We studied the involvement of the mitogen-acti- vated protein kinase MAPK pathway in regulating ChAT expression in a murine septal cell line.. Fax: + 1 617 638 5400, Tel.: + 1 617 638 59
Trang 1Mitogen-activated protein kinase kinase negatively modulates
ciliary neurotrophic factor-activated choline acetyltransferase
gene expression
Tiffany Mellott’, Ignacio Lopez-Coviella”, Jan Krzysztof Blusztajn'? and Brygida Berse’
' Department of Pathology and Laboratory Medicine and ? Department of Psychiatry, Boston University School of Medicine;
Boston, MA, USA
The expression of the choline acetyltransferase (ChAT)
enzyme that synthesizes the neurotransmitter acetylcholine
(ACh) is upregulated by ciliary neurotrophic factor
(CNTF) We studied the involvement of the mitogen-acti-
vated protein kinase (MAPK) pathway in regulating
ChAT expression in a murine septal cell line Surprisingly,
we found that PD98059 and U0126, two structurally dis-
tinct inhibitors of MAPK kinase (MEK1), increased both
basal and CNTF-induced ACh production Transient
transfections with ChAT promoter-luciferase reporter
construct demonstrated synergy between PD98059 and
CNTF at the transcriptional level Moreover, in cotrans-
fection studies, overexpression of constitutively activated
MEK 1 completely abrogated the CNTF-mediated induc- tion of the reporter Blocking MEK 1 did not significantly alter CNTF-induced Tyr705 phosphorylation of the prin- cipal mediator of the CNTF pathway, the transcription factor Stat3 However, PD98059 inhibited Ser727 phos- phorylation of Stat3, demonstrating that the latter is MEK I-dependent Taken together, these results indicate that activation of the MEK1/MAPK pathway inhibits the CNTF-mediated stimulation of ChAT expression, possibly
as a part of a feedback mechanism
Keywords: acetylcholine; choline acetyltransferase; MAP kinase; ciliary neurotrophic factor; Stat3
Biosynthesis of the neurotransmitter acetylcholine (ACh)
from choline and acetyl coenzyme A is catalyzed by the
enzyme choline acetyltransferase (ChAT, acetyl-CoA:cho-
line O-acetylotransferase, EC 2.3.1.6) [1] Following syn-
thesis, ACh is transported into synaptic vesicles by vesicular
acetylcholine transporter (VAChT) ChAT and VAChT are
encoded within one gene locus and share regulatory
elements for transcription [2] Recent studies have shown
that the two genes are coordinately regulated in response to
various extracellular factors, including nerve growth factor
(NGF) and ciliary neurotrophic factor (CNTF) [3-10]
Signal transduction pathways involved in this regulation are
largely unknown Quirin-Stricker ef al identified several
transcription factors involved in transcriptional activation
of the human cholinergic locus by NGF and demonstrated
that cyclic AMP-dependent protein kinase (PKA) is
Correspondence to B Berse, Department of Pathology and Laboratory
Medicine, Boston University School of Medicine, 85 East Newton
Street, Room M1006, Boston, MA 02118, USA
Fax: + 1 617 638 5400, Tel.: + 1 617 638 5960,
E-mail: berse@bu.edu
Abbreviations: ACh, acetylcholine; ChAT, choline acetyltransferase;
CNTF, ciliary neurotrophic factor; DMEM, Dulbecco’s modified
Eagle’s medium; ERK, extracellular signal-regulated kinase; JAK,
Janus kinase; LIF, leukemia inhibitory factor; MAPK, mitogen-acti-
vated protein kinase; MEK, mitogen-activated protein kinase kinase;
NGF, nerve growth factor; PKA, cyclic AMP-activated protein kin-
ase; STAT, signal transducer and activator of transcription; VAChT,
vesicular acetylcholine transporter; VIP, vasoactive intestinal peptide
(Received 6 June 2001, revised 12 November 2001, accepted 30
November 2001)
important for this process [11] Recently, it was shown that regulation of the rat cholinergic gene locus by cyclic AMP is mediated by PKA type II, but not by PKA type I [12]
In a previous study, we demonstrated that NGF, acting through receptor tyrosine kinase TrkA, can activate cholinergic gene expression and ACh production in the murine septal cell line SN56T17 [9] At the same time, NGF downregulates the CNTF-induced increases in ACh pro- duction, pointing to interference between the NGF and CNTF signaling pathways As the major signaling event evoked by NGF in many cell types, including SN56T17, is the activation of mitogen-activated protein kinase (MAPK),
we investigated the role of this pathway in regulating the cholinergic phenotype and specifically in modulating the CNTF effect
MAPK cascades are present in all eukaryotic cells and are utilized in many signal transduction pathways origi- nating from receptors at the cell surface [13] The Ras/Raf/ ERK pathway is activated by growth factors and cyto- kines, including NGF and CNTF [14,15] The transient formation of Ras-GTP and activation of Raf kinase at the cell membrane eventually leads to the phosphorylation of the two members of MAPK family, ERK1 (p44MAPK) and ERK2 (p42 MAPK) by MAPK kinase | (MAPKK1
or MEK1) [14] Activated ERKs phosphorylate and regulate numerous targets in the cytoplasm and in the nucleus, including other protein kinases, transcription factors, cytoskeletal proteins, growth factor receptors, and other regulatory proteins [13] Specificity of MAPK effects in different cell types and in response to different stimuli is achieved by the action of scaffolding proteins, which bring together the components of a given MAPK pathway [16]
Trang 2In this study we employed two distinct noncompetitive
MEK 1 inhibitors to prevent the phosphorylation of MAPK
by MEK1 in SNS56T17 cells We report that inhibiting the
MEK1/MAPK pathway increases both basal and CNTF-
induced ACh synthesis and ChAT promoter activity in
SN56T17 cells Moreover, introducing constitutively active
MEK 1 into the cells by transient transfection abrogates the
stimulatory effect of CNTF Experiments with the JAK/
STAT pathway inhibitor tyrphostin AG490 suggest that
CNTF-induced ACh production is JAK/STAT dependent
Blocking MAPK does not significantly influence tyrosine
phosphorylation of the transcription factor Stat3 evoked by
CNTF, but it reduces its serine phosphorylation We
propose that the MEK1/MAPK cascade negatively regu-
lates ACh synthesis, possibly as a part of a feedback
mechanism
EXPERIMENTAL PROCEDURES
Materials
PD98059 was obtained from Calbiochem-Novabiochem
(La Jolla, CA, USA) U0126, luciferase reporter vector
pGL3-Basic, and Luciferase Assay System were from
Promega (Madison, WI, USA) The expression plasmid
pUSE-MEKI(activated), as well as the control vector
pUSE were obtained from Upstate Biotechnology (Lake
Placid, NY, USA) CNTF was purchased from R & D
Systems (Minneapolis, MN, USA) and Pepro Tech Inc
supplement, and LipofectAMINE were obtained from
Gibco BRL (Bethesda, MD, USA) Polyclonal anti-
ChAT Ig ABI44P and ABI143 were purchased from
Chemicon (Temecula, CA, USA), and all other antibod-
ies [anti-(phospho p44/42 MAPK) Ig, anti-Stat3 Ig, anti-
(phosphoSer727 Stat3) Ig, anti-(phosphoTyr705 Stat3) Ig]
were from New England Biolabs (Beverly, MA, USA)
Immobilon P membrane for Western blotting was from
Millipore (Bedford, MA, USA) Renaissance reagent for
chemiluminescence was obtained from New England
Nuclear (Boston, MA, USA) Sigma Chemical Co
(St Louis, MO, USA) supplied all other chemicals and
reagents
Cell culture and treatments
The mouse septal cell line SN56 (generously provided by
B H Wainer, University of Chicago, IL, USA), is a
product of the fusion of septal neurons from postnatal
day 21 mice with murine neuroblastoma N18TG2 The
SN56T17 cell line was obtained by stable transfection of
SN56 cells with rat trkA cDNA [9] Both cell lines were
maintained at 37 °C in an atmosphere of 95% air, 5%
CO, in Dulbecco’s modified Eagle’s medium (DMEM)
containing | mm pyruvate and 10% fetal bovine serum
LA-N-?2 cells (a generous gift from R Seeger, University
of California, LA, USA) were grown in Leibowitz L-15
medium containing 10% fetal bovine serum at 37 °C in
an atmosphere of 100% air During treatment periods
with test compounds, cells were grown in DMEM
containing N2 supplement [17] instead of fetal bovine
serum, and the medium was changed every 24 h
Acetylcholine measurements The intracellular ACh content was measured by HPLC as described in Berse ef al [9], except 50 tum neostigmine was used in the physiological salt solution
Western blotting and immunoprecipitation For Western analysis, cell extracts were prepared by adding lysis buffer (50 mm Tris pH 7.5, 150mm NaCl, 1% Nonidet NP-40, 10% glycerol, 2mm 4-(2-aminoethyl)- benzenesulfonyl fluoride, 1 g-mL~ leupeptin, 2 pgemL! aprotinin, 2 ugmL' pepstatin) to a 60-mm culture dish, scraping the cells into a tube, mixing, incubating for 15 min
on ice, and briefly centrifuging to clear The extracts were normalized for total protein content and separated by SDS/ PAGE After blotting, membranes were blocked using 5% nonfat milk in a NaCl/P;/Tween solution pH 7.4 (contain-
ing 140 mm NaCl, 3 mm KCl, 10 mm Na,HPO,, 2 mm KH>POq, 0.15% Tween 20) The primary antibodies were
used at the dilutions recommended by the manufacturers The antibody—antigen complexes were detected with anti- (rabbit IgG) Ig peroxidase conjugates and visualized using the chemiluminescence method and Kodak X-Omat AR film Band intensities were quantified with a densitometric scanner and NIH mace or Molecular Dynamics IMAGE-
QUANT NT software
For immunoprecipitation, cell lysates were prepared as described above Cell extracts were incubated overnight with either polyclonal anti-ChAT Ig AB144P (2 pg per
500 ug of extract) or polyclonal anti-Stat3 Ig (1.5 ug per
500 ug of extract) and protein A Sepharose (3.5 mg
per sample) in a rotary mixer at 4 °C Samples were briefly
centrifuged and washed three times The pellet was resus- pended in lysis buffer and proteins were electrophoretically separated by SDS/PAGE
Reporter gene assay The ChAT luciferase reporter construct has been described previously [18] Briefly, a 4.8-kb Xhol—HindIII portion of the ChAT promoter region was excised from a 6.4-kb clone
of mouse genomic DNA (GenBank accession no AF019045) [19,20], kindly provided by J Naciff (University
of Cincinnati College of Medicine, Cincinnati, OH, USA)
The 4.8-kb fragment stretches from position —4817 (relative
to the ChAT translation start) to position +46, and contains the proximal N and M promoters of the ChAT gene It was inserted between the XhoI and HindIII sites upstream of the luciferase coding region of the vector pGL3-
Basic For cotransfection studies, the luciferase construct
was used together with the pUSE-based expression plasmid containing rat MEK 1 cDNA under the control of the CMV promoter MEK 1 cDNA in this construct carries mutations substituting two regulatory serine residues with aspartic acid, which results in constitutive kinase activity SNS6T17 cells were transfected using LipofectAMINE in serum-free
DMEM Three to four hours after transfection, the medium
was replaced with DMEM containing 10% fetal bovine serum and the test compounds The cells were treated for
48 h (with one medium change) and luciferase activity was measured with the Luciferase Assay System, according to
Trang 3the manufacturer’s protocol Light intensity was measured
in the Lumat LB 9501 semiautomatic luminometer (EG &
G Berthold, Bad Wildbad, Germany), and expressed in
arbitrary units
Statistical analysis
One- or two-way analysis of variance, as appropriate, and
Tukey’s test were performed with the help of SYSTAT
statistical software (SPSS Inc., Chicago, IL, USA) using
Macintosh computers
RESULTS
To address the relationship between MAPK activation and
ACh synthesis, we used PD98059, a specific MEKI
inhibitor [21] It has been previously shown that PD98059
decreases ERK1 (p44) and ERK2 (p42) activation (mea-
sured as the degree of threonine/tyrosine phosphorylation)
in a variety of cell lines following short incubations with the
inhibitor [21,22] First we tested the inhibitor’s effectiveness
at blocking basal and CNTF-enhanced MAPK phospho-
rylation in our model septal cell line SN56T17 We probed
Western blots with an antibody that recognizes the ERK1
and ERK2 only when phosphorylated on Tyr and Thr
residues The basal level of phosphorylated ERK1 and
ERK2 in SNS6T17 cells is easily detectable with this
antibody Incubation with CNTF for 15 min increased the
level of phosphorylated ERK1/2 by 40% (Fig 1A) In
similar experiments, we determined that treatment with
CNTF for time periods up to 30 min increased ERK1/2
phosphorylation in these cells up to twofold (data not
shown) Pretreatment with 10 um PD98059 for 15 min
almost completely obliterated MAPK phosphorylation in
both CNTF-treated and untreated cells (Fig 1A) Our
results are in agreement with those of Bartoe & Nathanson,
who also observed a high level of basal MAPK activation in
SN56 cells that could be further stimulated about twofold
by a related cytokine, leukemia inhibitory factor (LIF) [23]
Both basal and LIF-stimulated MAPK activity could be
eliminated by PD98059 [23]
A
CNTF 0 5 15 0 5 15
p42 >———
We then investigated whether the downregulation of MAPK phosphorylation was maintained following an extended treatment with PD98059 When SN56T17 cells were cultured in the presence of the inhibitor for 48 h, a decrease of up to 70% in MAPK phosphorylation level was visualized by immunoblotting (Figs 1B,C) After protracted serum withdrawal in a nonsupplemented medium, the basal MAPK phosphorylation level was greatly reduced and a 30- min treatment with CNTF resulted in an eightfold induction
of MAPK phosphorylation, which was almost completely prevented by pretreatment with the inhibitor (Fig 1B) In cells grown in N2-supplemented medium in the presence of CNTF for 48 h, there was no significant difference in MAPK _ phosphorylation between control and CNTF- treated cells, and PD98059 again decreased MAPK activa- tion by 50-70% Factors present in serum and insulin in the N2-supplemented medium could be responsible for the residual MAPK activation, since MAPK phosphorylation
is completely eliminated in these cells following a prolonged incubation with PD98059 in a nonsupplemented medium (data not shown)
In order to investigate the role of MEKI/MAPK pathway in ACh synthesis, we first measured the effect of inhibiting this pathway on the intracellular ACh level We examined the SNS6T17 cells and their parental cell line
SN56, as well as the human neuroblastoma cell line LA-N-2
In order to rule out unspecific effects of PD98059, we also tested another noncompetitive MEK1 inhibitor, U0126 The two compounds are structurally unrelated, although it remains unclear if they share a common binding site on MEK 1 [24] Both inhibitors are highly specific to the MEK 1/2/ERK pathway, as they do not affect other MAPK pathways MEKK1-3, MKK4/JNK, or MKK6/p38, or numerous other protein kinases tested [22,24,25] In each cell line, there was a statistically significant effect of both MEK 1 inhibitors on ACh production as compared to con- trols, and both inhibitors were equally effective (Fig 2A) The effect of the inhibitors on LA-N-2 cells was smaller than
on the mouse cell lines, probably because of lower basal MAPK activity in LA-N-2 cells as compared to SN56 cells (data not shown) A time-course study revealed that 10 tm
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Fig 1 Effect of PD98059 on MAP kinase phosphorylation in SN56T17 cells (A) The cells were serum-starved for 3 h, pretreated with 10 um PD98059 for 15 min, and then treated with 20 ngmL~' CNTF for the indicated periods of time Cell lysates (40 yg per lane) were analyzed by Western blotting with an antibody specific to doubly phosphorylated forms of ERK1 (p44) and ERK2 (p42) The results are representative of two experiments (B) The cells were grown for 48 h either in the presence or in the absence of 10 um PD98059 (PD), serum-starved for 6 h, and then treated with 20 ngmL~! CNTF for 30 min Western blotting was performed as described in (A) The results are representative of two experiments (C) The cells were grown for 48 h either in the presence or in the absence of 10 um PD98059 (PD), 20 nemL~! CNTF, or both Western blotting was performed as described in (A) The results are representative of four experiments.
Trang 4
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Control PD U Control PD U Control PD U
Fig 2 MEK1 inhibitors increase ACh production in cholinergic cell lines (A) SN56T17, SN56, and LA-N-2 cells were grown for 48 h in the presence
or absence of 10 tm PD98059 (PD) or 10 um U0126 (U) Intracellular ACh was measured in cell extracts as described in Experimental procedures The results are presented as means + SEM of two experiments, each performed in quadruplicate Acetylcholine levels in cells treated with PD98059 and U0126 were statistically different from control values for each cell line (SNS56T17 P < 0.001 and P < 0.005, respectively, SN56 P < 0.001 and P < 0.001, respectively; and LA-N-2 P < 0.01 and P < 0.01, respectively), and were not statistically different from each other (SN6T17
P = 0.834;SN56 P = 0.765; and LA-N-2 P = 0.977); Tukey’s test (B) Time Course: the SN56T17 cells were grown for 24, 48 or 72 h in the presence or absence of the MEK inhibitor PD98059 (10 um) The results are presented as means + SEM and are representative of three experiments (C) Concentration-response curve of PD98059: the SN56T17 cells were treated for 48 h in the presence of various PD98059 concentrations (0, 1, 3, 5, 10, 15, and 20 pm) The data are representative of three experiments A rectangular hyperbola was fitted to the data, permitting the estimation of the median effective concentration (ECsp = 3.7 pm)
PD98059 produced a twofold increase in ACh levels in
SN56T17 cells and that the full effect could be observed
after 48 h of treatment (Fig 2B) Following a 48-h treat-
ment, PD98059 increased the intracellular levels of ACh in
SN56T17 cells in a concentration-dependent and saturable
manner (Fig 2C), with an ECs of 3.7 um, comparable to
the reported ICsg of 4 um on MEK 1 activity im vitro [21,22]
Thus, these data suggest that the increase in intracellular
ACh levels is a result of down-regulating MEK 1-mediated
MAPK activation
Because our previous studies have shown that CNTF
upregulates the cholinergic phenotype and that NGF
strongly activates MAPK in SN56T17 cells while interfering
with the actions of CNTF [5,9], we examined the effect of
PD98059 and U0126 on the ability of CNTF to increase
ACh synthesis The results are presented in Fig 3A
Consistent with our previous findings, CNTF alone signi-
ficantly increased intracellular ACh level by more than
threefold PD98059 increased ACh level by twofold, while
the combined treatment with CNTF and PD98059
increased ACh content by sixfold Substituting U0126 for
PD98059 resulted in a similar effect on CNTF-stimulated
ACh production Similar results were obtained with SN56
and LA-N-2 cells (data not shown) This increase in
intracellular ACh levels was the result of an increased
amount of ChAT protein, as demonstrated by immunopre-
cipitation followed by Western blotting with two polyclonal
anti-ChAT Ig (Fig 3B) We measured ChAT protein in
SN56T17 cells which had been treated with PD98059
(10 pm), CNTF (20 ngmL7'), or the combination for 48 h
Both PD98059 and CNTF increased ChAT protein levels,
and the combined treatment with PD98059 and CNTF had
the greatest effect on the level of ChAT protein (Fig 2B)
In summary, both PD98059 and CNTF are able to increase
intracellular ACh levels via increasing ChAT protein levels
Furthermore, even though the MAPK signaling is required
for CNTF-mediated activation of certain genes in the CNS
[26], in this case, blocking MAPK activation enhances the
CNTF effect on ACh production
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Fig 3 Up-regulation of the cholinergic phenotype by CNTF and MEK inhibitors The SNS6T17 cells were grown for 48 h in the presence or absence of 10 um PD98059 (PD), 10 um U0126 (U), 20 ngmL! CNTF, or combinations (A) MEK inhibitors and CNTF increase intracellular ACh levels Intracellular ACh was measured in cell extracts as described in the Experimental procedures The results are presented as means + SEM of 12 samples (four experiments, each performed in triplicate) Two-way analysis of variance revealed a sig- nificant effect of PD98059 (P < 0.001), U0126 (P < 0.005), and CNTF (P < 0.001), and a significant interaction between PD98059 and CNTF (P < 0.05) and between U0126 and CNTF (P < 0.01) (B) ChAT protein levels are increased following PD98059 and CNTF treatment Cell lysates (250 ug per sample) were immunoprecipitated with anti-ChAT Ig AB144P, and then analyzed by Western blotting using anti-ChAT Ig AB143 Similar results were obtained in four additional experiments.
Trang 5In order to examine whether the effect of MAPK on the
cholinergic phenotype involves changes in gene expression,
we employed transient transfection assays to measure the
activation of the ChAT promoter by PD98059 and CNTF
We used a ChAT-luciferase construct containing the
proximal mouse ChAT promoter region linked to a
luciferase reporter gene as described previously [18]
Following transfection with the reporter construct,
SN56T17 cells were treated with PD98059, CNTF, or both,
for 48 h and assayed for luciferase reporter activity (Fig 4)
Luciferase activity increased ~ fourfold following CNTF
treatment and twofold with PD98059 Substituting U0126
for PD98059 resulted in a similar effect on luciferase activity
(data not shown) Moreover, there was a substantial
synergistic increase in luciferase activity (by 20-fold) when
both PD98059 and CNTF were employed (Fig 4B) This
result suggested that the MEK 1/MAPK pathway interferes
with the CNTF pathway and that inactivation of the former
is necessary to observe the full CNTF effect on the ChAT
promoter We tested this hypothesis directly in the cotrans-
fection experiments using the expression vector pUSE with
a cDNA insert producing constitutively active MEK1
When this plasmid was cotransfected into SN56T17 cells
together with the luciferase reporter construct, the effect of
CNTF on the luciferase expression was completely abro-
gated, whereas cotransfection with the empty pUSE vector
did not inhibit CNTF-induced luciferase expression (Fig 4C) Thus, we conclude that the MEK1 pathway interferes with the CNTF action in cholinergic cells The main signaling pathway utilized by CNTF involves activation of transcription factors of the STAT (signal transducers and activators of transcription) family Upon CNTF binding, the CNTF receptor/Janus protein tyrosine kinase (Jak) 2 complex preferentially phosphorylates Stat3
at residue Tyr705 [27] We verified the involvement of the Jak2/Stat3 pathway in regulating ACh synthesis using the Jak2 inhibitor tyrphostin AG490 This compound has been shown to be selective for JAK family kinases [28] Although AG490 at higher concentrations inhibits Jak3 in lympho- cytes [29], its primary specific target in most cell types is the Jak2/Stat3 pathway [28,30,31] We used the inhibitor at
10 um, the concentration at which it is specific for JAK kinases in vitro and significantly reduces the DNA binding activity of Stat3 in vivo [32] First, we tested the ability of the inhibitor to block the Jak2/Stat3 pathway in SN56T17 cells
Following a 15-min exposure to 10 um AG490, CNTF-
induced Tyr705 phosphorylation of Stat3 was significantly reduced (Fig 5A) After the cells were treated for 48 h with
the inhibitor, either alone or in combination with CNTF,
intracellular ACh levels were measured as described above (Fig 5B) Tyrphostin AG490 alone had no effect on basal
ACh synthesis; however, it reduced CNTF-stimulated ACh
ATG (Met) ATG (ev ChAT
{VAChT ORF ORF povaste coding exon
13 more ChAT
R orémoter hũ ` Charny ChAT M ; coding exons
\ promoter promoter ,
\4.8-kb ChAT genomic BNA Luciferase reporter
HinDIll
pGL3Basic
pp | }
cnTF |}
PD/CNTF L
0 5 10 15 20
Luciferase Activity, arbitrary units
ChAT-Luc + Vector
ChAT-Luc + MEK1 O Control
CNTF
0 05 1 15 2 25 3
Luciferase Activity, arbitrary units
Fig 4 MEK1 interferes with the up-regulation of ChAT promoter activity by CNTF in a reporter gene assay (A) ChAT—luciferase reporter construct The diagram illustrates the localization of the promoter region used in the construct within the cholinergic locus The positions of the distal R promoter, as well as VAChT-specific V1 and V2 promoters and ChAT-specific N and M promoters are marked with horizontal arrows ORF, open reading frame (B) SNS56T17 cells were transfected with the ChAT-luciferase reporter construct and treated with the compounds indicated for 48 h Luciferase activity was measured in cell extracts as described in Experimental procedures The results are presented as means + SEM and are representative of three experiments Two-way analysis of variance revealed a significant effect of each individual treatment (P < 0.001) and a significant interaction (P < 0.001) (C) SNS6T17 cells were transfected with the ChAT—luciferase reporter construct (ChAT-Luc) alone or in combination with the pUSE vector (Vector) or with the constitutive MEK 1 expression construct (MEK 1) The cells were treated with 20 ngmL"! CNTF for 48 h; luciferase activity was measured as above and normalized to untreated controls The results are presented as means + SEM and are representative of four experiments Two-way analysis of variance and Tukey’s test revealed a significant effect of CNTF treatment in cells transfected with the luciferase construct alone (P < 0.005) and in combination with pUSE (P < 0.05) The CNTF effects in those two groups of transfectants were not statistically different from each other (P = 0.97) There was no effect of CNTF in cells transfected with the luciferase construct together with the MEK1 expression vector (P = 0.99).
Trang 6A
—— ~-=— @®Tyr Stat3
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300
250
200
150
50
Control CNTF TyrAG 490 Tyr/CNTF
Fig 5 Inhibition of CNTF-induced effects by a Jak2 inhibitor
(A) Tyrphostin AG490, a Jak2 inhibitor, reduces CNTF-induced
tyrosine phosphorylation of Stat3 The SNS6T17 cells were serum-
starved for 3 h, pretreated with 10 pw AG490 for 15 min, and then
treated with 20 ngmL~' CNTF for 20 min Cell lysates (500 pg per
sample) were immunoprecipitated with a Stat3 antibody and then
analyzed by Western blotting using an antibody specific for phos-
phoTyr705 Stat3 or the Stat3 antibody (B) Tyrphostin AG490 blocks
the CNTF-induced effect on intracellular ACh The SNS6T17 cells
were grown for 48h in the presence or absence of 10 pm AG490
(TyrAG490), 20 ngmL! CNTF, or a combination of the two
Intracellular ACh was measured in cell extracts as described in
Experimental procedures The results are presented as means + SEM
of six samples (two experiments, each performed in triplicate) Two-
way analysis of variance and Tukey’s test revealed a significant effect of
CNTF (P < 0.001), no effect of AG490 alone (P = 0.6359), and a
significant difference between the combination and CNTF alone
(P < 0.01)
synthesis to approximately control levels Therefore, the
results suggest that CNTF-stimulated ACh production is
mediated by the activation of Stat3 by Jak2
We then examined how blocking the MEK1/MAPK
pathway affects the phosphorylation state of Stat3
SN56T17 cells were cultured in the presence or absence of
10 um PD98059 and then treated with CNTF for 20 min
Stat3 phosphorylation on Tyr705 and Ser727 was visualized
by immunoblotting Shorter (up to 4 h) pretreatment with
PD98059 did not alter the level of Stat3 Tyr705 phospho-
rylation evoked by CNTF, while longer (up to 48 h)
exposure to the inhibitor resulted in an increase in Tyr705
phosphorylation to a variable degree In some experiments,
the level of Tyr705 phosphorylation of Stat3 in PD98059-
pretreated cells was higher than in control cells, however,
statistical analysis revealed that the increase was not
significant (Fig 6) The CNTF treatment resulted in a
twofold to threefold increase in Stat3 Ser727 phosphoryl-
ation Pre-treatment with PD98059 reproducibly reduced
Stat3 Ser727 phosphorylation to control levels MAPK phosphorylation levels in the same cells, in the presence and
absence of PD98059 and CNTF, mirrored the Stat3 serine
phosphorylation status and were consistent with previous findings (Fig 1) CNTF increased MAPK phosphorylation
by 90%, and this phosphorylation was almost completely abrogated by PD98059 Thus, it appears that, in SN56T17 cells, the MEK1/MAPK pathway mediates CNTF-evoked Stat3 phosphorylation on the Ser727 residue, whereas tyrosine phosphorylation of Stat3 is at least partially MAPK -independent
DISCUSSION
We demonstrate here that exposure of cholinergic
U0126, induces an increase in ChAT gene expression, resulting in elevated ChAT protein levels and ACh production The effect of MEK1 inhibitors on ACh synthesis is not an exclusive property of the SN56T17 cell line, as it can also be observed in the parental murine cell
line SN56, as well as, albeit to a lesser extent, in the human
cholinergic LA-N-2 cells The data suggest that PD98059 and U0126 up-regulate ACh synthesis by inhibiting MAPK activation This finding is surprising, because the cholinergic phenotype is also known to be upregulated by several stimuli (notably NGF), that utilize the MAPK signaling pathway
In our model cell line, PD98059 increases both basal and
CNTF-stimulated expression of the luciferase reporter under the control of the murine ChAT promoter The results of the reporter gene experiments suggest a negative regulatory role for MAPK in cholinergic gene expression It
is worth noting that Espinos ef al observed no effect of PD98059 on the human ChAT promoter activity after 6 h
of treatment in human neuroepithelioma CHP126 cells [33]
However, we also noticed no effect of PD98059 on at 6 h
(data not shown), suggesting that prolonged inhibition of MEK 1 is neede to observe changes in cholinergic gene expression Differences in the cellular context of the cell lines and/or the exact ChAT promoter region, as well as species
of origin of the gene used in the studies, may also have contributed to the apparent discrepancy between our findings and those of Espinos et al
We observed a synergistic effect of the combination of PD98059 and CNTF on ChAT promoter-driven reporter expression (but not on ACh or ChAT activity) The synergy
on the gene expression level indicates that the two treatments do not work independently, but affect cross- talking pathways It appears that the MEK1/MAPK pathway blocks the full CNTF effect on the ChAT promoter This conclusion is supported by the cotransfec- tion experiments, where we demonstrated that constitutively active MEK 1 prevents the induction of the ChAT-luciferase construct by CNTF This action of MEK1 provides an interpretation of our previous study, where we have shown that while both CNTF and NGF separately enhance the cholinergic phenotype, NGF, paradoxically, interferes with the CNTF effect [9] We also have found that NGF evokes a rapid and profound increase in ERK 1/2 phosphorylation
in SNS6T17 cells, whereas CNTF-evoked activation of
MAPK is delayed and weaker ((9]; this study) Hence, the attenuation of the CNTF effect caused by NGF could be
Trang 7
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œ œ 7
0 Control PD “ Control PD Control PD
Fig 6 Reduction of Stat3 Ser727 phosphorylation by PD98059 (A) The cells were grown in the presence or absence of 10 um PD98059 for 48 h and then were serum-starved for 3 h The cells were then treated with 20 ngmL7! CNTF for 20 min Cell lysates (500 pg per sample) were immunoprecipitated with a Stat3 antibody and then analyzed by Western blotting using an antibody specific for phosphoSer727-Stat3, phos- phoTyr705-Stat3, or an antibody that recognizes Stat3 regardless of its phosphorylation state Western blotting with an antibody specific for the phosphorylated forms of MAP kinase was performed on the crude cell lysates (40 ug per lane) Similar results were obtained in 24 additional experiments (B) Band intensities from the blots shown in (A) were quantified by densitometry and the results are presented as means + SEM of 3-5 experiments, each performed in duplicate PhosphoSer727-Stat3 and phosphoTyr705-Stat3 levels were normalized to the total Stat3 level In the absence of PD98059, phosphoSer727-Stat3, phosphoTyr705-Stat3 and phosphorylated MAPK levels in cells treated with CNTF were statistically different from control values (P < 0.01, p < 0.001, and p < 0.005, respectively) In CNTF-treated cells, PD98059 had a statistically significant effect on phosphoSer727-Stat3 and phosphorylated MAPK levels (P < 0.01 and p < 0.005, respectively), but not on phosphoTyr705-Stat3 level
(P = 0.31)
explained by the inhibitory action of NGF-activated treatment of SNS56T17 cells [9] and in primary spinal MEK1/MAPK pathway on ChAT gene expression Simi- neurons (B Berse, unpublished data) As CNTF-induced larly, in a recent study, Bartoe & Nathanson observed that MAPK activation is delayed relative to the rapid Stat3 inhibiting MAPK in SN56 cells stimulated the expression of response, it could serve as a negative feedback mechanism to LIF-induced vasoactive intestinal peptide (VIP) promoter downregulate the effects of CNTF mediated by Stat3 The
by twofold [23] In that study, treatment with PD98059 was mechanism of this downregulation remains to be investi- performed | h before a 24-h stimulation with LIF That gated This study shows that Stat3 tyrosine phosphorylation particular treatment strategy was not sufficient to increase is not significantly affected by PD98059, although there LIF-induced ChAT—luciferase reporter The authors con- appears to be a limited increase in Stat3 Tyr705 phospho- cluded that MAPK activation is involved in negatively rylation after prolonged exposure to the inhibitor regulating LIF-mediated VIP induction, but not ChAT Curiously, in SN56T17 cells, the phosphorylation of Stat3 induction However, the ChAT -luciferase construct on Ser727 in response to CNTF appears to be MAPK- employed in that study contains a smaller fragment of dependent Recent studies on STATS point to the regulatory murine ChAT promoter than the one used here; it responds role of serine/threonine phosphorylation in addition to relatively weakly to LIF and CNTF [34] and it does not tyrosine phosphorylation (for a review see [37]) The Ser727 respond to overexpression of LIF receptor [23] Thus, the residue of Stat3 was shown to be a target for multiple kinase ChAT promoter region tested by Bartoe & Nathanson may pathways including MEK1/MAPK [38-43] There are not contain all the sequences needed for the attenuation of conflicting conclusions about the actual biological conse- CNTF-mediated gene expression by MAPK Furthermore, quences of serine phosphorylation of STATs The majority although CNTF and LIF share the transmembrane receptor of reports demonstrated that serine phosphorylation can subunits, possible differences between signaling by these two enhance Stat3 DNA binding and/or transcriptional activity cytokines cannot be excluded [37] Other studies point to a negative role for Stat3 serine The presence of potential STAT binding sites in the phosphorylation, through both MAPK-dependent and cholinergic locus promoter region indicates that the effect of -independent mechanisms [38,4346] Although it has been CNTF on ACh production in cholinergic cells is likely documented that ERKs can directly phosphorylate Stat3 on mediated by the transcription factor Stat3 In SN56 cells Ser727, the MAPK-mediated inhibition of the IL-6 signal- and their derivatives, the Stat3 homodimer is the prevalent ing pathway occurs upstream of Stat3, as it was unaffected DNA binding complex activated by CNTF [35,36] We by Ser727 to Ala mutation [47] It is becoming clear that the demonstrated that Stat3 is rapidly tyrosine-phosphorylated relationship between STAT- and MAPK-mediated path- (Figs 5 and 6) and translocated to the nucleus upon CNTF ways is complex and the final outcome depends on the
Trang 8specific genes being regulated, the cell type, and possibly on
the cross-talk with other pathways (e.g other serine
kinases)
ACKNOWLEDGEMENTS
The authors thank Dr Jorge M Naciff for the gift of the mouse
genomic DNA clone containing ChAT proximal promoters This work
was supported by National Institutes of Health Grant AG09525
(to J K B), National Science Foundation Grant IBN-9907572 (to
J.K.B and B.B.), a grant from the Whitehall Foundation
(to B B.), Alzheimer’s Association grant ITRG-00-2073 (to I L.-C)
and National Institutes of HealthTraining Grant AGO0115 (to T M)
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