Irene Gutie ´rrez-Can ˜as, Marı ´a G. Juarranz, Beatriz Collado, Nieves Rodrı ´guez-Henche,
Antonio Chiloeches, Juan C. Prieto,* and Marı ´a J. Carmena
UniversidaddeAlcal? a a,Alcal? a adeHenares,Madrid,Spain
BACKGROUND. Neuroendocrine (NE) differentiation in prostate cancer has been correlated
with unfavorable clinical outcome. The mechanisms by which prostate cancer acquires NE
previously observed that vasoactive intestinal peptide (VIP) stimulates cAMP production
mainly through VPAC1receptor, inducing NE differentiation in LNCaP cells. The aim of this
study was to analyze the mechanisms involved in this process.
METHODS. Reverse transcriptase (RT)-polymerase chain reaction (PCR), quantitative real-
time RT-PCR, Western blotting, and immunocytochemistry were performed.
VIP induced NE differentiation of LNCaP cells at a time as short as 1 hr of treatment, and the
same occurred with the expression and secretion of neuronal-specific enolase (NSE, a NE
differentiation marker). These effects were fasterthanthose exerted byserum-deprivation. VIP
induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and NE
differentiation by PKA-dependent and independent pathways, since the PKA inhibitor H89
partially blocked VIP-induced NE differentiation and did not affect ERK1/2 phosphorylation.
be also involved since the inhibitors PD98059 and wortmannin abolished ERK1/2 phosphor-
ylation and decreased NE differentiation induced by VIP. Moreover, VIP activated Ras
suggesting the involvement of a Ras-dependent pathway.
CONCLUSIONS. VIP behaves as autocrine/paracrine factor in LNCaP cells by inducing
NE differentiation through PKA, ERK1/2, and PI3K. Prostate 63: 44–55, 2005.
# 2004 Wiley-Liss, Inc.
KEY WORDS:VIP; protein kinases; LNCaP; NE differentiation; prostate cancer
Neuroendocrine (NE) cells are present within the
pancreas, respiratory, and gastrointestinal systems as
well as in other locations including the prostate gland.
growth and differentiation of normal prostate tissue
as chromogranin A or neuronal-specific enolase (NSE),
and several neuropeptides including neurotensin,
Grant sponsor: Ministerio de Ciencia y Tecnologı ´a; Grant number:
SAF2001-1025; Grant sponsor: Asociacio ´n Espan ˜ola de Urologı ´a
(Premio Leonardo de la Pen ˜a-Yamanouchi).
Marı ´a G. Juarranz’s present address is Departamento de Biologı ´a
Celular, Facultad de Biologı ´a, Universidad Complutense de Madrid,
28040 Madrid, Spain.
*Correspondence to: Juan C. Prieto, Unidad de Neuroendocrinologı ´a
Molecular, Departamento de Bioquı ´mica y Biologı ´a Molecular,
Universidad de Alcala ´, 28871 Alcala ´ de Henares, Spain.
Received 23 January 2004; Accepted 28 June 2004
Published online 5 October 2004 in Wiley InterScience
? 2004 Wiley-Liss,Inc.
calcitonin gene-related peptide (CGRP), bombesin, or
serotonin [2,3]. NE cells may represent a potential
pathway of differentiation of prostate stem cells.
Several clinical studies have suggested that NE cells
may also have a role in the progression of prostate
with unfavorable clinical outcome .
involved in the proliferation and/or differentiation of
various normal and cancer cell lines [5,6]. Regarding
the prostate, VIP and another structurally-related
neuropeptide that share receptors with it, pituitary-
adenylate cyclase activating polypeptide (PACAP),
stimulate rat prostatic epithelial cell proliferation,
induce NE differentiation in LNCaP cells (an andro-
gen-dependent prostate cell line), and protect from
apoptosis in PC-3 cells (an androgen-independent
prostate cell line) [7–10]. These two neuropeptides
exert their biological effects through specific receptors
that belong to family 2 of GPCRs: VPAC1and VPAC2
which recognize VIP and PACAP with the same
affinity, and PAC1that recognizes PACAP with higher
affinity than VIP . In human and rat prostate, VIP/
PACAP receptors are mainly VPAC1receptors [5,12],
and the same occurs in the prostate cancer cell lines
LNCaP and PC-3 [8,10]. Moreover, the expression of
VIP receptors in rat prostate increases during puberty,
a period with high rates of cell proliferation and
differentiation [13,14]. VIP receptors are mainly
coupled to adenylate cyclase as a signal transduction
pathway in normal and cancer prostate cells [7–
10,12,15]. In this context, there is an increasing interest
of cAMP levels is related to the acquisition of NE
phenotype by prostate cancer cells [16,17].
In the present study, we show for the first time that
VIP acts as an autocrine factor in a prostate cancer cell
line, LNCaP. We also demonstrate that VIP induces
NE differentiation and extracellular signal-regulated
kinase 1 and 2 (ERK1/2) phosphorylation by PKA-
dependent and PKA-independent mechanisms. More-
mitogen-activated protein kinase kinase (MEK) and
phosphoinositide 3-kinase (PI3K) are involved in these
effects induced by VIP.
The androgen-dependent human prostate cancer
cell line LNCaP was purchased from the American
Type Culture Collection (Rockville, MD) and routinely
cultured in RPMI-1640 medium (Life Technologies,
Barcelona, Spain) supplemented with 10% heat-inacti-
vated fetal bovine serum (FBS) and 1% penicillin/
streptomycin/amphotherycin B (Life Technologies)
and seeded at a density of 30,000–40,000 cells/cm2.
The culture medium was changed every 3 days. For
LNCaP differentiation, cells were allowed to attach
to plates for 24 hr and then they were washed twice
with PBS and medium was changed to serum-free
Total RNA was prepared from LNCaP cells using
standard techniques. Five micrograms of total RNA
were reverse-transcribed using 6 mg hexamer random
primers and 200 U M-MLV retrotranscriptase (Life
Technologies) in the buffer supplied with the enzyme
supplemented with 10 mM dithiothreitol, 40 U RNasin
(Promega, Madison, WI), and 0.5 mM of deoxyribonu-
cleotides(dNTPs).Two microliters of RT reaction were
then PCR-amplified with specific primers for VIP:
which should give a PCR product of 470 bp. The
GenBank accession number is M36634 and the 50and 30
ends of nucleotides of the fragment amplified are 227–
696.ThePCR conditionswere:denaturation at948Cfor
3 min followed by 40 cycles of 50 sec at 948C, 50 sec at
at 728C. The PCR products were visualized in 2%
agarose gels. The corresponding bands were cut from
the gel, eluted, and automatically sequenced with an
ABI 377 sequencer (Applied Biosystems, Foster City,
Cells were detached with trypsin/EDTA and pel-
As previously described , cell suspensions were
centrifuged onto glass slides (9?104cells/slide), dried
treated for 5 min with methanol/water/H2O2in order
to block endogenous peroxidase. Slides were again
rinsed in PBS and treated with normal rabbit serum
to block background staining. Immunocytochemical
demonstration of immunoreactive VIP (IR-VIP) was
carried out by successive incubation with monoclonal
anti-VIP antibody (1:1,000) for 1 hr, biotin-conjugated
rabbit anti-mouse IgG, and subsequently incubated
with streptavidin–peroxidase for 1 hr followed by
addition of the 3,30-diaminobenzidine-tetrachloride
(DAB) substrate and hydrogen peroxide in PBS.
Methyleneblue wasused forcounterstaining.Controls
for immunocytochemical studies were carried out by
replacing the mouse anti-VIP antibody with PBS and
staining with secondary antibodies according to
the above protocol. The specificity for this antibody
was previously demonstrated . The mouse anti-
VIP antibody was kindly supplied by Helen Wong
(University of California at Los Angeles, CA).
LNCaPcellswere allowed to attach to24-well plates
at a density of 4?105cells in complete medium for
24 hr. Thereafter, the medium was changed to either
10% serum-supplemented medium (control cells) with
or without 100 nM VIP (Neosystem, Strasbourg,
France) or serum-free medium with or without
100 nM VIP. Similar assays were carried out in the
instead of VIP. Medium and peptide were renewed
every 48 hr. After 1 or 96 hr of treatment cells were
photographed using an inverted and phase contrast
microscope Nikon Optiphot-2, and NE differentiated
cells were considered as those bearing neurites of at
least twice the length of cell body. Values lower than
for VPAC1and VPAC2receptors, [K15, R16, L27] VIP
(1–7)/GFR (8–27) and RO 25-1553, respectively, was a
gift from Patrick Robberecht (Universite ´ Libre de
For Western blotting of NSE and phospho-ERK1/2
PBS and 1 mM NaVO3, and pelleted. Then, cells were
lysed in 50 mM Tris-HCl pH 7.5 buffer containing
5 mM EDTA pH 8.0, 1 mM DTT, 0.1% SDS, 1 mM
leupeptin. Protein content was measured using bovine
serum albumin as standard . For NSE, the super-
natants were also collected. Equal amounts of protein
were subjected to SDS–PAGE and blotted onto a
tion, Ann Arbor, MI). Membranes were blocked with
Tris-bufferedsaline (pH7.6) containing5% non-fat dry
milk and 0.05% Tween-20 and then incubated with
rabbit anti-NSE (1:1,000; ICN Biomedical, Aurora,
OH) for 1 hr at room temperature, or mouse anti
phospho-ERK1/2 (1:500; Cell Signaling Technology,
Beverly, MA) at 48C overnight. Horseradish peroxi-
dase-conjugated secondary antibody was used for
detection. Proteins were visualized using an enhanced
chemiluminescence Western blotting analysis system
(Pierce Biotechnology, Rockford, IL). The membranes
of p-ERK1/2 were then stripped and reprobed with
rabbit anti-ERK2 (1:1,000; Santa Cruz Biotechnology,
Santa Cruz, CA) to assess total protein levels.
The amount of mRNA that encodes for NSE was
determined using the SYBR1Green PCR Master Mix
manufacturer (Applied Biosystems). Briefly, reactions
were performed in 20 ml containing 50 ng of total RNA,
10 ml 2? SYBR Green PCR Master Mix, 6.25 U Multi-
10 U RNase inhibitor (Invitrogen), and 0.1 mM specific
actin sense, 50-AGAAGGATTCCTATGTGGGCG-30,
antisense 50-CATGTCGTCCCAGTTGGTGAC-30; and
human NSE sense, 50-CCACATCAACTCCACCAT-
CG-30, antisense, 50-CAGTCCCATCCAACTCCAGC-
30. The GenBank accession numbers and numbers for
the 50and 30ends of nucleotides for the PCR products
are: b-actin, E00829, 1435–1535; and NSE, BC002745,
277–377. The amplification conditions were 30 min at
488C, 10 min at 958C, 40 cycles of denaturation at 958C
for 15 sec, and annealing/extension at 608C for 1 min.
All PCR reactions were performed using ABI-Prism
the identity of PCR products, they were separated by
agarose gel electrophoresis and the bands were cut,
eluted, and automatically sequenced with an ABI 377
sequencer (Applied Biosystems).
For relative quantization, we used a method that
compares the amount of target gene amplification,
NSE, normalized to an endogenous reference, b-actin.
The n-fold differential expression in a specific gene
of a treated sample compared to the control sample
was expressed as 2?DDCt, where Ctwas the mean of
threshold cycle (cycle at which the amplification of the
PCR product is first detected, the larger the starting
quantity of the target molecule, the earlier a significant
increase in fluorescence is observed), DCt was the
the reference gene, b-actin (in each sample assayed),
and DDCtrepresented the difference between the DCt
from the control and each data . Before using this
method, we performed a validation experiment com-
paring the standard curve of the reference and the
target to demonstrate that efficiencies are approxi-
The capacity of Ras-GTP to bind to RBD (Ras-
binding domain of Raf-1) was used to analyze the
46Gutie ¤ rrez-Can ‹ asetal.
100 mm dishes, serum starved for 24 hr and treated
with 100 nM VIP. Cells were washed with ice-cold PBS
and lysed in the culture dish with Ras extraction buffer
(20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl,
5 mM MgCl2, 1% (v/v) Triton X-100, 5 mM NaF, 10%
(v/v) glycerol, 0.5% (v/v) b-mercaptoethanol, 1 mM
NaVO3, 2 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml
leupeptin, and 10 mg/ml pepstatin). Lysates were
clarified by centrifugation at 13,000g for 10 min at
48C, and protein supernatants were incubated for 2 hr
at 48C with glutathione–Sepharose 4B beads pre-
coupled with GST-RBD (1 hr at 48C). The beads were
washed four times in the lysis buffer. Bound proteins
were eluted by the addition of gel loading buffer and
run on 12.5% SDS–PAGE gels. The amount of Ras was
analyzed by Western blotting. Membranes were
blocked with Tris-buffered saline (pH 7.6) containing
5% non-fat dry milk and 0.05% Tween-20 and then
incubated for 2 hr with mouse anti Ras (1:1,000; BD
Transduction Laboratories, Madrid, Spain). For detec-
tion, horseradish peroxidase conjugated secondary
antibody was used. The reaction was visualized by
Super-Signal (Pierce). Total Ras levels were evaluated
by Western blotting of whole cell extracts using the
The results are expressed either as the mean?SEM
or as representative experiments. When appropriate,
statistical significance was assessed by comparing data
with those obtained with starved cells, using the
Student’s t-test. The level of significance was regarded
The expression of mRNA coding for VIP in LNCaP
cells was analyzed by RT-PCR amplification. A single
band was observed in both control and starved cells at
470 bp (Fig. 1A). The integrity of the RNA/cDNA was
confirmed by means of RT-PCR for b-actin (data not
size for PCR amplification of VIP. Furthermore,
sequences were verified by sequencing the PCR
products. After the demonstration of the expression
of VIP mRNA in LNCaP cells, we next studied the
production of the neuropeptide in cell suspensions by
means of immunocytochemical methods. IR-VIP was
detected in cytocentrifuge preparations from control
and serum-deprived LNCaP cells (Fig. 1B, b and d). In
both situations, the reaction products were spread
throughout the cytoplasm. In addition, negative con-
trols were performed by treating the cytocentrifuge
preparations with PBS instead of the primary antibody
(Fig. 1B, a and c).
We have shown previously that the addition of VIP
during 4 days to the culture medium induced NE
morphology in LNCaP cells, even in the presence of
induce such NE differentiation at short times of
treatment. LNCaP cells were grown in serum-supple-
mented medium (control cells), and in serum-free
medium in the presence or absence of 100 nM VIP.
Serum-deprived cells showed neurite outgrowth after
96 hr (Fig. 2C). Cells treatment during 1 hr with VIP,
both in the presence or absence of serum, significantly
increased the percentage of neurite-bearing cells
(Fig. 2B,E, respectively) as compared with control cells
serum-deprived cells. By contrast, 1 hr of serum
deprivation was not enough to increase the number of
neurite-bearing cells as VIP did (Fig. 2D). Selective
VPAC1agonist exerted the same effect as VIP (Fig. 2F
and data not shown). However, selective VPAC2
agonist did not induce NE differentiation (Fig. 2G),
suggesting that the receptor involved in this VIP action
is the VPAC1receptor.
Figure 3A shows the expression of NSE mRNA, as
measured by quantitative real-time RT-PCR. VIP
for NSE after 1 or 96 hr of treatment, mainly in the
presence but also in the absence of serum in the culture
medium. Figure 3B shows immunoblotting results on
with 100 nM VIP treatment. VIP was able to increase
NSE levels in the lysates from LNCaP cells after 1 hr of
VIP treatment both in the presence or absence of serum
in the culture medium (Fig. 3B). After 96 hr in the
absence of serum, cells showed an important increase
of NSE level, similar to that obtained after 96 hr of VIP
treatment, both in the presence or absence of serum
(Fig. 3B). This increase in the NSE presence was
corresponded with an increase in NSE secretion to the
medium (Fig. 3C). We tested if the effect of VIP on NSE
production was also related with an effect at transcrip-
tional level. The NSE production induced by serum
deprivation during 1 hr reached a lesser extent than
that produced by VIP, indicating that VIP effect on NE
differentiation is faster than serum deprivation effects
in LNCaP cells.
VIPStimulates NEDifferentiationinLNCaPCells 47
To explore the signaling pathways that could be
involved in VIP-induced NE differentiation, LNCaP
cells were cultured, in the presence or in the absence of
or more of three different protein kinase inhibitors:
PKA inhibitor H89, MEK1/2 inhibitor PD098059, or
PI3K inhibitor wortmannin. After each treatment, we
measured the percentage of NE differentiated cells as
thosebearing neuritesof at least twice the length of cell
48 Gutie ¤ rrez-Can ‹ asetal.
a control of NE differentiation. Treatment of control
cells (LNCaP cells cultured in medium supplemented
with 10% FBS) or serum-deprived cells with 100 nM
VIP for 1 hr was enough to induce a NE differentiation
similar to that exerted by serum deprivation for 96 hr.
effect of VIP. Inclusion of PD098059 decreased VIP
effect on NE differentiation although the MEK1/2
inhibitor did not completely abolish the VIP effect.
Similar results were obtained with H89 or wortmannin
treatment, although inhibition of NE differentiation
observed with these kinases inhibitors was less
pronounced than that exerted by PD098059. In addi-
tion, the combination of two inhibitors was not enough
to abolish the VIP effect but all the inhibitors together
were able to do it. Some of these results obtained in the
absence of serum are shown in Figure 2 (H–K).
Fig. 2. VIPinducesneuroendocrine(NE)phenotypeinLNCaPcellsatshortperiodsoftreatment.Controland96hrserum-deprivedcellsare
VIPStimulates NEDifferentiationinLNCaPCells 49
2 phosphorylation in a time-dependent manner, the
maximal effect being at 10–15 min and then declining
to control levels after 1 hr of VIP treatment. The effect
was also observed in serum-deprived LNCaP cells, but
it reached a lesser extent. We used the same kinase
inhibitors as above to explore the different pathways
involved in ERK1/2 activation by VIP (Fig. 5B). Cell
treatment with 10 mM PD098059 fully abolished VIP-
induced ERK1/2 phosphorylation whereas 10 mM H89
50 Gutie ¤ rrez-Can ‹ aset al.
5 indicate that MEK1/2 activation is essential for VIP-
induced activation of ERK1/2 but it is not sufficient to
elicit VIP-induced NE differentiation in LNCaP cells.
By contrast, blocking PKA activation with H89 had no
effect on VIP-induced ERK1/2 phosphorylation but
decreased the morphological changes exerted by the
peptide. On the other hand, wortmannin partially
blocked both ERK1/2 phosphorylation and morpholo-
gical changes induced by VIP, suggesting a role for
PI3K in mediating the effects of VIP in LNCaP cells.
Since VIP-induced ERK1/2phosphorylationrequir-
ed MEK1/2 activation and this is one of the down-
stream targets of Ras, we analyzed whether VIP was
was able to increase Ras-GTP levels in LNCaP cells
lysates in a time-dependent manner, in the absence of
serum in the culture medium. Maximal activation of
Raslevelswere observed at5–10 minof VIPtreatment,
and then Ras-GTP content decreased (data not shown).
This result suggests that VIP-induced Ras activation
occur previously to ERK activation, since maximal
ERK1/2 activation was observed at 10–15 min of VIP
As we observed that PI3K is involved in this
signaling machinery and this kinase can be positioned
presentincontrolLNCaPcells after1hrof treatmentwith100 nM
H89 (PKAinhibitor),and100 nM wortmannin (PI3Kinhibitor).For
mediumareincluded.Resultsare themean?SEMof four separate
experiments performedin duplicate. **P<0.01, ***P<0.001com-
paringvaluesforeffectsofinhibitors ofproteinkinaseswith those
inVIP-treatedcells alone.B:Percentage ofneurite outgrowthpre-
sent in control LNCaP cells after1hr treatment with100 nM VIP,
For comparison, results after 96 hr of cell incubation in serum
deprived medium are included. Results are the mean?SEM of
four separate experiments performed in duplicate. **P<0.01,
***P<0.001 comparing values for effects of inhibitors of protein
Signal transduction pathwaysinvolvedinVIP-induced NE
trol and serum-deprived LNCaP cells after different periods of
lysis of ERK1/2 protein phosphorylation in cell lysates of control
of the sameprotein kinaseinhibitorsmentioned above.ERK2 was
A: Immunoblot analysis of extracellular signal-regulated
upstream or downstream of Ras, we analyzed if
wortmannin was able to inhibit the Ras activation
inducedbyVIP. Inthiscase,weshowthat wortmannin
presence or in the absence of serum in the culture
medium at 5 min, i.e., when the maximal activation
exerted by VIP was achieved (Fig. 6B).
Since wortmannin partially blocked both ERK1/2
phosphorylation and NE differentiation induced by
VIP, we tried to analyze whether VIP was able to
activate Akt/PKB, one of the downstream proteins of
PI3K. By means of immunoblotting, we did not detect
activated in this cancer cell line .
Mechanisms implicated in the multiple cell pheno-
types in human tumors are poorly understood. How-
ever, the microenvironment is likely to play a critical
role in growth and control of phenotypic properties in
a neuropeptide, VIP, can act as an autocrine/paracrine
product in an androgen-dependent human prostate
cancer cell line (LNCaP), and that VIP exerts an
important effect on the acquisition of NE morphology
in these cells through the activation of different path-
ways in which are involved the kinases PKA, ERK1/2,
Although the clinical significance of NE differentia-
tion in prostate cancer cells is still not well understood,
there is a clear relationship between the tumor grade
and a poor prognosis in prostate cancer, related with
the development of an androgen-resistant state .
Neuropeptides, growth factors, and cytokines operat-
ing either within the context of androgen-dependent
signaling mechanisms or in an androgen-independent
context, are likely to be critical in the process of
prostate cancer progression [1,2]. The presence of VIP
has been described in autonomic nerves surrounding
the human prostate acini  and we have recently
described that an androgen-independent prostate can-
cer cell line, PC-3, is able to produce VIP . Another
member of the VIP family of peptides, PACAP, is also
present in the epithelial layer of normal and carcino-
cell lines produce it [9,10]. This is important since VIP
and PACAP can act in the prostate gland as autocrine/
paracrine factors, modulating cell phenotypes.
the activity of adenylate cyclase enzyme [8,10,14]. We
have previously shown that VIP induced NE differ-
entiation in LNCaP cells after 96 hr of treatment at a
similar extent than that observed after serum-depriva-
tion . Present results show that VIP and VPAC1
agonist were able to induce similar morphological
changes in LNCaP cells at times as short as 1 hr of
treatment, both in the presence or absence of serum.
Moreover, those changes were accompanied with an
increased expression and secretion of NSE, a protein
accepted to be secreted by cells with a NE phenotype.
Both morphological changes and augmented NSE
independent on serum. However, it has been demon-
strated that agents that increase intracellular levels of
Thus, the production of cAMP associated with VPAC1
activation  seems to be enough to develop VIP-
induced NE differentiation in LNCaP cells.
We explored the VIP-activated molecular mechan-
isms that lead LNCaP cells to acquire the NE pheno-
type. It has been previously shown that elevation of
cAMP levels potentiates ERK activation by growth
factors  and by G-protein coupled receptors .
NE differentiation in LNCaP cells . Here, we de-
monstrate that VIP, probably through cAMP elevation,
is able to activate ERK1/2 at 3 min of treatment and
maintains such ERK1/2 phosphorylation until 1 hr of
peptide treatment, both in the presence or absence of
serum. However, the possibility exists that PKA could
Fig. 6. VIP increase of endogenous Ras-GTP in LNCaP cells.
A: Serum-deprivedLNCaPcellswere treatedwith100 nMVIP for
different times. Ras activation was measured by GST-RBD pull-
Rasproteinlevels. Arepresentative experimentof three othersis
with100 nM VIP in the presence of100 nM wortmannin and the
52 Gutie ¤ rrez-Can ‹ asetal.
serum to exert its effects on ERK1/2 phosphorylation
and NE differentiation, some of them could potentiate
it. Moreover, VIP-stimulated ERK1/2 phosphorylation
was completely abolished by the MEK inhibitor
PD098059 and it resulted unaffected by the PKA
inhibitor H89. These results indicate that the effect of
VIP on ERK1/2 phosphorylation is dependent of
MEK activation and PKA-independent. However, the
fact that H89 does not affect ERK activation by VIP
but decreases NE differentiation rises the possibility
that this PKA inhibitor could be acting at some point
downstream of ERK to inhibit NE differentiation. In
this regard, it has been shown that H89 inhibits the
mitogen- and stress-activated protein kinase 1 (MSK1),
a kinase associated with the nucleosomal response
also demonstrated that an increase of cAMP levels is
able to activate MEK through Rap-1/B-Raf in different
ERK1/2 phosphorylation could be mediated by this
pathway, as previously demonstrated for PACAP in
LNCaP and PC12 cells [9,31] and for VIP in HT-29
cells . Moreover, another cAMP binding protein
besides PKA could mediate ERK activation by VIP.
In this sense, a family of cAMP-responsive guanine
nucleotide exchange factors (GEFs) for Rap and Epac
has been identified and shown to be responsible for
cAMP effects independently of PKA activation .
Here, we demonstrate that VIP is able to increase the
levels of GTP-Ras, suggesting that Ras participates in
the signaling pathways induced by VIP. However, we
have not determined whether Rap is also involved
on NE differentiation even when the levels of cAMP
are increased by VIP. Accordingly with these results,
multiple Ras-dependent pathways converge in the
regulation of differentiation in PC12 cells, in which the
effect of Ras on neurite extension is mimicked by Raf-1
and PI3K .
We show in the present study that ERK phos-
phorylation and NE differentiation induced by VIP are
inhibited in wortmannin treated cells, indicating that
PI3K is required at some point upstream of ERK. In
this sense, PI3K has been shown to act as an early
intermediate of ERK1/2 activation in response to
GPCRs, PI3K being positioned upstream or down-
stream of Ras . In the present system, we have
can contribute to GPCR-mediated ERK1/2 activation
acting upstream of Ras by a mechanism involving Gbg
subunits,adaptor proteins,tyrosine phosphatases, and
as Ras effector and regulatory effects of PI3K on Raf-1
have been described in some but not other cell types
[39–41]. Although both possibilities can occur in our
system, it is more likely that PI3K contribute to VIP-
mediated ERK1/2 activation upstream of Ras by a
mechanism involving the Gbg subunits released from
Gas subunits upon VPAC1 receptor activation. How-
ever, the molecular events linking VIP to PI3K, and
PI3K to ERK1/2 activation and NE differentiation in
LNCaP cells remain to be established.
Taken together, our findings show that LNCaP cells
factor to induce NE differentiation in prostate cancer
cells. This is true after both short (this study) and long
 periods of VIP treatment, and in the presence or
absence of serum in the culture medium. The possibi-
lity exists that the short time course of VIP action
described in the present report may be related in some
extent to a priming effect of endogenous VIP produc-
tion by LNCaP cells for the effects of exogenously
added VIP. The effects of VIP involved different
signaling pathways, including PKA-dependent and
PKA-independent pathways. Furthermore, ERK1/2
activation induced by VIP is required to produce NE
differentiation through a signaling pathway that
includes PI3K kinase and probably Ras. Our observa-
tion on that a combination of inhibitors to MEK, PI3K,
and PKA completely inhibits NE differentiation in
LNCaP cells suggests that there are not yet other path-
ways involved. If we summarize all VIP effects on
prostate cells, proliferation on normal epithelial cells
, NE-differentiation in the androgen-dependent
cancer cell line LNCaP ( and this study), and
protection from apoptosis in the androgen-indepen-
dent cancer cell line PC-3 , we can propose that VIP
acts as an autocrine/paracrine neuropeptide which is
implicated in prostate cancer progression from an
androgen-dependent state to an androgen-indepen-
dent state, a pattern which is an index of increasing
aggressiveness of the tumor.
We thank Patrick Robberecht (Universite ´ Libre de
Bruxelles, Belgium) for the VPAC1and VPAC2ago-
nists; Helen Wong (UCLA School of Medicine, Los
Angeles, CA) for anti-VIP antibody; and Rosa P.
Gomariz (Universidad Complutense, Madrid, Spain)
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