A new antibody for an orphan
G protein-coupled receptor, AXOR12/GPR54/OT7T175
AXOR12 (OT7T175, GPR54) is a newly identified orphan G Protein-Coupled
Receptor for KiSS-1/Metastin. Mouse pancreas and placenta as well
as human placenta tissues can be stained with Phoenix Rabbit anti-AXOR12
(375-398) (Human) antibody (H-048-61). more
information of metastin/KiSS-1 (68-121) peptide
KiSS-1 and KiSS-1R mRNA and
protein expression in first trimester placenta. The
histology of the maternal-fetal interface and KiSS-1
(A) and KiSS-1R (B) expression patterns are shown schematically
from in situ hybridization and immunohistochemistry
staining (Fig. 4). AV, anchoring villus; evCT: extravillous
cytotrophoblast; ST, syncytiotrophoblast; vCT, villous
cytotrophoblast.
KiSS-1/Kp-54 and KiSS-1R localization
in first trimester placenta. Sections of human FT placental
tissue at week 6-10 of gestation showing the localization
of KiSS-1/Kp-54 (A,B) and KiSS-1R (E,F). KiSS-1 and
KiSS-1R mRNA were detected by in situ hybridization
as dark blue precipitates, whereas their respective
proteins were detected using affinity purified polyclonal
antibodies evident as dark red precipitate. Sense probes
(C,G) and nonimmune sera (D,H) produced no detectable
signal. KiSS-1 mRNA (A) and Kp-145/Kp-54 protein (B)
were detected mainly on the outer (syncytiotrophoblast)
surface of villi. Higher magnification showed that KiSS-1/Kp-54
expression is restricted to the syncytiotrophoblast
(insets; A and B; bars, 25 µm), whereas it was undetectable
in villous (vCT) and extravillous (evCT) cytotrophoblasts.
KiSS-1R mRNA is located in the syncytiotrophoblast (ST),
villous (vCT) and extravillous (evCT) cytotrophoblasts
(E). This mRNA staining pattern is paralleled by that
of KiSS-1R protein (F). Bars, 50 µm.
KiSS-1 sequence and cleavage
products resulting in various kisspeptins
KiSS-1 and KiSS-1R are differentially
expressed in FT and term human trophoblast cells. (A)
RT-PCR of total RNA of freshly isolated FT or term trophoblasts
(representative experiment: left panel). (B) Western
blotting of FT and term trophoblasts. Lysates were probed
with anti-Kp145/Kp-54 antiserum. With purified Kp-54
(5 ng) the antiserum produced a single band at 5-6 kDa,
which is in good agreement with the theoretical MW for
Kp-54 (5858.0 kDa; lane `M'). The antibody reacted only
with lysates from FT trophoblasts producing one prominent
band at 15-16 kDa, which is in good agreement with the
calculated Mr for Kp-145 (15.391 kDa). ß-actin served
as loading control. (C) FT trophoblast conditioned media
were subjected to MALDI-TOF analysis after reverse phase-HPLC
fractionation. The theoretical masses for C-terminally
amidated Kps-10, -13, -14 and -54 are 1304, 1626, 1704
and 5858, respectively. Kp-54 was identified also as
Na-adduct (m/z: 5876). The masses at around 2600 are
unidentified.
Kisspeptin-10 raises intracellular Ca2+
in isolated first trimester human trophoblasts. FT trophoblasts
were stimulated with different Kps and intracellular
[Ca2+] was measured (A). Only Kp-10 resulted in an increase
in intracellular [Ca2+] (n=9), whereas Kp-13, -14 and
-54 were ineffective (n=6). (B) Concentration-response
curve for Kp-10 on intracellular free Ca2+ concentration
in isolated FT trophoblasts. The intracellular [Ca2+]
is expressed as ratio (F340/F380) (mean±s.e.m.; n=6-9).
The EC50 was found to be 21 (16-29) nM (95% confidential
interval).
Kisspeptin-10 inhibits trophoblast outgrowth
and migration, but not proliferation in first trimester
human villous explant cultures. Villous explants from
6-9 weeks' gestation were maintained in culture for
72 hours in the absence (A,B) or presence of 0.3 µM
(C,D) or 1.0 µM (E,F) Kp-10. Identical villi were photographed
at 48 or 72 hours. The dark areas are tissue, and sheets
of outgrowing cytotrophoblast can readily be observed
in the untreated cultures (A,B; arrows mark the limits/boundaries
of the outgrowth). Kp-10 treatment profoundly decreases
trophoblast outgrowth from the distal end of the villous
tips when compared with control villous explants. Magnification:
x40. (G-J) Proliferative potential of placental villi
was determined by incorporation of BrdU after 24 hours
in culture in the absence (G,I) or presence (H,J) of
1 µM Kp-10. Villus sections were stained with an anti-BrdU
antibody, which detects cells in the S-phase (G,H),
or were labeled with the nuclear stain dapi (I,J). The
nonproliferating syncytiotrophoblast (ST) is devoid
of anti-BrdU staining (G,H). Cell column (CC) formation
by villus explants maintained in the absence or presence
of Kp-10 (0.3 µM) was similar. In addition, no significant
difference in the proportion of nuclei that incorporated
BrdU was detected. Six villi per treatment were examined
per placenta and the experiment was repeated three times.
Magnification: x400. The extent of migration was increased
between 48 and 72 hours under all conditions. Migration
distance was reduced already at 0.3 µM Kp-10 at both
48 and 72 hours (K). The higher concentration of 1 µM
did not augment the effect. *P<0.05 vs control. VS,
villous stroma.
Kisspeptin-10 inhibits migration and gelatinolytic
activity, but not proliferation of isolated first trimester
human trophoblasts. (A) Microscopic image of the abluminal
side of collagen I (Col I)- or fibronectin (FN)-coated
membranes after 48 hours during which isolated FT trophoblasts
(1x105 per filter, week 6-9 of gestation) migrated across
the membrane in the absence or presence of 0.5 µM Kp-10.
(B) Bars on left: Addition of Kp-10 (0.5 µM, black bars)
reduced the number of cells that migrated through the
filter pores. Results are expressed as mean±s.e.m. (n=3
different trophoblast isolations) of cell migration
relative to unstimulated cells set as 1 (white bars)
(*P<0.05 vs control). Bars on right: To assess cell
proliferation, 7x104 freshly isolated FT trophoblasts
were plated on collagen I in the absence (white bar)
or presence of Kp-10 (black bars) added at the time
of seeding. Cell numbers were counted after 48 hours.
Results are presented as cell number (mean±s.e.m.; n=3)
expressed relative to control (=100%). Kp-10 did not
affect FT trophoblast proliferation significantly. ns,
not significant vs control. (C) Conditioned medium from
isolated FT trophoblasts plated on collagen I for 24
or 48 hours was subjected to gelatin zymography. Proteolytic
activity was noted for the 72-kDa collagenase corresponding
to pro-MMP-2. Protein molceular weight markers are indicated
on the right. (D) Addition of Kp-10 suppressed 72-kDa
collagenase (MMP-2) activity (*P<0.01 vs control).
KiSS-1/GAPDH and hOT7T175/GAPDH
ratios of 8 normal livers (white bars), 60 non-cancerous
cirrhotic livers (white bars), and 60 carcinomas
(black bars)
Disease-free survival curves. The disease-free
5-year survival rate of the 27 in group A (51.8%) was
better than that of the 33 in groups B and C (24.2%;
p=0.065). Group A: normal expression of both KiSS-1
and hOT7T175 genes; group B: overexpression of one of
two genes; and group C: overexpression of both genes
Correlation
between the clinicopathological
findings in patients, and the KiSS-1
and hOT7T175 gene expression levels
in HCC
Number
Group A (n=27)
Group B (n=27)
Group C (n=6)
p
Histological
type
Well
8
4
3
1
0.959
Moderate
41
19
18
4
Poor
11
4
6
1
Venous
invasion
Negative
25
15
8
2
0.141
Positive
35
12
19
4
Stage
I and II
22
16
6
0
0.003
III and IV
38
11
21
6
Tumor recurrence
No
22
13
8
1
0.208
Yes
38
14
19
5
Group A:
normal expression of both genes; group
B: overexpression of one of the two genes;
group C: overexpression of both genes
