome of murine BaF cells with nuclear targeted of
hGH receptor, I have identified genes regulated by the human GHR independent of GH
itself, but dependent on nuclear localization of the receptor. Key genes identified were
Aldehyde dehydrogenase (Aldh1a1), CD59a and Carboxypeptidase family member A3
(Cpa3), and their dependence on nuclear GHR localization for expression was confirmed by
real-time PCR. The functions of these genes as stem cells and cancer markers strongly
suggests that the nuclear GHR regulates stem cell development and differentiation and is
also a potential factor for tumour progression. Using the MEME multiple alignment
bioinformatics tool, a motif of 6 conserved nucleotides was identified among the mouse and
human genomic sequences of Aldh1a1, CD59a and Cpa3. A search through the Tomtom
program using the JASPAR CORE database identified this motif with some similarity to that
of SNAI2, a transcription factor involved in stem cell regulation. However, it is most likelythat the identified sequence is unique and represents a binding site for the nuclear GH
receptor.
In agreement with a direct transcriptional action of nuclear GH receptor, I report the novel
function of the nuclear ICD of the GHR as a transcriptional activator. In support of this role,
I have used yeast two hybrid and luciferase reporter gene assays to show
that the ICD is a potent transactivator in which the transcriptional activity is confined to two
specific segments, with one comprising the C-terminal 50 residues of the GHR.
BaF cells stably expressing NLS-rbGHR with this C-terminal activation domain deleted
showed markedly decreased cell proliferation and decreased constitutive Stat5 activation.
BaF cells stably expressing NLS-rbGHR with mutation of the two tyrosine residues in this
C-terminal activation domain showed a more dramatic decrease in cell proliferation and
constitutive Stat5 activation. This demonstrated the importance of the C-terminal activation
domain and its tyrosine residues in affecting nuclear ICD function in promoting cell
proliferation.
Finally, I report efforts to create NLS-GHR overexpressing mice. Despite considerable
effort, no transgenic mouse expressing the full-length nuclear GHR was obtained. This was
possibly due to the deleterious embryonic lethality of the nuclear GHR expression,
postulated to exhibit hyperproliferative effects. A truncated form of the nuclear GHR almost
equivalent to that containing nuclear GHBP and TMD was identified in one transgenic
founder. Its offspring showed a phenotype similar to wild-type mice, suggesting that the
oncogenic effect of the nuclear GHR is not mediated by the extracellular domain of the GHR
and that the presence of the ICD is essential for its tumorigenic function.
Therefore, the autocrine GH activation of the nuclear GHR plays an important role in
mediating its proliferative action. The direct regulation of a subset of target genes by nuclear
GHR appears to be a mechanism for determining the specificity of GHR diverse actions
relative to other class 1 cytokines. The induction of Aldh1a1 may act to regulate the balance
between stem cell differentiation and cancer development. At the same time, the direct
induction of CD59a and Cpa3 by the nuclear GHR may act to initiate tumorigenesis in cells,
as observed in a range of cancers. Thus, transcriptional activation by the nuclear ICD may
regulate a different subset of genes in the nucleus compared to the usual set of cell surface
regulated genes.
In conclusion, this study thus provides important information on signalling pathways directly
regulated by nuclear GHR, which have not been previously studied in the paradigm of GHRsignal transduction. Potentially, this work provides insight into a new level of GHR signal
transduction.
Keyword Nuclear growth hormone receptor, autocrine growth hormone, cell proliferation, intracellular
Additional Notes Please print Coloured pages for Pages 30, 40, 42, 46, 55, 79, 84-89, 96-100, 106, 113, 117, 121, 123, 125, 127, 129, 132, 143, 145, 147, 154, 156-158, 160, 162, 163, 166, 173, 175, 178, 180-184, 186, 204, 295-300. Please print pages 242-265 in landscape format

 
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Created: Fri, 31 Oct 2008, 14:27:08 EST by Miss Jong Wei Wooh on behalf of Library - Information Access Service