Nerve Growth Factor Regulates Dopamine D2Receptor Expression in Prolactinoma Cell Lines viap75NGFR-Mediated Activation of Nuclear Factor-�B

CHIARA FIORENTINI*, NICOLETTA GUERRA*, 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 andImmunology (L.T., L.S.), Department of Biomedical Sciences and Biotechnology, University ofBrescia, 25123 Brescia, Italy

Two groups of prolactinoma cell lines were identi-fied. One group (responder) expresses both D2 do-pamine receptors and an autocrine loop mediatedby nerve growth factor (NGF) and one group (non-responder) lacks both D2 receptors and NGF pro-duction. D2 receptor expression in these cell linesis dependent on NGF. Indeed, NGF inactivation inresponder cells decreases D2 receptor density,while NGF treatment induces D2 receptor expres-sion in nonresponders.

Here we show that inactivation of p75NGFR, butnot of trkA, resulted in D2 receptor loss in re-sponder cells and prevented D2 receptor expres-sion induced by NGF in the nonresponder. Analysisof nuclear factor-�B (NF-�B) nuclear accumulationand binding to corresponding DNA consensus se-quences indicated that in NGF-secreting re-sponder cells, but not in nonresponders, NF-�B isconstitutively activated. Moreover, NGF treatment

of nonresponder cells induced both nuclear trans-location and DNA binding activity of NF-�B com-plexes containing p50, p65/RelA, and cRel subunits,an effect prevented by anti-p75NGFR antibodies. Dis-ruption of NF-�B nuclear translocation by SN50 re-markably impaired D2 receptor expression in re-sponder cells and prevented D2 gene expressioninduced by NGF in nonresponders. These data indi-cate that in prolactinoma cells the effect of NGF onD2 receptor expression is mediated by p75NGFR in atrkA-independent way and that NGF stimulation ofp75NGFR activates NF-�B, which is required for D2

gene expression. We thus suggest that NF-�B is akey transcriptional regulator of the D2 gene and thatthis mechanism may not be confined to pituitary tu-mors, but could also extend to other dopaminergicsystems. (Molecular Endocrinology 16: 353–366,2002)

NERVE GROWTH FACTOR (NGF) is the first de-scribed member of a family of neurotrophic fac-

tors, known as the neurotrophins, which are requiredfor differentiation and survival of specific neuronalpopulations.

NGF binds to two different receptors, trkA andp75NGFR (1–3). TrkA, a 140-kDa protein with intrinsictyrosine kinase activity, signals via a ras-dependentpathway leading to activation of the MAPKs (4–6) andalso through other enzymes, such as phosphatidylino-sitol-3 kinase (6, 7). p75NGFR is a member of the TNFcytokine receptor superfamily and activates ceramideproduction (8–10), nuclear factor-�B (NF-�B) (10–13),and c-Jun N-terminal kinase (JNK) (11, 13, 14). Whilethe role of trkA in mediating NGF action on cell survivaland differentiation is well established, the functions ofp75NGFR are still a matter of some debate. p75NGFR

has been proposed to act as a coreceptor for trkA(1–3), to modulate trkA signaling (1–3), or to initiate itsindependent transduction pathways. The best charac-

terized trk-independent activity of p75NGFR is regula-tion of neuronal cell death and survival (10–16). Otherp75NGFR-mediated effects have been proposed in dif-ferent neuronal systems, including stimulation of do-pamine 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 berestricted to few populations of neuronal cells, is nowknown to extend also to different neuroendocrine sys-tems such as the thyroid and parathyroid glands (21),the pancreas (22–24), the prostate (25–28), and thepituitary (29–32). In the pituitary, NGF and its receptorshave been identified in the anterior lobe (29–32), wherethey play a role in the control of maturation and pro-liferation of lactotrope cells (33). In addition, NGF isemerging as a regulator of proliferation and differenti-ation of various tumors of neuroendocrine origin (34–36) including pituitary PRL-secreting tumors (37).These are the most frequently occurring neoplasms inthe human pituitary, often express D2 receptors fordopamine (38), the physiological inhibitor of PRL se-cretion (39), and are currently treated with D2 receptoragonists. Ten to 15% of patients, however, due todecreased density (40) or loss (37) of D2 receptors, are

Abbreviations: DTT, Dithiothreitol; HRP, horseradish per-oxidase; JNK, c-Jun N-terminal kinase; NF-�B, nuclearfactor�B; NGF, nerve growth factor; PMSF, phenylmethylsul-fonylfluoride; TBS, Tris-buffered saline.

0888-8809/02/$15.00/0 Molecular Endocrinology 16(2):353–366Printed in U.S.A. Copyright © 2002 by The Endocrine Society

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refractory to this pharmacological therapy and requiresurgical intervention.

In previous studies we have developed and charac-terized two phenotypically different groups of humanprolactinoma cell lines. Those derived from tumorsrefractory to the pharmacological therapy (here re-ferred to as “nonresponder”) are more transformed,have a high tumorigenic potential, and lack D2 dopa-mine receptors, while those obtained from bromocrip-tine-sensitive tumors (here referred to as “responder”)are more differentiated, are not tumorigenic, and ex-press D2 receptors (37). One of the characteristics ofthese cell lines is that their phenotype is highly depen-dent on the NGF system. In particular, an autocrineloop that involves the secretion of NGF and the ex-pression of both trkA and p75NGFR has been identifiedin the responder prolactinomas, but not in nonre-sponders that do not produce NGF and express trkAbut not p75NGFR (41). The relevance of this mechanismis such that ablation of NGF production in respondercells leads to transformation and D2 receptor loss,while administration of NGF to nonresponder cellspromotes their differentiation and induces the expres-sion of both p75NGFR and D2 receptors (41). The mo-lecular and cellular mechanisms activated by NGF inthese cell lines, however, remain largely unknown. Inparticular, which NGF receptor subtype and whichintracellular signaling pathways are involved is still amatter of investigation.

In this study we used two previously characterizedprolactinoma cell lines, one responder and one non-responder (37, 41), to define the role of trkA andp75NGFR in NGF-mediated regulation of D2 receptorgene expression and to identify the molecular mech-anisms that are involved in this effect. The resultsshow that p75NGFR plays a critical role, which is inde-pendent of trkA, in triggering and maintaining D2 re-ceptor gene expression in prolactinoma cells. NGFstimulation of p75NGFR in these cells results in theactivation and nuclear translocation of NF-�B tran-scription factor, an effect that is necessary for theexpression of the D2 receptor gene. These data pointto NF-�B as a key transcriptional regulator of the D2

gene in neuroendocrine cells.

RESULTS

Inactivation of p75NGFR Results in the Loss of D2

Receptors in Responder Cells

We have shown previously that the expression of theD2 receptor gene in responder prolactinoma cells ishighly dependent on endogenous NGF (41). To eval-uate the role of trkA and p75NGFR in this effect, eachNGF receptor was individually inactivated, and D2 re-ceptor expression was evaluated by both RT-PCR andradioreceptor binding. In particular, p75NGFR-medi-ated NGF activity was inhibited by using two differentanti-p75NGFR antibodies (42, 43), and trkA was inacti-

vated by inhibiting its intrinsic tyrosine kinase activitywith either genistein or K-252a (44, 45). Control exper-iments were performed to evaluate the efficiency andselectivity of treatments. TrkA and its Tyr490-phos-phorylated form were measured by Western blot byusing an enhanced chemiluminescent system allowingprotein detection in the low femtogram range. Asshown in Fig. 1A (upper panel), the anti-trkA antibody(Cell Signaling Technology) recognized two majorbands in responder cells, one at 110 and one at 140kDa, consistent with the presence of differentially gly-cosylated forms of trkA (46). The specificity of thesebands was confirmed by immunoreacting the samemembrane with another anti-trkA antibody (Santa CruzBiotechnology, Inc., Heidelberg, Germany; not shown).The expression of trkA in responder cells was not mod-ified by treatments, as shown by the intensity of the 110-and 140-kDa bands. TrkA tyrosine phosphorylation wasevaluated with a phospho-specific antibody detecting itstyr490 phosphorylated form (47), which binds Shc (48)and initiates the Ras-MAPK cascade. In responder cellsthat produce and secrete NGF (41), trkA appears to beconstitutively Tyr490 phosphorylated (Fig. 1A, middlepanel, lanes 1 and 4). A 4-d exposure to genistein re-sulted in a significant decrease of Tyr490 trkA phosphor-ylation (lane 2). K-252a, however, was less efficient ininhibiting trkA phosphorylation (lane 3), and the anti-p75NGFR antibody did not affect it (lane 5). These obser-vations were further confirmed by evaluating the degreeof phosphorylation of p44/42 MAPK. The results, re-ported in Fig. 1B, show that both p44 and p42 MAPKwere constitutively phosphorylated in untreated re-sponder cells (upper panel, lane 1). A 4-d exposure togenistein significantly decreased p44/42 MAPK phos-phorylation (lane 2), while K-252a (lane 3) affected it onlyslightly. These observations suggest that in our experi-mental conditions, genistein, but not K-252a, effectivelyblocks trkA function. This result could be in line with theobservation that K-252a is a partial agonist and exertseither inhibitory or stimulatory effects, depending on thecell phenotype and the experimental conditions (49).Thus, in subsequent experiments we used genistein (1�g/ml) to efficiently block trkA function. The expressionof p75NGFR in responder cells exposed to the differentchemicals was measured by both RT-PCR and Westernblot. Neither p75NGFR mRNA (Fig. 1C, lane 3), nor thep75NGFR protein (Fig. 1D, lane 3) was modified by a 5-dtreatment with the anti-p75NGFR antibody. This antibody,however, completely inhibited p75NGFR signaling (seeFigs. 5, 6, and 7A), suggesting that it efficiently blocksp75NGFR-mediated effects. Exposure of responder cellsto 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 bymeasuring NF-�B DNA binding activity (see Fig. 7A),suggesting that in our experimental conditions this com-pound does not interfere with p75NGFR-mediatedmechanisms.

354 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

The results obtained by RT-PCR analysis of D2

mRNA expression are reported in Fig. 2. Amplificationof cDNA with D2-specific primers located within thefourth transmembrane domain and the third intracel-lular loop, downstream of the alternatively spliced do-main, revealed that only the D2S mRNA isoform ispresent in responder cells, as shown by a single334-bp band generated by the PCR reaction (Fig. 2A,lane 1). This finding was supported by the observationthat 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 theRNA, i.e. omitting the RT reaction, did not produce anyband (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 adramatic decrease of the D2S mRNA. By contrast, 1�g/ml genistein (Fig. 2B, lane 2) given for 4 d did notaffect D2S mRNA expression. Amplification with �-actin primers was performed as a control of theamount 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 cellcultures to MC8211 antibody (50 ng/ml) for 2–8 dresulted in a significant decrease of specific D2 bindingsites. This effect was evident after a 2-d treatment andwas maximal after 8 d, when specific [3H]spiperonebinding was about 90% decreased. In line with theresults obtained by RT-PCR, both 2- and 4-d exposureof responder cells to 1 �g/ml genistein did not modify[3H]spiperone binding. Thus, inhibition of endogenousNGF-mediated p75NGFR stimulation, but not trkA in-activation, resulted in D2 receptor loss in respondercells, suggesting a crucial role for p75NGFR in regulat-ing D2 receptor gene transcription in this cell line.

The Expression of D2 Receptors Induced by NGFin Nonresponder Cells Is Dependent on p75NGFR

Our data have shown that exposure of nonrespondercells to exogenous NGF resulted in two temporallydistinct events: the expression of p75NGFR, occurringafter 2–24 h of treatment (Fig. 3A and Ref. 41) and theexpression of D2 receptors that was detectable by

Fig. 1. Effects of Genistein, K-252a, and Anti-p75NGFR Antibody on trkA and p75NGFR Expression and Function in ProlactinomaCells

Responder cells were exposed to genistein (1 �g/ml) and K-252a (100 nM) for 4 d and to anti-p75NGFR monoclonal antibodyMC8211 (50 ng/ml) for 5 d. A, Effects of treatments on trkA expression and tyrosine phosphorylation. Upper panel, Cell proteins(30 �g/lane) were analyzed for trkA content as described in Materials and Methods. Two major bands of 110 and 140 kDa wereidentified. 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 �g/lane) were immunoreacted with the anti-phospho-trkA (tyr490) antibody (1:1,000dilution). Lanes 1 and 4, Untreated cells; lane 2, genistein treatment; lane 3, K-252a treatment; lane 5, anti-p75NGFR antibodytreatment. Lower panel, �-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 �g/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-�-tubulin antibody (lowerpanel). Lane 1, Untreated cells; lane 2, genistein treatment; lane 3, anti-p75NGFR antibody treatment. All experiments wererepeated three times with superimposable results.

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 355

radioreceptor binding after a 2-d treatment and wasmaximal after a 5-d treatment (37). Nonresponder cellswere exposed to 100 ng/ml NGF in the absence or inthe presence of anti-p75NGFR antibodies for 5 consec-utive days, and D2 receptors were measured in thedifferent experimental groups. As reported in Fig. 3B,D2 mRNA was virtually absent in untreated nonre-sponder cells (lane 1). Exposure of this cell line to NGFresulted in a remarkable and selective induction of theD2S isoform mRNA (lane 2), an effect that was pre-vented by anti-p75NGFR antibodies (lanes 3 and 4). Theresults obtained in binding studies with [3H]spiperoneare reported in Fig. 3C. While specific [3H]spiperonebinding was undetectable in untreated cells, exposureto NGF (100 ng/ml; 5 d) resulted in the appearance ofmeasurable levels of specific D2 receptor binding. Thiseffect was abolished when cells were cultured withNGF in the presence of MC8211 anti-p75NGFR anti-body (50 ng/ml). Thus, the newly expressed p75NGFR

receptor appears to be necessary for the stimulatoryeffect of NGF on D2 receptor expression in nonre-sponder cells.

To further prove that p75NGFR is the NGF receptorthat triggers D2 receptor expression, a fragment span-ning 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 to-gether with an expression vector containing the codingsequence of p75NGFR receptor. The transfected cellswere exposed to 100 ng/ml NGF for 4 h. As shown in Fig.4, NGF did not modify luciferase activity in cells express-ing the D2 promoter construct but not p75NGFR. How-ever, NGF administration to cells coexpressing p75NGFR

significantly stimulated D2 promoter-driven luciferase ac-tivity. Thus, the stimulation of p75NGFR in a host cellsystem triggers D2 promoter activation, strongly sup-porting the view that D2 receptor expression in prolacti-noma cells is dependent on p75NGFR.

NGF Selectively Activates NF-�B inProlactinoma Cells

One signaling mechanism transduced by p75NGFR isthe activation of NF-�B (11). This family of transcrip-tion factors is composed of several members that formhetero- and homodimers that are able to trigger sig-naling from cell membrane to the nucleus (50, 51).

Since responder and nonresponder cells differ forthe production of NGF, we evaluated both the distri-bution and the activity of NF-�B in these cell lines. Thecellular localization of p65/RelA was investigated byimmunostaining with a specific monoclonal antibody.As shown in Fig. 5, in responder cells p65/RelA im-munostaining appeared to be homogeneously distrib-uted within the nucleus (Fig. 5A, solid arrowheads) andthe cytoplasm (Fig. 5A, open arrowhead). By contrast,in nonresponder cells p65/RelA immunoreactivity ap-peared to be preferentially localized in the cytoplasm(Fig. 5B, solid arrowheads), with only a faint nuclearstaining (Fig. 5B, open arrowhead), suggesting that inresponder, but not in nonresponder, cells NF-�B maybe constitutively activated. As reported in Fig. 5C,exposure of nonresponder cells to 100 ng/ml NGFresulted in the nuclear translocation of p65/RelA (solidarrowheads). This translocation was detectable after a2-h incubation with NGF, was maximal after 4 h, and

Fig. 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 theircDNA was amplified by PCR (28 cycles) with D2-specific primers as described in Materials and Methods. The amount of cDNAin each sample was determined by PCR amplification with �-actin-specific primers (25 cycles). The results of a representativeexperiment 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 �g/ml) for 4 d andprocessed as in panel A. The results of a representative experiment are shown. Lane 1, Untreated cells; lane 2, genisteintreatment. The experiments were repeated three times with superimposable results. C, Cells were exposed to the MC8211antibody (50 ng/ml) for 2–8 d or to 1 �g/ml genistein for 2–4 d, and D2 receptor expression was evaluated by [3H]spiperone bindingas 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.

356 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

was blocked by the anti-p75NGFR antibody (Fig. 5D),indicating that p75NGFR is necessary for NF-�B acti-vation. Similar results were obtained in Western blotexperiments on isolated nuclear and cytoplasmic pro-teins. In untreated cells, p65/RelA immunoreactivitywas more concentrated in the cytosolic than in thenuclear fraction (Fig. 6, lanes 1 and 2), while in NGF-treated cells the nuclear fraction appeared to be highlyenriched in p65/RelA (lanes 3 and 4). p65/RelA nucleartranslocation was prevented by the anti-p75NGFR an-tibody (lanes 5 and 6).

To demonstrate that NGF-activated NF-�B was ableto bind to DNA, nuclear protein extracts were tested inEMSA using a double-stranded [32P]-labeled oligonu-cleotide containing the characterized NF-�B consen-sus sequence from the mouse Ig �-gene (52). Asshown in Fig. 7A, EMSA revealed high DNA bindingactivity in responder cells (lanes 1, 4, and 6) and a verylow DNA binding activity in nonresponder cells (lane2). The constitutive NF-�B binding activity detectablein responder cells was abolished by the anti-p75NGFR

antibody (lane 7), but not by genistein (lane 5), sug-gesting that it was entirely due to the interaction ofsecreted NGF with p75NGFR. Exposure of nonre-sponder cells to 100 ng/ml NGF for 4 h resulted in aremarkable up-regulation of the NF-�B DNA bindingactivity (lane 3). As shown in Fig. 7B, this effect wasdose-dependent over the range of 10–100 ng/ml. In-creasing NGF concentrations over 100 ng/ml did notresult in a further increase of NF-�B DNA binding

activity (not shown). The time dependence of the effectof NGF is reported in Fig. 7C. NGF induced up-regu-lation of NF-�B activity was detectable after a 2-htreatment (lane 2) and was maximal within 4 h (lane 3).After 8 h of stimulation, NF-�B binding activity was stillabove control (lane 4) and after a 24-h NGF treatment itwas similar to untreated cells (lane 5). To identify theNF-�B subunits activated by NGF in prolactinoma cells,antibodies specific for various NF-�B peptides weretested for their ability to either interfere with DNA bindingor supershift DNA-bound activity. Nuclear extracts fromNGF-treated nonresponder were incubated with the[32P]-labeled NF-�B probe in the absence or in the pres-ence of antibodies to p50, p65/RelA, and c-Rel subunits.As shown in Fig. 7D, all the antibodies interfered with theinteraction of the NF-�B probe with nuclear proteins. Inparticular, the anti-p50 (lane 2) and anti-c-Rel (lane 4)antibodies inhibited the protein-DNA binding, while theanti-p65/RelA antibody (lane 3) supershifted the DNA-protein band, thus suggesting that all three protein sub-units are present in the complexes activated by NGF. Bycontrast, a preimmune serum did not significantly modifythe DNA-protein binding.

NGF-Inducible NF-�B Is Required for theExpression of the D2 Receptor Gene

The data reported so far suggest that in human pro-lactinomas, p75NGFR is required for NGF-mediated ex-

Fig. 3. Inactivation of p75NGFR Prevents D2 mRNA Expression Induced by NGF in Nonresponder CellsA, 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 presenceof either MC8211 (50 ng/ml) or AB1554 (1:100 dilution) anti-p75NGFR antibody for 5 consecutive days. Their cDNA was PCRamplified with either D2 specific primers (35 cycles) or �-actin primers (25 cycles) as described in Materials and Methods. Theresults of a representative experiment are shown. Lane 1, Untreated cells; lane 2, NGF treatment; lane 3, NGF and MC8211antibody 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 thepresence of MC8211 antibody (50 ng/ml), and [3H]spiperone binding was performed as described in Materials and Methods. Barsrepresent the means � SEM of three independent experiments run in triplicate. *, P � 0.001 vs. untreated cells. Note that inuntreated cells and in cells exposed to NGF in the presence of the MC8211 antibody the specific [3H]spiperone binding wasundetectable.

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 357

pression of D2 receptors and that p75NGFR activationresults in the stimulation of NF-�B binding activity. Toinvestigate whether NF-�B is the cellular signal re-quired for D2 receptor gene transcription, we usedSN50, a cell-permeable peptide containing the nuclearlocalization sequence from the p50 subunit of NF-�B,which is known to inhibit translocation of the NF-�Bactive complex into the nucleus (53). Since in re-sponder cells, which spontaneously express D2 recep-tors, NF-�B appears to be constitutively activated, weevaluated whether SN50-induced inhibition of NF-�Bnuclear translocation may affect D2 receptor expres-sion. Responder cells were thus exposed to either 100�g/ml SN50 or 100 �g/ml SN50M, an inactive controlpeptide mutated within the nuclear localization se-quence motif, for 2–6 d. As a control of the efficiencyof these treatments, NF-�B cell localization was eval-uated by p65/RelA immunostaining. The resultsshowed that SN50, but not SN50M, completely inhib-ited p65/RelA nuclear translocation (data not shown).Moreover, as shown in Fig. 8A, exposure of respondercells to SN50 for 2 and 6 d resulted in a dramaticdown-regulation of D2S mRNA expression (lanes 2 and3). By contrast, the levels of D2S mRNA were un-changed in cells treated with SN50M for 6 d (lane 4).

Similarly, as reported in Fig. 8B, the stimulatory effectof NGF (100 ng/ml; 5 d) on D2S mRNA expression innonresponder cells (lane 2) was lost when NGF treat-ment was performed in the presence of 100 �g/mlSN50 (lane 3), but not SN50M (lane 4).

Fig. 4. NGF Induces the Transcriptional Activation of the D2

Promoter-Driven Luciferase Gene in COS-7 Cells Expressingp75NGFR

COS-7 cells were cotransfected either with the reportervector pGL3-D2 alone or in combination with the expressionvector pcDNA-p75NGFR as described in Materials and Meth-ods. Transfection efficiency in each sample was monitoredby cotransfection with the Renilla luciferase expression vec-tor. Forty eight hours after transfection cells were cultured inthe absence or in the presence of 100 ng/ml NGF for 4 h,lysed, and assayed for both firefly and Renilla luciferaseactivities. The firefly luciferase activity in each sample wasnormalized to Renilla luciferase activity. Open bars, Un-treated cells; solid bars, NGF-treated cells. Each bar repre-sents the mean � SEM of three independent experiments. *,P � 0.001 vs. untreated cells by t test.

Fig. 5. Distribution of NF-�B in Untreated and NGF-TreatedProlactinoma Cells

Untreated and NGF-treated (100 ng/ml; 4 h) cells werefixed with methanol at �20 C and processed for p65/RelAimmunostaining as described in Materials and Methods. A,distribution of p65/RelA immunoreactivity in responder cells.The nucleus (solid arrowheads) and the cytoplasm (openarrowheads) appear to be equally stained. B, Distribution ofp65/RelA immunoreactivity in nonresponder cells. The immu-nostaining appears to be segregated in the cytoplasm (solidarrowheads) with virtually no nuclear staining (open arrow-heads). C, In NGF-treated nonresponder cells p65/RelA im-munostaining appears to be more concentrated in the nu-cleus (solid arrowheads) than in the cytoplasm (openarrowheads); D, in nonresponder cells treated with NGF in thepresence of MC8211 anti-p75NGFR antibody (50 ng/ml), p65/RelA immunoreactivity was more concentrated in the cyto-plasm (open arrowheads) than in the nucleus (solid arrow-heads).

Fig. 6. Western Blot Analysis of p65/RelA in Nuclear andCytoplasmic Proteins from Nonresponder Cells

Nuclear (Nucl) and cytoplasmic (Cyt) proteins were immu-noblotted with the anti-p65/RelA antibody as described inMaterials and Methods. The results of a representative ex-periment are shown.

358 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

To definitely prove that NF-�B regulates D2 receptorexpression, the luciferase reporter vector containingthe D2 promoter was transiently transfected intoCOS-7 cells together with expression vectors contain-ing the �B-related species p50 and c-Rel. As shown inFig. 9, expression of p50 by itself or in combinationwith c-Rel led to a significant increase of D2 promoter-driven luciferase activity, while the expression of c-Relalone was devoid of effects on the reporter gene tran-

scription. These data indicate that NF-�B is a necessaryand sufficient signal to induce D2 gene expression.

DISCUSSION

The p75NGFR NGF receptor, although long believed toprimarily act as a coreceptor for trkA (1–3), is now

Fig. 7. NF-�B DNA Binding Activity in Untreated and NGF-Treated Prolactinoma CellsA, 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 �g/ml) for 4 d or anti-p75NGFR antibody (50 ng/ml)for 5 d. EMSA was performed with [32P]-labeled NF-�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-�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. Lane1, 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-�BDNA binding activity in nonresponder cells. Cells were exposed to 100 ng/ml NGF for 2–24 h, and the nuclear proteins wereisolated and processed for EMSA analysis. Lane 1, Untreated cells; lane 2, 2 h NGF treatment; lane 3, 4 h NGF treatment; lane4, 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 wasdetermined 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. Thepositions of the immunoreactive complexes (solid arrowhead) and the supershifted bands (open arrowhead) are indicated. Thefilm was overexposed to detect the supershifted bands.

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 359

emerging as being also capable of autonomous func-tions. On this line some independent activities ofp75NGFR have been proposed in the nervous system(10, 15–20). However, although ceramide (8–10),

NF-�B (10–13), and JNK (11, 13, 14) have been iden-tified as potential p75NGFR-dependent signaling effec-tors, only in a few cases has their direct involvement inspecific p75NGFR-mediated neuronal functions beenelucidated (9, 10, 12, 13, 16, 55). The role of trkA andp75NGFR in the neuroendocrine effects of NGF hasbeen only partially investigated. In particular, somestudies suggested the involvement of trkA in the reg-ulation of pancreas morphogenesis (22–24), one studycorrelated the activation of trkA with the control ofprostate cancer cell line proliferation (56), and frag-mentary data indirectly correlated the loss of p75NGFR

with the progression of prostate cancer (27, 28) and itsexpression with the antiproliferative effects of NGF onthyroid tumor cells (34). On the other hand, the signal-ing mechanisms activated by each NGF receptor sub-type in neuroendocrine tissues are still elusive.

In previous studies we have shown that the expres-sion of D2 receptors for dopamine in pituitary PRL-secreting tumor cell lines is correlated with their de-gree of transformation and is highly dependent on anendogenous 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 thep75NGFR NGF receptor in a trkA-independent way.Moreover, we identified the NF-�B transcription factoras the NGF-activated, p75NGFR-dependent intracellu-lar signaling molecule that is required for D2 receptorgene transcription.

The demonstration that p75NGFR is indeed the re-ceptor that mediates the effect of NGF on D2 geneexpression came from the results obtained with theanti-p75NGFR antibodies and in transfection experi-ments. In the more differentiated responder cells inhi-bition of endogenous NGF interaction with p75NGFR

resulted in D2 receptor loss. Similarly, when thep75NGFR receptor was inactivated during NGF admin-istration to the more transformed nonresponder cells,the stimulatory effect of NGF on D2 receptor expres-

Fig. 8. The NF-�B-Inhibitory Peptide SN50 Abolishes D2 Receptor Expression in Prolactinoma CellsA, Responder cells were treated with 100 �g/ml SN50 or 100 �g/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. Lane1, Untreated cells; lane 2, 2-d SN50 treatment; lane 3, 6-d SN50 treatment; lane 4, 6-d SN50M treatment. B, Nonresponder cellswere exposed to 100 ng/ml NGF in the absence or in the presence of either 100 �g/ml SN50 or 100 �g/ml SN50M for 5 d. ThecDNA obtained from untreated and treated cells was subjected to PCR amplification with D2-specific primers (35 cycles). Lane1, Untreated cells; lane 2, NGF-treated cells; lane 3, NGF/SN50-treated cells; lane 4, NGF/SN50M-treated cells. The amount ofcDNA in each sample was determined by PCR amplification with �-actin specific primers (25 cycles). The results of representativeexperiments are shown.

Fig. 9. Transcriptional Activation by NF-�B Subunits of theD2 Receptor Promoter

COS-7 cells were cotransfected with the reporter vectorpGL3-D2 and either the pSG-p50, pSG-c-Rel, or the combi-nation pSG-p50/pSG-c-Rel as described in Materials andMethods. Transfection efficiency in each sample was moni-tored by cotransfection with the Renilla luciferase expressionvector. Forty-eight hours after transfection cells were lysedand assayed for both firefly and Renilla luciferase activities.The firefly luciferase activity in each sample was normalizedto Renilla luciferase activity. Each bar represents the mean �SEM of three independent experiments. *, P � 0.001 vs. pGL3-D2, t test.

360 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

sion was lost. These data thus suggest that, whenp75NGFR signaling is negated, the selective activationof trkA-mediated pathways is not sufficient, per se, tosustain D2 receptor gene transcription. Moreover, thedata obtained with the tyrosine kinase inhibitorgenistein argue against the possibility of a functionalinteraction between trkA and p75NGFR in NGF-medi-ated regulation of D2 receptor expression. Disruptionof trkA signaling did not modify, in fact, the levels ofD2S mRNA and protein in responder cells, suggestingthat D2 receptor expression is under the exclusivecontrol of p75NGFR and its signaling effectors. Thisconclusion is further supported by the finding thatNGF triggers D2 promoter-directed luciferase tran-scription in COS-7 cells expressing p75NGFR, but notin those lacking it. In line with this is the observationthat in prolactinoma cells there is a close correlationbetween p75NGFR and D2, but not trkA and D2 receptorexpression levels (41). It should be noted, however,that genistein slightly down-regulates p75NGFR mRNAin responder cells. Although this decrease was notaccompanied by a correspondent decrease ofp75NGFR protein and transductional efficiency, it couldbe inferred that a longer inhibition of trkA could actu-ally down-regulate the p75NGFR receptor. On this linewe had previously shown that p75NGFR expression inprolactinomas is highly dependent on NGF (41) andthat a deprivation of secreted NGF for at least 6 d wasnecessary to induce p75NGFR protein loss in respondercells (41). These observations suggest that trkA couldindirectly contribute to the effects of NGF on D2 genetranscription by inducing and maintaining the expres-sion 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 otherspecific effects of NGF such as inhibition of cell pro-liferation and abrogation of tumorigenicity (37).

The D2 dopamine receptor is known to exist as twodifferent isoforms, called D2S and D2L, which are gen-erated by alternative splicing from the same gene andwhich 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 am-plification of D2 cDNA in responder prolactinomas re-vealed that, unlike their physiological counterpart,these cells express only the D2S receptor isoform.Furthermore, exposure of nonresponder prolactino-mas to NGF resulted in the selective expression of D2S

mRNA. Different mechanisms may be invoked to ex-plain this finding. It could be possible that geneticalterations occurring during lactotrope cell transfor-mation led to the loss of the D2L isoform. On the otherhand, it is also possible that NGF affects the splicingmechanisms leading to the selective expression of theD2S receptor isoform. In line with this view, subpopu-lations of lactotropes have been identified that mayexpress different D2S/D2L mRNA ratios in response todifferent stimuli (57), and activated sex steroid recep-tors have been reported to modulate the alternative

splicing of the D2 receptor mRNA in the MMQ pituitarycell line (58).

How does p75NGFR induce the expression of D2

receptors in prolactinoma cells? Our analysis of thesignaling molecules activated by NGF strongly pointedto NF-�B transcription factors as the most plausiblecandidates. The NF-�B family is composed of severaldistinct DNA binding subunits that can hetero- andhomodimerize, thereby forming complexes with dis-tinct cell type distribution, DNA sequence specificity,and transcriptional activity (51, 52). In its inactive state,NF-�B is sequestered into the cytoplasm by binding toI�B proteins (51, 52). Responder and nonresponderprolactinomas showed a different pattern of NF-�Bactivation, likely due to the presence or the absence ofsecreted NGF in the culture media. In particular, in theNGF-secreting responder cells, but not in the nonre-sponder cell lines, NF-�B appears to be constitutivelyactivated. Furthermore, exposure of nonrespondercells to NGF promoted the nuclear translocation andinduced the DNA binding activity of NF-�B complexescontaining p50, p65/RelA, and c-Rel subunits in adose- and time-dependent way, an effect mediated byp75NGFR with apparently no contribution of trkA. Whenp75NGFR activation was blocked by a specific anti-body, the selective NGF stimulation of trkA failed, infact, to promote NF-�B translocation. This finding is inline with the observation that, in cultured oligodendro-cytes, trkA does not modify p75NGFR-mediated induc-tion of NF-�B, while suppressing p75NGFR-inducedJNK activation (13). However, it is worth noting thatboth p75NGFR and trkA have been recently reported toactivate NF-�B in PC12 cells (12). Our data, showingthat trkA inactivation by genistein did not modify theconstitutive NF-�B activity in responder cells, whilethe p75NGFR antibody abolished it, suggest that in thiscell line NF-�B activation is entirely dependent onp75NGFR. Thus, the evidence is increasing that the cellphenotype strongly influences the type of signalingand of interaction between p75NGFR and trkA.

As in other cell systems, activation of NF-�B com-plexes in prolactinoma cells was inhibited by SN50, acell-permeable peptide that specifically inhibits nu-clear translocation of p50-containing NF-�B com-plexes (54). By using SN50, we were able to demon-strate that NF-�B activation is a critical step in theregulation of D2 receptor gene transcription. SN50, infact, not only prevented NF-�B activation, but alsoabolished D2 receptor expression in both responderand NGF-treated nonresponder prolactinoma cells.This conclusion is strongly supported by the results ofcotransfection experiments showing that NF-�B com-plexes including the p50 homodimer and the p50/c-Rel heterodimer increase the transcriptional activity ofa D2 promoter-driven reporter gene. Interestingly, dif-ferences in the transcriptional activity were observedwith the two different p50-containing complexes. Al-though the reason for these differences has not beenaddressed directly, this observation is in agreementwith previous data by other groups (59, 60). Thus,

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 361

p75NGFR-mediated stimulation of NF-�B activity pro-vides a molecular mechanism underlying the stimula-tory action of NGF on D2 receptor gene expression.

Analysis of the D2 receptor promoter revealed that ithas the characteristics of a housekeeping gene (61,62), suggesting that the specificity in the expressionpattern of this receptor must be dictated spatially andtemporally by cell-specific transcription factors. Onthis line various regulatory sequences, including Sp1,AP-1, AP-2, and RA-response element, have beenidentified in the rat D2 promoter (61–64). However, onlyfor retinoids has a role in the physiological regulationof D2 receptor expression been clearly demonstrated(65, 66). Our present data first point to NF-�B asanother key transcriptional regulator of the D2 gene inneuroendocrine cells. Our observation that the humanD2 promoter contains at least one consensus se-quence able to specifically bind NF-�B complexes(Guerra, N., C. Fiorentini, and C. Missale, manuscriptin preparation) supports this view and suggests thatthe role of NF-�B in the regulation of D2 receptor genetranscription may not be confined to pituitary tumors,but could extend also to other dopaminergic systems.

The regulation of D2 receptor gene transcription ap-pears to be a key element in the function of the do-paminergic systems. Deletion of the D2 receptor genein knockout animals results in both nervous and pitu-itary dysfunctions (67–69) and ablation of specific ret-inoid receptors, by impairing the control imposed byretinoids on D2 gene transcription, leads to specificneurological symptoms in the null animals (65).

Our data raise the possibility that dysregulations inthe expression or function of NF-�B transcription fac-tors, resulting in an aberrant control of D2 receptorgene expression, may lead to specific neuroendocrinedisorders.

MATERIALS AND METHODS

Prolactinoma Cell Cultures and Treatments

Two prolactinoma cell lines (one nonresponder and one re-sponder), previously developed and characterized in our lab-oratory (37, 41), were used in this study. Cells were grown inHam’s F10 medium supplemented with 2.5% FCS, 15%horse serum, 4 mM glutamine, and 100 U of penicillin-strep-tomycin (F10�).

Cells were cultured under the following conditions: re-sponder cells were grown in F10� containing 1) either 1�g/ml genistein (RBI, Natick, MA) or 100 nM K-252a (BIOMOLResearch Laboratories, Inc., Hamburg, Germany) for 2–4 dwithout further medium changes; 2) either the antihumanp75NGFR monoclonal antibody MC8211 (50–100 ng/ml;Roche Molecular Biochemicals, Milano, Italy) or a polyclonalanti-p75NGFR antibody (1:100 dilution, AB1554 Chemicon In-ternational, Roma, Italy) for 2–8 d with the antibodies addedto the culture medium every day; 3) either SN50 or SN50M(100 �g/ml; BIOMOL Research Laboratories, Inc.) for 2–6 dwith the peptides added to the cultures every day. Nonre-sponder cells were grown in F10� containing: 1) 100 ng/mlNGF (2.5S, mouse, Alomone Labs, Jerusalem, Israel) in theabsence or in the presence of the anti-p75NGFR antibodies for

either 2–24 h or 5 d. NGF was added once at the beginningof treatment, and the antibodies were added to the culturesevery day; 2) 100 ng/ml NGF in the absence or in the pres-ence of either SN50 or SN50M (100 �g/ml) for 5 d with NGFadded once at the beginning of treatment and the peptidesadded to the cultures every day.

Detection of D2 and p75NGFR Receptors by RT-PCR

Total RNA was isolated from cells using the SV Total RNAIsolation System (Promega Corp., Milano, Italy). Four micro-grams of each sample were transcribed into cDNA by usingthe 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�-TCCTGC-CCACTCCTCTTCGGACTC-3� encoding human D2 residuesSCPLLFGL and 5�-AGAGTCAGCTGGTGGTGGCTGGG-3�encoding human D2 residues PSHHQLTL (MWG Biotech,Firenze, Italy) were used. Reactions were performed for either28 or 35 cycles (95 C, 30 sec; 57 C, 30 sec; 72 C, 1 min) withinthe linear range of amplification. Omission of the reversetranscription reaction and amplification of cloned human D2ScDNA (hD2S) were performed as a control of the PCR spec-ificity. The p75NGFR receptor was amplified with 5�-AGC-CAACCAGACCGTGTGT-3� and 5�-TTGCAGCTGTTCCAC-CTCTT-3� primers encoding human p75NGFR residues GANQTVand EGEKLH, respectively. Reactions were performed for 28cycles (95 C, 30 sec; 58 C, 30 sec; 72 C, 1 min) within the linearrange of amplification. Omission of the reverse transcriptionreaction and amplification of cloned human p75NGFR cDNAwere performed as a control of the PCR specificity.

Amplification with 5�-TAAAGACCTCTATGCCAACACAGT-3�and 5�-CACGATGGAGGGCCGGACTCATC-3� primers encod-ing human �-actin residues KDLYANTV and DESGPSIV, re-spectively (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 eachsample. The PCR products were analyzed on 1% agarose gelsstained 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 cen-trifuged at 800 � g for 10 min. Cells were resuspended inice-cold 50 mM Tris-HCl (pH 7.4), homogenized with an Ultra-Turrax homogenizer (two 15-sec bursts), and centrifuged at100,000 � g for 15 min at 4 C. The resulting pellets wereresuspended in ice-cold 50 mM Tris-HCl containing 120 mM

NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbicacid, pH 7.4. Aliquots of the membrane suspension (80–100�g protein/sample) were incubated with 1 nM [3H]spiperone(108 Ci/mmol; Amersham International, Milano, Italy) for 10min at 37 C. The nonspecific binding was defined with 1 �M

l-sulpiride (RBI). Incubations were stopped by rapid filtrationunder reduced pressure through GF/B filters (Whatman,Clifton, NJ).

Immunocytochemistry

Cells were plated at low density on poly-L-lysine-coatedglass 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., Heidel-berg, Germany) for 10 min at room temperature to masknonspecific absorption sites, and then incubated overnight at4 C with a monoclonal antibody to p65/RelA (1:200 dilution inPBS containing 1% normal goat serum; Roche MolecularBiochemicals, Milano, Italy). Cells were then incubated withthe biotinylated goat antimouse secondary antibody (1:400dilution; Santa Cruz Biotechnology, Inc.) for 1 h at roomtemperature. After three rinses with PBS, cells were incu-

362 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

bated with the avidin-biotin complex (ABC kit, DAKO Corp.S.p.A., Milano, Italy) for 45 min at room temperature. Perox-idase 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 �M

pyrrolidine dithiocarbamate (Sigma-Aldrich Corp., Milano, It-aly) to block NF-�B activation during the harvest procedureand centrifuged at 800 � g for 10 min. The cell pellets wereresuspended 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 � g for15 sec at room temperature. The resulting supernatants con-taining the cytoplasmic proteins were stored at �20 C. Thepellets were resuspended in 10 mM Tris-HCl (pH 7.5) con-taining 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride(PMSF), 10 �g/ml leupeptin, and 10 �g/ml pepstatin, incu-bated on ice for 20 min, and centrifuged at 18,000 � g for 2min at 4 C. The resulting supernatants containing the nuclearproteins were stored at �20 C. Aliquots of cytoplasmic andnuclear proteins (50 �g protein/lane) were resolved on 12%SDS-PAGE and transferred onto polyvinylidene difluoridemembranes. After blotting for 1 h at room temperature inTris-buffered saline (TBS) containing 0.1% Tween 20 and 5%nonfat powdered milk (Blotto A), membranes were incubatedovernight 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 (Amer-sham International, Milano, Italy) was used with a horseradishperoxidase (HRP)-conjugated secondary antibody (1:2,000dilution; Santa Cruz Biotechnology, Inc.).

In another group of experiments, cells were resuspendedin 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 Bio-chemicals), incubated on ice for 20 min, and centrifuged at18,000 � g for 2 min at 4 C. The resulting cell extracts werestored at �20 C. To detect trkA and its phosphorylatedforms, aliquots of cell extracts (30 �g protein/lane) wereresolved on 6% SDS-PAGE, transferred onto nitrocellulosemembranes, and blotted for 1 h at room temperature in BlottoA. Membranes were then incubated overnight at 4 C with 1)anti-trkA antibody (1:1,000 dilution in Blotto A; Cell SignalingTechnology, Milano, Italy); 2) anti-phospho-trkA (Tyr490) an-tibody (1:1,000 dilution in TBS containing 5% BSA and 0.1%Tween 20; Cell Signaling Technology). For detection, an en-hanced chemiluminescent system allowing visualization ofproteins in the low femtogram range (SuperSignal West;Pierce Chemical Co., Milano, Italy) with a HRP-conjugatedsecondary antibody (Pierce Chemical Co.) was used. To de-tect MAPK, cell extracts (10 �g protein/lane) were resolvedon 10% SDS-PAGE and processed as previously described.Membranes were incubated overnight at 4 C with anti-p44/42MAPK antibody (1:1,000 dilution in TBS containing 5%BSA and 0.1% Tween 20; Cell Signaling Technology) or withthe anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody(1:1,000 dilution in TBS containing 5% BSA and 0.1% Tween20; Cell Signaling Technology). To detect p75NGFR, cell ex-tracts (30 �g 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 incubatedovernight at 4 C with the anti-p75NGFR antibody (1:1,000dilution in TBS containing 3% nonfat powdered milk and0.1% Tween 20; Promega Corp.). The amount of proteins ineach lane was checked by immunoreaction with �-tubulinantibody (1:1,500 dilution in TBS containing 5% nonfat pow-dered milk and 0.1% Triton X-100; Neo-Markers, Fremont,CA). For detection, an ECL chemiluminescence (AmershamInternational) system with HRP-conjugated secondary anti-

bodies (1:2,000 dilution; Santa Cruz Biotechnology, Inc.) wasused.

Nuclear Extracts and EMSA

Cells were rinsed with ice-cold PBS and harvested by scrap-ing with ice-cold PBS containing 100 �M pyrrolidine dithio-carbamate. Cells were pelleted at 4 C (800 � g, 5 min), lysedin 500 �l 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 MiniProtease Inhibitors, Roche Molecular Biochemicals) and cen-trifuged at 800 � g at 4 C for 5 min. The resulting pellet wasresuspended in 500 �l of the lysis buffer described abovewith the addition of 0.5% NP-40, homogenized with aDounce homogenizer, and centrifuged at 2,500 � g for 5 minat 4 C. The resulting pellet, containing the nuclei, was resus-pended in 30 �l of ice-cold 20 mM HEPES, pH 7.9, containing420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5mM DTT, 0.5 mM PMSF, and the complete set of proteaseinhibitors, incubated on ice for 20 min, and centrifuged at14,000 � g for 15 min at 4 C. The supernatant containing thenuclear proteins was stored at �80 C. Protein concentrationwas assessed by Bradford assay according to the manufac-turer’s instructions (Bio-Rad Laboratories, Inc., Hercules,CA). DNA binding reactions were initiated by combining 10�g of nuclear extracts with 100,000 cpm (0.5 ng) of �-[32P]-labeled oligonucleotide in 25 �l of 10 mM Tris-HCl, pH 7.5,containing 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 �gpoly(dIdC). Reactions were carried out for 20 min at roomtemperature, and protein-DNA complexes were resolved on anondenaturing 5% polyacrylamide gel in Tris/borate/EDTAbuffer. Gels were dried and subjected to autoradiography at�80 C by using Kodak Biomax MR films (Eastman KodakCo., Rochester, NY). In supershift experiments, 10 �g ofnuclear extracts were incubated for 1 h at 4 C with selectedantibodies before addition of the other components of thereaction mixture. Incubation was continued for an additional20 min at room temperature. The following antibodies wereused: a monoclonal anti-p50 antibody (Santa Cruz Biotech-nology, Inc.); a monoclonal anti-p65/RelA antibody (SantaCruz Biotechnology, Inc.); a monoclonal anti-p65/RelA anti-body (Roche Molecular Biochemicals); and a monoclonalanti-cRel antibody (Santa Cruz Biotechnology, Inc.). Preim-mune serum was used as a control of antibody specificity.

Synthetic DNA Oligonucleotides

The specific NF-�B oligonucleotide 5�-GGATCCTCAACA-GAGGGGACTTTCCGAGGCCA-3� and its complementarystrand were used. For gel shift analysis, 200 ng of the senseoligonucleotide were end labeled with �-[32P]ATP (3,000 Ci/mmol; NEN Life Science Products, Milano, Italy) and T4polynucleotide kinase (Promega Corp.) for 1 h at 37 C. Thelabeled oligonucleotide was annealed with its complemen-tary 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-strandedoligonucleotide was purified by denaturing 20% polyacryl-amide gel electrophoresis.

Plasmid Construction

A fragment consisting of 284 bp of the 5�-flanking sequenceand 20 bp of the first exon of the human D2 gene wasobtained by a two-step PCR with the sense primer 5�-ACT-GGCGAGCAGAGCGTGAGGACCC-3� and antisense primer5�-TGCGCGCGTGAGGCTGCCGGTTCGGC-3� according toArinami et al. (70). The KpnI linker was added to sense andthe HindIII linker was added to antisense primer. The reactionwas performed with native Pfu polymerase (Stratagene, Mi-lano, Italy) at 98 C for 1 min followed by 35 cycles of 98 C for

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 363

20 sec and 75 C for 5 min. PCR reaction buffer was supple-mented with 4% formamide. After sequencing, the generatedfragment was cloned into the luciferase reporter plasmidpGL3-basic (Promega Corp.). The �B/Rel expression plas-mids pSG-p50 and pSG-cRel have been described previ-ously (71). The p75NGFR coding sequence was subcloned intothe BamHI/XhoI restriction sites of the pcDNA3.1 expressionvector (Invitrogen, Milano, Italy).

Cell Transfection and Luciferase Activity

COS-7 cells (60–80% confluent) were transiently cotrans-fected for 3 h with 1) pGL3-D2 (1 �g) and the empty pSGvector (2 �g); 2) pGL3-D2 (1 �g), pSG-p50 (1 �g), and pSG (1�g); 3) pGL3-D2 (1 �g), pSG-p50 (1 �g), and pSG-cRel (1 �g);4) pGL3-D2 (1 �g), pSG-cRel (1 �g), and pSG (1 �g); 5)pGL3-D2 (1 �g) and pcDNA-p75NGFR (1 �g), using the lipo-fectine (LipofectAMINE Reagent, Invitrogen-Life Technolo-gies, Milano Italy) technique. Transfection efficiency through-out the experiments was monitored by cotransfection with aRenilla luciferase expression vector (50 ng). After transfec-tion, cells were cultured in the complete medium for 48 h.Cells cotransfected with pGL3-D2 and pcDNA-p75NGFR weretreated 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.) accord-ing to the manufacturer’s instructions. Firefly luciferase ac-tivity in each sample was normalized to Renilla luciferaseactivity.

Acknowledgments

We thank Dr. Marc Caron for his gift of the human D2ScDNA; Dr. Karen L. O’Malley, for her gift of the human D2promoter; Dr. Mariagrazia Grilli and Dr. Pierre Jalinot forkindly providing the pSG-p50 and pSG-c-Rel expressionvectors; Dr. Moses V. Chao, for providing the p75NGFR cDNA;Dr Monica Di Luca and Dr. Francesca Colciaghi for their helpand advice in the Western blot studies; Dr. Laura Bianchi forher gift of the pGL3 basic and Renilla vectors; and Dr. Rob-erto Bresciani for his gift of COS-7 cells and his help andadvice in construct preparation and transfection experiments.

Received December 6, 2000. Accepted October 10, 2001.Address all correspondence and requests for reprints to:

C. Missale, Division of Pharmacology, Department of Bio-medical Sciences and Biotechnology, University of Brescia,Via Valsabbina 19, 25123 Brescia, Italy. E-mail: cmissale@med.unibs.it.

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

* These authors contributed equally to this work.

REFERENCES

1. Bothwell M 1995 Functional interactions of neurotrophinsand neurotrophin receptors. Annu Rev Neurosci 18:223–253

2. Lewin GR, Barde YA 1996 Physiology of the neurotro-phins. Annu Rev Neurosci 19:289–317

3. Chao MV, Hempstead BL 1995 p75 and trk: a two-receptor system. Trends Neurosci 18:321–326

4. Kaplan DR, Stephens RM 1994 Neurotrophin signaltransduction by the trk receptor. J Neurobiol 25:1404–1417

5. Greene LA, Kaplan DR 1995 Early events in neurotrophinsignalling via trk and p75 receptors. Curr Top Neurobiol5:579–587

6. Schlessinger J 2000 Cell signaling by receptor tyrosinekinases. Cell 103:211–225

7. Yao R, Cooper GM 1995 Requirement for phosphatidyl-inositol-3 kinase in the prevention of apoptosis by nervegrowth factor. Science 267:2003–2006

8. Dobrowsky RT, Werher MM, Castellino AM, Chao MV,Hannun YA 1994 Activation of the sphingomyelin cyclethrough the low affinity neurotrophin receptor. Science265:1596–1599

9. Brann AB, Scott R, Neuberger Y, Abulafia D, Boldin S,Fainzilber M, Futerman AH 1999 Ceramide signallingdownstream p75 neurotrophin receptor mediates the ef-fects of nerve growth factor on outgrowth of culturedhippocampal neurons. J Neurosci 19:8199–8206

10. Carter BD, Lewin GR 1997 Neurotrophins live or let die:does p75NTR decide? Neuron 18:187–190

11. Carter B, Kaltschmidt C, Kaltschmidt B, Offenhauser N,Bohm-Matthaei R, Baeuerle PA, Barde YA 1996 Selec-tive activation of NF-�B by nerve growth factor throughthe neurotrophin receptor p75. Science 272:542–545

12. Foehr ED, Lin X, O’Mahony A, Geleziunas R, BradshowRA, Greene WC 2000 NF-�B signalling promotes bothcell survival and neurite process formation in nervegrowth factor-stimulated PC12 cells. J Neurosci 20:7556–7563

13. Yoon SO, Casaccia-Bonnefil P, Carter B, Chao MV 1998Competitive signalling between trkA and p75 nervegrowth factor receptors determines cell survival. J Neu-rosci 18:3273–3281

14. Casaccia-Bonnefil P, Carter BD, Dobrowsky RT, ChaoMV 1996 Death of oligodendrocytes mediated by theinteraction of nerve growth factor with its receptor p75.Nature 383:716–719

15. Bredesen DE, Rabizadeh S 1997 p75NTR and apoptosis:trk-dependent and trk-independent effects. Trends Neu-rosci 20:287–291

16. Gentry JJ, Casaccia-Bonnefil P, Carter BD 2000 Nervegrowth factor activation of nuclear factor kB through itsp75 receptor is an antiapoptotic signal in RN22 Schwan-noma cells. J Biol Chem 275:7558–7565

17. Blochl A, Stirrenberg C 1996 Neurotrophins stimulate therelease of dopamine from rat mesencephalic neurons viatrk and p75(lntr) receptors. J Biol Chem 271:21100–21107

18. Stucki CL, Koltzenburg M 1997 The low-affinity neuro-trophin receptor p75 regulates the function but not theselective survival of specific subpopulations of sensoryneurons. J Neurosci 17:4398–4405

19. Anton ES, Weskamp G, Reichardt LF, Mathew WD 1994Nerve growth factor and its low-affinity receptor promoteSchwann cell migration. Proc Natl Acad Sci USA 91:2795–2799

20. Bentley CA, Lee KF 2000 p75 Is important for axongrowth and Schwann cell migration during development.J Neurosci 20:7706–7715

21. Dicou E, Lee J, Brachet P 1986 Synthesis of nervegrowth factor mRNA and precursor protein in the thyroidand parathyroid glands of the rat. Proc Natl Acad SciUSA 83:7084–7088

22. Kanaka-Gantenbein C, Tazi A, Czernichow P, Scharf-mann R 1995 In vivo presence of the high affinity nervegrowth factor receptor TrkA in the rat pancreas: differ-ential localization during pancreatic development. Endo-crinology 136:761–769

23. Kanaka-Gantenbein C, Dicou E, Czernichow P, Scharf-mann R 1995 Presence of nerve growth factor and itsreceptors in an in vitro model of islet cell development:implication in normal islet morphogenesis. Endocrinol-ogy 136:3154–3162

364 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression

24. Miralles F, Philippe P, Czernichow P, Scharfmann R 1998Expression of nerve growth factor and its high-affinityreceptor trkA in the rat pancreas during embryonic andfetal life. J Endocrinol 156:431–439

25. Graham CW, Lynch JH, Djakiew D 1992 Distribution ofnerve growth factor-like protein and nerve growth factorreceptor in human benign prostatic hyperplasia andprostatic adenocarcinoma. J Urol 147:1444–1447

26. Macgrogan D, Saint-Andre JP, Dicou E 1992 Expressionof nerve growth factor and nerve growth factor receptorgenes in human tissues and prostatic adenocarcinomacell lines. J Neurochem 59:1381–1391

27. Paul AB, Grant ES, Habib FK 1996 The expression andlocalization of �-nerve growth factor (�-NGF) in benignand malignant human prostate tissue: relationshipto neuroendocrine differentiation. Br J Cancer 74:1990–1996

28. Pflug BR, Onoda M, Lynch JH, Djakiew D 1992 Reducedexpression of the low affinity nerve growth factor recep-tor in benign and malignant human prostate tissue andloss of expression in four human metastatic prostatetumor cell lines. Cancer Res 52:5403–5406

29. Burnham P, Conner JM, Varon S 1995 Co-localization ofNGF and TSH-like immunoreactivity in cultures of adultrat anterior pituitary cells. J Neurosci Res 41:73–78

30. Missale C, Boroni F, Sigala S, Buriani A, Fabris M, LeonA, Dal Toso R, Spano PF 1996 Nerve growth factor in theanterior pituitary: localization in mammotroph cells andco-secretion with prolactin by a dopamine-regulatedmechanism. Proc Natl Acad Sci USA 93:4240–4245

31. Patterson JC, Childs GW 1994 Nerve growth factor in theanterior pituitary: regulation of secretion. Endocrinology135:1697–1704

32. Patterson JC, Childs GW 1994 Nerve growth factor andits receptors in the anterior pituitary. Endocrinology 135:1689–1696

33. Missale C, Boroni F, Frassine M, Caruso A, Spano PF1995 Nerve growth factor promotes the differentiation ofpituitary mammotroph cells in vitro. Endocrinology 136:1205–1213

34. Paez-Pereda M, Missale C, Grubler Y, Artz E, Schaaf L,Stalla GK 2000 Nerve growth factor and retinoic acidinhibit proliferation and invasion in thyroid tumor cells.Mol Cell Endocrinol 167:99–106

35. Missale C, Codignola A, Sigala S, Finardi A, Paez-PeredaM, Sher E, Spano PF 1998 Nerve growth factor abro-gates tumorigenicity of human small cell lung cancer celllines. Proc Natl Acad Sci USA 95:5366–5371

36. Polak M, Scharfmann R, Seilheimer B, Eisenbarth G,Dressler D, Verma IM, Potter H 1993 Nerve growth factorinduces neuron-like differentiation of an insulin-secretingpancreatic � cell line. Proc Natl Acad Sci USA 90:5781–5785

37. Missale C, Boroni F, Losa M, Giovanelli M, Zanellato A,Dal Toso R, Balsari A, Spano PF 1993 Nerve growthfactor suppresses the transforming phenotype of humanprolactinomas. Proc Natl Acad Sci USA 90:7961–7965

38. De Camilli P, Macconi D, Spada A 1979 Dopamine in-hibits adenylate cyclase in human prolactin-secreting ad-enomas. Nature 278:252–254

39. Missale C, Nash SR, Robinson SW, Jaber M, Caron MG1998 Dopamine receptors: from structure to function.Physiol Rev 78:189–225

40. Pellegrini I, Rosolonjanahari R, Gunz G, Bertrand P, De-livet S, Jedynak CP, Kordon C, Peillon F, Jacquet P,Enjalbert A 1989 Resistance to bromocriptine in prolacti-nomas. J Clin Endocrinol Metab 69:500–509

41. Missale C, Losa M, Sigala S, Balsari A, Giovanelli M,Spano PF 1996 Nerve growth factor controls proliferationand progression of human prolactinoma cell linesthrough an autocrine mechanism. Mol Endocrinol 10:272–285

42. Ross AH, Grob P, Bothwell M, Elder DE, Ernst CS, Ma-rano N, Ghrist BFD, Slemp CC, Herlyn M, Atkinson B,Koprowski H 1984 Characterization of nerve growth fac-tor receptor in neural crest tumors using monoclonalantibodies. Proc Natl Acad Sci USA 81:6681–6685

43. Huber LJ, Chao MV 1995 Mesenchymal and neuronalcell expression of the p75 neurotrophin receptor geneoccur by different mechanisms. Dev Biol 167:227–238

44. Koizumi S, Contreras ML, Matsuda Y, Hama T, Lazarov-ich P, Guroff G 1988 K-252a: a specific inhibitor of theaction of nerve growth factor on PC12 cells. J Neurosci8:715–721

45. Berg MM, Sternberg DW, Parada LF, Chao MV 1992K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity.J Biol Chem 267:13–16

46. Martin-Zanca R, Oskam R, Mitra G, Copeland T, Bar-bacid M 1989 Molecular and biochemical characteriza-tion of the human trk proto-oncogene. Mol Cell Biol9:24–33

47. Maliartchouk S, Saragovi HU 1997 Optimal nerve growthfactor trophic signals mediated by synergy of trkA andp75 receptor-specific ligands. J Neurosci 17:6031–6037

48. Dikic I, Batzer AG, Blaikie P, Obermeier A, Ullrich A,Schlessinger J, Margolis B 1995 Shc binding to nervegrowth factor receptor is mediated by the phosphoty-rosine interaction domain. J Biol Chem 270:15125–15129

49. Knusel B, Hefti F 1992 K-252 compounds: modulators ofneurotrophin signal transduction. J Neurochem 59:1987–1996

50. Gandelman KY, Harmon S, Todd RD, O’Malley KL 1991Analysis of the structure and expression of the humandopamine D2A receptor gene. J Neurochem 56:1024–1029

51. Baeuerle PA, Baltimore D 1996 NF-�B: ten years after.Cell 87:13–20

52. O’Neill LAJ, Kaltschmidt C 1997 NF-�B: a crucial tran-scription factor for glial and neuronal cell function.Trends Neurosci 20:252–258

53. Sen R, Baltimore D 1986 Multiple factors interact with theimmunoglobulin enhancer sequences. Cell 46:706–716

54. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J1995 Inhibition of nuclear translocation of NF-�B by asynthetic peptide containing a cell membrane-permeablemotif and nuclear localization sequence. J Biol Chem270:14255–14258

55. Maggirvar SB, Sarmiere PD, Dewhurst, Freeman RS1998 Nerve growth factor-dependent activation ofNF-�B contributes to survival of sympathetic neurons.J Neurosci 18:10356–10365

56. Sortino MA, Condorelli F, Vancheri C, Chiarenza A, Ber-nardini R, Consoli U, Canonico PL 2000 Mitogenic effectof nerve growth factor (NGF) in LNCaP prostate adeno-carcinoma cells: role of the high- and low-affinity NGFreceptors. Mol Endocrinol 14:124–136

57. Kukstas LA, Domec C, Bascles L, Bonnet J, Venier D,Israel JM, Vincent JD 1991 Different expression of thetwo dopaminergic D2 receptors, D2415 and D2444, in twotypes of lactotroph each characterized by their responseto dopamine and modification of expression by sex ste-roids. Endocrinology 129:1101–1103

58. Guivarc’h D, Vincent JD, Vernier P 1998 Alternative splic-ing of the D2 dopamine receptor messenger ribonucleicacid is modulated by activated sex steroid receptors inthe MMQ prolactin cell line. Endocrinology 139:4213–4221

59. Grilli MG, Ribola M, Alberici A, Valerio A, Memo M, SpanoPF 1995 Identification and characterization of a �B/Relbinding site in the regulatory region of the amyloid pre-cursor protein gene. J Biol Chem 270:26774–26777

60. Liou HC, Baltimore D 1993 Regulation of the NF-�B/Reltranscription factor and I-�B inhibitory system. Curr OpinCell Biol 5:477–487

Fiorentini et al. • p75NGFR and D2 Receptor Expression Mol Endocrinol, February 2002, 16(2):353–366 365

61. Minowa T, Minowa MT, Mouradian MM 1993 Negativemodulator of the rat D2 dopamine receptor gene. J BiolChem 269:11656–11662

62. Valdenaire O, Vernier P, Maus M, Dumas Milne EdwardsJP, Mallet J 1994 Transcription of the rat dopamine-D2-receptor gene from two promoters. Eur J Biochem 220:577–584

63. Wang J, Miller JC, Friedhoff AJ 1997 Differential regula-tion of D2 receptor gene expression by transcription fac-tor AP-1 in cultured cells. J Neurosci Res 50:23–31

64. Yajima S, Lee SH, Minowa T, Mouradin MM 1998 Spfamily transcription factors regulate expression of rat D2dopamine receptor gene. DNA Cell Biol 17:471–479

65. Krezel W, Ghyselink N, Samad TA, Dupe V, Kastner P,Borrelli E, Chambon P Impaired locomotion and dopa-mine signalling in retinoid receptor mutant mice. Science279:863–867

66. Samad TA, Krezel W, Chambon P, Borrelli E 1997 Reg-ulation of dopaminergic pathways by retinoids: activationof the D2 gene promoter by members of the retinoic acidreceptor-retinoid X receptor family. Proc Natl Acad SciUSA 94:14349–14354

67. Baik JH, Picetti R, Saiardi A, Thiriet G, Dietrich A, De-paulis A, Le Meur M, Borrelli E 1995 Parkinsoninan-likelocomotor impairment in mice lacking dopamine D2 re-ceptors. Nature 377:424–428

68. Saiardi A, Bozzi Y, Baik JH, Borrelli E 1997 Antiprolifera-tive role of dopamine: loss of D2 receptors causes hor-monal dysfunction and pituitary hyperplasia. Neuron 19:115–126

69. Asa SL, Kelly MA, Grandy DR, Low MJ 1999 Pituitarylactotroph adenomas develop after prolonged lactotrophhyperplasia in dopamine D2 receptor-deficient mice. En-docrinology 140:5348–5355

70. Arinami T, Gao M, Hamaguchi H, Torn M 1997 A func-tional polymorphism in the promoter region of the dopa-mine D2 receptor gene is associated with schizophrenia.Hum Mol Genet 6:577–582

71. Crenon I, Beraud C, Simard P, Montagne J, VeschambreP, Jalinot P 1993 The transcriptionally active factors me-diating the effect of the HTLV-I Tax transactivator on theIL-2R� �B enhancer include the product of the c-relproto-oncogene. Oncogene 8:867–875

366 Mol Endocrinol, February 2002, 16(2):353–366 Fiorentini et al. • p75NGFR and D2 Receptor Expression