Nerve Growth Factor Regulates Dopamine D2 Receptor Expression in Prolactinoma Cell Lines via p75NGFR-Mediated Activation of Nuclear Factor-{kappa}B

Chiara Fiorentini1, Nicoletta Guerra1, Marco Facchetti, Alessandra Finardi, Laura Tiberio, Luisa Schiaffonati, PierFranco Spano and Cristina Missale

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two groups of prolactinoma cell lines were identified. One group (responder) expresses both D2 dopamine receptors and an autocrine loop mediated by nerve growth factor (NGF) and one group (nonresponder) lacks both D2 receptors and NGF production. D2 receptor expression in these cell lines is dependent on NGF. Indeed, NGF inactivation in responder cells decreases D2 receptor density, while NGF treatment induces D2 receptor expression in nonresponders.

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-{kappa}B (NF-{kappa}B) nuclear accumulation and binding to corresponding DNA consensus sequences indicated that in NGF-secreting responder cells, but not in nonresponders, NF-{kappa}B is constitutively activated. Moreover, NGF treatment of nonresponder cells induced both nuclear translocation and DNA binding activity of NF-{kappa}B complexes containing p50, p65/RelA, and cRel subunits, an effect prevented by anti-p75NGFR antibodies. Disruption of NF-{kappa}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-{kappa}B, which is required for D2 gene expression. We thus suggest that NF-{kappa}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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NERVE GROWTH FACTOR (NGF) is the first described member of a family of neurotrophic factors, known as the neurotrophins, which are required for differentiation and survival of specific neuronal populations.

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-{kappa}B (NF-{kappa}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-{kappa}B transcription factor, an effect that is necessary for the expression of the D2 receptor gene. These data point to NF-{kappa}B as a key transcriptional regulator of the D2 gene in neuroendocrine cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inactivation of p75NGFR Results in the Loss of D2 Receptors in Responder Cells
We have shown previously that the expression of the D2 receptor gene in responder prolactinoma cells is highly dependent on endogenous NGF (41). To evaluate the role of trkA and p75NGFR in this effect, each NGF receptor was individually inactivated, and D2 receptor expression was evaluated by both RT-PCR and radioreceptor binding. In particular, p75NGFR-mediated NGF activity was inhibited by using two different anti-p75NGFR antibodies (42, 43), and trkA was inactivated by inhibiting its intrinsic tyrosine kinase activity with either genistein or K-252a (44, 45). Control experiments were performed to evaluate the efficiency and selectivity of treatments. TrkA and its Tyr490-phosphorylated form were measured by Western blot by using an enhanced chemiluminescent system allowing protein detection in the low femtogram range. As shown in Fig. 1AGo (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. 1AGo, 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. 1BGo, 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. 1CGo, lane 3), nor the p75NGFR protein (Fig. 1DGo, lane 3) was modified by a 5-d treatment with the anti-p75NGFR antibody. This antibody, however, completely inhibited p75NGFR signaling (see Figs. 5Go, 6Go, and 7AGo), suggesting that it efficiently blocks p75NGFR-mediated effects. Exposure of responder cells to genistein resulted in a slight decrease of p75NGFR mRNA expression (Fig. 1CGo, lane 2). Genistein, however, did not affect either the levels of the p75NGFR protein (Fig. 1DGo, lane 2) or its transductional capability evaluated by measuring NF-{kappa}B DNA binding activity (see Fig. 7AGo), suggesting that in our experimental conditions this compound does not interfere with p75NGFR-mediated mechanisms.



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Figure 1. Effects of Genistein, K-252a, and Anti-p75NGFR Antibody on trkA and p75NGFR Expression and Function in Prolactinoma Cells

Responder cells were exposed to genistein (1 µg/ml) and K-252a (100 nM) for 4 d and to anti-p75NGFR monoclonal antibody MC8211 (50 ng/ml) for 5 d. A, Effects of treatments on trkA expression and tyrosine phosphorylation. Upper panel, Cell proteins (30 µg/lane) were analyzed for trkA content as described in Materials and Methods. Two major bands of 110 and 140 kDa were identified. Lane 1, Untreated cells; lane 2, genistein treatment; lane 3, K-252a treatment; lane 4, anti-p75NGFR antibody treatment. Middle panel, Aliquots of cell protein (30 µg/lane) were immunoreacted with the anti-phospho-trkA (tyr490) antibody (1:1,000 dilution). Lanes 1 and 4, Untreated cells; lane 2, genistein treatment; lane 3, K-252a treatment; lane 5, anti-p75NGFR antibody treatment. Lower panel, ß-Tubulin immunostaining: lane 1, untreated cells; lane 2, genistein treatment; lane 3, K-252a treatment; lane 4, anti-p75NGFR antibody treatment. B, Effects of treatments on MAPK phosphorylation. Aliquots of cell proteins (10 µg/lane) were immunoreacted with anti-phospho-p44/42 MAPK antibody (upper panel) or with anti-p44/42 MAPK antibody (lower panel). Lane 1, Untreated cells; lane 2, genistein treatment; lane 3, anti-p75NGFR antibody treatment. C, Effects of treatments on p75NGFR mRNA levels. The cDNA prepared from untreated and treated cells was PCR amplified as described in Materials and Methods. Lane 1, Untreated cells; lane 2, genistein treatment; lane 3, anti-p75NGFR antibody treatment. D, Effects of treatments on p75NGFR protein levels. Cell proteins were immunoreacted with anti-p75NGFR antibody (upper panel) or with anti-ß-tubulin antibody (lower panel). Lane 1, Untreated cells; lane 2, genistein treatment; lane 3, anti-p75NGFR antibody treatment. All experiments were repeated three times with superimposable results.

 


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Figure 5. Distribution of NF-{kappa}B in Untreated and NGF-Treated Prolactinoma Cells

Untreated and NGF-treated (100 ng/ml; 4 h) cells were fixed with methanol at -20 C and processed for p65/RelA immunostaining as described in Materials and Methods. A, distribution of p65/RelA immunoreactivity in responder cells. The nucleus (solid arrowheads) and the cytoplasm (open arrowheads) appear to be equally stained. B, Distribution of p65/RelA immunoreactivity in nonresponder cells. The immunostaining appears to be segregated in the cytoplasm (solid arrowheads) with virtually no nuclear staining (open arrowheads). C, In NGF-treated nonresponder cells p65/RelA immunostaining appears to be more concentrated in the nucleus (solid arrowheads) than in the cytoplasm (open arrowheads); D, in nonresponder cells treated with NGF in the presence of MC8211 anti-p75NGFR antibody (50 ng/ml), p65/RelA immunoreactivity was more concentrated in the cytoplasm (open arrowheads) than in the nucleus (solid arrowheads).

 


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Figure 6. Western Blot Analysis of p65/RelA in Nuclear and Cytoplasmic Proteins from Nonresponder Cells

Nuclear (Nucl) and cytoplasmic (Cyt) proteins were immunoblotted with the anti-p65/RelA antibody as described in Materials and Methods. The results of a representative experiment are shown.

 


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Figure 7. NF-{kappa}B DNA Binding Activity in Untreated and NGF-Treated Prolactinoma Cells

A, Nuclear proteins were isolated from untreated responder and nonresponder cells, from nonresponder cells treated with 100 ng/ml NGF for 4 h, and from responder cells treated with either genistein (1 µg/ml) for 4 d or anti-p75NGFR antibody (50 ng/ml) for 5 d. EMSA was performed with [32P]-labeled NF-{kappa}B oligonucleotide as a probe. Lanes 1, 4, and 6, Responder cells; lane 2, nonresponder cells; lane 3, NGF-treated nonresponder cells; lane 5, genistein-treated responder cells; lane 7, anti p75NGFR antibody-treated responder cells. B, Dose-response curve of NGF-induced NF-{kappa}B DNA binding activity in nonresponder cells. Cells were exposed to 10–100 ng/ml NGF for 4 h, and the nuclear proteins were isolated and processed for EMSA analysis. Lane 1, 10 ng/ml NGF; lane 2, 50 ng/ml NGF; lane 3, 100 ng/ml NGF; lane 4, untreated cells. C, Time course of NGF-induced NF-{kappa}B DNA binding activity in nonresponder cells. Cells were exposed to 100 ng/ml NGF for 2–24 h, and the nuclear proteins were isolated and processed for EMSA analysis. Lane 1, Untreated cells; lane 2, 2 h NGF treatment; lane 3, 4 h NGF treatment; lane 4, 8 h NGF treatment; lane 5, 24 h NGF treatment. The positions of immunoreactive DNA-protein complexes (solid arrowheads) and of the uncomplexed DNA (open arrowheads) are shown. D, The molecular composition of the DNA/protein complex was determined by incubating nuclear extracts in the presence of antibodies raised against p50 (lane 2), p65/RelA (lane 3), and c-Rel (lane 4) subunits. The specificity of the antibodies was determined by performing the supershift with preimmune serum. The positions of the immunoreactive complexes (solid arrowhead) and the supershifted bands (open arrowhead) are indicated. The film was overexposed to detect the supershifted bands.

 
The results obtained by RT-PCR analysis of D2 mRNA expression are reported in Fig. 2Go. 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. 2AGo, 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. 2AGo, 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. 2AGo, 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. 2BGo, 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. 2Go, A and B). Similar results were obtained in binding studies with [3H]spiperone. As shown in Fig. 2CGo, 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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Figure 2. The Expression of D2 Receptors in Responder Prolactinoma Cells Is Selectively Inhibited by Inactivation of p75NGFR

A, Responder cells were exposed to either MC8211 (50 and 100 ng/ml) or AB1554 (1:100 dilution) antibody for 5 d, and their cDNA was amplified by PCR (28 cycles) with D2-specific primers as described in Materials and Methods. The amount of cDNA in each sample was determined by PCR amplification with ß-actin-specific primers (25 cycles). The results of a representative experiment are shown. Lane 1, Untreated cells; lane 2, MC8211 antibody treatment (50 ng/ml); lane 3, MC8211 antibody treatment (100 ng/ml); lane 4, AB1554 antibody treatment; lane 5, hD2S. B, Responder cells were treated with genistein (1 µg/ml) for 4 d and processed as in panel A. The results of a representative experiment are shown. Lane 1, Untreated cells; lane 2, genistein treatment. The experiments were repeated three times with superimposable results. C, Cells were exposed to the MC8211 antibody (50 ng/ml) for 2–8 d or to 1 µg/ml genistein for 2–4 d, and D2 receptor expression was evaluated by [3H]spiperone binding as described in Materials and Methods. Bars represent the means ± SEM of three independent experiments run in triplicate. **, P < 0.005 vs. untreated cells; *, P < 0.001 vs. untreated cells by t test.

 
The Expression of D2 Receptors Induced by NGF in Nonresponder Cells Is Dependent on p75NGFR
Our data have shown that exposure of nonresponder cells to exogenous NGF resulted in two temporally distinct events: the expression of p75NGFR, occurring after 2–24 h of treatment (Fig. 3AGo 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. 3BGo, 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. 3CGo. 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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Figure 3. Inactivation of p75NGFR Prevents D2 mRNA Expression Induced by NGF in Nonresponder Cells

A, RT-PCR analysis of p75NGFR expression in nonresponder cells exposed to NGF for different times. B, RT-PCR analysis of D2 receptor mRNA expression in non responder cells. Cells were exposed to 100 ng/ml NGF in the absence or in the presence of either MC8211 (50 ng/ml) or AB1554 (1:100 dilution) anti-p75NGFR antibody for 5 consecutive days. Their cDNA was PCR amplified with either D2 specific primers (35 cycles) or ß-actin primers (25 cycles) as described in Materials and Methods. The results of a representative experiment are shown. Lane 1, Untreated cells; lane 2, NGF treatment; lane 3, NGF and MC8211 antibody treatment; lane 4, NGF and AB1554 antibody treatment. The experiments were repeated three times with similar results. C, [3H]spiperone binding in nonresponder cell membranes. Cells were exposed to NGF (100 ng/ml) in the absence or in the presence of MC8211 antibody (50 ng/ml), and [3H]spiperone binding was performed as described in Materials and Methods. Bars represent the means ± SEM of three independent experiments run in triplicate. *, P < 0.001 vs. untreated cells. Note that in untreated cells and in cells exposed to NGF in the presence of the MC8211 antibody the specific [3H]spiperone binding was undetectable.

 
To further prove that p75NGFR is the NGF receptor that triggers D2 receptor expression, a fragment spanning the 5'-flanking region of the human D2 gene (50) was inserted into a luciferase reporter vector (pGL3-D2) and transiently transfected into COS-7 cells together with an expression vector containing the coding sequence of p75NGFR receptor. The transfected cells were exposed to 100 ng/ml NGF for 4 h. As shown in Fig. 4Go, 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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Figure 4. NGF Induces the Transcriptional Activation of the D2 Promoter-Driven Luciferase Gene in COS-7 Cells Expressing p75NGFR

COS-7 cells were cotransfected either with the reporter vector pGL3-D2 alone or in combination with the expression vector pcDNA-p75NGFR as described in Materials and Methods. Transfection efficiency in each sample was monitored by cotransfection with the Renilla luciferase expression vector. Forty eight hours after transfection cells were cultured in the absence or in the presence of 100 ng/ml NGF for 4 h, lysed, and assayed for both firefly and Renilla luciferase activities. The firefly luciferase activity in each sample was normalized to Renilla luciferase activity. Open bars, Untreated cells; solid bars, NGF-treated cells. Each bar represents the mean ± SEM of three independent experiments. *, P < 0.001 vs. untreated cells by t test.

 
NGF Selectively Activates NF-{kappa}B in Prolactinoma Cells
One signaling mechanism transduced by p75NGFR is the activation of NF-{kappa}B (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-{kappa}B in these cell lines. The cellular localization of p65/RelA was investigated by immunostaining with a specific monoclonal antibody. As shown in Fig. 5Go, in responder cells p65/RelA immunostaining appeared to be homogeneously distributed within the nucleus (Fig. 5AGo, solid arrowheads) and the cytoplasm (Fig. 5AGo, open arrowhead). By contrast, in nonresponder cells p65/RelA immunoreactivity appeared to be preferentially localized in the cytoplasm (Fig. 5BGo, solid arrowheads), with only a faint nuclear staining (Fig. 5BGo, open arrowhead), suggesting that in responder, but not in nonresponder, cells NF-{kappa}B may be constitutively activated. As reported in Fig. 5CGo, 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. 5DGo), indicating that p75NGFR is necessary for NF-{kappa}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. 6Go, 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-{kappa}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-{kappa}B consensus sequence from the mouse Ig {kappa}-gene (52). As shown in Fig. 7AGo, 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-{kappa}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-{kappa}B DNA binding activity (lane 3). As shown in Fig. 7BGo, 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-{kappa}B DNA binding activity (not shown). The time dependence of the effect of NGF is reported in Fig. 7CGo. NGF induced up-regulation of NF-{kappa}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-{kappa}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-{kappa}B subunits activated by NGF in prolactinoma cells, antibodies specific for various NF-{kappa}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-{kappa}B probe in the absence or in the presence of antibodies to p50, p65/RelA, and c-Rel subunits. As shown in Fig. 7DGo, all the antibodies interfered with the interaction of the NF-{kappa}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-{kappa}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-{kappa}B binding activity. To investigate whether NF-{kappa}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-{kappa}B, which is known to inhibit translocation of the NF-{kappa}B active complex into the nucleus (53). Since in responder cells, which spontaneously express D2 receptors, NF-{kappa}B appears to be constitutively activated, we evaluated whether SN50-induced inhibition of NF-{kappa}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-{kappa}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. 8AGo, 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. 8BGo, 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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Figure 8. The NF-{kappa}B-Inhibitory Peptide SN50 Abolishes D2 Receptor Expression in Prolactinoma Cells

A, Responder cells were treated with 100 µg/ml SN50 or 100 µg/ml SN50M for 2 and 6 d, and the cDNA obtained from untreated and treated cells was PCR amplified with D2-specific primers (28 cycles) as described in Materials and Methods. Lane 1, Untreated cells; lane 2, 2-d SN50 treatment; lane 3, 6-d SN50 treatment; lane 4, 6-d SN50M treatment. B, Nonresponder cells were exposed to 100 ng/ml NGF in the absence or in the presence of either 100 µg/ml SN50 or 100 µg/ml SN50M for 5 d. The cDNA obtained from untreated and treated cells was subjected to PCR amplification with D2-specific primers (35 cycles). Lane 1, Untreated cells; lane 2, NGF-treated cells; lane 3, NGF/SN50-treated cells; lane 4, NGF/SN50M-treated cells. The amount of cDNA in each sample was determined by PCR amplification with ß-actin specific primers (25 cycles). The results of representative experiments are shown.

 
To definitely prove that NF-{kappa}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 {kappa}B-related species p50 and c-Rel. As shown in Fig. 9Go, 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-{kappa}B is a necessary and sufficient signal to induce D2 gene expression.



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Figure 9. Transcriptional Activation by NF-{kappa}B Subunits of the D2 Receptor Promoter

COS-7 cells were cotransfected with the reporter vector pGL3-D2 and either the pSG-p50, pSG-c-Rel, or the combination pSG-p50/pSG-c-Rel as described in Materials and Methods. Transfection efficiency in each sample was monitored by cotransfection with the Renilla luciferase expression vector. Forty-eight hours after transfection cells were lysed and assayed for both firefly and Renilla luciferase activities. The firefly luciferase activity in each sample was normalized to Renilla luciferase activity. Each bar represents the mean ± SEM of three independent experiments. *, P < 0.001 vs. pGL3-D2, t test.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The p75NGFR NGF receptor, although long believed to primarily act as a coreceptor for trkA (1, 2, 3), is now emerging as being also capable of autonomous functions. On this line some independent activities of p75NGFR have been proposed in the nervous system (10, 15, 16, 17, 18, 19, 20). However, although ceramide (8, 9, 10), NF-{kappa}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-{kappa}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-{kappa}B transcription factors as the most plausible candidates. The NF-{kappa}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-{kappa}B is sequestered into the cytoplasm by binding to I{kappa}B proteins (51, 52). Responder and nonresponder prolactinomas showed a different pattern of NF-{kappa}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-{kappa}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-{kappa}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-{kappa}B translocation. This finding is in line with the observation that, in cultured oligodendrocytes, trkA does not modify p75NGFR-mediated induction of NF-{kappa}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-{kappa}B in PC12 cells (12). Our data, showing that trkA inactivation by genistein did not modify the constitutive NF-{kappa}B activity in responder cells, while the p75NGFR antibody abolished it, suggest that in this cell line NF-{kappa}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-{kappa}B complexes in prolactinoma cells was inhibited by SN50, a cell-permeable peptide that specifically inhibits nuclear translocation of p50-containing NF-{kappa}B complexes (54). By using SN50, we were able to demonstrate that NF-{kappa}B activation is a critical step in the regulation of D2 receptor gene transcription. SN50, in fact, not only prevented NF-{kappa}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-{kappa}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-{kappa}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-{kappa}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-{kappa}B complexes (Guerra, N., C. Fiorentini, and C. Missale, manuscript in preparation) supports this view and suggests that the role of NF-{kappa}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-{kappa}B transcription factors, resulting in an aberrant control of D2 receptor gene expression, may lead to specific neuroendocrine disorders.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prolactinoma Cell Cultures and Treatments
Two prolactinoma cell lines (one nonresponder and one responder), previously developed and characterized in our laboratory (37, 41), were used in this study. Cells were grown in Ham’s F10 medium supplemented with 2.5% FCS, 15% horse serum, 4 mM glutamine, and 100 U of penicillin-streptomycin (F10+).

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-{kappa}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 {gamma}-[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-{kappa}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 {gamma}-[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 {kappa}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.


    ACKNOWLEDGMENTS
 
We thank Dr. Marc Caron for his gift of the human D2S cDNA; Dr. Karen L. O’Malley, for her gift of the human D2 promoter; Dr. Mariagrazia Grilli and Dr. Pierre Jalinot for kindly providing the pSG-p50 and pSG-c-Rel expression vectors; Dr. Moses V. Chao, for providing the p75NGFR cDNA; Dr Monica Di Luca and Dr. Francesca Colciaghi for their help and advice in the Western blot studies; Dr. Laura Bianchi for her gift of the pGL3 basic and Renilla vectors; and Dr. Roberto Bresciani for his gift of COS-7 cells and his help and advice in construct preparation and transfection experiments.


    FOOTNOTES
 
This work was supported by grants from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (9706151106), Consiglio Nazionale delle Ricerche (99.02532.CT04) and AIRC Associazione Italiana per la Ricerca sul Cancro (to C.M.).

1 These authors contributed equally to this work. Back

Abbreviations: DTT, Dithiothreitol; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; NF-{kappa}B, nuclear factor{kappa}B; 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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 DISCUSSION
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