Functional analyses of the role of kisspeptins and their receptor, gpr-54 in the biology of reproductive tissues
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Date
2010Author
Roseweir, Antonia Kathryn
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Abstract
GnRH neurons represent the final common pathway for the regulation of the
reproductive axis and they are modulated by multiple signals. It has recently been
shown that a potent effector of GnRH neuron function is an afferent network of
kisspeptin-producing neurons. Kisspeptin released from these neurons acts upon a
specific receptor (gpr-54) expressed on GnRH neurons, and increases the secretion
of GnRH from the hypothalamus. The kisspeptin system has since been implicated
as a downstream mediator for regulation of the Hypothalamic-Pituitary-Gonadal
(HPG) axis by steroid hormones, metabolic signals and photoperiod, potentially
placing it at the centre of reproductive physiology. However, the supporting
evidence to date has been indirect, relying on interpretation of changes in mRNA
levels and immuno-histochemical staining to infer the actions of kisspeptin upon
the central control of reproduction. The detailed mechanisms of kisspeptin action
are yet to be fully elucidated.
The research within this thesis elucidates the effect of kisspeptin on the HPG axis
via the development of kisspeptin-10 (kp-10) analogues with antagonistic
properties. Functionally important residues within the peptide were delineated.
Structure-activity studies of kp-10 analogues indicated that residues Asn2, Trp3,
Phe6, Arg9 and Phe10 interact with gpr-54 to facilitate receptor binding. Two other
residues, Tyr1 and Leu8 were shown to be critical for receptor activation by
kisspeptin. Four synthetic peptide antagonists were selected according to a
consensus sequence for good antagonism: X1-N-W-N-X5-F-G-X8-R-F-NH2 where X1
= D-Ala or D-Tyr, X5 = Gly or D-Ser and X8 = D-Trp or D-Leu. One of the
antagonists, peptide 234, was used in in vivo studies, where it inhibited the
amplitude of GnRH and LH pulses without affecting basal secretion of GnRH or
LH. These results indicate for the first time that basal and pulsatile secretion of
these factors is regulated by separate pathways. Use of the antagonist also demonstrated the direct involvement of endogenous kisspeptin in steroid hormone
negative feedback, positive regulation of the pre-ovulatory LH surge and in
regulating the onset of puberty in rodents, as had been suggested via indirect
methods.
Although a major role of the kisspeptin system is in the regulation of the HPG axis,
the system may also be important in the inhibition of cancer cell metastasis and in
placental development (trophoblast cell invasion) but little is known about the
mechanisms involving kisspeptin in these processes. This thesis describes novel
signalling mechanisms for the regulation cell migration by kisspeptin, involving the
MAPK and GSK3β signalling pathways. Using a stably transfected CHO cell line,
kisspeptin-gpr-54 signalling can activate all members of the MAPK pathway, the β-
catenin/GSK3β pathway, NFκB and FAK. These factors are involved in inhibiting
the migration of these cells via an ERK1/2-p90rsk-GSK3β-β catenin pathway to
potentially up- regulate formation of adherens junctions at the plasma membrane.
This pathway was also shown to be involved in the inhibition of migration within
an immortalised human first trimester placental trophoblast cell line and in human
umbilical vein endothelial cells. Some of these pathways were also active within a
mouse GnRH neuronal cell line, where ERK1/2, NFκB and GSK3β were activated
by kisspeptin with no effect on migration. However, the role of these pathways in
the GnRH neuronal cells requires further investigation.
In summary, the research presented within this thesis defines receptor-binding and
activating residues within kisspeptin-10, which should enable more details of
ligand-receptor binding interactions to be fully elucidated. Novel gpr-54
antagonists have been identified and used in in vivo studies. The thesis
demonstrates the direct involvement of endogenous kisspeptin in the regulation of
GnRH/LH secretion at the onset of puberty and throughout the reproductive cycle
in mature animals. The antagonists developed within this thesis represent useful
tools to further delineate mechanisms of kisspeptin action within the HPG axis and peripheral tissues. Other findings describe kisspeptin signalling mechanisms for
the inhibition of cell migration, potentially important in a variety of normal and
pathological processes, including for the first time a description of the regulation of
GSK3β and β-catenin signalling factors by kisspeptin and gpr-54.