From the North Shore-Long Island Jewish Health
System, Manhasset, New York 11030, § Yale
University School of Medicine, New Haven, Connecticut 06520, and the
¶ James Graham Brown Cancer Center, University of Louisville,
Louisville, Kentucky 40202
Received for publication, August 28, 2002
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ABSTRACT |
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Proper stimulation of cell cycle progression and
DNA synthesis requires cooperating signals from integrin and growth
factor receptors. We previously found that the proinflammatory peptide, macrophage migration inhibitory factor (MIF), functions as an autocrine
mediator of growth factor-dependent ERK MAP kinase
activation and cell cycle progression. We now report that MIF secretion
is induced by cell adhesion to fibronectin in quiescent mouse
fibroblasts. Adhesion-mediated release of MIF subsequently promotes
integrin-dependent activation of MAP kinase, cyclin D1
expression, and DNA synthesis. Secretion of MIF requires protein
kinase C activity, and recombinant MIF reconstitutes the activation of
MAP kinases in the presence of protein kinase C inhibition. Finally, we
show that cells deficient in MIF have significantly higher
retinoblastoma tumor suppressor and lower E2F transcriptional
activities. These results suggest that MIF is an important autocrine
mediator of adhesion-dependent signaling events and may
provide mechanistic insight into how MIF regulates proliferative
and oncogenic processes.
The microenvironment of a cell plays an important role in
the maintenance of normal cell morphology and gene expression. Members of the integrin family of cell surface adhesion molecules are now known
to relay signals between extracellular matrix proteins in the
microenvironment and intracellular signaling pathways in cells (1). The
requirement for integrin-mediated signaling in cell cycle progression
is also well established (2). However, the exact nature of these events
and how they impinge upon growth factor signaling is unresolved.
A significant amount of research has focused on the role of the
extracellular signal-regulated kinase
(ERK)1 MAP kinase pathway in
integrin-dependent regulation of the cell cycle (2-4). The
requirement for MAP kinase activation in adhesion-dependent cell cycle control is due to modulation of the expression of cyclin D
and thus the activity of specific cyclin-dependent kinases
(Cdks) (5, 6). The regulation of G1 phase progression
relies on cyclins, Cdks, and Cdk inhibitors (7, 8). Cyclin
D-Cdk4/6 and cyclin E-Cdk2 activities phosphorylate the
retinoblastoma (Rb) tumor suppressor, which in turn releases free E2F
transcription factors, resulting in the transcription of critical S
phase enzymes and regulators (9).
Despite extensive studies, the mechanism of integrin-mediated MAP
kinase activation remains somewhat controversial. Although several
reports have shown that activation of Ras is required for
adhesion-dependent MAP kinase stimulation (10, 11), others have suggested that Ras is not an essential component of this activation (12, 13). Howe and Juliano (14) have demonstrated how these
inconsistencies might be resolved. They report that there are two
phases of ERK activation, the first being Ras-dependent and
the second being Ras-independent. The first is the initial, acute
phase, which requires Ras-mediated Raf-1 membrane localization, whereas
a sustained phase of ERK activation is independent of Ras but requires
protein kinase C (PKC) activation of Raf-1 (14). Roovers
et al. (15) similarly found that the sustained activation of
MAP kinase is required for cyclin D1 expression, Rb phosphorylation, and G1 phase progression in normal, untransformed cells.
The authors go on to show that in order for a cell to efficiently
promote sustained MAP kinase activation and cyclin D1 expression,
signals from both integrins and growth factors are needed (15).
Our previous studies have established that MIF, a protein historically
associated with inflammation and immune regulation, stimulates the
proliferation of quiescent mouse fibroblasts (16). This response is
associated with the activation of the p44/p42 ERK MAP kinases. We
further demonstrated that growth factors stimulate the rapid release of
preformed MIF from adherent, quiescent fibroblasts. Importantly, the
sustained activation of MAP kinase in serum-stimulated fibroblasts was
dependent upon MIF autocrine action (16). We now report that MIF is
secreted in a PKC-dependent fashion as a consequence of
cell adhesion to the extracellular matrix and plays a significant role
in integrin-mediated signaling to sustained MAP kinase, cyclin D1
expression, and cell cycle progression.
Cell Culture--
MIF Immunoblotting Studies--
Whole cell extracts were prepared
from cells after the indicated treatments. Cells first were washed in
cold phosphate-buffered saline, and then ice-cold radioimmune
precipitation assay buffer (containing 1 mM NaVO4, 2 mM NaF, and a protease inhibitor mixture (Roche Molecular
Biochemicals)) was added. The cells were disrupted by repeated
aspiration through a 21-gauge needle. After incubation on ice for 10 min and microcentrifugation at 3000 rpm for 15 min (4 °C), the
supernatants were removed, the protein concentration was determined,
and lysates were stored at In Vitro Kinase Assays--
Whole cell extracts were prepared
from 1 × 106 cells as described above. The p44/p42
MAP kinase assay was performed according to the manufacturer's
directions (Cell Signaling Technology). Briefly, equal amounts of
lysate were incubated with 15 µl of an immobilized
anti-phospho-p44/p42 MAP kinase monoclonal antibody, and the samples
were rotated overnight at 4 °C. The pellet was collected by
centrifugation and washed with 500 µl of radioimmune precipitation
assay buffer followed by three washes with 1× kinase buffer. The
pellet then was resuspended in 50 µl of 1× kinase buffer
supplemented with 200 µM ATP and 2 µg of Elk-1 fusion
protein (Cell Signaling Technology). After incubation at 37 °C for
30 min, the reaction was terminated by adding 25 µl of 3× Laemmli sample buffer. Thirty µl of each sample was electrophoresed on a 10%
SDS-PAGE and transferred to polyvinylidene difluoride membrane. The
blot was then probed for phospho-Elk-1 protein by utilizing an
anti-phospho-Elk-1 antibody. Cdk4 kinase assay was performed similarly
except that the primary antibody used was against cdk4 (Santa Cruz
Biotechnology) and protein A/G beads were used to immunoprecipitate. A
final concentration of 10 µM cold ATP, 10 µCi of
[ Retroviral and Plasmid Constructs--
The
p53His-175 retroviral expression vectors was a gift of Dr.
Oleksi Petrenko (State University of New York (SUNY) Stonybrook, NY) and is described elsewhere (16).2
Viral supernatants were collected following transfection of viral packaging cells. 1 × 105 fibroblasts were infected
with the indicated retroviral supernatants for 24 h, washed,
refed, and split 2 days later. The expression of p53His-175
by primary fibroblasts resulted in efficient immortalization of both
MIF+/+ and MIF Luciferase Promoter Assay--
MIF+/+ and
MIF Cell Adhesion to Extracellular Matrix Stimulates the Release of MIF
from Fibroblasts--
The requirement for two distinct signaling
mechanisms for cell cycle progression in non-transformed cell lines is
well documented (22). The first such signal requires growth factor
receptor stimulation whereas the second is dependent upon cell
attachment to extracellular matrix proteins via integrin receptors. It
is thought that this dual signaling scheme functions to stimulate convergent signal transduction pathways resulting in maximal enzyme activation, gene expression, and cell growth (23).
We chose to investigate the role of MIF in adhesion-induced signal
transduction because of our previous observation that quiescent fibroblasts held in suspension are refractory to growth factor-induced MIF release (16). To determine whether cell adhesion to the extracellular matrix induces the secretion of preformed MIF, quiescent cells, in the absence of growth factors, were either plated onto fibronectin or held in suspension (low cluster plates). Western blotting analysis of supernatants from adherent cells showed a significant amount of MIF release within 30 min of plating (Fig. 1A). In contrast, cells in
suspension secreted no detectable MIF over the course of 4 h.
Autocrine Action of MIF Contributes to Integrin-induced ERK MAP
Kinase Activation--
The recent finding that MIF modulates MAP
kinase activation in response to growth factors suggested to us that
secreted MIF may also participate in signaling to MAP kinase in
response to integrin ligation. The activation of MAP kinase by
extracellular matrix was examined in NIH-3T3 fibroblasts as described
previously (12, 14, 24). Quiescent cells plated onto fibronectin show a
rapid activation of MAP kinase, which remains active for at least 40 min as demonstrated by immunoblotting using a phospho-specific antibody
against ERK MAP kinase (Fig. 1B) and by a MAP kinase enzymatic assay (data not shown) (12, 24).
It has been proposed that integrin ligation to extracellular matrix
induces two distinct phases of MAP kinase activation: an acute and a
sustained phase. Our previous observation demonstrated that MIF
autocrine action was important for growth factor-induced sustained, but
not acute, MAP kinase activation (16). To determine whether secreted
MIF contributes to the sustained phase of integrin-induced ERK
activation, we assessed MAP kinase activation by integrins in the
presence of a well characterized, neutralizing monoclonal anti-MIF
antibody. As shown in Fig. 1C, lysates from cells stimulated with fibronectin for 40 min in the presence of an IgG1
isotype control antibody display similar levels of phosphorylated
(active) MAP kinase as cells incubated without antibody. By contrast,
treatment of fibronectin-plated cells with anti-MIF antibody results in a dose-dependent inhibition of integrin-stimulated
sustained MAP kinase activation. These data suggest that MIF plays a
role in the modulation of integrin-induced MAP kinase activation.
MIF
To exclude the possibility that the difference in integrin-induced MAP
kinase activation observed between MIF+/+ and
MIF Inhibition of PKC Suppresses Adhesion-dependent MIF
Secretion and Sustained MAP Kinase--
The involvement of PKC in
coupling integrin-mediated signaling events to MAP kinase is well
documented (14, 25, 26). It has been proposed that PKC is required for
the delayed, or sustained, phase of integrin-stimulated MAP kinase
(14). Moreover, several studies have suggested that PKC activity is
critical for the production and secretion of MIF (27, 28). To test the hypothesis that integrin-dependent PKC activation results
in MIF secretion and subsequent autocrine activation of MAP kinase, a broad spectrum PKC inhibitor was employed. Pretreatment of NIH-3T3 fibroblasts with the PKC inhibitor, Ro-31-8220,
dose-dependently inhibited adhesion-induced MIF secretion
with maximal effect, displaying almost 100% inhibition (Fig.
3A). As mentioned above, PKC
inhibition has been shown to disrupt the delayed phase of integrin-induced signaling to MAP kinase. If PKC- dependent
release of MIF is responsible for modulating the sustained activation of MAP kinase, then rMIF addition to PKC-inhibited cells should restore
adhesion-stimulated MAP kinase activation. Fig. 3B
illustrates the dose-dependent inhibition of MAP kinase
phosphorylation by PKC inhibition, consistent with previous
observations (14). Supplementation of rMIF to Ro-31-8220-treated cells
adhering to fibronectin fully restored the capacity of integrins to
activate signaling to MAP kinase (Fig. 3B). Note that the
inhibition of MAP kinase by Ro-31-8220 very closely mimics the degree
of inhibition of MIF secretion (Fig. 3, A and B),
further supporting the conclusion that PKC-dependent MIF
secretion is partially responsible for integrin-stimulated sustained
MAP kinase activation.
MIF
It was suggested recently that MIF is post-translationally modified and
that this modification might influence bioactivity (29). To ensure
bioactivity of MIF and more closely mimic a physiologic setting of MIF
expression and secretion, we transiently transfected
MIF
As discussed above, signals generated from cell adhesion to integrins
coupled with growth factor-derived signals mediate sustained signaling
to MAP kinase, cyclin D expression and, ultimately, DNA synthesis (15).
Because our results suggest that MIF is important for growth
factor/integrin-induced signaling to sustained MAP kinase, we
speculated that cyclin D1 accumulation and DNA synthesis may be
similarly disrupted in the absence of MIF. To determine whether MIF
deficiency adversely affects DNA synthesis and cell proliferation,
primary MIF+/+ and MIF MIF Participates in Growth Factor Plus Adhesion-induced Cyclin D1
Accumulation, Rb Inactivation, and E2F-dependent
Transcription--
To further delineate the requirements for MIF in
cell cycle-associated events, we next sought to determine whether MIF
deficiency influenced downstream targets of MAP kinase such as cyclin
D1 expression and Rb/E2F activities. We recently found that
immortalization of MIF+/+ and MIF
Rb-mediated transcriptional repression results from decreased
G1 Cdk enzyme activities normally responsible for
phosphorylating and inactivating Rb (9). Rb-dependent
transcriptional repression was compared in primary and
p53His-175-immortalized MIF+/+ and
MIF Our prior studies revealed that MIF secretion and autocrine action
contribute to growth factor-dependent sustained MAP kinase activation and DNA synthesis (16). We now show that integrin ligation
to extracellular matrix stimulates MIF secretion,
MIF-dependent sustained MAP kinase activation, and cell
proliferation. Furthermore, cells deficient in MIF are defective in
growth factor plus integrin-induced cyclin D1 expression, Rb
inactivation, and E2F-dependent transcription.
Sustained signaling to MAP kinase contributes to cyclin D1 expression,
Rb inactivation, and progression through the cell cycle (22). Although
we demonstrate that fibroblasts derived from MIF Despite extensive efforts, a classical, membrane-bound receptor for MIF
has not yet been described. An intriguing mechanism by which MIF may
carry out its cellular actions was recently proposed by Kleemann
et al. (35). They describe that extracellular MIF has the
unique ability to traverse the plasma membrane and interact with
cytosolic c-jun-activating binding protein (Jab1). Jab1
effectors include the transcription factors AP1, HIF1 Welsh et al. (39) recently described a crucial role for Rho
GTPase in modulating sustained activation of MAP kinase and the
appropriately timed expression of cyclin D1 in mid-G1
phase. This study reaffirmed the importance of MAP kinase and the
timing of cyclin expression for progression through the cell cycle
while highlighting a novel functional role for Rho in cell growth
regulation. Although beyond the scope of this manuscript, elucidation
of a role for MIF in the modulation of Rho or vice versa
would yield critical insight into the biology of MIF in cell cycle regulation.
Hudson et al. (40) recently reported that MIF has the
ability to negatively regulate p53-dependent processes. In
a cell-based genetic screen for modulators of p53 function, MIF was
identified and found to suppress p53-dependent cell
senescence and apoptosis. Although our studies support this finding,
they additionally support the idea that MIF function modulates
Rb/E2F-dependent processes and that MIF regulation of these
two pathways may, in fact, be linked.3
In a separate study, we find that MIF action in cellular transformation
impinges upon the Rb/E2F pathway, but the net cause and effect of this
remain unclear.2 From these
studies we cannot conclude that the blunted signaling to MAP kinase and
cyclin D1 expression in MIF The importance of cytokines and growth factors in maintaining tumor
growth and viability is well established (41). This field of study has
recently become the focus of several novel targeting strategies for
anticancer chemotherapies (42). In particular, the family of epidermal
growth factor ligands and receptors has gained much attention because
inhibitors of the epidermal growth factor receptor tyrosine kinase and
ligand-neutralizing antibodies have shown therapeutic efficacy in
preclinical and clinical trials (43). Interestingly, dysregulation of
this ligand and/or the expression of its receptor results in persistent
activation of MAP kinase, cyclin D1 expression, and tumor promotion
(44). Together with prior studies showing the antitumorigenic effects of MIF inhibition (45, 46), the present data suggest that MIF may
represent an important new target for cancer chemotherapeutics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice were kindly provided
by Dr. John David (Harvard School of Public Health, Boston, MA) and
have been described previously (19, 33). MIF
/
mice and
their wild-type littermates were maintained on a mixed 129Sv × C57Bl/6 background (F3). MEFs were generated from embryos at day 14.5 and grown in DMEM with 10% FCS (Hyclone, Logan, UT) and
penicillin/streptomycin. NIH-3T3 fibroblasts were maintained in low
glucose DMEM with 10% FCS and penicillin/streptomycin. For adhesion
experiments, NIH-3T3 and MEFs were G0 synchronized for 1 and 2 days, respectively, in serum-free media. Quiescent cells were
lifted from plates with trypsin-EDTA and neutralized with DMEM/2%
fatty acid free BSA and 2 mg/ml soybean trypsin inhibitor. Cells then
were then spun down and resuspended in DMEM with 0.2% FCS and
allowed to sit for 30 min (in the presence of PKC inhibitor Ro-31-8220
(Calbiochem) where indicated). Cells were plated onto fibronectin-coated (BD Biosciences) or low cluster plates for suspension conditions (Corning Costar, Harrodsburg, KY) for the indicated times. In select experiments, recombinant MIF (rMIF) (16, 17)
was added to cells just prior to plating. For growth factor/adhesion
experiments, cells were resuspended in DMEM with 10% FCS and plated
immediately. For antibody neutralization experiments, anti-murine MIF
monoclonal antibody (14.15.5, IgG1 subclass) (18) or an
isotype control monoclonal antibody was added at the indicated concentrations prior to plating of the cells. The 14.15.5 monoclonal antibody has been shown previously to neutralize both endogenously released (native) MIF and rMIF in a variety of in vitro and
in vivo studies (16, 19, 20). DNA synthesis experiments were performed by plating quiescent, transfected MEFs in DMEM with 10% FCS
or DMEM with 0.2% FCS and 20 ng/ml platelet-derived growth factor/1
µM insulin (R&D Diagnostics, Minneapolis, MN) onto
fibronectin-coated 96-well plates (BD Biosciences). Cells were
pulsed with [3H]thymidine (1 µCi/ml)
(PerkinElmer Life Sciences) for 20 h. The cells then were
harvested, and the incorporation of [3H]thymidine into
DNA was quantified by liquid scintillation counting (Packard Instrument
Co.).
80 °C. Equal amounts of cellular
proteins were fractionated on SDS-PAGE gels (Bio-Rad) and transferred
to polyvinylidene difluoride membranes (Millipore, Bedford,
MA). Immunoblotting was performed with antibodies directed against
phospho-ERK MAP kinase, total ERK MAP kinase (Cell Signaling Technology, Beverly, MA), p16Ink4a, p21Cip1,
p27Kip1, Cdk4 (Santa Cruz Biotechnology, Santa
Cruz, CA), cyclin D1 (Upstate Biotechnology, Waltham, MA),
p53 (Pharmingen), and MIF (prepared by our laboratory) (16).
Densitometric analysis of Western blots was performed using the NIH
Image software package.
32P]ATP, and 2 µg of the C terminus of Rb
protein were added to each kinase reaction. Samples were
electrophoresed, and the gel was dried and exposed to autoradiography
for 12 h.
/
cells as evidenced by
passaging for greater than 25 times with no evidence of cell
senescence. The MIF eukaryotic expression vector (pcDNA3.1 GS/MIF)
has been described elsewhere (19).
/
primary and immortalized cells were transfected
(24 h) with 0.8 µg of pmyc-TA-Luc, pRb-TA-Luc, pE2F-TA-Luc
(Clontech), or p53-Luc (Stratagene, La Jolla,
CA)-sensitive luciferase promoter constructs using FuGENE 6 transfection reagent (Roche Molecular Biochemicals). 0.2 µg of
Renilla pRL-TK vector (Promega, Madison, WI) was
co-transfected with each of the above vectors. Luciferase and
Renilla luciferase activities were measured by the
Dual-Luciferase reporter assay system (Promega) on a TD-20/20
luminometer (Turner Designs). Results are expressed as fold increase
over control (MIF+/+ or MIF+/+ p53m) after
normalizing ratios of luciferase/Renilla luciferase and
averaging quadruplicate samples.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cell adhesion to
fibronectin stimulates the secretion of MIF. As shown in
A, quiescent NIH-3T3 cells were plated onto either
fibronectin (FN)-coated or low cluster 6-well plates.
Supernatants were removed at the indicated times, concentrated, and
analyzed by Western blot using mouse-specific polyclonal anti-MIF
antibody. The data shown are representative of four independent
experiments. As shown in B, cells were plated as in
panel A and collected and lysed at the indicated times. The
levels of total and phosphorylated MAP kinase (MAPK) were determined as
described under "Experimental Procedures." As shown in
C, inhibition of late phase adhesion induced MAP kinase
activation by anti-MIF. Quiescent NIH-3T3 cells were plated onto
fibronectin-coated dishes for 40 min in the absence or presence of
anti-MIF or an isotype control antibody as indicated. The data shown
are representative of three independent experiments. mAb,
monoclonal antibody.
/
Fibroblasts Are Partially Resistant to
Adhesion-dependent Sustained MAP Kinase Activation--
To
better understand the role of MIF in integrin-mediated signaling to MAP
kinases, embryonic fibroblasts (MEFs) were derived from
MIF
/
mice and their wild-type littermates.
MIF+/+ and MIF
/
fibroblasts were examined
for their relative abilities to conduct adhesion-mediated signaling to
MAP kinase. As shown in Fig.
2A, MIF+/+
fibroblasts efficiently induced MAP kinase phosphorylation at both the
acute (~20 min) and the sustained phase (~40 min) time points (13).
In contrast, adhesion-dependent MAP kinase activation in
MIF-deficient cells was much less efficient than in wild-type cells.
The phosphorylation status of MAP kinase in MIF
/
cells
closely correlated with a decrease in ERK enzymatic activity as
assessed by an in vitro kinase assay for ERK (Fig.
2A and data not shown). The effect of MIF deficiency was
less pronounced at the acute than at the sustained time point,
suggesting that the contribution of MIF to integrin-mediated MAP kinase
signaling is predominantly to the delayed phase of MAP kinase
activation.
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Fig. 2.
MIF /
fibroblasts are
defective in adhesion-mediated signaling to MAP kinase. As shown
in A, quiescent MIF+/+ and MIF
/
primary fibroblasts were plated onto fibronectin (FN)-coated
dishes for the indicated times with 0 time representing cells not
plated. Lysates were analyzed for MAP kinase activation by Western
blotting with total and phospho-specific antibodies against ERK MAP
kinase. As shown in B, rMIF add-back restores sustained MAP
kinase activation by cell adhesion. Cells were plated as in panel
A for 40 min (sustained phase of MAP kinase activation), and rMIF
was added at the time of plating to the indicated samples.
C, as in panel B except that increasing
concentrations of rMIF were added at the time of plating. In addition
to total and phospho-MAP kinase evaluation, MAP kinase kinase, the
upstream activator of MAP kinase, was also assessed for its activation
state by a phospho-specific antibody. The data shown are representative
of at least two independent experiments.
/
cells was independent of MIF loss, we reintroduced
rMIF into the culture system. The addition of 50 ng/ml rMIF to
MIF-deficient cells restored integrin signaling to MAP kinase (Fig.
2B) and its upstream activator, MAP kinase kinase (MEK)
(Fig. 2C). We occasionally observed that the addition of
rMIF to MIF+/+ fibroblasts during integrin ligation
inhibits MAP kinase activation by cell adhesion to fibronectin (Fig.
2C). We believe that this is due to a "plateau" effect:
i.e. levels of integrin-induced MIF secretion are optimal
for MAP kinase activation in wild-type cells, and excess rMIF increases
total MIF levels into inhibitory concentrations. This effect is similar
to the bell-shaped activity curves reported previously for the
migration inhibitory activity of MIF (17).
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Fig. 3.
PKC inhibition suppresses
adhesion-dependent MIF secretion and MAP kinase
activation. As shown in A, the PKC inhibitor,
Ro-31-8220, disrupts integrin-dependent MIF secretion.
Quiescent NIH-3T3 fibroblasts were treated for 30 min with the
indicated concentrations of Ro-31-8220 or Me2SO
alone (0). Cells then were then plated onto fibronectin-coated dishes
for 40 min, and supernatants were collected and analyzed for MIF
secretion as described under "Experimental Procedures." As shown in
B, PKC inhibition suppresses adhesion-dependent
sustained MAP kinase activation and rMIF rescues. Cells were treated as
in panel A except that where noted, rMIF was added at the
indicated concentrations just before plating. Data are representative
of four independent experiments.
/
Embryonic Fibroblasts Are Deficient in Growth
Factor Plus Adhesion-induced MAP Kinase Activation and DNA
Synthesis--
The results described above together with our previous
observation that MIF is a growth factor-induced autocrine signaling factor (16) prompted us to examine the role of MIF as a mediator of
cell cycle regulators and DNA synthesis. As mentioned previously, signals derived from integrins and growth factors synergize to maximally stimulate cell cycle progression and DNA synthesis (22). It
is thought that the sustained activation of MAP kinase is required for
efficient G1/S phase progression (5, 15). Because MIF is
regulated by both growth factor and integrin activation and serves as
an autocrine activator of MAP kinase, we hypothesized that MIF is
important for growth factor/integrin-dependent signaling and cell cycle progression.
/
fibroblasts with an MIF expression plasmid to
restore the phenotypic effects of MIF (19). Shortly after
transfections, cells were serum-starved and then replated in the
presence of 10% FCS for either 2 or 12 h. As shown in Fig.
4A, MIF status (deficiency or
transfections) had no effect on growth factor/adhesion-induced MAP
kinase activity at the earliest time point investigated. In contrast,
growth factor/adhesion-mediated sustained MAP kinase activation was
compromised in cells lacking MIF, and reconstitution of MIF in these
cells restored the defect (Fig. 4A). Note that in cells that
are actively producing MIF, ectopic expression of MIF blunts the
sustained MAP kinase activation, similar to what we observed after the
addition of rMIF (Fig. 2C and discussed above). It should
also be noted that cell supernatants from MIF-transfected MIF
/
fibroblasts stimulated with growth factors and
adhesion consistently contained between 20 and 45 ng/ml of MIF, whereas
MIF
/
cells transfected with the empty plasmid had MIF
levels that were undetectable by immunoprecipitation/Western blotting
(data not shown).
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Fig. 4.
MIF is required for optimal growth factor
(GF) plus adhesion-stimulated sustained MAP kinase
activation and DNA synthesis. As shown in A, primary
fibroblasts were transiently transfected with the indicated plasmids
for 8 h and then rendered quiescent by serum starvation for an
additional 36 h. Quiescent MIF+/+ and
MIF /
transfected cells were plated onto
fibronectin-coated dishes in the presence of 10% FCS for the indicated
times. After harvesting, cell lysates were examined for levels of total
and phosphorylated MAP kinase. As shown in B, DNA synthesis
in MIF-deficient cells is impaired. Cells were transfected and rendered
quiescent as in panel A. Cells were then plated on
fibronectin-coated 96-well plates in DMEM with 10% FCS or DMEM with
0.2% FCS plus 20 ng/ml platelet-derived growth factor
(PDGF)/1 µM insulin and pulsed with
[3H]thymidine. After 20 h, cells were harvested and
assessed for [3H]thymidine incorporation by liquid
scintillation counting. Results are expressed as fold DNA synthesis
(cpm averages from three experiments) relative to controls
(MIF+/+ vector transfected).
/
fibroblasts were
transfected and synchronized as in Fig. 4A. We used two
different sources of growth factors to determine differences between
defined and serum-derived growth factors. The extent of DNA synthesis
(assessed by [3H]thymidine incorporation) is shown in
Fig. 4B. Growth factor/adhesion-stimulated DNA synthesis of
MIF
/
cells or MIF
/
cells transfected
with vector alone was between 40 and 50% less than that of the
MIF+/+ cells. Importantly, reconstitution of MIF by
transient transfection fully reversed the defect in DNA synthesis
associated with MIF deficiency. As the defect in growth
factor/adhesion-stimulated sustained MAP kinase activation is also
reversed by MIF reconstitution and sustained MAP kinase activation has
been shown to be important for S phase progression, these results
support the conclusion that MIF contributes to MAP kinase signaling and
S phase progression.
/
primary
fibroblasts with a dominant-negative mutant of p53 significantly enhances the growth differences between MIF-deficient and
MIF-containing cells.2
MIF+/+ and MIF
/
primary
fibroblasts were infected with a replication-defective virus encoding a
well characterized mutant allele of p53 (p53His-175) (30,
31). As shown in Fig. 5A,
introduction of dominant-negative p53 had no effect on the relative
levels of the cyclin-dependent kinase inhibitors
p21Cip1, p27Kip1 (Fig. 5A), and
p16Ink4a (not shown) in normally cycling cells regardless
of MIF status. In contrast, p53His-175 immortalized
MIF
/
fibroblasts displayed an impaired ability to
accumulate cyclin D1 in response to growth factors and
anchorage-dependent signals (Fig. 5B,
upper panel). Moreover, the inability to efficiently elevate
cyclin D1 levels in MIF
/
cells resulted in a
proportionately reduced Cdk4 kinase activity that was not
associated with alteration of Cdk4 protein levels (Fig.
5B, middle and lower panel).
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Fig. 5.
MIF deficiency results in impaired cyclin D1
expression, Cdk4 activity and Rb/E2F function. As shown in
A, primary MIF+/+ and MIF /
embryonic fibroblasts were immortalized by infection with a
replication-defective retrovirus encoding a dominant interfering mutant
of p53 (p53His-175) as described under "Experimental
Procedures." Three days after infection, cells were analyzed for
relative levels of p53, p21Cip1, p27Kip1, and
Cdk4 by immunoblotting. wt, wild-type; ko,
knockout. As shown in B, quiescent, p53His-175
immortalized MIF+/+ and MIF
/
MEFs were
plated onto fibronectin-coated dishes with DMEM and 10% FCS for 6, 9, 12, and 15 h. Cell lysates were subjected to immunoblotting
(IB) for cyclin D1 and Cdk4. A parallel set of samples was
lysed, and Cdk4/cyclin D complexes were immunoprecipitated and
incubated in a kinase reaction with glutathione
S-transferase (GST)-Rb peptide as a substrate as described
under "Experimental Procedures." As shown in C, primary
MIF+/+ and MIF
/
embryonic fibroblasts and
p53His-175 immortalized MIF+/+ and
MIF
/
fibroblasts were transiently co-transfected with
the indicated transcription factor-responsive luciferase plasmids and
the Renilla pRL-TK vector for 40 h. Results are
expressed as fold increase over control after normalizing ratios of
luciferase/Renilla luciferase from quadruplicate samples.
All experiments were repeated at least twice with similar
results.
/
fibroblasts by an Rb-sensitive luciferase promoter
construct. Cycling, asynchronous cell populations displayed significant
differences in Rb-transcriptional repression (where the degree of
repression is inversely proportional to promoter activity) (Fig.
5C). MIF-deficient primary or immortalized cells
consistently had higher Rb repressor activity with the
p53His-175-immortalized cells, showing the largest
differences between MIF+/+ and MIF
/
cells.
Conversely, levels of c-myc or p53-dependent
transcription were unaffected by MIF status (Fig. 5C). As
would be expected in cells with higher Rb tumor suppressor activity,
E2F-dependent transcription was decreased in MIF-deficient
cells, and the degree of reduction correlates with increased
Rb-dependent repression.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice
are partially refractory to growth factor/adhesion-stimulated MAP
kinase activation and cyclin D1 expression, it should be noted that MIF
only partially regulates this phenomenon. For instance, whereas
deletion of the Mif gene in mice has been achieved with two
different targeting constructs, none of the resulting mice appear to
suffer from developmental or growth-related abnormalities (33, 34). We
propose that the correct dosage of MIF in normal cells may be important
for optimal cell growth under specific conditions and that in
immortalized or transformed cells, there is a greater requirement for
an MIF-dependent contribution to MAP kinase activation and
cyclin D1 expression.
, and SMAD4,
among others (36, 37). Another interesting effector reported for Jab1
is the
2 subunit of integrin LFA-1 (38). This
raises the possibility that MIF binding to intracellular Jab1 could
influence the dynamics or strength of integrin signaling cascades.
Whether the MIF/Jab1 interaction is the sole mechanism by which MIF
exerts its pluripotent activities will require more thorough investigation.
/
cells is, in fact, causal to
the transformation resistance we have observed. Although it is
premature to conclude that MIF promotes cell transformation by
modulating cyclin D1 expression and Rb/E2F activities, it is not
unreasonable to speculate that MIF-dependent regulation of
sustained MAP kinase activity may be an important contributing factor
in oncogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Steve Jennings (Charles River Laboratories) for help in isolating embryonic fibroblasts. We also express our gratitude to Drs. Marc Symons and Maria Elena Bottazzi for very helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant 1RO1-AR049610 from the National Institutes of Health (to R. B.).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: James Graham Brown
Cancer Center, University of Louisville, 529 S. Jackson St., Louisville, KY. Tel.: 502-852-7698; Fax: 502-852-5679. E-mail: robert.mitchell@louisville.edu.
Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.M208820200
2 O. Petrenko, G. Fingerle-Rowson, R. A. Mitchell, and C. A. Metz, submitted for publication.
3 Unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; MAPK, MAP kinase; MEK, MAPK/ERK kinase; Cdk, cyclin-dependent kinase; MIF, macrophage migration inhibitory factor; rMIF, recombinant MIF; PKC, protein kinase C; Luc, luciferase; TK, thymidine kinase; Rb, retinoblastoma; MEF, murine embryonic fibroblasts; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.
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