(Received for publication, October 11, 1996, and in revised form, January 6, 1997)
From INSERM Unité 339, Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France
The binding of neurotensin (NT) to specific receptors triggers the multiple functions that NT exerts in both periphery and brain. By studying the effect of the concentration and time of NT agonist exposure, two separate regulatory mechanisms were detected for the neurotensin receptor (NTR) gene in human colonic adenocarcinoma cells (HT-29).
The incubation of cells for 6 h with the NT agonist, JMV 449, resulted in an increase of 270% in NTR mRNA levels. These changes were the direct result of new NTR gene transcription, as indicated by run-on and half-life experiments. In addition, the transcriptional activation of the NTR gene was dependent on NT-receptor complex internalization and de novo protein synthesis.
A second response was detected with prolonged exposure to JMV 449. In this case, a decrease of 70% was detected in NTR mRNA levels. Unlike the initial phase, this change was mediated by a post-transcriptional event as the half-life of NTR mRNA from treated cells decreased by 50% as compared with control cells.
NT agonist appears to regulate the synthesis of NTR mRNA. In HT-29 cells, this feedback is exerted by a biphasic response. These phases are apparently independent and mediated by two separate mechanisms.
Neurotensin (NT)1 is a tridecapeptide, widely distributed in the central nervous system and peripheral tissues, exerting multiple functions (1). In the central nervous system, NT is a neurotransmitter as well as a neuromodulator of other neurotransmitters such as dopamine, acetylcholine, serotonin, and noradrenaline (2, 3). NT also possesses neuroendocrine actions inducing the release of several pituitary hormones (4). In the periphery, NT is secreted from mucosal endocrine cells of the small intestine into the circulation (5). In the gastrointestinal tract, NT causes many physiological effects including the stimulation of pancreatic secretion, the facilitation of colonic motility and fatty acid translocation, and tissue growth (6).
In rat, NT actions are mediated by the stimulation of several specific receptors exhibiting high or low affinity for NT (7, 8). The high affinity neurotensin receptor (NTR) is composed of 424 amino acids and belongs to the seven-transmembrane domain receptor family coupled to the G-proteins (7). The human NTR counterpart has also been cloned from human colonic adenocarcinoma cells (HT-29) (9). When HT-29 cells are challenged with a NT agonist, phosphatidylinositols are hydrolyzed leading to Ca2+ mobilization (10). In contrast to N1E-115 cells, stimulation by NT in HT-29 cells is not associated with protein kinase C activation (10, 11).
In addition to triggering cellular responses by specific ligands, receptors are often themselves regulated by their own agonists. In the case of NT, several studies have shown that variations in NTR expression were caused by changes in NT levels. For example, acute agonist stimulation of NTR induces desensitization and down-regulation of receptor in primary cultures of rat forebrain and HT-29 cells (12, 13). Prolonged exposure of N1E-115 cells to NT resulted in the disappearance of most NT-binding sites, and de novo synthesis of NTR was required for the recovery of receptor-binding sites and function (14). When hypothalamic neurons from primary cultures were chronically exposed to forskolin and dexamethasone, an increase in NT synthesis and release into the culture media were observed. Concomitantly, a decrease in NT binding and in NTR mRNA levels was observed (15). Moreover, chronic treatment with the NTR-specific antagonist, SR 48692, produced substantial increases in NT-binding sites and in NTR mRNA levels in rat brain (16). These results suggest that endogenous NT may exert a negative control upon its own receptors.
Transcriptional and post-transcriptional regulation mechanisms have
been described for several G-protein coupled receptors including,
2-adrenergic (17),
-adrenergic (18), angiotensin (19), muscarinic (20), and thyrotropin receptors (21). The best
described of these receptors is the
2-adrenergic
receptor, which was reported to be down-regulated by long-term agonist
exposure via destabilization of its own mRNA (22, 23). In contrast, shorter exposure to agents that elevated cAMP levels resulted in an
increase in the transcription rate of the
2-adrenergic receptor gene (17). However, in another system, a short exposure to a
serotoninergic agonist was recently shown to cause the up-regulation of
5-HT2 receptor mRNA by a post-transcriptional mechanism
(24).
The objective of the current study was to investigate the molecular mechanisms of NTR synthesis regulation in HT-29 cells. A time course using different doses of agonist was performed, while applying a quantitative RT-PCR method to measure NTR mRNA levels. We demonstrate that high doses of NT agonist induce a short-term transcriptional up-regulation of NTR mRNA requiring receptor internalization. Furthermore, a post-transcriptional down-regulation of NTR mRNA was detected upon long-term exposure to agonist. This mechanism included the destabilization of NTR mRNA, even at low agonist concentrations.
HT-29 human colon adenocarcinoma cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 2 mM glutamine, in a humidified atmosphere of 5% CO2, 95% air. At confluence, cells were routinely dispersed in trypsin-EDTA and subcultured at a 1:15 dilution. The cells were used for experiments on the fourth day after plating. The media was changed every other day. Cells were treated with 0.3 or 100 nM JMV 449 (Neosystem), a potent and stable pseudopeptide NT agonist (25). These concentrations were chosen because they represent 85 and 100% binding site occupancy, respectively, based on the determined Ki value for JMV 449 of 0.06 nM at 20 °C.2 For some experiments cells were treated with the NTR antagonist SR 48692, with a Ki value of 24 nM (26).
Binding StudiesRadioligand binding studies were carried
out on membranes prepared as described previously in Boudin et
al. (27). Binding studies were performed as followed, 60 µg of
protein was incubated with 0.1 nM 125I-NT in a
final volume of 250 µl of buffer A (50 mM Tris, pH 7.4, 0.2% bovine serum albumin, and 0.8 mM
1,10-orthophenanthroline). Nonspecific binding was measured in the
presence of 1 µM unlabeled NT. Binding assays were
performed for 60 min at 4 °C and terminated by centrifugation at
4 °C for 4 min at 12,000 × g. The supernatant was
removed, the membrane pellets were rinsed twice with 500 µl of buffer
A and centrifuged again. The pellets were counted in a -counter
(Wallac model 1470 Wizard). The saturation experiments were carried out
under the same conditions, using a range of 125I-NT
concentrations (0.015-1 nM). The saturation kinetics of
125I-NT binding was analyzed by Scatchard plot and the
apparent Kd and Bmax were
estimated.
To verify that JMV 449 was completely washed away before the membrane was prepared, cells were incubated for 30 min on ice with 100 nM JMV 449. Cells were washed three times with cold phosphate-buffered saline, membrane preparation and binding were performed as described previously. Under these conditions, the recovery of binding from cells incubated with JMV 449 was 85 ± 15% compared with the control cells.
RNA ExtractionTotal RNA was extracted from cells by the
acidic phenol/chloroform guanidine thyocianate method (28). An
additional ethanol precipitation was performed in NET buffer (150 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1 mM EDTA). The RNA pellet was resuspended in 50 µl of
sterile deionized diethyl pyrocarbonate-treated H2O. Aliquots were prepared and stored at 80 °C. Total RNA recovery was
measured by spectrophotometric absorbance at 260 nm.
Neurotensin receptor
cDNA was kindly supplied by Dr. Nakanishi (Kyoto University,
Japan). The plasmid p96 was constructed by deleting a 96-nucleotide
fragment (HincII-NcoI) from the rat NTR cDNA
(
7 to 1301), which had been previously inserted into the
SmaI-BamHI site of pT7/T3
18. An
oligonucleotide containing poly(dA)45 was inserted at the
SalI-BamHI site. The internal control used in
this study, cRNA
96, was prepared by in vitro
transcription of the linearized plasmid p
96 at the SalI
site with T7 RNA Polymerase (Life Technologies, Inc.) and then purified
on oligo(dT) columns (Sigma) (29). After elution from oligo(dT)
columns, the cRNA
96 was ethanol precipitated, then diluted in
diethyl pyrocarbonate/H2O containing 1 unit/µl RNasin
(Promega). The quality of cRNA
96 was checked by electrophoresis (30)
and the concentration estimated by spectrophotometric absorbance at 260 nm. The cRNA
96 solution was diluted to 1 × 107
molecules/µl in diethyl pyrocarbonate/H2O containing 0.5 unit/µl of RNasin, aliquoted, and stored at
80 °C.
Fifty pmol of antisense PCR primer were
5-32P-end-labeled with 20 units of T4 polynucleotide
kinase (New England Biolab, 10,000 units/ml) in a final volume of 50 µl of buffer (70 mM Tris-HCl, pH 7.6, 10 mM
MgCl2, 5 mM dithiothreitol) containing 100 pmol of [
-32P]ATP (Amersham, 3000 Ci/mmol) at 37 °C, for
30 min. The end-labeled oligonucleotide was subsequently purified on a
Sephadex G50-150 spin column (30), and 1 µl of eluent was counted on
a GF/C filter (Whatman) in 3 ml of dry extract scintillation fluid
(Optiphase 178 HiSafe 178 2, Wallac-Pharmacia).
Quantitative RT-PCR was carried out as in the conditions
described by Souazé et al. (31). The primer RT-NTR
(5-GCTGACGTAGAAGAG-3
) was used for reverse transcription of
endogenous and internal control molecules. The primers S-NTR
(5
-CCTTCAAGGCCAAGACCCTC-3
) and AS-NTR (5
-CAGCCAGCAGACCACAAAGG-3
)
were used in PCR, giving a PCR product of 349 nucleotides for the
internal control, cRNA
96, and 433 nucleotides for endogenous NTR
mRNA. The assay consisted of two steps. In the first step, the
estimation of mRNA molecules in each group was made by a titration
assay. A 100 ng of total HT-29 RNA and various dilutions of cRNA
96
were reverse transcribed for 1 h at 37 °C with 200 units of
Moloney murine leukemia virus reverse transcriptase (Life Technologies)
in a mixture containing 20 mM Tris-HCl, pH 8.3, 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 1 unit/µl RNasin, 50 pmol of the
specific primer (RT-NTR), and 1 mM of each dNTP in a
30-µl final volume. The reaction was terminated by heating at
95 °C for 5 min and the samples were quick-chilled on ice. The PCR
amplification was performed on 1:5 (v/v) of the RT reaction in a
mixture containing 16 mM Tris-HCl, pH 8.3, 40 mM KCl, 1.5 mM MgCl2, 0.2 mM concentration of each dNTP, 25 pmol of each primer
(NTR-S and NTR-AS), 1 × 106 cpm of a 5
end-labeled
[
-32P]ATP NTR-AS, and 1 unit of Taq
polymerase (Perkin Elmer). The amplification profile consisted of
denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min 30 s. The 26 cycles of
PCR were preceded by denaturation at 95 °C for 5 min and were
followed by a final extension at 72 °C for 10 min. Amplification was
performed in a DNA thermal cycler 480 (Perkin Elmer). In the second
step, a precise quantification was performed using the quantitative
assay (32). Depending on the level of NTR mRNA, 100 or 500 ng of
total RNA was mixed with an exact number of cRNA
96 molecules which
were previously estimated from the titration assay. This mixture was
reverse transcribed and six tubes of a 3-fold dilution of this reaction
were amplified by PCR under the same conditions as described above. In
all experiments, the difference between the internal control,
cRNA
96, and NTR mRNA never exceeded 1.5-fold, providing an
accuracy of at least 90% (31).
In both titration and quantitative
assays, 20 µl of PCR samples were electrophoresed on 5%
polyacrylamide gels in 90 mM Tris borate, 2 mM
EDTA buffer. We routinely introduced a 100-base pair DNA ladder (Life
Technologies, Inc.) size marker. Gels were stained with ethidium
bromide and the bands cut out from the gel and counted in a
-scintillation counter (Beckman, Model LS6000SC) with 3 ml of
scintillation fluid. The amount of radioactivity (cpm) recovered from
the excised gel bands was plotted against the number of known cRNA
96
control molecules or the quantity of total RNA. Linear regressions of
both curves were calculated and the absolute number of target molecules
(number of NTR mRNA molecules) was estimated by extrapolating the
value of 1 µg of total RNA to the internal control. Results are
expressed as number of target molecules/µg of total RNA.
A negative control was routinely introduced for
all titration and quantitative assays to confirm the absence of
contamination. For these controls, RNA was omitted from the RT reaction
mixture and the reverse transcription was carried as described above. The PCR amplification was performed in the same conditions as the
samples and the radioactivity present at the equivalent position of the
positive band was counted. The radioactivity in this band, guided by
ethidium bromide (EtBr) staining, was used as background. The
experiment was rejected if the negative control contained visible bands
or background greater than 100 cpm. The absence of contaminating DNA in
the cRNA preparation was tested by performing a PCR on 1 × 107 cRNA96 molecules under standard conditions.
To estimate the stability of the NTR mRNA, HT-29 cells were exposed to 100 nM JMV 449 for 1, 3, 6, or 72 h before the addition of 5 µg/ml actinomycin D. Total cellular RNA was extracted at each time point and the level of NTR mRNA measured by the quantitative RT-PCR assay.
Nuclear Run-on AssaysNuclei were isolated according to the
alternate protocol described by Greenberg and Bender (33). Isolated
nuclei were aliquoted by 8 × 107 in 200 µl of
glycerol buffer (50 mM Tris-HCl, pH 8.3, 40% (v/v) glycerol, 5 mM MgCl2, 0.1 mM EDTA)
and frozen in liquid nitrogen. To detect nascent transcripts, 200 µl
of nuclei preparation in glycerol buffer were added to 200 µl of a
reaction buffer containing 10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 0.3 mM KCl, 5 mM dithiothreitol, 0.5 mM unlabeled GTP, ATP,
and CTP each, 1 µM UTP, and 40 µl of [-32P]UTP (400 Ci/mmol) for 30 min at 30 °C. Twenty
nmol of each dNTP was then added to the reaction for 15 min at
30 °C. The transcription mixture was digested with 50 µg of
RNase-free DNase followed by 200 µg of proteinase K. Newly
transcribed labeled RNA was extracted and subsequently hybridized for
65 h at 45 °C with a fragment (
7 to 1301) of NTR cDNA (3 µg/slot) or
-tubulin (1 µg/slot) immobilized on nitrocellulose.
After hybridization, each sample was washed twice with 5 × SSC
containing 50% formamide, 0.1% SDS for 45 min at 50 °C and twice
with 2 × SSC for 15 min at room temperature. The samples were
then treated with 200 µg of RNase A for 45 min at 50 °C, followed
by a wash with 1 × SSC containing 0.1% SDS at 50 °C for
30 min.
The filters were dried and subjected to autoradiography for 24 h with an intensifying screen. Relative changes in transcription were assessed from autoradiograms which were analyzed by scanning densitometry using the software program RAG (Biocom France).
StatisticsStatistical analysis were performed using the Student's t test. Data are expressed as the mean ± S.E.
The number of molecules of NTR
mRNA was measured by quantitative RT-PCR as described by
Souazé et al. (31). Chronic exposure of HT-29 cells to
the NT agonist, JMV 449, resulted in a biphasic response as detected by
the variations in NTR mRNA. As shown in Fig. 1, the
levels of receptor mRNA increased to a maximum of 270% between 6 and 8 h of treatment for cells chronically treated with 100 nM JMV 449. This effect on NTR mRNA was equally
observed at 10 nM and 1 µM JMV 449 (data not
shown).
NTR mRNA expression returned to control values after 48 h of
continuous treatment with 100 nM JMV 449 (Fig.
1A). Continued exposure to JMV 449 for up to 96 h
caused a decrease of 70% in the quantity of NTR mRNA molecules, as
compared with NTR mRNA level observed under basal conditions (Fig.
1A). In contrast, when cells were treated with nonsaturating
concentrations of JMV 449, 0.3 nM, no changes in receptor
mRNA expression was detected during the initial 24 h. However,
prolonged exposures between 48 and 96 h resulted in a similar
decrease of NTR mRNA, as was seen with treatment at 100 nM (Fig. 1B). As shown in Fig. 2,
treatment with JMV 449 concentrations as low as 3 × 1012 M was sufficient to induce the decrease
in NTR mRNA levels observed after 72 h of exposure, indicating that
this down-regulation can be produced by the activation of a small
number of NT-binding sites.
To determine if the two phases of NTR mRNA expression involved independent mechanisms, NTR mRNA augmentation was induced during the down-regulation period. As shown in Table I, the up-regulation observed with 100 nM JMV 449 was still obtained under conditions where maximal NTR mRNA down-regulation was produced. This increase, however, was lower in the pretreated cells (173%) as compared with the non-pretreated cells (270%).
|
To further identify any potential relationship between these apparently
separate NTR mRNA responses, cells were concomitantly treated with
JMV 449 and the NTR antagonist, SR 48692 (26). At a concentration of
100 nM agonist, the NTR mRNA peak habitually detected
at 6 h was completely inhibited with 1 µM NTR
antagonist (Fig. 3A), whereas SR 48692 had no
effect on the diminution of NTR mRNA observed at a longer exposure
(Fig. 3B). These experiments suggest that the NTR mRNA
changes are mediated through two different intracellular and
independent mechanisms. Nevertheless, as expected according to the
difference of Ki of the two components (see
"Experimental Procedures"), the decline of NTR mRNA observed with 0.3 nM at 72 h was completely antagonized by 1 µM SR 48692 (Fig. 3B). Those results confirmed
that the effects caused by JMV 449 treatment act through NTR.
Destabilization of NTR mRNA by Long-term Exposure to JMV 449
NTR mRNA turnover was studied to determine the molecular
mechanisms underlying the variations observed in NTR mRNA levels. Transcription was inhibited with actinomycin D in control cells or
cells preincubated with agonist for various durations. A similar NTR
mRNA half-life was observed in cells treated for 6 h with 100 nM JMV 449 (56.1 ± 6.9 min) and in control cells
(58.8 ± 10.8 min). In addition, cells treated with 100 nM JMV 449 also had the same half-life at 1 and 3 h
(data not shown). In contrast, pretreatment of cells with 100 nM JMV 449 for 72 h resulted in a rapid decrease in
NTR mRNA half-life (24.8 ± 2.2 min). This effect was also
observed with 0.3 nM JMV 449 treatment (data not shown). A
semi-logarithmic plot of the data revealed that JMV 449 treatment for
72 h decreased the half-life of NTR receptor mRNA by
approximately 60% (Fig. 4). Thus, a
post-transcriptional event is directly implicated in the
down-regulation of the NTR mRNA induced by long-term NT agonist
treatment, whereas, mRNA stabilization is not responsible for the
NTR mRNA induction.
Transcriptional Activation of the NTR Gene
To confirm this
hypothesis, nuclear run-on assays were performed on cells to evaluate
the cause of NTR mRNA induction. The transcription rate of control
cells was compared with the rate determined from cells pretreated with
100 nM JMV 449 for 4 h. As shown in Fig.
5, a 220% increase of newly synthesized mRNA was
detected in JMV 449-treated cells as compared with control cells.
Therefore, the increase in NTR mRNA observed after short-term exposure to 100 nM JMV 449 is mediated by changes in the
NTR transcription rate.
NTR Gene Activation Requires Protein Synthesis
In an effort to further discern the nature of the NTR gene activation caused by treatment with 100 nM JMV 449, HT-29 cells were treated for 3 h with 100 nM JMV 449 in the presence of the protein inhibitor synthesis, cycloheximide. Incubation with 2.5 µg/ml cycloheximide alone resulted in an increase in NTR mRNA levels (15.2 × 106 ± 1.3) equivalent to those caused by incubation with JMV 449 alone (14.3 × 106 ± 1.6). JMV 449 had no effect in the presence of cycloheximide, since cotreatment with both agents did not result in any further increases in NTR mRNA levels (14.3 106 ± 1.0) (Table II). Thus, protein synthesis is required prior to the induction of NTR gene transcription by JMV 449.
|
It is
known that NT induces the internalization of NTR in a number of cell
lines including HT-29 (12, 14). Previous experiments have shown that in
rat basal forebrain slices and in septal neuroblastoma cells (SN17),
NT-NTR internalized complex is transported from the cell periphery to
the perinuclear region by endosomes (34, 35). This result suggested
that NTR internalization may play a role in NT signaling. To determine
if the internalization process was important for NTR gene activation,
cells were treated with phenylarzine oxide (PAO) or concanavalin A,
components which have previously been shown to inhibit the
sequestration of 2-adrenergic receptors (36, 37). As
seen in Table III, concomitant exposure of JMV 449 and
PAO or JMV 449 and concanavalin A for 3 h strongly inhibited the
transcriptional activation of the NTR gene. The level of NTR mRNA
in the presence of these inhibitors was equal to that found in control
cells. Interestingly, the steady state level of NTR mRNA was not
modified by PAO or concanavalin A treatment. In parallel, it was
confirmed that PAO and concanavalin A inhibit NTR internalization by
performing the experiments as described by Chabry et al.
(38). When cells were incubated with 0.1 nM 125I-NT for 30 min, 70 ± 5% of total NT was
internalized. When cells were preincubated with PAO 92 ± 1% of
the radioactivity remained bound to the membranes and could be
completely washed away with phosphate-buffered saline, pH 2.5, indicating that PAO completely inhibited NTR internalization. When the
same experiment was repeated with concanavalin A, a similar result was
seen. However, only 62 ± 5% of the bound radioactivity could be
washed away suggesting that concanavalin A is less effective, compared
with PAO, in inhibiting NTR internalization.
|
To
place the functional significance of NTR mRNA variations into
context, the NT binding was analyzed. When cells were challenged with
100 nM JMV 449, 125I-NT binding rapidly
decreased. This effect was maximal between 1 and 8 h and
corresponded to 85% of the control values (Fig. 6A, inset). After a prolonged exposure to JMV
449, membranes exhibited a 60% 125I-NT binding recovery
after 24 h as compared with the control values (Fig.
6A). Saturation experiments carried out with
125I-NT at 4 °C on HT-29 cell membranes demonstrated a
single population of high-affinity binding sites, with an apparent
dissociation constant (Kd) of 0.70 ± 0.20 nM and a maximal number of sites
(Bmax) of 269 ± 35 fmol/mg of protein
(Table IV). These binding characteristics were
equivalent to those previously described for the same cell line (10).
The reduced binding capacity of the HT-29 membranes caused by a 6-h
exposure of 100 nM JMV 449 corresponded to a decrease of
70% in the Bmax value compared with the
control, without any significant changes in the Kd values. The recovery of 125I-NT binding after prolonged
treatment with JMV 449 similarly corresponded to an increase of 40% in
the number of NTR sites between 6 and 72 h with no change in the
affinity for 125I-NT (Table IV). This increase of
NT-binding sites immediately ensued the transcription peak (at 6 h) suggesting that the restoration of NT-binding sites is the
consequence of de novo protein synthesis. However, only a
partial restoration of NTR was detected at the cell membrane. This
result can be explained by a dynamic situation, where newly synthesized
NTR is made available to the cell membrane while the NTRs are
internalized due to the continued agonist exposure.
|
When cells were treated with 0.3 nM JMV 449, the 125I-NT binding profile was altered and significantly shifted in time. A 45% decrease in 125I-NT binding was observed after 1 h of treatment. 125I-NT binding stabilized after 6 h of JMV 449 treatment at 80% of the control value (Fig. 6B, inset). This is in contrast with incubations at 100 nM JMV 449 where HT-29 cells required 24 h for the stabilization of 125I-NT binding.
For prolonged agonist exposures (48-96 h), a decrease of 40 or 20% in NT binding was detected when cells were incubated with either high 100 or 0.3 nM JMV 449 (Fig. 6, A and B). NTR mRNA destabilization was activated during this period and could explain why the level of receptor at the cell surface remained below control values.
In this study we have shown that the binding of NT agonist induced a biphasic response in the regulation of NTR mRNA. Initial exposure to 100 nM agonist generated a large increase in NTR mRNA (~270%). This initial response was optimal at approximately 6 h of agonist exposure, as shown in Fig. 1A. Another separate response occurred with continued NT agonist exposure. This response stabilized at 72 h, corresponding to a net decrease of 70% in NTR mRNA quantities.
These two phases were mediated by distinct and apparently independent mechanisms. NTR mRNA induction was the direct result of transcriptional activation as determined by both run-on and half-life experiments (Figs. 4 and 5). The decrease in NTR mRNA is primarily mediated through a post-transcriptional mechanism as determined by the change in its half-life during this second phase. These two phases appear to be independent because it is possible to induce new transcription during the NTR mRNA destabilization phase. Experiments with the NTR-specific antagonist, SR 48692, corroborate this hypothesis because concomitant exposure of 100 nM JMV 449 and SR 48692 resulted in a marked inhibition of NTR mRNA induction. In contrast, the same treatment had no effect on the decrease of NTR mRNA when compared with incubation of JMV alone. These results would suggest that the two types of NTR mRNA responses, detected in this study, originate from the same receptor but require different degrees of stimulation.
It has been previously described that high doses of agonist exposure
leads a transient increase in receptor mRNA levels, as is the case
for the 2-adrenergic and 5-HT2 receptors. In
both cases the result was due to the transcriptional activation of the
cognate receptor gene (17, 41). In the case of NTR mRNA, the
activation of NTR gene expression could only be induced with high
concentrations of NT agonist. Indeed, the effect occurring at 99% site
occupancy (10 nM) was not observed when 83% of NTR sites
were occupied (0.3 nM). Apparently, maximal receptor
stimulation is required to produce this effect.
The physiological effects produced from the activation of G-protein
coupled receptors are the direct result of events ensuing from signal
transduction in the second messenger pathways. One important class of
responses is the modification of homologous receptor mRNA signals.
For example, activation of the cAMP system by agonist exposure induces
the stimulation of the 2-adrenergic receptor
transcription rate (17, 39). Likewise, protein kinase C activation
resulting from the stimulation of 5-HT2a receptor by
serotonin binding is responsible for the stabilization of
5-HT2a receptor mRNA (24). In the case of NTR, gene
activation was not detectable at 6 h when cells were treated for
1 h of agonist (data not shown). Therefore, transcriptional
activation of the NTR gene did not occur at the outset of agonist
binding. These observations connote that second messenger activation by
NT agonist is not sufficient to induce NTR transcription activation,
and additional events requiring a longer exposure to agonist are
implicated.
NTR mRNA up-regulation only occurred after at least 2 h of JMV 449 treatment (Fig. 1). During this period, 125I-neurotensin binding decreased to 20% of the control. Previous results have shown that NTR internalization requires continued exposure to agonist and, in HT-29 cells, internalization is a protracted process since only 25% of NTRs are internalized after 30 min of agonist exposure (13). Moreover, we noticed that when cells were treated with 0.3 nM JMV 449 the phase corresponding to the stage of receptor internalization is very short (1 h) compared with 100 nM JMV 449 treatment (8 h). The decrease of 125I-neurotensin binding is also less extended, 45% for 0.3 nM versus 85% for 100 nM. At a concentration of 0.3 nM JMV 449, NTR gene activation is not observed indicating that a threshold must be reached to turn on the transcription process. Therefore, we were interested to test the hypothesis that the trigger for NTR gene activation was the internalization of NTR, as the time required to generate NTR gene activation corresponded to the delay necessary for total receptor internalization.
Two internalization inhibitors, PAO and concanavalin A, were employed to validate this premise (36, 37). As shown in Table III, a 3-h treatment with either inhibitor completely blocked the increase of NTR mRNA induced by JMV 449. This effect could have been due to the blockade of receptor internalization or to a secondary effect. However, it was previously shown that PAO did not alter the binding characteristics of the NTR receptor (38) and therefore did not affect agonist-receptor interaction. Furthermore, in the case of angiotensin II receptor, the initial phospholipase C-mediated signaling event was not affected by PAO (19), suggesting that this inhibitor does not disturb this early transduction response. In addition, a truncated NTR possessing diminished internalization capacity, maintained the ability to activate phospholipase C (40). The inhibition of NTR gene activation observed with PAO and concanavalin A treatment appeared to be the direct consequence of blocking NTR internalization.
Protein synthesis was blocked with cycloheximide to determine if the factors responsible for NTR gene activation were already present in the cells, or required de novo synthesis, when activated by NT agonist treatment. NTR mRNA levels detected after cotreatment with JMV 449 and cycloheximide is in the same range as cells treated with cycloheximide alone, indicating that newly transcribed factors are necessary to activate NTR gene. Similar results have also been noted for the up-regulation of 5-HT2 receptor mRNA caused by agonist treatment in smooth muscle cells (41). Cycloheximide alone also induced an increase in NTR mRNA levels. However, this phenomenon has been previously detected and is believed to occur through the inhibition of the synthesis of labile proteins engaged in the natural turnover of the mRNA (42).
The second phase of NTR mRNA regulation was seen after chronic
exposure to agonist and was the result of a post-transcriptional event,
since the half-life of NTR mRNA was decreased. Decrease in mRNA
stability after prolonged exposure to agonist has already been
documented for 2-adrenergic receptor (23). A 35,000-kDa protein displaying an ARE binding activity of
1- and
2-adrenergic receptor mRNA was reported following
treatment with
-adrenergic agonist (43). Further studies on proteins
responsible for NTR mRNA destabilization would be necessary to
determine if mRNA destabilization is a common mechanism to alter
NTR mRNA levels after long-term agonist exposure.
Interestingly, we have observed that the decline of NTR was observed at an extremely low concentration of agonist, 3 pM, corresponding to 5% site occupancy. This result implies the existence of a very high affinity site in HT-29 cells which could be localized on the cloned "high NTR affinity site" (9), or in an unknown receptor subtype. In either case, the detection of this low abundant and supposed site are beyond the sensitivity limits of binding experiments and easily explains why this site has never been described. Previous studies from our laboratory showed that in vivo and in vitro, NT exerts a negative control upon its own receptors (15, 16). A regulatory mechanism such as the destabilization of NTR mRNA, induced by extremely low concentration of agonist, might be expected to have an effect on receptor synthesis in vivo where the concentration of agonist is limited by the short half-life of endogenous NT (44). Further characterization of the proteins involved in the degradation of NTR mRNA in HT-29 cells and in the tissue extracts will determine whether this mechanism is pertinent in vivo.
In summary, the present study demonstrates that high doses of a NT agonist activate NTR gene transcription, an effect linked to the internalization of the receptor and de novo protein synthesis. Long-term agonist exposure induces a post-transcriptional response resulting in the down-regulation of NTR mRNA. A dynamic process can be observed between NTR mRNA and 125I-NT-binding sites because both regulatory events are associated with changes in 125I-NT binding. These regulatory events most likely participate in maintaining a precise level of NTR at the cell surface dependent on the quantity of NT released.
We express our many thanks to Dr. Neil Insdorf for his precious help in writing the manuscript and for helpful discussions. We also thank Dr. D. Pélaprat for helpful discussions on binding studies, Dr. D. Gully for providing SR 48692 (Sanofi Recherches), Dr. Christian Gespach for providing the HT-29 cells, and Anne Marie Lhiaubet for providing 125I-neurotensin.