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J Biol Chem, Vol. 274, Issue 27, 19294-19300, July 2, 1999
From the The inhibitory effect of the neuropeptide
somatostatin on the expression of growth hormone was measured by
quantitative polymerase chain reaction in the pituitary cell line
AtT-20. We demonstrate that this effect is dependent on the
internalization of somatostatin-receptor complexes and that it is
totally independent from the peptide-induced inhibition of adenylate
cyclase. Indeed, the inhibitory effect of the peptide on growth hormone
mRNA levels was totally insensitive to pertussis toxin treatment
but was totally abolished under conditions which block somatostatin
receptor internalization. Comparative confocal microscopic imaging of
fluorescent somatostatin sequestration and fluorescence immunolabeling
of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably
responsible of the inhibitory effect of somatostatin on growth hormone expression.
The neuropeptide somatostatin (somatotrophin release inhibitory
factor; SRIF)1 is known to
play a critical role for the regulation of hormone secretion by the
anterior pituitary and peripheral glands as well as to act as
neuromediator in the central nervous system. Biological actions of SRIF
are exerted through multiple receptors subtypes, which have recently
been cloned and are referred to as sst1, sst2, sst3, sst4, and sst5
(for review, see Refs. 1 and 2). These receptors are widely expressed
in the anterior pituitary, peripheral tissues, and brain (3, 4). In the
pituitary, the predominant effects of SRIF are the inhibition of the
secretion of growth hormone (GH) and thyroid-stimulating hormone,
although inhibitory effects of SRIF on the release of luteinizing
hormone and prolactin have also been documented (5, 6). GH secretion is
inhibited by SRIF through both cAMP-dependent and
cAMP-independent pathways (7). In both cases, these effects have been
described as being pertussis toxin-sensitive, suggesting the
implication of a SRIF receptor coupled to Gi or
Go (8). SRIF has also been shown to reduce transcription of
the GH gene in vivo (9). However, this effect was
interpreted as being indirect, involving central inhibition of growth
hormone-releasing hormone release (10-12). There has been no evidence
thus far that SRIF may directly inhibit GH expression.
Recent studies have shown that interactions of SRIF with its receptors
resulted in a temperature- and receptor-dependent
internalization of receptor-ligand complexes (13, 14). The efficacy of
this internalization process, as well as the pattern of intracellular trafficking of internalized ligand and receptors, were shown to vary
according to the receptor subtypes involved (13-16). As for other G
protein-coupled receptors, ligand-induced sst receptor internalization
has been proposed to play a role in receptor desensitization through
cell surface down-regulation (17, 18). Early observations of nuclear
translocation of internalized 125I-SRIF in AtT-20 cells
have also raised the possibility that internalization may affect
transcriptional activity in this cell line (19). The possibility that
internalization of receptor-ligand complexes may play a role in
transmembrane signaling has so far mainly been explored for growth
factor and cytokine receptors (for a review, see Ref. 20). Recent
studies, however, have suggested that internalization of G
protein-coupled receptors may also be mandatory for certain types of
cell signaling. Thus, changes in the duration of inositol phosphate
accumulation and associated calcium responses (21, 22) or in the
transcription of receptor mRNA (23) have been reported in target
cells under conditions of impaired receptor internalization.
Furthermore, ligand-induced receptor internalization of The mouse AtT-20 pituitary cell line has been widely used as a model to
study the physiological, pharmacological, and biophysical properties of
SRIF receptors in anterior pituitary cells (25-27). Molecular
biological and biochemical studies have shown that AtT-20 cells express
four of the five cloned SRIF receptors subtypes (sst1, sst2, sst4, and
sst5) (28) and internalize radioactive SRIF with high efficiency (29).
Although AtT-20 cells are mainly documented to express POMC and to
secrete ACTH (30, 31), preliminary studies from our laboratory suggest
that AtT-20 cells may also express
GH.2 The aim of the present
study was to confirm that AtT-20 cells truly express GH, to determine
whether this expression is regulated by SRIF, and to investigate the
role played by receptor/ligand internalization in this regulatory function.
Materials
Mouse AtT-20/D16-16 tumor cell line was a gift from Dr. Nabil
Seidah (Clinical Research Institute, Montréal, Québec,
Canada). Dulbecco's modified Eagle's medium and gentamycin were
purchased from Life Technologies Inc., fetal calf serum and
1,10-phenanthroline from Roche Molecular Biochemicals, and horse serum
from Boehringer Ingelheim. The polyclonal rabbit anti-sst5 antibody was
kindly provided by Dr. Hans-Jürgen Kreienkamp (Institut für
Zellbiochemie und Klinische Neurobiologie, University of Hamburg,
Hamburg, Germany) and polyclonal rabbit anti-sst1 and -sst2A antibodies
were gifts from Agnes Schonbrunn (Department of Integrative Biology and
Pharmacology, University of Texas, Houston, TX). Other products were
from the following sources: Texas Red-conjugated goat anti-rabbit
antibody, Jackson Immunoresearch Laboratory Inc. (West Grove, PA);
forskolin, isobutylmethylxanthine, and pertussis toxin, Sigma; Biotrak
cAMP enzyme immunoassay system, Amersham Pharmacia Biotech;
Erase-a-Base and Reverse Transcription Systems, Promega; TA cloning
vector, Invitrogen; RNAble kit, Eurobio; Opti-Prime PCR optimization
kit, Stratagene.
Tyr0-[D-Trp8]SRIF-14
was iodinated and purified as described previously (32). Bodipy-[D-Trp8]SRIF-14 (fluo-SRIF)
was synthesized and purified as published (14).
Cell Culture
Mouse AtT-20/D16-16 tumor cells were grown in Dulbecco's
modified Eagle's medium with 10% fetal calf serum and 10% horse
serum supplemented with gentamycin (50 µg/ml). Cells were plated in 12-mm multi-well dishes for binding experiments, in 60-mm culture dishes for cAMP measurements, and in 100-mm culture dishes for GH
mRNA detection.
Binding and Internalization of
125I-Tyr0-[D-Trp8]SS14
and Fluo-SRIF on AtT-20 Cells
Cell Pretreatments--
The culture medium was removed, and
cells were equilibrated with 500 µl of Earle's Tris-Hepes buffer, pH
7.4, containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 3.6 mM MgCl2,
supplemented with 0.1% glucose and 1% bovine serum albumin (binding
buffer) in the presence or in the absence of 0.45 M sucrose
for 30 min or of 100 µg/ml pertussis toxin for 15 h.
Association Kinetics--
The incubation medium was replaced by
250 µl of fresh buffer containing 0.1 nM
125I-labeled
Tyr0-[D-Trp8]SRIF
(2000 Ci/mmol) in the presence of 0.8 mM
1,10-phenanthroline. At the indicated times, cells were washed twice
with 500 µl of binding buffer. To discriminate between surface-bound
and sequestered 125I-Tyr0-[D-Trp8]SRIF,
cells were left unwashed or washed for 2 min with 500 µl of a
hypertonic acid buffer (binding buffer containing 0.2 M
acetic acid and 0.5 M NaCl, pH 4). Cells were then
harvested in 1 ml of 0.1 M NaOH, and the radioactivity was
counted in a Equilibrium Binding Experiments--
Saturation experiments were
performed by incubating pretreated cells with increasing concentrations
(from 0.5 to 16 nM) of 125I-Tyr0-[D-Trp8]SRIF
isotopically diluted with unlabeled
Tyr0-[D-Trp8]SRIF in
the binding buffer. Dissociation constant (Kd) and
maximal binding capacity (Bmax) were derived
from Scatchard analysis of the data.
Internalization of Fluo-SRIF in AtT-20 Cells--
AtT-20 cells
were grown on 12-mm glass coverslips coated with polylysine (10 µg/ml). Fluo-SRIF (20 nM) was added on preincubated cells
for the indicated times, and cells were washed twice with 500 µl of
binding buffer. Nonspecific labeling was determined in parallel by
carrying out the incubation in the presence of 1 µM
[D-Trp8]SRIF-14. For selective
visualization of internalized fluo-SRIF, cells were washed with
hypertonic acid buffer as described above. Labeled cells were deposited
on glass microscope slides, air-dried, and examined under a Zeiss laser
scanning confocal microscope equipped with an Axiovert 100 inverted
microscope and an argon-krypton laser. Samples were scanned under 568 nm wavelength excitation; images were acquired as single transcellular
optical sections and averaged over 32 scans/frame. Images were then
processed using the Carl Zeiss CLSM software 3.1 version, stored on
Jazz disks, and mounted using Photoshop.
Immunodetection of sst1, sst2A, and sst5 Receptors in AtT-20
Cells
AtT-20 cells grown on 12-mm glass coverslips coated with
polylysine (10 µg/ml) were preincubated for 10 min at 37 °C in
Earle's buffer containing 0.2% bovine serum albumin and 0.1% glucose
and then incubated with or without 20 nM
[D-Trp8]SRIF-14 at 37 °C. After
washing twice with 500 µl of equilibration buffer, cells were
immediately fixed with 4% paraformaldehyde in phosphate-buffered
saline for 20 min at room temperature. Fixed cells were washed twice
with Tris-buffered saline (TBS) (2 × 10 min) and incubated
overnight at 4 °C in TBS containing 0.05% Triton X-100 and one of
the following: 1) a 1:50 dilution of a rabbit polyclonal sst5 antibody,
2) a 1:2500 dilution of a rabbit polyclonal sst2A antibody, 3) a 1:2000
dilution of a rabbit polyclonal sst1 antibody. The specificity of each
of these receptor antibodies has been fully established elsewhere (15,
18, 33, 34). Cells were then rinsed two times for 10 min each in TBS,
incubated with a Texas Red-conjugated goat anti-rabbit antibody diluted 1:100 in TBS for 45 min, rinsed in TBS (two 10-min rinses), and mounted
on glass slides with Aquamount for confocal microscopic examination.
Images were acquired, stored, and archived as described above.
GH mRNA Measurement in AtT-20 Cells
Cell Pretreatments--
Cells were preincubated in the culture
medium either alone or in the presence of 100 ng/ml pertussis toxin for
15 h or with 0.45 M sucrose in Earle's buffer for 30 min to inhibit internalization. The culture medium was then removed,
and cells were incubated with 100 nM
[D-Trp8]SRIF-14 for 60 min at
37 °C or 4 °C. Cells were then washed twice with TBS, and total
RNAs were prepared at various times.
Competitive PCR--
The method of analysis was based on the
competition between known amounts of a cDNA competitor and the GH
cDNA to be measured for the annealing with common primers during
the PCR.
Preparation of the cDNA Competitor--
The cDNA
competitor was prepared from the TA-cloning plasmid containing the
wild-type GH cDNA. The cDNA encoding GH was obtained from the
reverse transcript product of untreated cells by PCR using a sense
oligonucleotide (5'-ACCTCCTGGCTCCTGACCGTC-3') and an antisense
oligonucleotide (5'-GGTCTCCGCTTTGTGCAGGTC-3'), which corresponded to
nucleotide sequences 19-39 and 577-597 of the open reading frame of
GH mRNA (35). The PCR product (579 bp) was purified from a 2% low
gelling temperature agarose gel and subcloned into the TA-cloning
vector. The GH sequence was confirmed by sequencing using the ABI-PRISM
DNA sequencing kit (Applied Biosystems, Foster City, CA). The plasmid
was linearized with BsmI, which cleaves the GH cDNA.
After partial digestion with exonuclease III (Erase-a-Base) and
treatment with nuclease S1, extremities were blunted with the Klenow
fragment of polymerase I and ligated. We selected a 384-bp fragment
that has been used as the competitor in the RT-PCR quantitative experiments.
Quantitation of GH cDNA by Competitive RT-PCR--
Total
RNAs were extracted from AtT-20 cells following various experimental
conditions using the RNAble kit (Eurobio). The first cDNA strand
was obtained from RNAs (6 µg) using the Reverse Transcription System
kit (Promega). After cDNA synthesis for 1 h at 42 °C,
samples were denaturated for 5 min at 99 °C and chilled on ice.
Before analysis of GH mRNA content from AtT-20 cells submitted to
various effectors, PCR conditions were optimized with the Opti-Prime PCR optimization kit. The PCR buffer was then: 10 mM
Tris-HCl, pH 8.8, 1.5 mM MgCl2, 75 mM KCl.
One tenth of the first-strand cDNA was amplified in a volume of 50 µl with 200 ng of each primer, 2 µl of Taq polymerase
(0.5 unit/µl) (Appligene), and decreasing concentrations of the
cDNA competitor. Amplification was carried out with a first cycle
at 94 °C for 3 min, 55 °C for 2 min, 72 °C for 1 min 30 s, followed by 34 cycles, 94 °C for 45 s, 55 °C for 1 min,
72 °C for 1 min 30 s, and a final extension step at 72 °C
for 8 min. PCR products were analyzed on a 2% agarose gel. As
controls, each RT sample was submitted to PCR either with a sense or a
reverse primer. In some cases, the PCR was carried out without the
competitor cDNA, or without the RT product.
PCR band intensities detected with ethidium bromide were analyzed by
laser densitometry. When the ratio between the density of the cDNA
competitor band and that of the GH band was expressed as a function of
the amount of competitor, the representation was linear (Fig. 6) (36,
37). The amount of cDNA in the sample was calculated from the
experimental condition that gave a ratio = 1.
Measurement of cAMP Content in AtT-20 Cells
AtT-20 cells were preincubated with 1 mM
isobutylmethylxanthine for 15 min in culture medium under the various
experimental conditions (control, incubation in the presence of
hyperosmolar sucrose, or pretreatment with 100 ng/ml pertussis toxin;
see above). Cells were then incubated in culture medium containing 1 mM isobutylmethylxanthine with or without 0.1 µM [D-Trp8]SRIF-14
or 10 µM forskolin. The reaction was terminated by
removing the medium and scraping off cells in 1.5 ml of 65% ethanol.
After centrifugation, the dried pellet was dissolved in 0.05 M sodium acetate buffer, pH 5.8, containing 0.02% bovine
serum albumin and 0.01% preservative. The cAMP content was then
measured using an enzyme immunoassay kit according to the
manufacturer's recommendations.
Binding and Internalization of sst Receptors in AtT20
Cells--
In order to characterize the properties of SRIF
internalization in AtT-20 cells and to define optimal conditions for
blocking the internalization process, we performed association kinetics of
125I-Tyr0-[D-Trp8]SRIF-14
(125I-SRIF) on whole AtT-20 cells under various
experimental conditions. In the absence of any pretreatment,
125I-SRIF bound specifically to AtT-20 cells at 37 °C in
a time-dependent manner (Fig.
1A). The radioactivity
associated with the cells reached a plateau within 20 min. Removal of
surface-bound radioactivity by acid-NaCl wash revealed that 80% of the
total 125I-SRIF bound at this time was intracellular (Fig.
1A). By comparison, when the incubation was carried out at
4 °C, maximal binding reached a plateau somewhat later (at
approximately 40 min). The bulk of this binding (85% of total at 60 min) was acid-washable at all times, indicating that the ligand had not
been internalized into the cells (Fig. 1B). In the presence
of 0.45 M sucrose, the association of 125I-SRIF
was similar to that observed at 37 °C (plateau value obtained at 20 min; Fig. 1C). However, no remaining radioactivity was
detected following acid-NaCl wash, indicating that hyperosmolar sucrose efficiently blocked internalization of the bound ligand (Fig. 1C). Finally, when cells were preincubated with pertussis
toxin, the amount of 125I-SRIF sequestered into the cells
(i.e. acid wash-resistant) was similar to that seen in
controls, indicating that pertussis toxin had no effect on the
internalization process (Fig. 1D).
In order to verify that the parameters of 125I-SRIF
association with AtT-20 cells were not modified by the addition of
hyperosmolar sucrose or by preincubation with pertussis toxin,
saturation experiments were performed at 37 °C under equilibrium
conditions. As illustrated by the Scatchard plot in Fig.
2, the maximal amount of cell-associated 125I-SRIF was affected neither by hyperosmolar sucrose nor
by pertussis toxin treatment. However, the addition of 0.45 M sucrose decreased the EC50 value toward
125I-SRIF (4.42 ± 0.38 nM,
n = 2) as compared with control experiments (1.49 ± 0.21 nM, n = 4) or to experiments
performed in the presence of pertussis toxin (1.34 ± 0.17 nM, n = 3) (Fig. 2). These data clearly
indicate that the maximal amount of 125I-SRIF associated
with AtT-20 cells is not affected by sucrose or pertussis toxin
treatment.
Confocal Microscopic Visualization of Internalized SRIF Molecules
and sst Receptors--
To visualize the intracellular trafficking of
internalized SRIF, AtT-20 cells were incubated at 37 °C with 20 nM Fluo-SRIF and cell surface labeling was stripped off
with hypertonic acid wash. After 5 min of incubation, the internalized
ligand formed small intracellular fluorescent hot spots distributed
throughout the cytoplasm (Fig. 3). By 30 min, these hot spots had increased in number and intensity and were
heavily clustered next to the nucleus (Fig. 3). In cells incubated in
the presence of 0.45 M sucrose, the fluorescent label
remained sequestered at the periphery of the cells at all times (Fig.
4A). This labeling was
exclusively surface-bound since it was entirely strippable by
hypertonic acid wash (Fig. 4B).
Immunohistochemistry revealed very distinct distributional patterns for
sst1, sst2A, and sst5 receptors. In the absence of SRIF, all of these
receptors were essentially localized at the cell surface (Fig.
5). Following 5 or 20 min of incubation
with SRIF, both sst1 and sst5 immunolabeling remained confined to the periphery of the cells, where it formed a more or less continuous ring
(Fig. 5, left and right). By contrast, after 5 min of incubation with SRIF, sst2A immunolabeling was clearly
translocated from the cell surface to intracellular endosome-like
compartments (Fig. 5, center). After 20 min of incubation
with SRIF, sst2A-immunoreactive receptors formed a single concentrated
hot spot in the perinuclear region (Fig. 5). By that time, sst2 cell
surface labeling had almost completely disappeared (Fig. 5).
GH mRNA Measurement--
The amount of GH mRNA present in
AtT-20 cells following incubation with SRIF under various experimental
conditions was determined by quantitative RT-PCR using competing
primers against the target cDNA and a competitor DNA. An example is
given in Fig. 6A, in which the
amount of the target GH cDNA (579 bp) was determined in AtT-20
cells in the absence of SRIF (control) and after 90 min of incubation
with the peptide. This amount of GH mRNA was measured in a series
of PCR runs containing various amounts of the competitor DNA (384 bp).
All PCR products were quantified by laser scanning, and the ratios of
competitor over target band densities were plotted for each PCR
condition as a function of the amount of competitor DNA (Fig.
6B). The amount of GH cDNA was then extrapolated from
the straight line for a ratio of 1 and expressed in attomoles
(amol)/µg of total RNA taking into account that the DNA competitor
was double-stranded and that 1/10th of the RT product was used for PCR.
Once the method had been validated, the amount of GH mRNA was
measured in AtT-20 cells at various times following incubation with
SRIF in conditions of unimpaired (controls and pertussis toxin-treated)
and impaired (incubation at 4 °C or in the presence of sucrose)
internalization (Fig. 7A).
Incubation with 100 nM SRIF dramatically decreased the
amount of GH mRNA from 93 amol/µg of RNA to a minimum of 7.5 amol/µg within 90 min. The basal amount slowly recovered after
48 h (Fig. 7A). In cells preincubated with pertussis
toxin, SRIF produced identical inhibitory effect on GH mRNA levels.
By contrast, when the internalization process was blocked by 0.45 M sucrose or by incubation at 4 °C, no modification of
GH mRNA content was measured (Fig. 7A). To ensure that
the regulatory effect of SRIF on GH mRNA was specific, parallel PCR experiments were performed to demonstrate that incubation with SRIF did
not modify glyceraldehyde-3-phosphate dehydrogenase mRNA content in
AtT-20 cells between 0 min and 48 h (Fig. 7B). Taken together, the data indicate that the inhibitory effect of SRIF on GH
mRNA level are prevented when the internalization process is
blocked.
cAMP Measurement--
To confirm that the effects of receptor
internalization on GH mRNA levels were independent of adenylate
cyclase activity, we measured the ability of SRIF to stimulate the
production of cAMP in AtT-20 cells in the absence or in the presence of
hyperosmolar sucrose or of pertussis toxin. Incubation of AtT-20 cells
with 10 µM forskolin increased the intracellular content
of cAMP from 0.4 pmol/105 cells to 2.2 pmol/105
cells within 75 min (Fig. 8A).
SRIF-14 (100 nM) inhibited this forskolin-stimulated cAMP
content after 20 min and up to 75 min of incubation with the peptide at
37 °C (Fig. 8A). There also was an inhibitory effect of
SRIF on forskolin-stimulated cAMP formation when the experiments were
carried out at 4 °C, although the level of stimulation by forskolin
was weaker than at 37 °C (Fig. 8B). When the incubations
were carried out at 37 °C but in the presence of hyperosmolar
sucrose, the inhibitory effect of SRIF was similar to that observed in
the absence of sucrose (Fig. 8C). By contrast, when cells
were preincubated with pertussis toxin, SRIF was without effect on the
stimulatory action of forskolin (Fig. 8D). These results
demonstrate that the inhibitory action of SRIF on adenylate cyclase is
blocked by preincubation with pertussis toxin but not by co-incubation
with hyperosmolar sucrose and that the effect of SRIF on adenylate
cyclase activity is independent of internalization.
The present study provides the first demonstration that SRIF can
directly decrease GH mRNA levels in anterior pituitary cells. It
also demonstrates that this inhibitory effect is independent from the
inhibition of adenylate cyclase also produced by SRIF in these cells
but that it is dependent upon the internalization of SRIF/sst receptor complexes.
The parameters of
125I-Tyr0-[D-Trp8]SRIF-14
(125I-SRIF) binding to AtT-20 cells at 37 °C were
comparable to those reported previously in this cell line using
different SRIF agonists (19, 29). As documented previously for other
SRIF analogs in these cells (19, 29),
125I-[D-Trp8]SRIF-14
was internalized in a specific time- and
temperature-dependent manner. However, in contrast to what
was reported for 125I-Tyr3-octreotide
internalization in AtT-20 cells (29), we found that 125I-[D-Trp8]SRIF-14
internalization was totally insensitive to pretreatment of the cells
with pertussis toxin. This result suggests that signaling and endocytic
processes are two independent, albeit interlinked, mechanisms, an
interpretation consistent with previous reports of dissociation between
internalization and G protein-mediated signaling in the case of m2
muscarinic (38) and NT1 neurotensin (39, 40) receptors.
The internalization of both 125I-SRIF and fluo-SRIF in
AtT-20 cells was inhibited by hyperosmolar sucrose, suggesting that the internalization process is initiated at the level of clathrin-coated pits. These results are congruent with those obtained previously in
other cell lines naturally expressing SST receptors (41) or in cells
transfected with cDNA encoding either one of the five cloned sst
receptor subtypes (13-16). Confocal microscopy revealed that the
internalized ligand was first concentrated within small endosome-like
organelles, a finding consistent with earlier confocal observations in
transfected cells using the same type of fluorescent probe (13, 14) and
with the report of an association of internalized gold-conjugated SRIF
with coated vesicles in primary cultures of rat anterior pituitary
cells (42). At longer time intervals, the ligand was concentrated
deeper within the cell, in the juxta-nuclear region. This localization
is reminiscent of that of the so-called late recycling pathway
described for transferrin receptors (43), an interpretation in
accordance with the recent demonstration of abundant sst receptor
recycling in both naturally expressing and transfected cell lines (16,
41).
A major finding of the present study is the observation that SRIF
directly inhibits the expression of GH mRNA in AtT-20 cells. This
finding is novel on two counts. First, it demonstrates that GH mRNA
is expressed in AtT-20 cells, contrary to the common belief that the GH
gene is silent in this cell line (44). Second, it demonstrates that
SRIF may directly regulate GH gene transcription, and not merely
indirectly, through central inhibitions of growth hormone-releasing
hormone, again contrary to current assumptions (9).
The fact that GH mRNA expression had previously been overlooked in
AtT-20 cells is probably due to methodological differences between the
present and earlier studies. Indeed, our results were obtained using
RT-PCR, a method that is considerably more sensitive than the Northern
blotting approach on which earlier results were based. The inhibitory
effect of SRIF on the expression of GH is in keeping with the wide body
of evidence for an inhibitory role of SRIF on the secretion of GH
in vivo as well as in vitro (7). This effect of
the peptide on hormonal secretion has been correlated with the coupling
of sst receptors to Gi or Go and was shown to be pertussis toxin-sensitive, i.e. to be linked to the
adenylate cyclase signaling cascade (8). By contrast, the present study clearly demonstrates that the inhibitory effects of SRIF on GH mRNA
levels are totally insensitive to pertussis toxin treatment. They are,
however, highly dependent on SRIF-induced internalization of sst
receptors. Indeed, conditions that blocked sst receptor internalization
also blocked the inhibitory effect of SRIF on GH mRNA levels.
Furthermore, these procedures were ineffective against the effects of
the peptide on adenylate cyclase activity, confirming that the
expression and release of GH by SRIF are mediated by distinct
regulatory pathways.
Until recently, ligand-induced endocytosis had been considered to
mainly subserve receptor regulatory functions such as desensitization (45, 46) and resensitization (47, 48). Evidence has been accumulating,
however, to suggest that this endocytic process may also be critical
for transmembrane signaling. Most of the information available to date
concerns growth factors and cytokine receptors, for which there is good
evidence that ligand-induced internalization is essential for full
signaling activity (20, 49). There is also evidence that in the case of
G protein-coupled receptors, ligand-induced internalization may be
mandatory for the expression of certain signaling functions ranging
from the activation of mitogen-activated protein kinase (24) to the
regulation of target genes (23). Proposed mechanisms for this type of G protein-independent signaling include mobilization of adaptor proteins
such as As AtT-20 cells were demonstrated to express four (sst1, the two
variants of sst2, sst4, and sst5) of the five cloned sst receptors (28,
54), it is impossible to precisely determine which receptor(s) is (are)
involved in the internalization-dependent effects observed
in the present study. However, several lines of evidence point to sst2A
as playing a predominant role in this regard. Of all sst receptor
subtypes, sst2A is the one that is most abundantly expressed in the
AtT-20 cells (28). Also, the pattern of SRIF-induced sst2A receptor
trafficking, as visualized here by sst2 immunohistochemistry, is the
one that most closely resembled that of the fluorescent ligand.
Finally, in all cell lines in which sst receptor internalization has
been studied to date, sst2A is the one that was found to be the most
efficiently internalized (14, 55). Neither sst1 nor sst4 are likely to contribute significantly to the internalization of
[D-Trp8]SRIF-14 observed in the
present study since neither is expressed very abundantly in AtT-20
cells (28) or is found to internalize efficiently SRIF analogs in
heterologous transfection systems (13-16). However, the participation
of the sst5 receptor subtype cannot be as readily excluded since
[D-Trp8]SRIF-14 exposure has been
shown to promote sst5 internalization in transfected cells (56, 57) and
to translocate intracellular receptor stores to the membrane according
to a pattern comparable to the one observed in the present
immunohistochemical experiments (57). Furthermore, sst5 receptors are
known to transduce as efficiently as sst2 the effects of SRIF on GH
release from anterior pituitary cells (58).
In conclusion, the present work demonstrates that SRIF inhibits GH
mRNA transcription in AtT-20 cells and that this effect is mediated
by internalization of receptor/ligand complexes, independently from
activation of the adenylate cyclase pathway. Further studies are in
order to identify the receptor(s) responsible for this important effect
and to unravel the signaling cascade implicated in its transduction.
We are grateful to Drs. Agnes Schonbrunn and
Hans-Jürgen Kreienkamp for their generous provisions of sst2A and
sst5 antibodies, respectively. We thank Franck Aguila for photographic work.
*
This work was supported by the CNRS and by an INSERM Fonds
de la Recherche en Santé du Quebec exchange program (to J. M. and A. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Institut de
Pharmacologie Moléculaire et Cellulaire, CNRS, UPR 411, 660 Route des Lucioles, 06560 Valbonne, France. Tel.: 33-4-93-95-77-61; Fax:
33-4-93-95-77-08; E-mail: mazella{at}ipmc.cnrs.fr.
2
P. Sarret, D. Nouel, C. Dal Farra, J.-P.
Vincent, A. Beaudet, and J. Mazella, unpublished results.
The abbreviations used are:
SRIF, somatotrophin
release inhibitory factor, GH, growth hormone;
PCR, polymerase chain
reaction;
RT, reverse transcription;
TBS, Tris-buffered saline;
bp, base pair(s).
Receptor-mediated Internalization Is Critical for the
Inhibition of the Expression of Growth Hormone by Somatostatin in
the Pituitary Cell Line AtT-20*
,,,¶
Institut de Pharmacologie Moléculaire
et Cellulaire, CNRS, UPR 411, 660 Route des Lucioles, 06560 Valbonne,
France and the § Montreal Neurological Institute, McGill
University, Montréal, Québec H3A 2B4, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-adrenergic
receptors was recently shown to be directly involved in the activation
of mitogen-activated protein kinase pathway (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
counter. Nonspecific binding was determined in the
presence of 1 µM unlabeled [D-Trp8]SRIF and represented less
than 5% of the total binding.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Association kinetics of
125I-Tyr0-[D-Trp8]SRIF-14
binding to AtT-20 cells. Experiments were performed either at
37 °C (A, C, and D) or at 4 °C
(B), in the presence of 0.45 M sucrose
(C) or after pretreatment of cells for 16 h with 100 ng/ml pertussis toxin (D). At the indicated times, cells
were either washed twice with 500 µl of Earle's/HEPES/Tris buffer
(closed symbols) or treated twice with 500 µl
of acid-NaCl buffer for 2 min (open symbols).
Cell-associated radioactivity was counted with a counter. Each
point is the mean ± S.E. of at least three different experiments
performed in duplicate.
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Fig. 2.
Saturation of
125I-Tyr0-[D-Trp8]SRIF-14
binding to AtT-20 cells at 37 °C. Experiments were performed
with increasing concentrations of
125I-Tyr0-[D-Trp8]SRIF-14
(0.5-16 nM) in the absence of any drug (open
squares) or in the presence of hyperosmolar sucrose
(open circles) or of pertussis toxin
(closed squares). Data are representative of a
typical experiment. Inset, Scatchard representation of the
data. B, bound ligand; F, free ligand.
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Fig. 3.
Confocal microscopic images of AtT-20 cells
incubated with 20 nM fluo-SRIF at 37 °C and subjected to
hypertonic acid wash. Single trans-nuclear optical sections
scanned after 5 and 30 min of ligand application before wash. At both
time points, the internalized ligand is detected in the form of small
intracytoplasmic hot spots sparing the nucleus (N). Note
that these hot spots are both more numerous and more heavily
concentrated in the perinuclear region at 30 min than at 5 min. This
labeling is completely abolished by co-incubation with 1 µM nonfluorescent SRIF (data not shown). Scale
bar, 5 µm.
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Fig. 4.
Effect of hyperosmolar sucrose on fluo-SRIF
internalization in AtT-20 cells. Cells were incubated for 30 min
at 37 °C with 20 nM SRIF in the presence of hyperosmolar
sucrose and either air-dried (A) or acid-washed
(B). In the air-dried specimen (A), the label is
confined to the periphery of the cell, in a pattern distinct from that
observed in the absence of hyperosmolar sucrose (compare with Fig. 3).
This pericellular labeling is entirely surface-bound since it totally
disappears after hypertonic acid wash (B). Scale
bar, 5 µm
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Fig. 5.
Fluorescence immunolabeling of sst1, sst2A,
and sst5 receptors in AtT-20 cells incubated (middle
and bottom rows) or not
(top row) with 10 nM
[D-Trp8]SRIF-14. In the absence of
ligand, all three receptor subtypes are concentrated at the level of
the cell membrane, forming a pericellular ring. After 5 min of
incubation with SRIF, both sst1 and sst5 immunoreactivity remains
mainly pericellular whereas sst2A immunoreactivity forms small
intra-cytoplasmic hot spots. After 20 min of exposure to SRIF, sst1 and
sst5 receptors are still at the cell surface whereas sst2A
imunoreactivity is concentrated in the cytoplasmic core, next to the
nucleus (N). Note the total disappearance of sst2A cell
surface immunolabeling at this time. Scale bar, 5 µm.
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Fig. 6.
Measurement of the amount of GH mRNA
expression in AtT-20 cells. A, typical analysis by
agarose gel electrophoresis stained by ethidium bromide (2%) of RT-PCR
products obtained in the absence (control) or after 90 min of
incubation with SRIF. A constant amount of RT product and a decreasing
amount of competitor were mixed in a series of 11 different PCRs
(lanes 3-11). Lane 1 corresponds to the RT product alone, and lane 2 to the competitor alone. B, the intensity of each band
obtained by each PCR was laser-scanned and the ratio of competitor over
GH bands was expressed as a function of the concentration of competitor
(in attomoles). The amount of GH mRNA was calculated from the point
giving a ratio = 1 on the straight line. This representation was
made for each duration of incubation with SRIF in all experimental
conditions. Experiments were repeated at least three times.
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Fig. 7.
Effect of SRIF on GH mRNA expression in
AtT-20 cells. A, amount of GH mRNA measured during
and following incubation with 10 
7 M SRIF, the
peptide being removed after 60 min. Experiments performed with SRIF
alone (closed circles) or with SRIF in the
presence of pertussis toxin (closed squares), of
0.45 M sucrose (open squares), or at
4 °C (open circles). B, amount of
glyceraldehyde-3-phosphate dehydrogenase mRNA measured by RT-PCR as
a control of the amount of RT product (target size: 358 bp). All values
are the mean ± S.E. of triplicate determinations from three
independent experiments.
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Fig. 8.
Effect of SRIF on forskolin-stimulated cAMP
levels in AtT-20 cells. Cells were incubated with solvent
(hatched bars), with 10 µM
forskolin (white bars), or with forskolin and
10 7 M SRIF (black bars)
for 2, 20, and 75 min. Experiments were carried out at 37 °C
(A, C, and D) in the presence of 0.45 M sucrose (C) or of 100 ng/ml pertussis toxin
(D) or at 4 °C (B). All values are the
mean ± S.E. of triplicate determinations from two independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestins (24), endosome signaling (21, 22), or nuclear
translocation of internalized receptors and/or of sequences thereof
into the nucleus (49, 50). Either of these mechanisms could
theoretically be involved in mediating the effects of SRIF documented
here on GH mRNA. One might even speculate that it involves
interaction of the internalized ligand, provided that it remains
intact, with the 86-kDa subunit of autoantigen Ku, which has been shown
to correspond to an intracellular SRIF receptor regulating protein
phosphatase 2A activity and has been postulated to play a role in gene
transcription (51). It is unlikely that the
internalization-dependent signaling cascade unraveled in the
present study involves the pituitary-specific transcription factor GH
factor-1 (GHF-1; Pit-1) since treatment of pituitary cells with SRIF
for either 2 or 48 h failed to modify basal or growth
hormone-releasing hormone-induced GHF-1 mRNA levels (52). It could,
however, implicate one or more other pituitary-specific transcription
factors yet to be identified (53).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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