From the Research Unit Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, 24105 Kiel, Germany
Received for publication, January 2, 2003
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ABSTRACT |
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Several signaling pathways
have been implicated in mediating TGF- TGF- Among the proteoglycans that are up-regulated by TGF- In the pancreatic carcinoma cell line PANC-1 the dramatic (up to
50-fold) TGF- Materials--
The p38 MAPK pathway inhibitors SB203580,
SB202190, and SKF86002 and the MEK1 inhibitors PD98059 and U0126 were
purchased from Calbiochem and dissolved in water (SB203580) or
dimethyl sulfoxide (all others). The selective p38 MAPK inhibitor
SB239063 was obtained from GlaxoSmithKline. Human recombinant TGF- Cell Lines and Cell Culture--
The human
pancreatic adenocarcinoma cell lines PANC-1 and CFPAC-1 were purchased
from the American Type Culture Collection (Manassas, VA) or obtained
from Dr. W. von Bernstorff (University of Kiel), respectively. Both
cell lines were maintained in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, and 1 mM sodium pyruvate (all from Invitrogen). PANC-1 and CFPAC-1 cells stably transduced with recombinant retrovirus were cultured in the presence of
700 µg/ml (PANC-1) or 350 µg/ml (CFPAC-1) geneticin (biologically active concentration; Invitrogen) or 2.5 µg/ml puromycin (Sigma).
Antibodies--
The following antibodies were used:
anti-phospho-p38 MAP kinase (Thr180/Tyr182),
anti-p38 MAP kinase, anti-phospho-SAPK/JNK
(Thr183/Tyr185), anti-SAPK/JNK,
anti-phospho-MKK3/MKK6 (Ser189/Ser207),
anti-phospho ATF-2 (Thr71) (all from Cell Signaling
Technology, Heidelberg, Germany), anti-MKK6 (V-20; Santa Cruz
Biotechnology, Inc.), anti-MKK3 (N-20; Santa Cruz Biotechnology, Inc.),
anti-phospho-Smad2 (Ser465/Ser467) (Upstate
Biotechnology, Inc., Lake Placid, NY/Biomol, Hamburg, Germany),
anti-Smad2 (Zymed Laboratories Inc., Berlin, Germany), anti-Smad4 (B-8; Santa Cruz Biotechnology, Inc.), anti-Smad7 (N-19; Santa Cruz Biotechnology, Inc.), anti- RNA Isolation and Semiquantitative RT-PCR--
Total RNA was
isolated from cells with RNA Clean (AGS, Heidelberg, Germany) according
to the manufacturer's instructions. The general RT-PCR protocol and
the oligonucleotide primers used for amplification of BGN and GAPDH
mRNAs were described in detail earlier (33, 36). For
semiquantification of BGN, PAI-1, and GAPDH mRNAs we carried out a
competitive approach using gene-specific internal standards (33).
Briefly, for each target mRNA multiple reactions were run in
parallel containing identical amounts of cDNA (corresponding to 100 ng of total RNA) but different concentrations of internal standard DNA.
For this purpose, the standard DNA was serially diluted (0.9, 0.8, ... , 0.09, 0.08, ... and so forth). To keep reactions in
the exponential phase, the number of cycles with an annealing
temperature of 59 °C was adjusted to 16 cycles for BGN and 8 cycles
for GAPDH. Following electrophoretic separation of PCR products on
agarose gels and staining with ethidium bromide photographs were taken
and densitometrically scanned using the NIH Image software (version
1.62). TGF- Construction of Retroviral Expression Vectors and Generation of
Stable and Transient Transductants of the PANC-1 Cell Line--
For
retroviral transduction, cDNAs for murine flag epitope-tagged
kinase-inactive versions of p38 Transient Transfections and Reporter Gene Assays--
For
transient transfections followed by quantification of BGN mRNA,
PANC-1 cells (4 × 105) were seeded in 6-well plates
on day 1 and transfected on day 2 with an expression vector for MKK6
(pcDNA3-FLAG-MKK6; a kind gift of Dr. S. Ludwig) using
LipofectAMINE Plus (Invitrogen) according to the manufacturer's
instructions. Following a period of 24 h in normal growth medium
to allow expression of proteins from the transfected plasmids, cells
were starved (0.5% FCS) for another 24 h and stimulated with
TGF- Immunoprecipitation and Immunoblot Analysis--
For
immunoprecipitation cells were washed twice with ice-cold
phosphate-buffered saline (PBS) and lysed in 25 mM HEPES
buffer, pH 7.5, 100 mM NaCl, 10% glycerol, 5 mM EDTA, and 1% Triton X-100 supplemented with
CompleteTM protease inhibitor mixture (Roche
Diagnostics). Cleared lysates were subjected to immunoprecipitation
using anti FLAG-antibody, followed by adsorption with protein
G-Sepharose. Protein G beads were then washed in lysis buffer five
times, boiled in 2× Laemmli buffer (125 mM Tris-Cl, pH
6.8, 100 mM dithiothreitol, 20% glycerol, 4% SDS) and
subjected to immunoblot analysis. For direct immunoblot analysis, cells
were either lysed in radioimmune precipitation assay buffer (0.1% SDS,
1% Nonidet P-40, and 0.5% sodium deoxycholate and
CompleteTM in PBS) or, for detection of
phosphorylated proteins, were lysed directly in 2× Laemmli buffer. 40 µg of total cellular protein (measured with BCA protein assay
reagent; Pierce) from cleared lysates or equal volumes of Laemmli
lysates were separated by 12.5% SDS-PAGE and blotted on to Immobilon-P
polyvinylidene difluoride membranes (Millipore). Membranes were blocked
with PBS containing 5% non-fat dry milk, washed several times with PBS
containing 0.1% Tween 20, and then incubated with the primary
antibody. For detection of phosphorylated proteins, TBST (Tris-buffered
saline + 0.1% Tween 20) plus 5% bovine serum albumin was used for
membrane blocking, and TBST was used for washing. After washing, blots were incubated with the appropriate peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (ECL or ECL+Plus; Amersham Biosciences). Immunoblots for the detection of activated MAP kinases were first incubated with the respective phosphospecific antibody and then stripped and reprobed with an antibody against the corresponding total protein or Effect of p38 MAPK Blockade on TGF-
SB203580 and SB202190 were shown recently (38, 39) to inhibit ALK5
function at higher concentrations because of similar critical amino
acid sequences in the kinase domains (ATP binding pocket) of ALK5 and
p38 MAPK. To further exclude the possibility that the inhibitory effect
of these compounds on TGF- TGF- TGF- TGF- Activation of the Smad Pathway Is Required for TGF- In this study we have demonstrated that activation of
the p38 MAPK pathway, in addition to Smad signaling (33), is crucial for TGF- Recent studies showed that additional signals may be required for
proper activation of p38 by TGF- TGF- As mentioned above, TGF-1-induced extracellular matrix
production and fibrosis. We have shown recently that induction of
biglycan (BGN) expression by TGF-
1 depended on a functional Smad
pathway (Chen, W.-B., Lenschow, W., Tiede, K., Fischer, J. W.,
Kalthoff, H., and Ungefroren, H. (2002) J. Biol. Chem.
277, 36118-36128). Here, we present evidence that the ability of
TGF-
1 to induce BGN mRNA, in addition to Smads, requires p38
MAPK signaling, because 1) pharmacological inhibitors of p38
dose-dependently inhibited the TGF-
effect without
significantly affecting the transcriptional activity of a
constitutively active mutant of the TGF-
type I receptor or Smad2
phosphorylation at concentrations up to 10 µM, 2) the
up-regulation of BGN mRNA was preceded by a delayed increase in the
phosphorylation of p38 and its upstream activator MKK6 in
TGF-
1-treated PANC-1 cells, 3) inhibition of the p38 pathway by
stable retroviral transduction with a dominant negative mutant of
either p38 or MKK6 reduced TGF-
1-induced BGN mRNA expression,
and 4) overexpression of wild-type p38 or MKK6, but not MKK3, augmented
the TGF-
1 effect on BGN mRNA. We further demonstrate that the
(delayed) p38 activation by TGF-
1 is downstream of Smads and
requires a functional Smad pathway, because blocking TGF-
-induced
p38 activity with SB202190 had no effect on Smad2 phosphorylation, but
blocking Smad signaling by forced expression of Smad7 abolished
TGF-
1 induction of p38 activation and, as shown earlier, BGN
mRNA expression; finally, re-expression of Smad4 in Smad4-null
CFPAC-1 cells restored TGF-
-induced p38 phosphorylation and,
as demonstrated previously, BGN mRNA accumulation. These results
clearly show that TGF-
induction of BGN expression in pancreatic
cells requires activation of MKK6-p38 MAPK signaling downstream of Smad
signaling and provide a mechanistic clue to the up-regulation of BGN
seen in inflammatory response-related fibrosis and desmoplasia.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 has emerged as
a multifunctional cytokine involved in autocrine and paracrine
regulation of proliferation, differentiation, wound healing, apoptosis,
and immunomodulation (1, 2). TGF-
1, one of three mammalian TGF-
isoforms, TGF-
1-3, is a potent inducer of extracellular matrix
formation and has been implicated as the key mediator of fibrogenesis
and desmoplasia in a variety of tissues (3). In epithelial cells,
TGF-
, besides its powerful antiproliferative function, induces
morphological and biochemical changes toward a mesenchymal phenotype
designated epithelial to mesenchymal transdifferentiation (EMT) (4). A
hallmark of both fibrogenesis and EMT is the TGF-
-induced up-regulation of matrix-associated proteins, such as certain integrins and their extracellular matrix ligands (collagens, fibronectin, proteoglycans). The TGF-
responses are initiated by the interaction of the TGF-
ligand with cell surface receptors that form a
heterotetrameric complex of two type I and two type II serine/threonine
kinase receptors (5, 6). The activated ligand-receptor complex, in
turn, activates one or more downstream signaling pathway, the most
prominent one being the Smad pathway (7, 8). This pathway is initiated
with the phosphorylation of Smad2 or Smad3, which then heterodimerize
with the co-Smad, Smad4, followed by translocation of the Smad2/3-Smad4
complex to the nucleus; here, it binds directly or via other
DNA-binding proteins to the promoters of TGF-
-responsive genes to
stimulate or repress their transcription (7, 8). Activation of Smad
signaling is blocked by Smad7, which inhibits the phosphorylation of
Smad2/3 by the type I receptor thereby preventing their association
with Smad4 (9, 10). Evidence over the past few years suggests that
TGF-
may also stimulate other downstream pathways, involving RhoA
(11), as well as the MAP kinases, extracellular signal-regulated
kinases (ERKs) (12), c-Jun N-terminal kinases (JNKs) (13, 14), and p38
MAPKs (15-17). However, the question whether MAP kinases depend on a
functional Smad pathway and, if so, how both pathways interact, has
only been addressed in a few cases (13-15, 18-20). Nevertheless,
there is compelling evidence to suggest a functional separation among the individual signaling pathways (for review see Ref. 21). Smads, but
not p38 MAPKs, appear to be required for TGF-
-induced growth
inhibition of epithelial cells through their ability to transcriptionally up-regulate the promoters of p15INK4b,
p21CIP1, and c-myc (22-24). In contrast,
TGF-
-induced apoptosis in mouse mammary gland epithelial cells is
Smad-independent but requires activation of p38 (18). Still other
biological outcomes, e.g. EMT, seem to require activation of
both pathways (18).
in
vitro is biglycan (BGN), a prototype member of the small
leucine-rich proteoglycan family (reviewed in Refs. 25-27). BGN
can be considered a marker gene for TGF-
activity that is reflected
in vivo by the close spatial and temporal association of
both proteins under physiological and various pathophysiological
conditions. BGN has been implicated in the regulation of matrix
assembly, cellular adhesion (28), migration (29), and growth factor,
e.g. TGF-
, activity (30). Recently, BGN has been shown to
directly inhibit the growth of cancer cells in a TGF-
-independent
manner (31), a biological effect that it shares with its close
homologue decorin (32). In conjunction with our recent observations
that pancreatic tumor cells themselves synthesize and secrete BGN in
response to TGF-
in vitro and that a functional Smad
pathway is crucial for this to occur (33), we have proposed a novel
tumor suppressor function for Smad4: growth inhibition via
autoinhibitory BGN.
-induced increase in BGN mRNA and a concomitant, albeit smaller, rise in core-protein synthesis and release occurred in
a BGN promoter-independent fashion and by far exceeded the Smad-dependent transcriptional activation of a heterologous
reporter in these cells (33). This observation implies the existence of
an additional signaling pathway(s) that function(s) to amplify Smad-mediated signaling. PANC-1 cells undergo EMT in response to
TGF-
(34), which is characterized by adoption of a fibroblastoid morphology, a down-regulation of epithelial markers, and an
up-regulation of mesenchymal markers, e.g. BGN. As mentioned
above, EMT requires activation of both Smad and p38, and it is thus
conceivable that this may be reflected at the level of particular genes
involved in EMT. Finally, formation of fibrotic tissue with enhanced
BGN production often results from a previous inflammatory reaction known to be associated with increased p38 MAPK signaling (35). Together, these observations led us to hypothesize that efficient BGN induction by TGF-
, in addition to the well characterized Smad
pathway, requires activation of p38 MAPK. Using a combination of
specific pharmacologic inhibitors, as well as overexpression of
wild-type proteins from the p38 MAPK cascade and their dominant negative mutants, we present first-hand evidence that the p38 pathway
is essential for TGF-
-mediated induction of BGN and that it
cooperates with the Smad pathway. We further demonstrate that activation of p38 is secondary to and dependent on activation of Smad
proteins. This is the first report demonstrating the involvement of the
p38 MAPK pathway in the TGF-
control of BGN and small leucine-rich
proteoglycan gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
was obtained from R & D Systems, and anisomycin was from Sigma.
-actin (AC-15; Sigma). FLAG-tagged proteins were detected with anti-FLAG monoclonal
antibody M2 (Sigma).
induction of BGN and PAI-1 mRNA was assessed from
those reactions that showed an equimolar concentration of target and
internal standard. The corresponding amount of target mRNA in these
reactions was considered to be accurately determined when this ratio
and the target to standard ratios of at least two neighboring reactions
plotted against the corresponding standard dilutions on a
semilogarithmic scale formed a linear relationship. Relative values for
BGN and PAI-1 mRNA concentrations were normalized to GAPDH mRNA
levels. For each stimulation experiment at least two independent
competitive RT-PCR assays were performed yielding the same results.
(p38AF, harboring T180A and Y182F amino acid substitutions) and MKK6 (MKK6Ala) in
pcDNA3 (both generously provided by Dr. S. Ludwig, Würzburg,
Germany) were amplified with T7 and SP6 primers using
Turbo-Pfu polymerase (Stratagene) and subcloned in sense
orientation into the PmeI site of the retroviral vector
TJBA5bMoLink-neo (33). The coding sequences for wild-type p38
and MKK3 were generated by RT-PCR with primers p38-forward,
5'-AAAATGTCTCAGGAGAGGCCCACG-3' (start codon underlined) and
p38-reverse, 5'-TCAGGACTCCATCTCTTCTTGGTC-3' (stop codon
underlined) and MKK3-forward,
5'-ACCATGTCCAAGCCACCCGCACC-3' and MKK3-reverse,
5'-CCCTATGAGTCTTCTCCCAGGATC-3', respectively, using
Turbo-Pfu polymerase and PANC-1 cDNA as template. The
resulting fragments were gel-purified and ligated in sense orientation
into PmeI-restricted TJBA5bMoLink-neo vector (MKK3) or
SnaBI-restricted pBABEpuro (p38). In both cases sequencing
revealed authenticity with the published mRNA sequences. The
generation of a retrovirus encoding Smad4 was described in detail
earlier (33). A retrovirus for Smad7 was prepared by releasing the
Smad7 cDNA from pcDNA3 (a generous gift from Dr. C.-H. Heldin,
Institute for Cancer Research, Uppsala, Sweden) followed by directional
cloning into BamHI/PmeI-restricted TJBA5bMoLink-neo. Positive clones (evaluated by PCR, restriction analysis, and sequencing of the plasmid-cDNA junctions) were
cotransfected into human embryonic kidney 293T producer cells, along
with retroviral packaging vectors, as described previously (37).
Retroviral particles released by human embryonic kidney 293T cells were
used to infect PANC-1 and CFPAC-1 cells. Pools or individual clones (obtained by limited dilution) of productively infected cells were obtained by selection with geneticin (TJBA5bMoLink-neo) or puromycin (pBABEpuro) and were analyzed for expression of the desired
proteins by RT-PCR analysis and immunoblotting. In the case of Smad7
only, transduced cells were used for experimentation 48 h after
infection without prior geneticin selection.
1 (5 ng/ml) in the same medium for 24 h. Subsequently,
cells were processed for RNA isolation and RT-PCR. For detection of
luciferase activity, PANC-1 cells were seeded in 96-well plates at
1 × 104 cells/well. On the next day cells were
cotransfected with the TGF-
responsive reporter p3TP-lux and a
plasmid encoding a constitutively active mutant of TGF-
receptor
type I/ALK5 (ALK5T204D; both plasmids kindly provided by
Dr. J. Massagué, Memorial Sloan-Kettering Cancer Center, New
York) using LipofectAMINE Plus. After removal of the transfection
mixture, cells were incubated in normal growth medium for 24 h.
For analysis of the effect of the MAPK inhibitors on
ALK5T204D activity, different amounts of SB203580 were then added to the medium for another incubation period of 24 h.
Following lysis in Glo lysis buffer luciferase activities were
determined with the Bright Glo luciferase assay system (Promega) in the
MicroBeta TriLux 1450 system (Wallac, Inc., Gaithersburg, MD) for
2 s. The mean ± S.D. for each sample and treatment were
determined from 6-8 wells processed in parallel. Because control
experiments showed that the overall results were not affected by
unequal transfection efficiencies, normalization to
-galactosidase
activity was omitted.
-actin to confirm equal loading.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Induction of BGN
Expression--
Several observations suggested that in PANC-1 cells an
additional pathway is activated by TGF-
that, together with the Smad pathway, functions to amplify the TGF-
signal for the BGN mRNA up-regulation (see Introduction). To analyze whether p38 MAP kinases or
ERKs are involved in the TGF-
effect on BGN expression, we used
pharmacological inhibitors that specifically block these pathways. The
pyridinylimidazole-based compounds SB203580 and SB202190 inhibit the
activity of p38 for its substrate but do not block its phosphorylation.
Pre-incubation of the cells with SB203580 or SB202190 strongly
suppressed the TGF-
effect on BGN mRNA (Fig.
1A) and proteoglycan synthesis
(data not shown) in a dose-dependent manner. Successful
inhibition of p38 was verified for SB202190 by reduced phosphorylation
of ATF-2, a nuclear target of the p38 pathway (Fig. 1A,
inset). SB202190 inhibition of TGF-
-induced BGN mRNA
was also seen in other cell types, e.g. osteoblastic MG-63
cells (data not shown). Pre-incubation of PANC-1 cells with PD98059 or
U0126, two inhibitors of MEK1, the upstream kinase that activates
ERK1/2, did not inhibit TGF-
induction of BGN mRNA. Rather, the
MEK1 inhibitors slightly enhanced the TGF-
effect (Fig.
1B), which would be compatible with an inhibitory effect of
the ERKs on TGF-
-induced BGN expression (see "Discussion").
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Fig. 1.
The TGF- effect on
BGN mRNA is blocked by inhibitors of p38 MAPK. PANC-1 cells
were seeded on day 1 in normal growth medium and switched to medium
containing 0.5% FCS on day 2 for 24 h, followed by treatment with
TGF-
1 (5 ng/ml) for another 24 h in the absence or presence of
various MAPK inhibitors or vehicle (0.5 h of pre-incubation prior to
the addition of TGF-
). Subsequently, cells were analyzed for BGN or
PAI-1 expression by quantitative RT-PCR (A, B,
and D) or reporter gene activity in a luciferase assay
(C). A, effect of the p38 MAPK inhibitors
SB203580 and SB202190. At the end of the incubation period total RNA
was isolated from the cells and subjected to reverse transcription.
Equal amounts of cDNA were amplified with specific primers for BGN
and GAPDH, along with different concentrations of internal standard as
described under "Experimental Procedures." Following agarose gel
fractionation and ethidium bromide staining, relative concentra- tions (Conc.) of BGN and GAPDH mRNAs were
determined from those reactions in which the ratio of target to
standard amplification products approximated 1 and was equal between
the TGF-
-treated sample and the control. Induction of BGN mRNA
by TGF-
was calculated as the ratio of BGN transcripts in
TGF-
-stimulated over unstimulated control cells after normalization
to GAPDH mRNA. All values were expressed relative to TGF-
and
MAPK inhibitor-untreated control cells set arbitrarily at 1. A
phospho-ATF-2 (p-ATF-2) immunoblot of PANC-1 cells treated
for 2 h with TGF-
1 or anisomycin as control (Co) in
the absence or presence of SB202190 is also shown. B, effect
of inhibition of MEK1 on TGF-
induction of BGN expression. PANC-1
cells were treated as described above, except that the p38 inhibitors
were replaced with the MEK1 inhibitors PD98059 (10 µM)
and U0126 (50 µM). The experiments in A and
B were repeated three times with similar results.
C, effect of SB203580 on the activity of the TGF-
type I
receptor (ALK5). PANC-1 cells were transiently transfected by
lipofection with a constitutively active mutant of ALK5,
ALK5T204D, along with the TGF-
-responsive reporter
p3TP-lux. Following transfection, cells were cultured in normal growth
medium for 24 h and incubated in the presence or absence of the
indicated concentrations of SB203580 for another 24 h. Cell
extracts were then assayed for luciferase activity. One representative
experiment of three experiments performed in total is shown. Results
are the mean ± S.D. of six wells processed in parallel and are
expressed relative to the value in untreated cells set arbitrarily at
100. Two independent experiments were performed with very similar
results. D, effect of the selective p38 MAPK inhibitor
SB239063. PANC-1 cells were treated with TGF-
1 (5 ng/ml) for 24 h in the absence or presence of the indicated concentrations of
SB239063 and processed for RT-PCR quantification of BGN (left
panel) and PAI-1 (right panel).
-induced up-regulation of BGN mRNA was
because of inhibition of ALK5 rather than p38, we sought to assess the
functional effect of SB203580 on ALK5 more directly. To do this,
we transiently cotransfected PANC-1 cells with ALK5T204D, a
constitutively active ALK5 mutant, along with the TGF-
responsive
reporter p3TP-lux and measured ALK5T204D activity in the
presence or absence of SB203580. As shown in Fig. 1C,
concentrations of up to 10 µM SB203580 did not significantly affect ALK5T204D transcriptional activity.
Finally, we used the chemically related
compound SB239063, which is considered to
be selective for p38 (40).2 Like SB203580 and
SB202190, SB239063 potently inhibited the TGF-
effect on BGN with an
IC50 of ~ 2 µM (Fig. 1D).
Notably, SB239063 only marginally affected TGF-
induction of
PAI-1 mRNA (Fig. 1D) indicating that SB239063 is
not a general inhibitor of TGF-
signaling. From these results we
conclude that the inhibition of TGF-
1-induced BGN mRNA
expression by SB203580 and SB202190 was not caused by inhibition of
ALK5 activity. Instead, the data presented suggest that TGF-
mediated part of its effect on BGN expression via the p38 MAPK pathway.
Activates p38 and the Upstream Kinases MKK3/6 in
a Delayed Fashion--
Several MAPK pathways have been shown to be
activated in response to TGF-
in PANC-1 cells (41). Because
pharmacological inhibitors of the p38 MAPK cascade blunted the TGF-
effect, we investigated whether p38 was activated in response to
TGF-
1 by analyzing the phosphorylation state of p38 by
immunoblotting using a phosphospecific antibody. From Fig.
2 it is evident that TGF-
1 induced an
increase in p38 activation that was first noticed 1 h after
TGF-
1 addition, peaked at 2-4 h, and returned to baseline levels
thereafter. Interestingly, another member of the stress-activated protein kinases, SAPK/JNK, was not activated following TGF-
treatment in PANC-1 cells, although it was readily activated by
anisomycin, a known activator for both JNK and p38 (Fig. 2). Because
p38 is activated by the two upstream MAPK kinases, MKK3 and MKK6 (42), we predicted that these, too, were activated by TGF-
in a temporal fashion similar to that of p38. This was confirmed by immunoblot analyses using a phosphospecific antibody against these two kinases (Fig. 2). In accordance with activation of p38 but not SAPK/JNK, MKK4,
the upstream kinase of SAPK/JNK, was not activated in response to
TGF-
1 (data not shown).
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Fig. 2.
Activation of p38 MAPK and MKK3/6 by
TGF- 1. PANC-1 cells were starved for
24 h in medium containing 0.5% FCS and subsequently treated in
the same medium with 5 ng/ml TGF-
1 or 150 nM anisomycin
(Co) for the indicated time periods. Cell lysates were
analyzed by immunoblotting for the phosphorylation status of various
endogenous MAP kinases using phosphospecific antibodies. Upper
panel, detection of phosphorylated p38
(p-p38) and
total p38 protein (t-p38) in cell lysates. Middle
panel, detection of phosphorylated SAPK/JNK (p-JNK) and
total SAPK/JNK protein (t-JNK). Note the absence of
constitutive and TGF-
-induced JNK phosphorylation. Lower
panel, detection of phosphorylated MKK3/6
(p-MKK3/6) and total MKK6 protein (t-MKK6). Note
the delayed activation that is similar to that of p38.
Induction of BGN mRNA Was Reduced upon Dominant
Negative Inhibition of p38 MAPK and Enhanced by Overexpression of
Wild-type p38--
To more specifically demonstrate the involvement of
p38 MAPK in TGF-
-regulation of BGN, we inhibited p38 function by
stable retrovirus-mediated expression of a phosphorylation-resistant mutant of p38
, p38AF, that has been shown to act in a
dominant negative fashion (43). Analysis of a pool of PANC-1
transductants, as well as several individual clones by
immunoprecipitation with anti-FLAG antibody followed by anti-p38
immunoblotting, indicated expression of the mutant construct (Fig.
3A, inset). The
amount of immunoprecipitated protein correlated well with the
degree of inhibition of the TGF-
effect on BGN mRNA (Fig.
3A). Moreover, overexpression of wild-type p38 in a clone
pool of transduced cells augmented the TGF-
effect on BGN (Fig.
3B). These data confirm the results of pharmacologic
inhibition and emphasize the pivotal role of p38 in TGF-
regulation
of BGN.
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Fig. 3.
Effect of dominant negative inhibition and
enforced exogenous expression of p38 MAPK on TGF-
induction of BGN mRNA. A, determination of
TGF-
-induced BGN mRNA expression in PANC-1 cells stably
overexpressing a p38 mutant (p38AF) known to act in a
dominant negative fashion or the empty retroviral vector. Cells were
stimulated with TGF-
1 (5 ng/ml) for 24 h and analyzed by
semiquantitative RT-PCR for BGN expression as described in the legend
to Fig. 1 and under "Experimental Procedures." BGN mRNA
concentrations were expressed relative to unstimulated vector controls
set arbitrarily at 1. Inset, combined immunoprecipitation
(IP) and immunoblot (IB) analysis of a pool of a
large number of stable transductants (pool), as well as of
two individual clones (12 and 14). Note that
higher expression of the p38 mutant correlates with a stronger
inhibitory effect. B, TGF-
effect on BGN transcript
levels in a pool of PANC-1 cells stably overexpressing wild-type p38
from the retroviral vector pBABEpuro. Inset, immunoblot
analysis of total p38 protein expression in lysates from p38-transduced
PANC-1 cells. An antibody to
-actin was used to control for equal
loading.
Regulation of BGN Expression Involves MKK6--
p38 is
activated by the MAPK kinases MKK3 or MKK6, which were activated in a
similar temporal pattern as p38 in response to TGF-
(see Fig. 2). To
test whether these MAPK kinases were part of the TGF-
signaling
pathway targeting BGN, we used SKF86002, which has been reported to
inhibit the activity of MKK6 for p38 (19). Notably, SKF86002 blocked
the TGF-
effect on BGN mRNA as efficiently as the p38 inhibitors
(Fig. 4A). To more selectively interfere with MKK6 activity, we stably overexpressed a kinase-negative dominant negative MKK6 mutant (MKK6Ala). This approach
efficiently blocked the TGF-
effect on BGN mRNA in a
dose-dependent fashion (Fig. 4B). Like for p38,
(transient) overexpression of the wild-type MKK6 protein enhanced the
TGF-
effect on BGN (Fig. 4C, upper panel).
Given the fact that transfection efficiency was only ~30% (as
determined with an expression vector encoding green fluorescent protein) (data not shown), an even stronger effect of MKK6 on TGF-
signaling can be anticipated if 100% of the cells were transfected. In
contrast, (stable) overexpression of wild-type MKK3 had no effect (Fig.
4C, lower panel). Together, these data strongly
implicate MKK6, rather than MKK3, as the relevant MAPK kinase in the
TGF-
control of BGN expression.
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Fig. 4.
Effect of pharmacologic and gene
expression-mediated inhibition of MKK6 activity or ectopic expression
of MKK6 or MKK3 on TGF- induction of BGN
expression. A, PANC-1 cells were treated with TGF-
1
(5 ng/ml) for 24 h in the absence or presence of the MKK6
inhibitor SKF86002 (0.5 h of pre-incubation prior to the addition of
TGF-
) and analyzed for BGN expression by quantitative RT-PCR as
described in Fig. 1 and "Experimental Procedures." This experiment
was repeated two times with almost identical results. B,
RT-PCR analysis of BGN expression in three individual clones of PANC-1
cells stably overexpressing a dominant negative MKK6 mutant
(MKK6Ala) or the empty retroviral vector. BGN mRNA
concentrations were expressed relative to unstimulated vector controls
set arbitrarily at 1. Inset, immunoprecipitation
(IP)/immunoblot (IB) analysis of
MKK6Ala expression. Duplicate samples are shown for each
clone and the controls. Note that higher expression of the MKK6 mutant
correlates with a stronger inhibitory effect. wt, wild-type.
C, upper panel, PANC-1 cells were transiently
transfected with a wild-type (wt), FLAG-tagged MKK6
cDNA, or empty pcDNA3 vector (vector) followed by
incubation in normal growth medium for 24 h. After another 24-h
incubation in starve medium, cells were stimulated with TGF-
1 (5 ng/ml) for 24 h and subsequently processed for BGN RT-PCR.
Lower panel, PANC-1 cells infected with a retrovirus
encoding wild-type MKK3 or empty retrovirus (vector) were
stimulated with TGF-
1 for 24 h followed by measurement of BGN
mRNA using RT-PCR. The MKK3 protein level in the transduced cells
was determined by immunoblot analysis.
-induced p38
Activation in Pancreatic Cells--
We have shown previously (33) that
a functional Smad pathway is crucial for the TGF-
effect on BGN
expression in pancreatic cells. The question therefore arises how the
Smad and p38 pathway interact to bring about the rise in BGN
expression. The results obtained so far are consistent with either of
the following two models. In model 1, TGF-
(through its type I
receptor) concomitantly but independently activates Smad complex and
p38 MAPK, which subsequently converge on a common
(nuclear) target, e.g. ATF-2 (16). In model 2, the activated
Smad complex induces expression of an (unknown) protein, which
subsequently activates p38 MAPK. As shown earlier, phosphorylation of
Smad2 commenced ~15 min after TGF-
addition (33) whereas
appreciable phosphorylation of p38 was only realized 1 h after
TGF-
addition suggesting that Smad activation precedes activation of
p38 (model 2). In concordance with this assumption, inhibition of p38
by SB202190 was unable to block TGF-
-induced phosphorylation of
Smad2 (Fig. 5A); even at
concentrations as high as 50 µM no major decrease in
Smad2 phosphorylation was seen when compared with cells that were
treated with TGF-
alone, further dismissing the possibility that
inhibition of ALK5, rather than p38, accounted for the TGF-
effect
on BGN expression. A corollary of model 2 is that p38 activation, like
BGN mRNA up-regulation, is dependent on functional Smad4
expression. To test this prediction, we initially analyzed the p38
response in the Smad4-deficient, TGF-
-non-responsive, pancreatic
carcinoma cell line CFPAC-1, which expresses functional TGF-
receptors. As predicted by this model, TGF-
failed to activate p38
in CFPAC-1 cells (Fig. 5B), as well as in control cells
expressing an empty retrovirus (Fig. 5C), though a robust
activation of p38 by anisomycin (Fig. 5B) indicated that
there was no defect in the p38 MAPK pathway per se. In
contrast, in CFPAC-1 stably transduced with the same retrovirus encoding wild-type Smad4, TGF-
-induced activation of p38 was restored (Fig. 5C). As demonstrated earlier the capability
to up-regulate BGN mRNA in response to TGF-
was also restored in these cells (33) (Fig. 6B).
Finally, we analyzed p38 activation in PANC-1 cells in which Smad
signaling has been blocked by ectopic expression of the antagonistic
Smad7. Overexpression of Smad7 was shown previously by us to
efficiently suppress TGF-
-induced BGN mRNA accumulation
(33). As predicted from the above experiments, TGF-
-induced
p38 phosphorylation, too, was inhibited by Smad7 (Fig. 5D).
Collectively, these data prove that intact Smad signaling is required
for p38 activation and that activation of the p38 pathway occurs
downstream of Smad signaling, hence that model 2 is correct. Fig. 6
summarizes and integrates current and previous data on signaling events
involved in TGF-
regulation of BGN expression.
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Fig. 5.
Ordering the p38 MAPK and the Smad
pathways. A, effect of the p38 MAPK inhibitor SB202190
on activation of the Smad pathway as assessed by phosphorylation of
Smad2. PANC-1 cells were treated with 5 ng/ml TGF- 1 in the absence
or presence of the indicated concentrations of SB202190 for 8 h.
Cells were lysed in Laemmli buffer and analyzed by immunoblotting with
anti-phospho-Smad2 (p-Smad2; upper panel) and
anti-Smad2 antibody (lower panel) for total Smad2 protein
(t-Smad2). B, TGF-
-induced p38 activation in
the TGF-
-unresponsive Smad4-deficient pancreatic carcinoma cell line
CFPAC-1. CFPAC-1 cells were treated for various times with TGF-
1 (5 ng/ml) or for 2 h with anisomycin (150 ng/ml) as control
(Co), and cell extracts were prepared. The phosphorylation
status of endogenous p38 was analyzed by immunoblotting as described in
the legend to Fig. 2. C, TGF-
-induced p38 activation in
CFPAC-1 cells stably transduced with Smad4 or empty vector. Pools of
transduced cells were stimulated with TGF-
1 (5 ng/ml) for 4 h
and processed as described for B. The total level of Smad4
is also shown. D, effect of exogenous expression of Smad7 on
the TGF-
-mediated phosphorylation of p38 MAPK. PANC-1 cells were
transiently transduced with a Smad7-encoding retrovirus or empty
retrovirus (vector). 24 h post-infection cells were
starved and then treated or not with TGF-
1 (5 ng/ml) for 4 h.
Subsequently, cells were lysed in Laemmli buffer and subjected to
immunoblot analysis for phospho-p38 and total p38. Two independent
experiments were performed (C and D) with similar
results.
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Fig. 6.
Proposed model of Smad and p38
signaling in TGF- induction of BGN
expression. This scheme summarizes available data on the signaling
events involved in conveying the TGF-
signal from the receptor
complex to the nucleus, ultimately resulting in the accumulation of
cytoplasmic BGN mRNA transcripts. Upon TGF-
binding TGF-
receptor I/ALK5 is activated and subsequently phosphorylates Smad2/3.
Smad2 or Smad3 then forms an active heterodimeric complex with Smad4,
which is translocated into the nucleus to induce expression of an as
yet unknown protein (protein X). This protein activates p38
MAPK via activation of the MAPKKK upstream of MKK6 or of a protein
further upstream of the MAPKKK level. Active p38 translocates to the
nucleus and induces BGN mRNA accumulation through nuclear mRNA
transcript processing, stability, and/or export. Recent results from
Takekawa et al. (20) suggest that the gene/protein X may be
GADD45
, which binds and activates the MAPKKK MTK1/MEK kinase 4 (see
"Discussion"). Signaling routes that in other cellular systems have
been shown to be involved in TGF-
-mediated activation of p38 are
marked by dashed arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
regulation of BGN expression in pancreatic carcinoma cells.
This conclusion was derived from experiments in which the expression of
known components of the p38 signaling pathway, namely p38
MAPK and
its upstream activators MKK3 and MKK6, was either enhanced or blocked
by specific drugs or dominant inhibitory mutants. In addition, we have
presented evidence that Smad proteins are required for p38 activation
and that their activation temporally precedes p38 activation. The
finding that the Smad and the p38 pathways are serially connected is
probably the most intriguing aspect of this study. It was confirmed by
inhibition and gene replacement experiments in which one pathway was
blocked or activated, respectively, followed by a monitoring of the
activation state of the other; whereas inhibition of p38 with SB202190
had no effect on Smad2 activation, inhibition of Smad signaling by
ectopic expression of Smad7 inhibited TGF-
-induced p38 activation
(this study) and BGN mRNA up-regulation (33), and reconstitution of
Smad signaling by re-expression of Smad4 in Smad4-deficient,
TGF-
-, and p38-unresponsive CFPAC-1 cells restored the TGF-
response of p38 (this study) and BGN mRNA induction (33). This
scenario was initially suspected from the observation that Smad2 is
phosphorylated much more rapidly (33) than p38 (this study) in response
to TGF-
1. With respect to the kinetics of p38 activation by
TGF-
, two different signaling mechanisms seem to exist;
in some cells, e.g. C2C12 mouse myoblasts, activation of p38
is rapid (within the first 30 min) and transient (basal levels restored
by 1 h) (15, 16, 44). This rapid activation of p38 has been
associated with TAK1, a member of the MAPKKK family (45, 46),
which is connected to ALK5 via bridging proteins XIAP and TAB1
and has been suggested to mediate TGF-
-induced p38 activation
without participation of Smad proteins (16, 45, 47). In other cell
types (e.g. keratinocytes, osteoblasts, gingival
fibroblasts, and pancreatic acinar cells) maximal p38 activation occurs
only 1-2 h after TGF-
stimulation and persists for several hours
(48-53). Takekawa et al. (20), in a publication that
appeared shortly before submission of our manuscript, analyzed
TGF-
-induced p38 activation in various cell types including
pancreatic carcinoma cells, and their findings on the time course of
p38 activation are in good agreement with our data. These authors
convincingly demonstrated that delayed activation of p38 by TGF-
was
independent of TAK1 but instead involves activation of the MAPKKK
MTK1/MEK kinase 4 through Smad-dependent intermediate GADD45
expression (20). TGF-
/Smad-stimulated GADD45
expression was found to be necessary for p38 activation in
PANC-1 cells. Although not directly shown in this cell type, inhibition
of MTK1 by a dominant interfering mutant abolished GADD45
-induced
p38 activation triggered by constitutive active ALK5. Also, in
cotransfection experiments carried out by these authors in COS-7 cells,
GADD45
only stimulated MTK1 but not TAK1 kinase activity. This is in
line with experiments from our group employing PANC-1 cells that
overexpress a dominant negative TAK1 mutant, TAK1K63W,
which failed to block TGF-
induction of BGN mRNA.3 Also, we
showed earlier that the TGF-
effect on BGN expression was
cycloheximide-sensitive and was not mediated via enhanced transcriptional activity from the BGN promoter (33). With regard to the
kinetics of p38 activation and BGN mRNA up-regulation it is
noteworthy that the initial rise in BGN mRNA occurs between 4 and
8 h after TGF-
addition (31), which is similar to that observed
for thrombospondin-1 (20) and nicely matches the observation that
activation of p38 was maximal at 2-4 h following TGF-
addition. These data are consistent with a model in which the TGF-
-induced activation of Smads results in the transcriptional induction of an
(hitherto unknown) protein that subsequently activates the p38 pathway
(Fig. 6). The very recent results from Takekawa et al. (20)
independently confirmed and extended this scenario by identifying
GADD45
as a Smad-dependent protein responsible for
activation of p38, most likely via direct activation of MTK1 (see
above). Experiments are underway to test whether GADD45
and MTK1 are
indeed involved in TGF-
regulation of BGN.
. Functional integrin
1 and
v signaling was necessary for
TGF-
-dependent (17) and -independent (54) p38 MAPK
activation, respectively. Interestingly,
v integrin-mediated p38
activation and urokinase plasminogen activator up-regulation required
MKK3 but not MKK6. Consequently, the possibility has been considered
that integrin-induced p38 activation may be mediated strictly by MKK3
(54). Because we showed that overexpression of wild-type MKK6, but not
wild-type MKK3, was capable of enhancing TGF-
-induced BGN
up-regulation (and supposedly p38 activation), the reverse conclusion
would argue against a role of integrins in TGF-
control of BGN
expression. In agreement with this assumption, blocking integrin
1 function in PANC-1 cells did not affect
TGF-
-mediated BGN mRNA up-regulation (data not shown). Although
MKK3 and MKK6 are 80% homologous to each other and, in many cases,
mediate the same signals for p38 MAPK activation, they have been
reported to exhibit differential involvement in certain cellular
events. Besides integrin-mediated p38 activation (54), MKK6, rather
than MKK3, is required for FasL and c-Abl-induced apoptosis in Jurkat T
and NIH3T3 cells, respectively (55, 56). Also, MKK6 is important for
interleukin-12-induced p38 and STAT4 activation in T and NK
cells (57). However, MKK3, rather than MKK6, mediates tumor necrosis
factor-
and lipopolysaccharide-induced p38 MAPK activation and
cytokine expression in both fibroblasts and macrophages (58, 59).
Although additional experiments, e.g. with specific
inhibitors of MKK3 activity, are required, the results nevertheless
provide further evidence for distinct functions of MKK3 and MKK6 for
p38 MAPK-mediated cellular responses.
-stimulated activation of p38 (and ERK) MAPK activity has been
demonstrated previously in various cell types; however, the
contributions of these pathways to the TGF-
-mediated regulation of
endogenous gene expression have only been characterized in a few cases.
Among the genes for which TGF-
regulation involves the p38 MAPK
pathway are matrix proteins such as aggrecan (19), fibronectin, as
suggested from the use of the selective p38 MAPK inhibitor SB242235
(39), and thrombospondin-1 (20). However, whereas TGF-
up-regulation
of aggrecan in chondrocytes involves an interplay of the Smad and both
MAPK pathways (19), BGN induction by TGF-
is inhibited, rather than
enhanced, by ERKs as suggested from experiments using the MEK1
inhibitors PD98059 and U0126. The small stimulatory effect of these
inhibitors may reflect an inhibitory effect of MEK/ERK on the p38
pathway. A reverse interaction, negative regulation of MEK/ERK by p38
MAPK, has been described recently (60) in PANC-1 cells with respect to
cell proliferation. Because TGF-
has been shown to repress ERK
activation induced by mitogenic stimuli in this cell type (41) it
appears that the TGF-
effect on BGN results from both direct
activation of p38 and, to a smaller extent, simultaneous inhibition
of ERKs.
stimulation of PANC-1 and other
TGF-
-responsive pancreatic cancer cell lines cells led to EMT, a
biological response that requires activation of both the Smad and the
p38 pathway. Up-regulation of BGN, too, depends on sequential activation of both pathways as shown here and may contribute to the
phenotypic changes associated with EMT. EMT involves an increase in
tumor cell migration, invasion, and scattering (34). Interestingly, up-regulation of BGN (28, 29) and p38 activation (61) have independently been implicated in cellular migration and adhesion. The
realization that the synthesis of BGN, like fibronectin (39), is
induced by TGF-
via p38 may thus point to an important role of this
proteoglycan in tumor spread and metastasis and may contribute to the
tumor-promoting effect of TGF-
in later stages of carcinogenesis (62). In conclusion, we have shown for the first time that TGF-
regulation of the small interstitial proteoglycan BGN requires activation of the p38 MAPK signaling pathway, which, in turn, depends
on functional Smad signaling.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to C.-H. Heldin, S. Ludwig, and J. Massagué for expression plasmids and GlaxoSmithKline for supplying SB239063.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant UN 128/1-1 and UN 128/1-2 (to H. U.). Some of the results from this study form part of the doctoral thesis of W. C. and W. L.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Research Unit
Molecular Oncology, Clinic for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Arnold-Heller Strasse 7, D-24105 Kiel,
Germany. Tel.: 49-0-431-597-2039; Fax: 49-0-431-597-1939; E-mail:
hungefroren@email.uni-kiel.de.
§ Present address: The First Affiliated Hospital, Medical College, Zhejiang University, Zhejiang, 310003 Hangzhou, People's Republic of China.
Published, JBC Papers in Press, January 20, 2003, DOI 10.1074/jbc.M300035200
2 N. Laping, personal communication.
3 H. Ungefroren, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: TGF, transforming growth factor; ALK5, activin receptor-like kinase 5; BGN, biglycan; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAP, mitogen-activated protein; MAPK, MAP kinase; PAI-1, plasminogen activator inhibitor-1; EMT, epithelial to mesenchymal transdifferentiation; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPKKK, mitogen-activated protein kinase kinase kinase; MEK, MAPK/ERK kinase; SAPK, stress-activated protein kinase; RT, reverse transcriptase; PBS, phosphate-buffered saline.
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