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B
Division of Pharmacology (C.F., N.G., M.F., A.F., P.S., C.M.) and Division of General Pathology and Immunology (L.T., L.S.), Department of Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy
Address all correspondence and requests for reprints to: C. Missale, Division of Pharmacology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Here we show that inactivation of p75NGFR, but not of trkA,
resulted in D2 receptor loss in responder cells and
prevented D2 receptor expression induced by NGF in the
nonresponder. Analysis of nuclear factor-
B (NF-B) nuclear
accumulation and binding to corresponding DNA consensus sequences
indicated that in NGF-secreting responder cells, but not in
nonresponders, NF-B is constitutively activated. Moreover, NGF
treatment of nonresponder cells induced both nuclear translocation and
DNA binding activity of NF-B complexes containing p50,
p65/RelA, and cRel subunits, an effect prevented by
anti-p75NGFR antibodies. Disruption of NF-B nuclear
translocation by SN50 remarkably impaired D2 receptor
expression in responder cells and prevented D2 gene
expression induced by NGF in nonresponders. These data indicate that in
prolactinoma cells the effect of NGF on D2 receptor
expression is mediated by p75NGFR in a trkA-independent way
and that NGF stimulation of p75NGFR activates NF-B,
which is required for D2 gene expression. We thus suggest
that NF-B is a key transcriptional regulator of the D2
gene and that this mechanism may not be confined to pituitary tumors,
but could also extend to other dopaminergic systems.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NGF binds to two different receptors, trkA and
p75NGFR (1, 2, 3). TrkA, a 140-kDa
protein with intrinsic tyrosine kinase activity, signals via a
ras-dependent pathway leading to activation of the MAPKs
(4, 5, 6) and also through other enzymes, such as
phosphatidylinositol-3 kinase (6, 7).
p75NGFR is a member of the TNF cytokine receptor
superfamily and activates ceramide production (8, 9, 10),
nuclear factor-B (NF-B) (10, 11, 12, 13), and c-Jun
N-terminal kinase (JNK) (11, 13, 14). While the role of
trkA in mediating NGF action on cell survival and differentiation is
well established, the functions of p75NGFR are
still a matter of some debate. p75NGFR has been
proposed to act as a coreceptor for trkA (1, 2, 3), to
modulate trkA signaling (1, 2, 3), or to initiate its
independent transduction pathways. The best characterized
trk-independent activity of p75NGFR is regulation
of neuronal cell death and survival (10, 11, 12, 13, 14, 15, 16). Other
p75NGFR-mediated effects have been proposed in
different neuronal systems, including stimulation of dopamine release
from rat mesencephalic neurons (17), regulation of sensory
neuron function and axon growth (18), and Schwann cell
migration (19, 20).
The action of NGF, which was initially found to be restricted to few populations of neuronal cells, is now known to extend also to different neuroendocrine systems such as the thyroid and parathyroid glands (21), the pancreas (22, 23, 24), the prostate (25, 26, 27, 28), and the pituitary (29, 30, 31, 32). In the pituitary, NGF and its receptors have been identified in the anterior lobe (29, 30, 31, 32), where they play a role in the control of maturation and proliferation of lactotrope cells (33). In addition, NGF is emerging as a regulator of proliferation and differentiation of various tumors of neuroendocrine origin (34, 35, 36) including pituitary PRL-secreting tumors (37). These are the most frequently occurring neoplasms in the human pituitary, often express D2 receptors for dopamine (38), the physiological inhibitor of PRL secretion (39), and are currently treated with D2 receptor agonists. Ten to 15% of patients, however, due to decreased density (40) or loss (37) of D2 receptors, are refractory to this pharmacological therapy and require surgical intervention.
In previous studies we have developed and characterized two phenotypically different groups of human prolactinoma cell lines. Those derived from tumors refractory to the pharmacological therapy (here referred to as "nonresponder") are more transformed, have a high tumorigenic potential, and lack D2 dopamine receptors, while those obtained from bromocriptine-sensitive tumors (here referred to as "responder") are more differentiated, are not tumorigenic, and express D2 receptors (37). One of the characteristics of these cell lines is that their phenotype is highly dependent on the NGF system. In particular, an autocrine loop that involves the secretion of NGF and the expression of both trkA and p75NGFR has been identified in the responder prolactinomas, but not in nonresponders that do not produce NGF and express trkA but not p75NGFR (41). The relevance of this mechanism is such that ablation of NGF production in responder cells leads to transformation and D2 receptor loss, while administration of NGF to nonresponder cells promotes their differentiation and induces the expression of both p75NGFR and D2 receptors (41). The molecular and cellular mechanisms activated by NGF in these cell lines, however, remain largely unknown. In particular, which NGF receptor subtype and which intracellular signaling pathways are involved is still a matter of investigation.
In this study we used two previously characterized prolactinoma cell
lines, one responder and one nonresponder (37, 41), to
define the role of trkA and p75NGFR in
NGF-mediated regulation of D2 receptor gene
expression and to identify the molecular mechanisms that are involved
in this effect. The results show that p75NGFR
plays a critical role, which is independent of trkA, in triggering and
maintaining D2 receptor gene expression in
prolactinoma cells. NGF stimulation of p75NGFR in
these cells results in the activation and nuclear translocation of
NF-B transcription factor, an effect that is necessary for the
expression of the D2 receptor gene. These data
point to NF-B as a key transcriptional regulator of the
D2 gene in neuroendocrine cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(upper panel), the
anti-trkA antibody (Cell Signaling Technology) recognized two major
bands in responder cells, one at 110 and one at 140 kDa, consistent
with the presence of differentially glycosylated forms of trkA
(46). The specificity of these bands was confirmed by
immunoreacting the same membrane with another anti-trkA antibody
(Santa Cruz Biotechnology, Inc., Heidelberg,
Germany; not shown). The expression of trkA in responder cells was not
modified by treatments, as shown by the intensity of the 110- and
140-kDa bands. TrkA tyrosine phosphorylation was evaluated with a
phospho-specific antibody detecting its tyr490 phosphorylated form
(47), which binds Shc (48) and initiates the
Ras-MAPK cascade. In responder cells that produce and secrete NGF
(41), trkA appears to be constitutively Tyr490
phosphorylated (Fig. 1A, middle panel, lanes 1 and 4). A 4-d
exposure to genistein resulted in a significant decrease of Tyr490 trkA
phosphorylation (lane 2). K-252a, however, was less efficient in
inhibiting trkA phosphorylation (lane 3), and the
anti-p75NGFR antibody did not affect it (lane 5).
These observations were further confirmed by evaluating the degree of
phosphorylation of p44/42 MAPK. The results, reported in Fig. 1B, show
that both p44 and p42 MAPK were constitutively phosphorylated in
untreated responder cells (upper panel, lane 1). A 4-d
exposure to genistein significantly decreased p44/42 MAPK
phosphorylation (lane 2), while K-252a (lane 3) affected it only
slightly. These observations suggest that in our experimental
conditions, genistein, but not K-252a, effectively blocks trkA
function. This result could be in line with the observation that K-252a
is a partial agonist and exerts either inhibitory or stimulatory
effects, depending on the cell phenotype and the experimental
conditions (49). Thus, in subsequent experiments we used
genistein (1 µg/ml) to efficiently block trkA function. The
expression of p75NGFR in responder cells exposed
to the different chemicals was measured by both RT-PCR and Western
blot. Neither p75NGFR mRNA (Fig. 1C, lane 3), nor
the p75NGFR protein (Fig. 1D, lane 3) was
modified by a 5-d treatment with the anti-p75NGFR
antibody. This antibody, however, completely inhibited
p75NGFR signaling (see Figs. 5, 6, and 7A),
suggesting that it efficiently blocks
p75NGFR-mediated effects. Exposure of responder
cells to genistein resulted in a slight decrease of
p75NGFR mRNA expression (Fig. 1C, lane 2).
Genistein, however, did not affect either the levels of the
p75NGFR protein (Fig. 1D, lane 2) or its
transductional capability evaluated by measuring NF-B DNA binding
activity (see Fig. 7A), suggesting that in our experimental conditions
this compound does not interfere with
p75NGFR-mediated mechanisms.
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Amplification of cDNA with D2-specific primers
located within the fourth transmembrane domain and the third
intracellular loop, downstream of the alternatively spliced domain,
revealed that only the D2S mRNA isoform is
present in responder cells, as shown by a single 334-bp band generated
by the PCR reaction (Fig. 2A, lane 1). This finding was supported by
the observation that the cloned hD2S, used as a
control template, generated a single PCR product of 334 bp (Fig. 2A, lane 5) and that the direct PCR amplification of the RNA,
i.e. omitting the RT reaction, did not produce any band (not
shown). Exposure of responder cells to
anti-p75NGFR antibodies (Fig. 2A, lanes 2, 3, and
4) for 5 d, but not to preimmune serum (not shown), resulted in a
dramatic decrease of the D2S mRNA. By contrast, 1
µg/ml genistein (Fig. 2B, lane 2) given for 4 d did not affect
D2S mRNA expression. Amplification with ß-
actin primers was performed as a control of the amount of cDNA in
each sample (Fig. 2, A and B). Similar results were obtained in binding
studies with [3H]spiperone. As shown in Fig. 2C, exposure of cell cultures to MC8211 antibody (50 ng/ml) for 2–8 d
resulted in a significant decrease of specific D2
binding sites. This effect was evident after a 2-d treatment and was
maximal after 8 d, when specific
[3H]spiperone binding was about 90% decreased.
In line with the results obtained by RT-PCR, both 2- and 4-d exposure
of responder cells to 1 µg/ml genistein did not modify
[3H]spiperone binding. Thus, inhibition of
endogenous NGF-mediated p75NGFR stimulation, but
not trkA inactivation, resulted in D2 receptor
loss in responder cells, suggesting a crucial role for
p75NGFR in regulating D2
receptor gene transcription in this cell line.
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and Ref.
41) and the expression of D2
receptors that was detectable by radioreceptor binding after a 2-d
treatment and was maximal after a 5-d treatment (37).
Nonresponder cells were exposed to 100 ng/ml NGF in the absence or in
the presence of anti-p75NGFR antibodies for 5
consecutive days, and D2 receptors were measured
in the different experimental groups. As reported in Fig. 3B, D2 mRNA was virtually absent in untreated
nonresponder cells (lane 1). Exposure of this cell line to NGF resulted
in a remarkable and selective induction of the
D2S isoform mRNA (lane 2), an effect that was
prevented by anti-p75NGFR antibodies (lanes 3 and
4). The results obtained in binding studies with
[3H]spiperone are reported in Fig. 3C. While
specific [3H]spiperone binding was undetectable
in untreated cells, exposure to NGF (100 ng/ml; 5 d) resulted in
the appearance of measurable levels of specific
D2 receptor binding. This effect was abolished
when cells were cultured with NGF in the presence of MC8211
anti-p75NGFR antibody (50 ng/ml). Thus, the newly
expressed p75NGFR receptor appears to be
necessary for the stimulatory effect of NGF on D2
receptor expression in nonresponder cells.
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, NGF did not modify luciferase activity
in cells expressing the D2 promoter construct but
not p75NGFR. However, NGF administration to cells
coexpressing p75NGFR significantly stimulated
D2 promoter-driven luciferase activity. Thus, the
stimulation of p75NGFR in a host cell system
triggers D2 promoter activation, strongly
supporting the view that D2 receptor expression
in prolactinoma cells is dependent on
p75NGFR.
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B in Prolactinoma CellsB
(11). This family of transcription factors is composed of
several members that form hetero- and homodimers that are able to
trigger signaling from cell membrane to the nucleus (50, 51).
Since responder and nonresponder cells differ for the production of
NGF, we evaluated both the distribution and the activity of NF-B in
these cell lines. The cellular localization of p65/RelA was
investigated by immunostaining with a specific monoclonal antibody. As
shown in Fig. 5
, in responder cells
p65/RelA immunostaining appeared to be homogeneously distributed within
the nucleus (Fig. 5A, solid arrowheads) and the cytoplasm
(Fig. 5A, open arrowhead). By contrast, in nonresponder
cells p65/RelA immunoreactivity appeared to be preferentially localized
in the cytoplasm (Fig. 5B, solid arrowheads), with only a
faint nuclear staining (Fig. 5B, open arrowhead), suggesting
that in responder, but not in nonresponder, cells NF-B may be
constitutively activated. As reported in Fig. 5C, exposure of
nonresponder cells to 100 ng/ml NGF resulted in the nuclear
translocation of p65/RelA (solid arrowheads). This
translocation was detectable after a 2-h incubation with NGF, was
maximal after 4 h, and was blocked by the
anti-p75NGFR antibody (Fig. 5D), indicating that
p75NGFR is necessary for NF-B activation.
Similar results were obtained in Western blot experiments on isolated
nuclear and cytoplasmic proteins. In untreated cells, p65/RelA
immunoreactivity was more concentrated in the cytosolic than in the
nuclear fraction (Fig. 6, lanes 1 and 2),
while in NGF-treated cells the nuclear fraction appeared to be highly
enriched in p65/RelA (lanes 3 and 4). p65/RelA nuclear translocation
was prevented by the anti-p75NGFR antibody (lanes
5 and 6).
To demonstrate that NGF-activated NF-B was able to bind to DNA,
nuclear protein extracts were tested in EMSA using a double-stranded
[32P]-labeled oligonucleotide containing the
characterized NF-B consensus sequence from the mouse Ig -gene
(52). As shown in Fig. 7A, EMSA revealed high DNA binding activity in responder cells (lanes 1, 4,
and 6) and a very low DNA binding activity in nonresponder cells (lane
2). The constitutive NF-B binding activity detectable in responder
cells was abolished by the anti-p75NGFR antibody
(lane 7), but not by genistein (lane 5), suggesting that it was
entirely due to the interaction of secreted NGF with
p75NGFR. Exposure of nonresponder cells to 100
ng/ml NGF for 4 h resulted in a remarkable up-regulation of the
NF-B DNA binding activity (lane 3). As shown in Fig. 7B, this effect
was dose-dependent over the range of 10–100 ng/ml. Increasing NGF
concentrations over 100 ng/ml did not result in a further increase of
NF-B DNA binding activity (not shown). The time dependence of the
effect of NGF is reported in Fig. 7C. NGF induced up-regulation
of NF-B activity was detectable after a 2-h treatment (lane 2)
and was maximal within 4 h (lane 3). After 8 h of
stimulation, NF-B binding activity was still above control (lane 4)
and after a 24-h NGF treatment it was similar to untreated cells (lane
5). To identify the NF-B subunits activated by NGF in prolactinoma
cells, antibodies specific for various NF-B peptides were
tested for their ability to either interfere with DNA binding or
supershift DNA-bound activity. Nuclear extracts from NGF-treated
nonresponder were incubated with the
[32P]-labeled NF-B probe in the absence or
in the presence of antibodies to p50, p65/RelA, and c-Rel subunits. As
shown in Fig. 7D, all the antibodies interfered with the interaction of
the NF-B probe with nuclear proteins. In particular, the anti-p50
(lane 2) and anti-c-Rel (lane 4) antibodies inhibited the protein-DNA
binding, while the anti-p65/RelA antibody (lane 3) supershifted the
DNA-protein band, thus suggesting that all three protein subunits are
present in the complexes activated by NGF. By contrast, a preimmune
serum did not significantly modify the DNA-protein binding.
NGF-Inducible NF-B Is Required for the Expression of the
D2 Receptor Gene
The data reported so far suggest that in human prolactinomas,
p75NGFR is required for NGF-mediated expression
of D2 receptors and that
p75NGFR activation results in the stimulation
of NF-B binding activity. To investigate whether NF-B is the
cellular signal required for D2 receptor gene
transcription, we used SN50, a cell-permeable peptide containing the
nuclear localization sequence from the p50 subunit of NF-B,
which is known to inhibit translocation of the NF-B active complex
into the nucleus (53). Since in responder cells, which
spontaneously express D2 receptors, NF-B
appears to be constitutively activated, we evaluated whether
SN50-induced inhibition of NF-B nuclear translocation may affect
D2 receptor expression. Responder cells were thus
exposed to either 100 µg/ml SN50 or 100 µg/ml SN50M, an inactive
control peptide mutated within the nuclear localization sequence motif,
for 2–6 d. As a control of the efficiency of these treatments, NF-B
cell localization was evaluated by p65/RelA immunostaining. The results
showed that SN50, but not SN50M, completely inhibited p65/RelA nuclear
translocation (data not shown). Moreover, as shown in Fig. 8A, exposure of responder cells to SN50
for 2 and 6 d resulted in a dramatic down-regulation of
D2S mRNA expression (lanes 2 and 3). By contrast,
the levels of D2S mRNA were unchanged in cells
treated with SN50M for 6 d (lane 4). Similarly, as reported in
Fig. 8B, the stimulatory effect of NGF (100 ng/ml; 5 d) on
D2S mRNA expression in nonresponder cells (lane
2) was lost when NGF treatment was performed in the presence of 100
µg/ml SN50 (lane 3), but not SN50M (lane 4).
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B regulates D2
receptor expression, the luciferase reporter vector containing the
D2 promoter was transiently transfected into
COS-7 cells together with expression vectors containing the
B-related species p50 and c-Rel. As shown in Fig. 9, expression of p50 by itself or in
combination with c-Rel led to a significant increase of
D2 promoter-driven luciferase activity, while the
expression of c-Rel alone was devoid of effects on the reporter
gene transcription. These data indicate that NF-B is a necessary and
sufficient signal to induce D2 gene
expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (10, 11, 12, 13), and JNK (11, 13, 14) have been identified as potential
p75NGFR-dependent signaling effectors, only in a
few cases has their direct involvement in specific
p75NGFR-mediated neuronal functions been
elucidated (9, 10, 12, 13, 16, 55). The role of trkA and
p75NGFR in the neuroendocrine effects of NGF has
been only partially investigated. In particular, some studies suggested
the involvement of trkA in the regulation of pancreas morphogenesis
(22, 23, 24), one study correlated the activation of trkA with
the control of prostate cancer cell line proliferation
(56), and fragmentary data indirectly correlated the loss
of p75NGFR with the progression of prostate
cancer (27, 28) and its expression with the
antiproliferative effects of NGF on thyroid tumor cells
(34). On the other hand, the signaling mechanisms
activated by each NGF receptor subtype in neuroendocrine tissues are
still elusive.
In previous studies we have shown that the expression of
D2 receptors for dopamine in pituitary
PRL-secreting tumor cell lines is correlated with their degree of
transformation and is highly dependent on an endogenous autocrine loop
mediated by NGF (37, 41). We now report that, in these
prolactinoma cell lines, the expression of the D2
gene is regulated by the p75NGFR NGF receptor in
a trkA-independent way. Moreover, we identified the NF-B
transcription factor as the NGF-activated,
p75NGFR-dependent intracellular signaling
molecule that is required for D2 receptor gene
transcription.
The demonstration that p75NGFR is indeed the receptor that mediates the effect of NGF on D2 gene expression came from the results obtained with the anti-p75NGFR antibodies and in transfection experiments. In the more differentiated responder cells inhibition of endogenous NGF interaction with p75NGFR resulted in D2 receptor loss. Similarly, when the p75NGFR receptor was inactivated during NGF administration to the more transformed nonresponder cells, the stimulatory effect of NGF on D2 receptor expression was lost. These data thus suggest that, when p75NGFR signaling is negated, the selective activation of trkA-mediated pathways is not sufficient, per se, to sustain D2 receptor gene transcription. Moreover, the data obtained with the tyrosine kinase inhibitor genistein argue against the possibility of a functional interaction between trkA and p75NGFR in NGF-mediated regulation of D2 receptor expression. Disruption of trkA signaling did not modify, in fact, the levels of D2S mRNA and protein in responder cells, suggesting that D2 receptor expression is under the exclusive control of p75NGFR and its signaling effectors. This conclusion is further supported by the finding that NGF triggers D2 promoter-directed luciferase transcription in COS-7 cells expressing p75NGFR, but not in those lacking it. In line with this is the observation that in prolactinoma cells there is a close correlation between p75NGFR and D2, but not trkA and D2 receptor expression levels (41). It should be noted, however, that genistein slightly down-regulates p75NGFR mRNA in responder cells. Although this decrease was not accompanied by a correspondent decrease of p75NGFR protein and transductional efficiency, it could be inferred that a longer inhibition of trkA could actually down-regulate the p75NGFR receptor. On this line we had previously shown that p75NGFR expression in prolactinomas is highly dependent on NGF (41) and that a deprivation of secreted NGF for at least 6 d was necessary to induce p75NGFR protein loss in responder cells (41). These observations suggest that trkA could indirectly contribute to the effects of NGF on D2 gene transcription by inducing and maintaining the expression of p75NGFR. Moreover, it cannot be excluded that, as in other cell models, in prolactinoma cells p75NGFR and trkA may also directly interact to mediate other specific effects of NGF such as inhibition of cell proliferation and abrogation of tumorigenicity (37).
The D2 dopamine receptor is known to exist as two different isoforms, called D2S and D2L, which are generated by alternative splicing from the same gene and which are mostly colocalized in the same tissues (39). In particular, in the anterior pituitary, D2S and D2L receptor isoforms are expressed in lactotrope cells, where the longer form is predominant (39). PCR amplification of D2 cDNA in responder prolactinomas revealed that, unlike their physiological counterpart, these cells express only the D2S receptor isoform. Furthermore, exposure of nonresponder prolactinomas to NGF resulted in the selective expression of D2S mRNA. Different mechanisms may be invoked to explain this finding. It could be possible that genetic alterations occurring during lactotrope cell transformation led to the loss of the D2L isoform. On the other hand, it is also possible that NGF affects the splicing mechanisms leading to the selective expression of the D2S receptor isoform. In line with this view, subpopulations of lactotropes have been identified that may express different D2S/D2L mRNA ratios in response to different stimuli (57), and activated sex steroid receptors have been reported to modulate the alternative splicing of the D2 receptor mRNA in the MMQ pituitary cell line (58).
How does p75NGFR induce the expression of
D2 receptors in prolactinoma cells? Our analysis
of the signaling molecules activated by NGF strongly pointed to NF-B
transcription factors as the most plausible candidates. The NF-B
family is composed of several distinct DNA binding subunits that can
hetero- and homodimerize, thereby forming complexes with distinct cell
type distribution, DNA sequence specificity, and transcriptional
activity (51, 52). In its inactive state, NF-B is
sequestered into the cytoplasm by binding to IB proteins (51, 52). Responder and nonresponder prolactinomas showed a different
pattern of NF-B activation, likely due to the presence or the
absence of secreted NGF in the culture media. In particular, in the
NGF-secreting responder cells, but not in the nonresponder cell lines,
NF-B appears to be constitutively activated. Furthermore, exposure
of nonresponder cells to NGF promoted the nuclear translocation and
induced the DNA binding activity of NF-B complexes containing p50,
p65/RelA, and c-Rel subunits in a dose- and time-dependent way, an
effect mediated by p75NGFR with apparently no
contribution of trkA. When p75NGFR activation was
blocked by a specific antibody, the selective NGF stimulation of trkA
failed, in fact, to promote NF-B translocation. This finding is in
line with the observation that, in cultured oligodendrocytes, trkA does
not modify p75NGFR-mediated induction of NF-B,
while suppressing p75NGFR-induced JNK activation
(13). However, it is worth noting that both
p75NGFR and trkA have been recently reported to
activate NF-B in PC12 cells (12). Our data, showing
that trkA inactivation by genistein did not modify the constitutive
NF-B activity in responder cells, while the
p75NGFR antibody abolished it, suggest that in
this cell line NF-B activation is entirely dependent on
p75NGFR. Thus, the evidence is increasing that
the cell phenotype strongly influences the type of signaling and of
interaction between p75NGFR and trkA.
As in other cell systems, activation of NF-B complexes in
prolactinoma cells was inhibited by SN50, a cell-permeable peptide that
specifically inhibits nuclear translocation of p50-containing NF-B
complexes (54). By using SN50, we were able to demonstrate
that NF-B activation is a critical step in the regulation of
D2 receptor gene transcription. SN50, in fact,
not only prevented NF-B activation, but also abolished
D2 receptor expression in both responder and
NGF-treated nonresponder prolactinoma cells. This conclusion is
strongly supported by the results of cotransfection experiments showing
that NF-B complexes including the p50 homodimer and the p50/c-Rel
heterodimer increase the transcriptional activity of a
D2 promoter-driven reporter gene. Interestingly,
differences in the transcriptional activity were observed with the two
different p50-containing complexes. Although the reason for these
differences has not been addressed directly, this observation is in
agreement with previous data by other groups (59, 60).
Thus, p75NGFR-mediated stimulation of NF-B
activity provides a molecular mechanism underlying the stimulatory
action of NGF on D2 receptor gene expression.
Analysis of the D2 receptor promoter revealed
that it has the characteristics of a housekeeping gene (61, 62), suggesting that the specificity in the expression pattern
of this receptor must be dictated spatially and temporally by
cell-specific transcription factors. On this line various regulatory
sequences, including Sp1, AP-1, AP-2, and RA-response element,
have been identified in the rat D2 promoter
(61, 62, 63, 64). However, only for retinoids has a role in the
physiological regulation of D2 receptor
expression been clearly demonstrated (65, 66). Our present
data first point to NF-B as another key transcriptional regulator of
the D2 gene in neuroendocrine cells. Our
observation that the human D2 promoter contains
at least one consensus sequence able to specifically bind NF-B
complexes (Guerra, N., C. Fiorentini, and C. Missale,
manuscript in preparation) supports this view and suggests that
the role of NF-B in the regulation of D2
receptor gene transcription may not be confined to pituitary tumors,
but could extend also to other dopaminergic systems.
The regulation of D2 receptor gene transcription appears to be a key element in the function of the dopaminergic systems. Deletion of the D2 receptor gene in knockout animals results in both nervous and pituitary dysfunctions (67, 68, 69) and ablation of specific retinoid receptors, by impairing the control imposed by retinoids on D2 gene transcription, leads to specific neurological symptoms in the null animals (65).
Our data raise the possibility that dysregulations in the expression or
function of NF-B transcription factors, resulting in an aberrant
control of D2 receptor gene expression, may lead
to specific neuroendocrine disorders.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cells were cultured under the following conditions: responder cells were grown in F10+ containing 1) either 1 µg/ml genistein (RBI, Natick, MA) or 100 nM K-252a (BIOMOL Research Laboratories, Inc., Hamburg, Germany) for 2–4 d without further medium changes; 2) either the antihuman p75NGFR monoclonal antibody MC8211 (50–100 ng/ml; Roche Molecular Biochemicals, Milano, Italy) or a polyclonal anti-p75NGFR antibody (1:100 dilution, AB1554 Chemicon International, Roma, Italy) for 2–8 d with the antibodies added to the culture medium every day; 3) either SN50 or SN50M (100 µg/ml; BIOMOL Research Laboratories, Inc.) for 2–6 d with the peptides added to the cultures every day. Nonresponder cells were grown in F10+ containing: 1) 100 ng/ml NGF (2.5S, mouse, Alomone Labs, Jerusalem, Israel) in the absence or in the presence of the anti-p75NGFR antibodies for either 2–24 h or 5 d. NGF was added once at the beginning of treatment, and the antibodies were added to the cultures every day; 2) 100 ng/ml NGF in the absence or in the presence of either SN50 or SN50M (100 µg/ml) for 5 d with NGF added once at the beginning of treatment and the peptides added to the cultures every day.
Detection of D2 and p75NGFR Receptors by
RT-PCR
Total RNA was isolated from cells using the SV Total RNA
Isolation System (Promega Corp., Milano, Italy). Four
micrograms of each sample were transcribed into cDNA by using the
murine Moloney leukemia virus reverse transcriptase (Promega Corp.) and oligo(dT)18 (Promega Corp.) as a primer. To amplify the D2
receptor the oligonucleotides 5'-TCCTGCCCACTCCTCTTCGGACTC-3' encoding
human D2 residues SCPLLFGL and
5'-AGAGTCAGCTGGTGGTGGCTGGG-3' encoding human D2
residues PSHHQLTL (MWG Biotech, Firenze, Italy) were used.
Reactions were performed for either 28 or 35 cycles (95 C, 30 sec; 57
C, 30 sec; 72 C, 1 min) within the linear range of amplification.
Omission of the reverse transcription reaction and amplification of
cloned human D2S cDNA
(hD2S) were performed as a control of the PCR
specificity. The p75NGFR receptor was amplified
with 5'-AGCCAACCAGACCGTGTGT-3' and 5'-TTGCAGCTGTTCCACCTCTT-3'
primers encoding human p75NGFR residues GANQTV
and EGEKLH, respectively. Reactions were performed for 28 cycles (95 C,
30 sec; 58 C, 30 sec; 72 C, 1 min) within the linear range of
amplification. Omission of the reverse transcription reaction and
amplification of cloned human p75NGFR cDNA were
performed as a control of the PCR specificity.
Amplification with 5'-TAAAGACCTCTATGCCAACACAGT-3' and 5'-CACGATGGAGGGCCGGACTCATC-3' primers encoding human ß-actin residues KDLYANTV and DESGPSIV, respectively (95 C, 30 sec; 60 C, 30 sec; 72 C, 1 min; 25 cycles), was performed as a control of the amount of cDNA used in each sample. The PCR products were analyzed on 1% agarose gels stained with ethydium bromide.
Detection of D2 Receptors by
[3H]Spiperone Binding
The F10+ medium was replaced with ice-cold 50 mM
Tris-HCl (pH 7.4), and cells were detached from plates and centrifuged
at 800 x g for 10 min. Cells were resuspended in
ice-cold 50 mM Tris-HCl (pH 7.4), homogenized
with an Ultra-Turrax homogenizer (two 15-sec bursts), and centrifuged
at 100,000 x g for 15 min at 4 C. The resulting
pellets were resuspended in ice-cold 50 mM
Tris-HCl containing 120 mM NaCl, 5
mM KCl, 2 mM
CaCl2, 1 mM
MgCl2, 0.1% ascorbic acid, pH 7.4. Aliquots of
the membrane suspension (80–100 µg protein/sample) were incubated
with 1 nM [3H]spiperone
(108 Ci/mmol; Amersham International, Milano, Italy) for
10 min at 37 C. The nonspecific binding was defined with 1
µM l-sulpiride (RBI). Incubations were stopped
by rapid filtration under reduced pressure through GF/B filters
(Whatman, Clifton, NJ).
Immunocytochemistry
Cells were plated at low density on
poly-L-lysine-coated glass coverslips, fixed with methanol
at -20 C for 5 min, incubated in PBS containing 0.2% Triton X-100 and
10% normal goat serum (Santa Cruz Biotechnology, Inc.,
Heidelberg, Germany) for 10 min at room temperature to mask nonspecific
absorption sites, and then incubated overnight at 4 C with a monoclonal
antibody to p65/RelA (1:200 dilution in PBS containing 1% normal goat
serum; Roche Molecular Biochemicals, Milano, Italy). Cells
were then incubated with the biotinylated goat antimouse secondary
antibody (1:400 dilution; Santa Cruz Biotechnology, Inc.)
for 1 h at room temperature. After three rinses with PBS, cells
were incubated with the avidin-biotin complex (ABC kit, DAKO Corp. S.p.A., Milano, Italy) for 45 min at room temperature.
Peroxidase staining was obtained by incubation in 0.06%
3,3'-diaminobenzidine and 0.01%
H2O2 in PBS buffer.
SDS-PAGE and Immunoblotting
Cells were detached from plates in PBS containing 100
µM pyrrolidine dithiocarbamate (Sigma-Aldrich Corp., Milano, Italy) to block NF-B activation during the
harvest procedure and centrifuged at 800 x g for 10
min. The cell pellets were resuspended in ice-cold 40
mM Tris-HCl containing 150
mM NaCl and 10 mM EDTA (pH
7.5), incubated on ice for 10 min, mixed by vortex for 10 sec, and
centrifuged at 12,000 x g for 15 sec at room
temperature. The resulting supernatants containing the cytoplasmic
proteins were stored at -20 C. The pellets were resuspended in 10
mM Tris-HCl (pH 7.5) containing 5
mM EDTA, 1 mM
phenylmethylsulfonylfluoride (PMSF), 10 µg/ml leupeptin, and 10
µg/ml pepstatin, incubated on ice for 20 min, and centrifuged at
18,000 x g for 2 min at 4 C. The resulting
supernatants containing the nuclear proteins were stored at -20 C.
Aliquots of cytoplasmic and nuclear proteins (50 µg protein/lane)
were resolved on 12% SDS-PAGE and transferred onto polyvinylidene
difluoride membranes. After blotting for 1 h at room temperature
in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5%
nonfat powdered milk (Blotto A), membranes were incubated overnight at
4 C with the monoclonal anti-p65/RelA antibody (1:100 dilution in
Blotto A; Santa Cruz Biotechnology, Inc.). For detection,
an ECL chemiluminescence system (Amersham International,
Milano, Italy) was used with a horseradish peroxidase (HRP)-conjugated
secondary antibody (1:2,000 dilution; Santa Cruz Biotechnology, Inc.).
In another group of experiments, cells were resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, 1 mM NaF, 1 mM Na3VO4, and a complete set of protease inhibitors (Complete Mini Protease Inhibitors, Roche Molecular Biochemicals), incubated on ice for 20 min, and centrifuged at 18,000 x g for 2 min at 4 C. The resulting cell extracts were stored at -20 C. To detect trkA and its phosphorylated forms, aliquots of cell extracts (30 µg protein/lane) were resolved on 6% SDS-PAGE, transferred onto nitrocellulose membranes, and blotted for 1 h at room temperature in Blotto A. Membranes were then incubated overnight at 4 C with 1) anti-trkA antibody (1:1,000 dilution in Blotto A; Cell Signaling Technology, Milano, Italy); 2) anti-phospho-trkA (Tyr490) antibody (1:1,000 dilution in TBS containing 5% BSA and 0.1% Tween 20; Cell Signaling Technology). For detection, an enhanced chemiluminescent system allowing visualization of proteins in the low femtogram range (SuperSignal West; Pierce Chemical Co., Milano, Italy) with a HRP-conjugated secondary antibody (Pierce Chemical Co.) was used. To detect MAPK, cell extracts (10 µg protein/lane) were resolved on 10% SDS-PAGE and processed as previously described. Membranes were incubated overnight at 4 C with anti-p44/42 MAPK antibody (1:1,000 dilution in TBS containing 5% BSA and 0.1% Tween 20; Cell Signaling Technology) or with the anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (1:1,000 dilution in TBS containing 5% BSA and 0.1% Tween 20; Cell Signaling Technology). To detect p75NGFR, cell extracts (30 µg protein/lane) were resolved on 7.5% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, blotted for 1 h at room temperature in Blotto A, and incubated overnight at 4 C with the anti-p75NGFR antibody (1:1,000 dilution in TBS containing 3% nonfat powdered milk and 0.1% Tween 20; Promega Corp.). The amount of proteins in each lane was checked by immunoreaction with ß-tubulin antibody (1:1,500 dilution in TBS containing 5% nonfat powdered milk and 0.1% Triton X-100; Neo-Markers, Fremont, CA). For detection, an ECL chemiluminescence (Amersham International) system with HRP-conjugated secondary antibodies (1:2,000 dilution; Santa Cruz Biotechnology, Inc.) was used.
Nuclear Extracts and EMSA
Cells were rinsed with ice-cold PBS and harvested by scraping
with ice-cold PBS containing 100 µM pyrrolidine
dithiocarbamate. Cells were pelleted at 4 C (800 x g,
5 min), lysed in 500 µl of ice-cold 10 mM
HEPES, pH 7.9, containing 10 mM KCl, 1.5
mM MgCl2, 0.5
mM dithiothreitol (DTT), 0.5
mM PMSF, and a complete set of protease
inhibitors (Complete Mini Protease Inhibitors, Roche Molecular Biochemicals) and centrifuged at 800 x g at 4 C
for 5 min. The resulting pellet was resuspended in 500 µl of the
lysis buffer described above with the addition of 0.5% NP-40,
homogenized with a Dounce homogenizer, and centrifuged at 2,500 x
g for 5 min at 4 C. The resulting pellet, containing the
nuclei, was resuspended in 30 µl of ice-cold 20
mM HEPES, pH 7.9, containing 420
mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 25%
glycerol, 0.5 mM DTT, 0.5
mM PMSF, and the complete set of protease
inhibitors, incubated on ice for 20 min, and centrifuged at 14,000
x g for 15 min at 4 C. The supernatant containing the
nuclear proteins was stored at -80 C. Protein concentration was
assessed by Bradford assay according to the manufacturer’s
instructions (Bio-Rad Laboratories, Inc., Hercules, CA).
DNA binding reactions were initiated by combining 10 µg of nuclear
extracts with 100,000 cpm (0.5 ng) of
-[32P]-labeled oligonucleotide in 25 µl of
10 mM Tris-HCl, pH 7.5, containing 50
mM NaCl, 1 mM DTT, 1
mM EDTA, 0.5 µg poly(dIdC). Reactions were
carried out for 20 min at room temperature, and protein-DNA complexes
were resolved on a nondenaturing 5% polyacrylamide gel in
Tris/borate/EDTA buffer. Gels were dried and subjected to
autoradiography at -80 C by using Kodak Biomax MR films
(Eastman Kodak Co., Rochester, NY). In supershift
experiments, 10 µg of nuclear extracts were incubated for 1 h at
4 C with selected antibodies before addition of the other components of
the reaction mixture. Incubation was continued for an additional 20 min
at room temperature. The following antibodies were used: a monoclonal
anti-p50 antibody (Santa Cruz Biotechnology, Inc.); a
monoclonal anti-p65/RelA antibody (Santa Cruz Biotechnology, Inc.); a monoclonal anti-p65/RelA antibody (Roche Molecular Biochemicals); and a monoclonal anti-cRel antibody
(Santa Cruz Biotechnology, Inc.). Preimmune serum was used
as a control of antibody specificity.
Synthetic DNA Oligonucleotides
The specific NF-B oligonucleotide
5'-GGATCCTCAACAGAGGGGACTTTCCGAGGCCA-3' and its complementary strand
were used. For gel shift analysis, 200 ng of the sense oligonucleotide
were end labeled with -[32P]ATP (3,000
Ci/mmol; NEN Life Science Products, Milano, Italy) and T4
polynucleotide kinase (Promega Corp.) for 1 h at 37
C. The labeled oligonucleotide was annealed with its complementary
strand for 3 min at 90 C, 10 min at 65 C, 10 min at 37 C, and 5 min at
room temperature, and the double-stranded oligonucleotide was purified
by denaturing 20% polyacrylamide gel electrophoresis.
Plasmid Construction
A fragment consisting of 284 bp of the 5'-flanking sequence and
20 bp of the first exon of the human D2 gene was
obtained by a two-step PCR with the sense primer
5'-ACTGGCGAGCAGAGCGTGAGGACCC-3' and antisense primer
5'-TGCGCGCGTGAGGCTGCCGGTTCGGC-3' according to Arinami et al.
(70). The KpnI linker was added to sense and
the HindIII linker was added to antisense primer. The
reaction was performed with native Pfu polymerase
(Stratagene, Milano, Italy) at 98 C for 1 min followed by
35 cycles of 98 C for 20 sec and 75 C for 5 min. PCR reaction buffer
was supplemented with 4% formamide. After sequencing, the generated
fragment was cloned into the luciferase reporter plasmid pGL3-basic
(Promega Corp.). The B/Rel expression plasmids pSG-p50
and pSG-cRel have been described previously (71). The
p75NGFR coding sequence was subcloned into the
BamHI/XhoI restriction sites of the pcDNA3.1
expression vector (Invitrogen, Milano, Italy).
Cell Transfection and Luciferase Activity
COS-7 cells (60–80% confluent) were transiently cotransfected
for 3 h with 1) pGL3-D2 (1 µg) and the
empty pSG vector (2 µg); 2) pGL3-D2 (1 µg),
pSG-p50 (1 µg), and pSG (1 µg); 3) pGL3-D2 (1
µg), pSG-p50 (1 µg), and pSG-cRel (1 µg); 4) pGL3-D2 (1 µg),
pSG-cRel (1 µg), and pSG (1 µg); 5) pGL3-D2
(1 µg) and pcDNA-p75NGFR (1 µg), using the
lipofectine (LipofectAMINE Reagent, Invitrogen-Life
Technologies, Milano Italy) technique. Transfection efficiency
throughout the experiments was monitored by cotransfection with a
Renilla luciferase expression vector (50 ng). After
transfection, cells were cultured in the complete medium for 48 h.
Cells cotransfected with pGL3-D2 and
pcDNA-p75NGFR were treated with 100 ng/ml NGF for
4 h. Cells were harvested, lysed, and assayed for luciferase
activities by using the Dual-Luciferase Reporter Assay System
(Promega Corp.) according to the manufacturer’s
instructions. Firefly luciferase activity in each sample was normalized
to Renilla luciferase activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These authors contributed equally to this work. 
Abbreviations: DTT, Dithiothreitol; HRP, horseradish
peroxidase; JNK, c-Jun N-terminal kinase; NF-B, nuclear factorB;
NGF, nerve growth factor; PMSF, phenylmethylsulfonylfluoride; TBS,
Tris-buffered saline.
Received for publication December 6, 2000. Accepted for publication October 10, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
B by nerve growth factor through the neurotrophin receptor p75.
Science 272:542–545[Abstract]
B signalling promotes both cell survival and
neurite process formation in nerve growth factor-stimulated PC12 cells.
J Neurosci 20:7556–7563
