Model Systems for Studying Kisspeptin Signalling: Mice and Cells - Kisspeptin Signaling in Reproductive Biology - page 482

(FAK) and paxillin proteins, which would increase cell adhesion. Migratory
inhibition of the trophoblast-derived cell line HTR8SVneo occurs through an
ERK1/2-p90rsk pathway to inhibit GSK3 β activity and increase β -catenin levels in

the cytoplasm [ 64 ]. -Catenin can bind to transmembrane cadherins to promote cell-

cell binding [ 67 ]. In addition, inhibition of migration of the human ovarian cancer
cell line SKOV3 can be overcome by phorbol ester stimulation of protein kinase C

β

α

[ 68 ], suggesting that continuous kisspeptin signalling could reduce cell migration by
inhibiting PKC signalling is associated with increased

cell adhesion in several different systems [ 69 - 71 ]. In addition to promoting cell
adhesion, kisspeptins may decrease cell invasion by reducing the expression of pro-
teases such as MMP-2 [ 72 , 73 ]. Kisspeptins also inhibit the intracellular signalling
cascade of the pro-metastatic G-protein coupled receptor CXCR4 [ 74 ].

α activity. Disruption of PKC α

Immortalized GnRH Neurons

Since GnRH neurons are the principal target for kisspeptin, immortalized cell lines
derived from these neurons are useful for studying intracellular signalling pathways
after kisspeptin stimulation. One of the most widely used GnRH cell lines is GT1-7,
which was derived from a hypothalamic tumour in a female mouse induced by
transgenic expression of the SV40 large T-antigen expressed from the GnRH pro-
moter [ 75 ]. Similarly, immortalized GnRH neuronal cell lines (GN and NLT series
sub-clones) have been derived from an olfactory bulb tumour induced in male mice
[ 76 , 77 ] by large T-antigen expression. Since the GN and NLT lines were derived
from GnRH neurons during their normal migration from the olfactory bulb, they are
generally considered to represent immature GnRH neurons.
To control for the developmental stage at which GnRH neurons are immortal-
ized, Wolfe et al. [ 78 ] generated transgenic mice in which a doxycycline-regulated
T-antigen transgene was used to select for cell lines (GRT cells) from adult mouse
hypothalamic explants. Salvi et al. [ 79 ] derived several GnRH cell lines (Gnv
clones) by immortalizing cells from isolated hypothalami by lentiviral delivery of a
tetracycline—inducible v - myc oncogene. An advantage of these approaches is that
the immortalized GnRH cell lines are selected in culture, which should limit the
acquisition of mutations associated with tumour formation in vivo.
GT1-7 cells retain markers of GnRH neurons including expression of the kiss-
peptin receptor [ 80 - 83 ] and the ability to secrete GnRH in an autonomous pulsatile
manner [ 84 , 85 ]. It should also be noted, however, that the GT1-7 cell line also
shows gene expression not normally found in GnRH neurons including the oestro-
gen receptor alpha (ERa) and Kiss1 [ 80 , 83 , 86 ]. Kisspeptin stimulation of GT1-7
cells causes an increase in intracellular Ca 2+ [ 87 ], ERK1/2 phosphorylation and
GnRH secretion [ 80 , 82 ], similar to the responses observed in GnRH neurons.
GT1-7 cells have also been used to study the mechanisms of transcriptional regu-
lation of the Kiss1 gene. Transient transfection of luciferase reporter plasmids with
different lengths of the Kiss1 promoter has shown that oestrogen can stimulate