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Dev Growth Differ
2002 Apr 01;442:103-12. doi: 10.1046/j.1440-169x.2002.00626.x.
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Synthesis and release of activin and noggin by cultured human amniotic epithelial cells.
Koyano S
,
Fukui A
,
Uchida S
,
Yamada K
,
Asashima M
,
Sakuragawa N
.
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Recent studies suggest that extra-embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.
Fig. 1. Expression of activin A and four activin receptor subtype
gene mRNA in human amniotic epithelial tissue (HAET) and
human amniotic epithelial cells (HAEC) by reverse transcription
(RT)–polymerase chain reaction (PCR). Lane 1, HAEC; lane 2,
HAET; lane 3, human fetal brain. Negative control experiments
were done without reverse transcriptase: lane 4, HAEC; lane 5,
HAET; lane 6, human fetal brain. ACVRIA, activin receptor type
IA; ACVRIB, activin receptor type IB; ACVRIIA, activin receptor
type IIA; ACVRIIB, activin receptor type IIB.
Fig. 2. Histological examination
of the explants. (A,B) control
medium; (C,D) conditioned
medium; and (E,F) activin A.
(B,D,F) show addition of excess
follistatin. (A,B,F) show differentiated
atypical epidermis. bl,
blood-like cells; coe, coelomic
epithelium; epi, epidermis; mes,
mesenchyme; mus, muscle; neu,
neural tissues; not, notochord.
Bar, 0.1 mm.
Fig. 3. Reverse transcription (RT)–polymerase chain reaction
(PCR) analysis of the explants. Lane 1, control medium; lane 2,
control medium + follistatin; lane 3, conditioned medium; lane 4,
conditioned medium + follistatin; lane 5, activin A; lane 6, activin
A + follistatin; lane 7, whole embryo. GSC and Xlim-1 are
used as activin-response gene markers, Xbrachyury as a
pan-mesodermal marker, Xnot-1 as a notochord marker, m-actin
as a muscle marker, NCAM as a pan neural marker, and ODC
as a loading control. RT(–), negative control.
Fig. 4. Immunostaining of cultured
human amniotic epithelial
cells (HAEC). Immunoreactiviy to
(a) noggin antibody, (b) CK-19
antibody and (c) double staining
of noggin and CK-19 antibodies.
(d) Negative control using
6.8 μg/mL IgG. Bar, 100 μm.
Fig. 5. (a) Noggin protein expression in conditioned medium
(CM) from cultured human amniotic epithelial cells (HAEC). Lane
1, 20 μL medium; lane 2, concentrated CM from 48 h HAEC
cultures; lane 3, 150 ng human recombinant noggin protein;
lane 4, 200 ng human recombinant noggin protein; lane 5, 250 ng
human recombinant noggin protein. Separation was by sodium
dodecylsulfate (SDS)–12% polyacrylamide gel electrophoresis
(PAGE), followed by western blot analysis with the antinoggin
antibody. Positions of the molecular mass makers (kDa) are
indicated on the left. (b) Expression of organizing factors
(noggin, chordin and follistatin mRNA) in human amniotic epithelial
tissue (HAET) and cultured HAEC by reverse transcription
(RT)–polymerase chain reaction (PCR). Lane 1, HAEC; lane 2,
HAET; lane 3, human fetal brain. Negative control experiments
were done without reverse transcriptase: lane 4, HAEC; lane 5,
HAET; lane 6, human fetal brain.
Fig. 6. (a) Dose-dependent induction of noggin mRNA by treatment of cultured human amniotic epithelial cells (HAEC) with activin
A. Cultured HAEC were treated with 0, 10, 30 or 50 ng/mL of recombinant bovine activin A for 40 h. Reverse transcription (RT)–
polymerase chain (PCR) was carried out using primers specific to the genes and with the following conditions: 30 cycles (noggin)
or 25 cycles (GAPDH) at 94°C for 1 min; 58°C for 30 s; and 72°C for 1 min. RT reactions were done with (lanes 1–4) or without
(lanes 5–8) reverse transcriptase. (b) Effect of cyclohexamide (CHX) treatment on cultured HAEC treated with activin A. Cultured
HAEC were incubated with or without 5 μg of CHX for 30 min before treatment with activin A. Lane 1, 0 ng/mL activin A and without
CHX; lane 2, 50 ng/mL activin A and without CHX; lane 3, 0 ng/mL activin A and with CHX; lane 4, 50 ng/mL activin A and with CHX.
Northern blot analysis was done using PCR-amplified human noggin cDNA as probe. Twenty micrograms of total RNA was loaded in
each lane. Quantity and integrity of RNA was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA (lower panel).
(c) Effect of anti-activin A antibody on noggin induction by activin A in cultured HAEC. HAEC were incubated with or without 50 μg/mL
rabbit polyclonal anti-activin antibody during the same period of treatment with activin A. Lane 1, without activin A and anti-activin A
antibody; lane 2, 50 ng/mL activin A and without anti-activin A antibody; lane 3, without activin A and with anti-activin A antibody; lane
4, 50 ng/mL of activin A and with anti-activin A antibody. Upper two panels show northern blotting and lower two panels show RT–PCR.
RT–PCR was done as described in (a). At least three independent experiments were done in each panel.
