Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Dev Neurobiol
2007 Jan 01;671:81-96. doi: 10.1002/dneu.20330.
Show Gene links
Show Anatomy links
Transgene expression of prion protein induces crinophagy in intermediate pituitary cells.
van Rosmalen JW
,
Martens GJ
.
???displayArticle.abstract???
The cellular form of the prion protein (PrP(C)) is a plasma membrane-anchored glycoprotein whose physiological function is poorly understood. Here we report the effect of transgene expression of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP-PrP(C)) specifically in the neuroendocrine intermediate pituitarymelanotrope cells of Xenopus laevis. In the transgenic melanotrope cells, the level of the prohormone proopiomelanocortin (POMC) in the secretory pathway was reduced when the cells were (i) exposed for a relatively long time to the transgene product (by physiologically inducing transgene expression), (ii) metabolically stressed, or (iii) forced to produce unfolded POMC. Intriguingly, although the overall ultrastructure was normal, electron microscopy revealed the induction of lysosomes taking up POMC secretory granules (crinophagy) in the transgenic melanotrope cells, likely causing the reduced POMC levels. Together, our results indicate that in neuroendocrine cells transgene expression of PrP(C) affects the functioning of the secretory pathway and induces crinophagy.
Figure 1 Intermediate pituitary-specific fluorescence and
transgene expression in Xenopus embryos transgenic for
GFP-PrP C or GFP-GPI. (A) Schematic representation of the
linear injection fragments pPOMC-GFP-PrP and pPOMC-
GFP-GPI containing the Xenopus POMC gene A promoter fragment (pPOMC), and the GFP-PrP and GFP-GPI fusion protein-coding sequence, which were used to generate transgenic Xenopus lines 102 and 150, respectively; SS, sig-
nal peptide sequence; GPI, glycosylphosphatidylinositol
signal sequence. (B) Pituitary-specific fluorescence in living Xenopus embryos (stage *40, staging according to (Nieuwkoop and Faber, 1967); left panels) and in black-
adapted 6-month-old frogs transgenic for GFP-PrP C or GFP-GPI (ventrocaudal view; right panels). In adult Xenopus, the brain was lifted to reveal intense fluorescence in
the intermediate lobe (IL), but not in the anterior lobe (AL) of the pituitary. Arrows indicate the localizations of the fluorescent intermediate pituitaries expressing the fusion product; the positions of the eye (E), nose (N), and gut (G) are also indicated. The fluorescence in the gut represents auto-fluorescence. Bars equal 0.5 mm. The upper two panels are
taken from van Rosmalen and Martens (2006). (C) Tissue lysates of neurointermediate lobes (NILs) and ALs from wild-type (wt) animals and animals transgenic for the GFP-PrP C
(102) or GFP-GPI (150) fusion protein were subjected to SDS-PAGE. Western blot analysis was performed using an anti-GFP antibody (alpha-GFP).
Figure 2 Steady-state levels of a number of intermediate pituitary proteins from wild-type and
transgenic black-adapted Xenopus. (A) Tissue lysates of neurointermediate lobes (NILs) derived
from wild-type (wt) animals and animals transgenic for the GFP-PrP
C
(102) or GFP-GPI (150) fusion
protein were subjected to SDS-PAGE. Western blot analysis was performed using anti-POMC, anti-
PC2, anticalnexin, anti-BiP, anti-p24
1/2
, and antitubulin antibodies. Tubulin was used as a control
for protein loading. Animals were adapted to a black background for 3 or >8 weeks. (B) The steady-
state protein levels of POMC, PC2, calnexin, BiP, and p24
1/2
in NILs from wild-type and transgenic
animals adapted to a black background for >8 weeks were quantified by densitometric scanning and
are presented in arbitrary units (AU), relative to the steady-state levels of tubulin. Shown are the
means 6 SEM (n ¼ 3). A significant difference is indicated by ** (p < 0.01) or *** (p < 0.001).
Figure 3 Biosynthesis and processing of newly synthesized POMC in wild-type Xenopus inter-
mediate pituitary cells and cells transgenic for GFP-PrP
C
or GFP-GPI. (A) Neurointermediate lobes
(NILs) from wild-type (wt) animals and animals transgenic for the GFP-PrP
C
(102) or GFP-GPI
(150) fusion protein were pulse labeled with [
35
S]-Met/Cys for 30 min and subsequently chased for
3 h. Newly synthesized proteins extracted from the lobes (5%) or secreted into the incubation me-
dium (20%) were resolved by SDS-PAGE on 15% gels and visualized by autoradiography. Animals
were adapted to a black background for 3 weeks (short-term adaptation) or >8 weeks (long-term
adaptation). The experiments were performed in triplicate and a representative example is shown.
(B) The amounts of newly synthesized 37-kDa POMC (black bars) and the 18-kDa POMC-derived
product (gray bars) were quantified by densitometric scanning, and are presented in arbitrary units
(AU), relative to the amounts of newly synthesized actin. Shown are the means 6 SEM (n ¼ 3).
Significant differences are indicated by ** (p < 0.01) or *** (p < 0.001
Figure 4 Effect of glucose depletion on the biosynthesis and processing of newly synthesized
POMC in wild-type Xenopus intermediate pituitary cells and cells transgenic for GFP-PrP
C
. (A) Wild-
type (wt) neurointermediate lobes (NILs) and NILs transgenic for GFP-PrP
C
(102) were preincubated
for 2 h and pulse label ed with [
35
S]-Met/Cys for 30 min in the presence or absence of glucose. Newly
synthesized proteins were extracted from the lobes, directly resolved by SDS-PAGE on 15% gels, and
visualized by auto radiography. The ex perim ents were per formed in triplicat e and a representative
exam ple is shown . (B) The amounts of newly synthesize d 37-kDa POMC were qua ntified by densi to-
metric scanning and are presented in arbitrary units (AU), relative to the amounts of newly synthesized
actin. Shown are the means 6 SEM (n ¼ 3). (C) Neurointermediate lobes (NILs) from wild-type (wt)
animals and animals transgenic for the GFP-PrP
C
fusion protein were pulse labeled with [
35
S]-Met/
Cys for 30 min and subsequently chased for 3 h in the presence or absence of glucose. Newly synthe-
sized proteins extracted from the lobes (5%) or secreted into the incubation medium (20%) were
resolved by SDS-PAGE on 15% gels an d visualiz ed by autoradiography. (D) The amounts of newly
synthesized 37-kDa POMC and the 18-kDa POMC-derived product were quantified by densitometric
scanning and are presented in arbitrary units (AU), relative to the amounts of newly synthesized actin.
Shown are the means 6 SEM (n ¼ 3). A significant difference is indicated by *** (p < 0.001). In all
cases, animals were adapted to a black background for 3 weeks (short-term adaptation).
Figure 5 Effect of L- or 2-deoxy-D-glucose on the biosynthesis and processing of newly synthe-
sized POMC in wild-type Xenopus intermediate pituitary cells and cells transgenic for GFP-PrP
C
.
(A) Neurointermediate lobes (NILs) from wild-type (wt) animals and animals transgenic for the
GFP-PrP
C
fusion protein (102) were preincubated for 2 h and pulse labeled with [
35
S]-Met/Cys for
30 min and subsequently chased for 3 h in the presence of D-, L-, or 2-deoxy-D-glucose. Newly syn-
thesized proteins extracted from the lobes (5%) or secreted into the incubation medium (20%) were
resolved by SDS-PAGE on 15% gels and visualized by autoradiography. The experiments were
performed in triplicate and a representative example is shown. (B) The amounts of newly synthe-
sized 37-kDa POMC and the 18-kDa POMC-derived product were quantified by densitometric
scanning and are presented in arbitrary units (AU), relative to the amounts of newly synthesized
actin. Shown are the means 6 SEM (n ¼ 3). Significant differences are indicated by ** (p < 0.01)
or *** (p < 0.001). In all cases, animals were adapted to a black background for 3 weeks (short-
term adaptation)
Figure 6 Effect of a reducing agent on the biosynthesis and processing of newly synthesized
POMC in wild-type Xenopus intermediate pituitary cells and cells transgenic for GFP-PrP
C
. (A)
Neurointermediate lobes (NILs) from wild-type (wt) animals and animals transgenic for GFP-PrP C
(102) were preincubated for 30 min, pulse labeled with [35
S]-Met/Cys for 30 min and subsequently chased for 3 h in the absence (-) or presence (+) of 5 mM dithiothreitol (DTT). Newly synthesized
proteins extracted from the lobes (5%) or secreted into the incubation medium (20%) were resolved
by SDS-PAGE on 15% gels and visualized by autoradiography. The experiments were performed
in triplicate and a representative example is shown. (B) The amounts of newly synthesized 37-kDa
POMC and the 18-kDa POMC-derived product were quantified by densitometric scanning and are presented in arbitrary units (AU), relative to the amounts of newly synthesized actin. Shown are the
means 6 SEM (n ¼ 3). A significant difference is indicated by ** (p < 0.01). In all cases, animals were adapted to a black background for 3 weeks (short-term adaptation).
Figure 7 Electron microscopy on wild-type Xenopus melanotrope cells and cells transgenic for GFP-PrP C or GFP-GPI. (A–C) Electron micrographs of wild-type (wt), GFP-PrP C-transgenic (102)
and GFP-GPI-transgenic (150) melanotrope cells. Animals were adapted to a black background for
>8 weeks. Wild-type and transgenic melanotrope cells show well-developed rough endoplasmic
reticulum and extensive Golgi areas. Only transgenic melanotrope cells from line 102 contain pleio-
morph electron-dense lysosomes (crinosomes). (D–G) Details of various stages of crinophagy in
transgenic melanotrope cells from line 102. (D) Accumulation of a number of autophagic vacuoles
(encircled) containing a variety of membraneous and electron-gray material. Arrows indicate large
electron-dense lysosomal structures. (E) Higher magnification of autophagic vacuoles and electron-
dense secretory granules. Fusing autophagic vacuoles are encircled. (F) A large electron-dense crino-
some containing electron-gray debris and invaginating membraneous cytoplasmic material. (G)
Aggregating dense crinosomes with capricious extensions. Av, Autophagic vacuoles; Cr, crinosomes;
ER, endoplasmic reticulum; Go, Golgi area; N, nucleus; Sg, secretory granules. Bars equal 1 um
