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Fig. 3. PN-1 negatively modulates SHH signal transduction in CGNPs.
(A,B) CGNPs of P5 wild-type mice cerebella were cultured on
polylysine (10 µg/ml) for 48 hours alone or with recombinant SHH and
recombinant PN-1, and were then pulsed with BrdU for 16 hours. (A) The ratio
of BrdU-positive cells/total number of CGNPs was determined and the
proliferation index normalized with respect to the control value, which was
set at 100% (corresponding to 1.4% BrdU-positive CGNPs). Addition of 50 or 200
ng/ml SHH stimulates BrdU incorporation (series 1). Addition of 30 nM (series
2) or 210 nM (series 3) PN-1 together with SHH significantly inhibits the
stimulation of cell proliferation. Notice that the addition of PN-1 alone
inhibits proliferation in comparison to the control. Error bars indicate
s.e.m. (*P<0.05; **P<0.01;
***P<0.001; Student's t-test). (B) CGNPs were
treated for 48 hours with SHH alone (50, 100 or 200 ng/ml; series 1), together
with 30 nM PN-1 (series 2) or SHH, which was added 5 hours prior to 30 nM PN-1
(series 3). Prior addition of SHH does not significantly alter the inhibitory
effect of PN-1. (C) Following 48 hours of treatment on polylysine
substrate (500 µg/ml), CGNPs were lysed and cyclin D1 expression levels
were determined. Notice that cyclin D1 protein levels increase in the presence
of 3 µg/ml SHH. This increase is inhibited by the addition of 30 nM PN-1 or
1 µg/ml RAP. (D) SHH signal transduction was investigated by RT-PCR
analysis of Gli1 expression in CGNPs cultured for 48 hours on a
proliferation-permissive substrate (polylysine; 10 µg/ml) in presence of
SHH (50 ng/ml) alone or with PN-1 (30 or 210 nM). The addition of SHH
increases Gli1 transcription, which can also be reversed by adding
PN-1. (E) The antagonistic effect of PN-1 on SHH signal transduction
was further studied using co-transfection assays in NIH3T3 cells. A CAT
reporter plasmid containing GLI-binding sites and a beta-galactosidase
expression plasmid were co-transfected into cells, and, 24 hours later, 3
µg/ml SHH and 30 nM PN-1 were added. A CAT ELISA assay was performed on 100
µg of cell extract prepared after 48 hours. The addition of SHH stimulates
the transcriptional activity more than fourfold. This effect is blocked by
PN-1. PN-1 alone has no effect. Values represent the mean relative activity of
the CAT reporter enzyme after adjustment for transfection efficiency and
normalization with mock-transfected controls. (*P<0.05,
Student's t-test). (F) Inactivation of the Pn-1 gene
enhances the proliferation rates of CGNPs. Mixed cultures of P5 wild-type
(grey bars) and Pn-1-/- cerebellar cells (black bars) were
treated for 48 hours with 3 µg/ml SHH, 30 nM PN-1 (PN-1 30), 210 nM PN-1
(PN-1 210) or KAAD-cyclopamine 1 µg/ml (Cp). The proliferation rates of the
CGNPs were determined and results normalized to wild type
(Pn-1+/+ controls). The proliferation rates are expressed
as percentages (100% corresponding to 2.98% of BrdU-positive CGNPs). Values
shown represent mean±s.e.m. (***P<0.0001;
two-ways ANOVA test). CGNPs from Pn-1-/- mice display
enhanced proliferation and increased sensitivity to SHH. The proliferation
rates are reduced to wild-type levels by adding a high concentration of PN-1
or by blocking SHH signal transduction with KAAD-cyclopamine.
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