Insulin-Induced Mitogen-Activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Attenuates Insulin-Stimulated MAP Kinase Activity: A Mechanism for the Feedback Inhibition of Insulin Signaling
Anasua B. Kusari,
John Byon,
Debdutta Bandyopadhyay,
Kathleen A. Kenner and
Jyotirmoy Kusari
The Department of Physiology (A.B.K., J.B., D.B., J.K) and,
Molecular and Cellular Biology Program (J.K.), Tulane University
Medical Center, New Orleans, Louisiana 70112-2699,
Department of Pediatrics (K.A.K.), University of California
San Diego, La Jolla, California 92093
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ABSTRACT
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Insulin signaling involves the transient
activation/inactivation of various proteins by a cycle of
phosphorylation/dephosphorylation. This dynamic process is regulated by
the action of protein kinases and protein phosphatases. One family of
protein kinases that is important in insulin signaling is the
mitogen-activated protein (MAP) kinases, whose action is reversed by
specific MAP kinase phosphatases (MKPs). Insulin stimulation of Hirc B
cells overexpressing the human insulin receptor resulted in increased
MKP-1 mRNA levels. MKP-1 mRNA increased in a dose-dependent manner to a
maximum of 3- to 4-fold over basal levels within 30 min, followed by a
gradual return to basal. The mRNA induction did not require the
continuous presence of insulin. The induction of MKP-1 protein
synthesis followed MKP-1 mRNA induction; MKP-1 protein was maximally
expressed after 120 min of insulin stimulation. MKP-1 mRNA induction by
insulin required insulin receptor tyrosine kinase activity, since
overexpression of an altered insulin receptor with impaired intrinsic
tyrosine kinase activity prevented mRNA induction. Forskolin,
(Bu)2-cAMP, 8-bromo-cAMP, and
8-(4-chlorophenylthio)-cAMP increased the MKP-1 mRNA content moderately
above basal. These agents also augmented the insulin-stimulated
expression of MKP-1 mRNA. However, in some cases the increase in MKP-1
mRNA expression was less than additive. Nevertheless, these results
indicate that multiple signaling motifs might regulate MKP-1 expression
and suggest another mechanism for the attenuation of insulin-stimulated
MAP kinase activity by cAMP. Overexpression of MKP-1 in Hirc B cells
inhibited both insulin-stimulated MAP kinase activity and MAP
kinase-dependent gene transcription. The results of these studies led
us to conclude that insulin regulates MKP-1 and strongly suggest that
MKP-1 acts as a negative regulator of insulin signaling.
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INTRODUCTION
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Insulin stimulates a spectrum of physiological effects, including
the transport of glucose and amino acids, the increased synthesis of
glycogen and certain metabolic enzymes, and enhanced DNA and RNA
synthesis (1, 2, 3). The initial step in insulin action is the association
of insulin with its cell surface receptor. The insulin receptor is a
heterotetrameric glycoprotein consisting of two extracellular
-subunits and two transmembrane ß-subunits. Insulin binds to the
-subunits and activates the ß-subunit tyrosine kinase, resulting
in receptor autophosphorylation and the phosphorylation of two major
endogenous substrates, insulin receptor substrate-1 and Shc. Tyrosine
phosphorylated insulin receptor substrate-1 and Shc associate with
Grb2. Grb2, in turn, associates with the Ras guanylnucleotide exchange
factor Son of Sevenless (SOS), resulting in an increase in GTP-bound
Ras. Ras in the GTP-bound state can then interact with Raf, which
functions as an upstream kinase for mitogen-activated protein (MAP)
kinase kinase (MEK) and results in MAP kinase activation (1, 2, 3).
The activation of MAP kinase requires the specific phosphorylation of
the threonine and tyrosine residues within the TEY motif by MEK (4, 5, 6).
MAP kinase phosphorylation is a reversible process, indicating that
protein phosphatases play a crucial role in controlling enzyme activity
(7). Among the large number of protein-tyrosine phosphatases currently
identified (8, 9, 10), an emerging class of dual-specificity phosphatases
may directly and specifically regulate MAP kinase family members (11).
The dual-specificity phosphatase family is exemplified by MKP-1 (also
known as CL100, 3CH134, Erp, and hVH-1), which dephosphorylates MAP
kinases at both the Tyr and Thr residues necessary for enzymatic
activity (7, 12, 13, 14, 15, 16). Other mammalian dual-specificity phosphatases
include VHR (17), PAC-1 (18, 19), B23 (also termed hVH-3) (20, 21),
hVH-2 (also known as MKP-2 and TYP-1) (22, 23), hVH-5 (24), and MKP-3
(25). Members of this phosphatase family have been defined as immediate
early genes, since their mRNA is undetectable or present at a low level
in quiescent cells and is rapidly induced by a range of stimuli
including growth factors, oxidative stress, and heat shock. In each
case, induction of the phosphatase is transient. In the case of MKP-1
the protein has a relatively short half-life of 4060 min (12). MKP-1
and PAC-1 are predominantly nuclear proteins (18, 26). However, the
cellular localization of other members of this dual-specificity
phosphatase family remains unclear.
Constitutive overexpression or microinjection of MKP-1 blocks
G1-specific gene expression and S phase entry in
fibroblasts (26), suppresses normal and oncogene-driven proliferation
(7, 14, 15, 27), inhibits neurite outgrowth in differentiating PC12
cells (28), and blocks MAP kinase-dependent mesoderm formation in
Xenopus embryo (29). On the other hand, antisense
oligonucleotides targeted to MKP-1 block angiotensin II-dependent MKP-1
induction in vascular smooth muscle cells and prolong MAP kinases
activation state (30). An antisense oligonucleotide to MKP-1 also
inhibits angiotensin II type 2 receptor-mediated MAP kinase
dephosphorylation and programmed cell death (31). Overexpression of
MKP-2 and PAC-1 in vivo inhibits MAP kinase-dependent gene
transcription (19, 23). Similarly, expression of MKP-3 in COS-7 cells
blocks both the phosphorylation and enzymatic activation of
extracellular-regulated kinase-2 (ERK-2) by mitogens (25). Clearly,
these observations indicate a critical role for dual-specificity
phosphatases in the control of the MAP kinase activation state and
associated cell functions. However, no information is available about
dual specificity phosphatase(s) involved in the regulation of
insulin-induced MAP kinase activity.
We report here that insulin induces a time- and concentration-dependent
increase in MKP-1 mRNA and protein expression in cells overexpressing
the human insulin receptor (Hirc B). Insulin receptor tyrosine kinase
activity is essential for the induction of the MKP-1 message. The
overexpression of MKP-1 inhibits insulin-stimulated MAP kinase activity
and MAP kinase-dependent gene expression. cAMP analogs and inducers
increase MKP-1 mRNA to approximately the same levels seen with insulin.
Treatment with both a cAMP analog/inducer and insulin induces MKP-1
mRNA to levels higher than with either insulin or a cAMP analog/inducer
alone and results in a corresponding decrease in MAP kinase activation.
Our findings provide new insight into the mechanisms of insulin action
and may furnish a new mechanism for the cAMP-dependent inhibition of
insulin-induced MAP kinase activity.
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RESULTS AND DISCUSSION
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Effects of Insulin on MKP-1 Expression in Hirc B Cells
MAP kinases are thought to play a key role in the signaling
process of growth factors, such as epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), nerve growth factor, and of
insulin (32). Although the precise role of MAP kinase activation in
insulin action remains unclear, however, the hormone has been shown to
increase MAP kinase activity in several cell types in a time-dependent
manner (33, 34). Maximal activity was achieved in between 5 and 10 min
after insulin treatment and declined thereafter. The decrease in MAP
kinase activity after few minutes of the hormone treatment could be due
to increased expression and/or activity of MKP-1 in response to
insulin. MKP-1 has been implicated in the inactivation of MAP kinase
(11). To test the hypothesis that the insulin signal, which leads to
MAP kinase activation, also leads to the induction of the specific
kinase attenuator, MKP-1, the expression of MKP-1 was measured in Hirc
B cells that had been incubated in the absence and presence of 100
nM insulin for various periods of time. Total RNA was
isolated, electrophoresed under denaturing conditions, and transferred
to nitrocellulose filters for Northern blotting. The filters were
probed with radiolabeled rat MKP-1 cDNA (35). As a control, the same
filters were reprobed with rat glyceraldehyde phosphate dehydrogenase
(GAPDH) cDNA, since we have seen previously that GAPDH mRNA levels are
not altered by insulin in Hirc B cells (A. B. Kusari and J.
Kusari, unpublished observation). A representative Northern blot is
shown in Fig. 1A
. The MKP-1 cDNA
recognized an mRNA of approximately 2.4 kb, corresponding in size to
rat MKP-1 mRNA (23). A second minor band of approximately 4.2 kb was
also apparent. The minor band could represent the transcript of a
related gene, or an alternatively spliced version of a single primary
MKP-1 message. The MKP-1 and GAPDH mRNA levels were quantitated
densitometrically from autoradiographs of the Northern blots. The
relative amount of MKP-1 mRNA at each time point was normalized to the
amount of GAPDH mRNA. The results of the densitometric scans are shown
in Fig. 1B
, expressed relative to the amount of MKP-1 mRNA present in
basal cells. MKP-1 mRNA was present at a low level in resting cells.
Increased MKP-1 mRNA levels were apparent within 15 min of insulin
stimulation, became maximal within 30 min (3.74-fold over basal), and
rapidly declined thereafter. During prolonged exposure to insulin (5
h), the abundance of MKP-1 mRNA fell to levels lower than in untreated
cells (0.5-fold basal). The time course of the induction of MKP-1 mRNA
by insulin was comparable with that observed previously with 10%
serum, angiotensin II, atrial natriuretic peptide, and EGF (12, 14, 30, 36). Insulin increased MKP-1 mRNA expression levels with a similar time
course in the insulin-responsive rat hepatoma cell type, McA-RH7777
(not shown). This important control demonstrated that the
insulin-stimulated expression of MKP-1 mRNA is not related to the
overexpression of insulin receptors in Hirc B cells but, rather, is a
heretofore unrecognized response to insulin in insulin-responsive
cells. The rapid accumulation of MKP-1 mRNA in Hirc B cells in response
to insulin may be due to increased transcription of the MKP-1 gene
and/or to stabilization of the mRNA. However, the increase in MKP-1
mRNA in response to serum and PDGF is due to rapid transcriptional
activation of the gene (37).

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Figure 1. Time Course for the Induction of MKP-1 mRNA by
Insulin in Hirc B Cells
Quiescent Hirc B cells were treated with 100 nM insulin for
the indicated time interval. Total RNA (50 µg) was subjected to
Northern blot analysis. The filter was hybridized with a
32P-labeled MKP-1 probe, stripped, and then rehybridized
with a GAPDH probe. A representative autoradiogram is shown in panel A,
and the results of the densitometric analysis of MKP-1 mRNA levels
normalized to GAPDH mRNA are shown in panel B (mean ±
SD, n = 3). Presented values in panel B represent fold
induction over untreated cells (0 time point).
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As mentioned above, incubation of Hirc B cells for 30 min with insulin
was necessary for the maximum induction of MKP-1 mRNA. To determine
whether the continuous presence of insulin was necessary during this
period, Hirc B cells were treated with 100 nM insulin for
various periods of time, insulin was then washed off, and the cells
were incubated further in insulin-free medium for a total treatment
period of 30 min. After the cells were harvested, the relative MKP-1
mRNA levels were ascertained by dot blot analysis as described above.
Cells that had been treated with insulin for 5 min and then incubated
for an additional 25 min in the absence of insulin contained
approximately 90% of the MKP-1 present in cells that were treated with
insulin continuously for 30 min (Fig. 2
).
This observation suggests that continuous exposure of Hirc B cells to
insulin is not necessary for the maximum induction of MKP-1 mRNA.

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Figure 2. The Kinetics of MKP-1 mRNA Accumulation in Response
to Insulin Treatment
Growth-arrested Hirc B cells were treated with 100 nM
insulin for the indicated times, washed, and further incubated in media
without insulin for a total of 30 min. Cells were harvested and total
RNA was prepared and examined by dot blot analysis, using a
32P-labeled MKP-1 cDNA probe. The autoradiograms were
quantitated by scanning densitometry, and the data were normalized
against GAPDH mRNA. The results are expressed relative to the amount of
MKP-1 mRNA present in untreated cells. This figure provides the mean ±
SE for three experiments.
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To determine the dose dependence of the stimulation of MKP-1 mRNA
expression by insulin, growth-arrested Hirc B cells were incubated in
the absence and presence of increasing concentrations of insulin for 30
min at 37 C. Total RNA was prepared and MKP-1 mRNA levels were
determined by dot blot analysis of total RNA as described above. A
representative dot blot is shown in Fig. 3A
. Induction of MKP-1 mRNA by insulin
was concentration-dependent, with a threshold response at 1
nM and a maximal response at 10100 nM (Fig. 3B
). The EC50 value was 3.6 nM, similar to that
observed for insulin (5 nM) to stimulate protein tyrosine
phosphatase activity in insulin-responsive rat skeletal muscle cells
(38). The low concentrations required to generate the effect suggest
that insulin stimulates MKP-1 mRNA expression through interaction with
its own receptor.

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Figure 3. Dose Response for the Induction of MKP-1 mRNA by
Insulin in Hirc B Cells
Quiescent Hirc B cells were treated with various concentrations of
insulin for 30 min. Total RNA was subjected to dot blot analysis as
described in the legend to Fig. 2 , and RNA samples were analyzed at two
serial 2-fold dilutions. A representative autoradiogram is shown in
panel A. Serial dilutions from left to right contained 20 and 10 µg
of total RNA. The results of the densitometric analysis of MKP-1 mRNA
levels normalized to GAPDH mRNA are shown in panel B (mean ±
SD, n = 3). Values represent fold induction over
untreated cells (0 nM).
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To study the effect of insulin on MKP-1 protein expression,
growth-arrested Hirc B cells were incubated at 37 C without and with
100 nM insulin for various periods of time. After cell
solubilization, 100 µg of total cell protein were fractionated by
SDS-PAGE and transferred to nitrocellulose. MKP-1 was detected by
immunoblotting using a polyclonal anti-MKP-1 antibody. Figure 4
shows a representative Western blot of
MKP-1 protein levels in Hirc B cells before and after incubation with
insulin for the indicated periods of time. A 40-kDa protein, similar in
molecular mass to MKP-1 protein, as reported in PC12 and rat mesangial
cells (23, 36), was detected in insulin- stimulated Hirc B cell
lysates. Insulin induced a time-dependent increase in MKP-1 protein
expression. The ligand-induced expression of MKP-1 protein was detected
at 30 min. Maximal expression was observed at 120 min, after which the
protein levels decreased. This correlated well with the time course of
insulin-induced MKP-1 mRNA expression as shown in Fig. 1
. In addition
to MKP-1, insulin also increased the expression of another protein with
a molecular mass of 4244 kDa in Hirc B cells. This protein might be
MKP-2 as reported in PC12 cells (23).

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Figure 4. The Effect of Insulin on MKP-1 Protein Expression
in Hirc B Cells
Quiescent Hirc B cells were treated with 100 nM insulin for
the indicated time intervals. Immunoblot analysis was performed on
whole cell lysates, using anti MKP-1 antibody. A representative result
from four experiments is shown.
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Several lines of evidence suggest that insulin receptor tyrosine kinase
activity may be involved in insulin action. To test whether the
receptor tyrosine kinase is essential for the induction of MKP-1 mRNA
by insulin, we compared the time course for the stimulation of MKP-1
mRNA expression in kinase-inactive A/K1018 cells with that in Hirc B
cells. A/K1018 cells express an insulin receptor variant, in which the
lysine residue at amino acid position 1018 has been mutated to alanine.
Although insulin binding to this mutant receptor is unaffected, kinase
activity is completely abolished (39). Growth-arrested Hirc B and
AK1018 cells were incubated at 37 C without and with 100 nM
insulin for various periods of time, total RNA was isolated, and the
amount of the MKP-1 transcript was determined by dot-blot analysis.
Insulin induced a time-dependent increase in MKP-1 mRNA levels in Hirc
B cells. However, there was almost no increase in insulin-stimulated
MKP-1 mRNA expression in AK1018 cells (not shown). These results
suggest that insulinstimulated receptor tyrosine kinase activity is
necessary for the ligand-induced expression of MKP-1 mRNA.
Effects of cAMP Derivatives and Agents That Increase cAMP
on Insulin-Induced MKP-1 mRNA Expression
Increasing intracellular cAMP levels attenuate
insulin-induced MAP kinase activity, at least in part, by inhibiting
hormone-dependent Raf-1 kinase activity (40). However, the increased
cAMP levels may also enhance the expression and activity of MKP-1,
inhibiting insulin-stimulated MAP kinase activity through enhanced
tyrosine and threonine dephosphorylation. To test this hypothesis,
serum-starved Hirc B cells were treated without and with various cAMP
analogs or inducers and 100 nM insulin for 30 min. Total
RNA was isolated, and the expression of MKP-1 mRNA was examined by dot
blot analysis, using a 32P-labeled MKP-1 cDNA probe. The
autoradiograms were quantitated by scanning densitometry, and the data
were normalized against GAPDH mRNA. Values represent the fold increase
over untreated cells. As shown in Fig. 5
, insulin increased MKP-1 mRNA levels to 3.5-fold over basal within 30
min (lane 2). (Bu)2cAMP (lanes 3 and 4), 8-bromo-cAMP
(8-Br-cAMP) (lanes 5 and 6), and 8-(4-chlorophenylthio)-cAMP (lanes 7
and 8) increased both basal and insulin-stimulated MKP-1 mRNA
expression. The basal and insulin-stimulated values determined for
these effects are as follows: (Bu)2cAMP, 3.8-fold and
4.3-fold control; 8-Br-cAMP, 3.64-fold and 6.95-fold control; and
8-(4-chlorophenylthio)-cAMP, 4.25-fold and 4.68-fold control,
respectively. Similarly, forskolin, an agent that increases
intracellular cAMP by directly activating adenylate cyclase, also
increased both basal (4.91-fold control) and insulin-stimulated
(6.42-fold control) MKP-1 mRNA expression. The strongest inducers were
insulin and 8-Br-cAMP (6.95-fold untreated control) and, to a lesser
extent, insulin and forskolin (6.42-fold untreated control). Although
in some cases the increase in MKP-1 mRNA expression by insulin and a
cAMP analog/inducer was less than additive, however, these results
indicate that multiple signaling motifs might regulate MKP-1 mRNA
expression. The enhancement of insulin-stimulated MKP-1 mRNA expression
levels by cAMP analogs and inducers could be another mechanism for the
inhibition of insulin-induced MAP kinase activation by cAMP. To test
this possibility, MAP kinase activity and MKP-1 mRNA expression levels
were measured in Hirc B cells that had been incubated in the absence
and presence of insulin (100 nM), and insulin (100
nM) in combination with 8-Br-cAMP (5 mM) for
various periods of time. As shown in Fig. 6A
, insulin induced a time-dependent
increase in MAP kinase activity in Hirc B cells. Increased MAP kinase
activity was evident within 5 min of insulin stimulation, became
maximal within 10 min (6.50-fold over basal), and declined thereafter.
8-Br-cAMP significantly inhibited insulin-induced MAP kinase activation
(2-fold over basal after 10 min, P < 0.001 in
comparison with insulin-treated control cells). Determination of the
time course of MKP-1 mRNA expression by insulin and 8-Br-cAMP (Fig. 6B
)
indicated that both insulin and insulin plus 8-Br-cAMP increased MKP-1
mRNA levels in a time-dependent manner. Increased MKP-1 mRNA levels
were apparent within 5 min of stimulation by insulin alone or in
combination with 8-Br-cAMP. Furthermore, the induction of MKP-1 mRNA
expression by insulin and 8-Br-cAMP was greater than by insulin alone.
The time course for the insulin-mediated induction of MKP-1 mRNA
expression correlated with the time course for the inactivation of
insulin-stimulated MAP kinase. The inhibition of insulin-induced MAP
kinase activity by 8-Br-cAMP is most likely via attenuated Raf kinase
activity (40). However, the augmented expression of MKP-1 mRNA in the
presence of insulin and 8-Br-cAMP may be responsible, at least in part,
for the inhibition of insulin-induced MAP kinase activation by
cAMP.

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Figure 5. Effects of cAMP Derivatives and Agents That
Increase cAMP on Insulin- Induced MKP-1 mRNA Expression
Growth-arrested Hirc B cells were treated without and with various
combinations of cAMP analogs/derivatives and insulin for 30 min. Total
RNA was prepared and examined by dot blot analysis using a
32P-labeled MKP-1 cDNA probe. The autoradiograms were
quantitated by scanning densitometry, and the data were normalized
against GAPDH mRNA. Values represent the fold increase over untreated
control cells (mean ± SD, n = 4). CTRL, Control;
INS, insulin, Bt2-cAMP, (Bu)2cAMP, 8-Br-cAMP,
8-bromo-cAMP; 8-CP-cAMP, 8-(4-chlorophenylthio)-cAMP; FOR, forskolin.
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Figure 6. The Time Course for Insulin and Insulin plus
8-Br-cAMP-Stimulated MAP Kinase Activity and MKP-1 mRNA Expression in
Hirc B Cells
Growth-arrested cells were treated at 37 C without and with
insulin (100 nM) or insulin (100 nM) and
8-Br-cAMP (5 mM) for various periods of time. A, MAP kinase
activity was measured in cell extracts as described in Materials
and Methods. This figure provides ± SE for
three experiments. *, P < 0.01 in comparison with
insulin- treated control cells. B, Total RNA was prepared and examined
by dot blot analysis using a 32P-labeled MKP-1 cDNA probe.
The autoradiograms were quantitated by scanning densitometry, and the
data were normalized against GAPDH mRNA. The results are expressed in
arbitrary units (mean ± SD, n = 3).
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MKP-1 Expression Blocks MAP Kinase Activity and the Transcriptional
Activation of MAP Kinase-Regulated Reporter Genes
It has been shown that MKP-1 dephosphorylates and inactivates MAP
kinase both in vitro and in vivo (15).
Furthermore, depleting cells of CL 100, a human homolog of MKP-1, and
preventing its induction using cycloheximide stopped the inactivation
of MAP kinase in swiss 3T3 fibroblasts after stimulation with EGF.
However, attenuation of CL 100 induction had no effect on the rapid
inactivation of MAP kinase in response to EGF in adipose (3T3-L1) or
chromaffin (PC12) cells or in response to PDGF in endothelial (PAE)
cells. Moreover, maximal induction of CL 100 mRNA and a CL 100-like
activity did not trigger inactivation of MAP kinase, which was
sustained at a high level after stimulation of PC 12 cells with nerve
growth factor, PAE cells with serum, or Swiss 3T3 cells with PDGF (41).
These results indicate that in multiple situations, induction of MKP-1
was not accompanied by the inactivation of MAP kinase. To test the
hypothesis that MKP-1 inhibits insulin-stimulated MAP kinase activity,
MKP-1 was overexpressed in Hirc B cells, and MAP kinase activity was
measured in the absence and presence of insulin. The phosphatase was
overexpressed in Hirc B cells by transfecting the expression plasmid
(pSG5), containing full-length MKP-1 cDNA. To ensure overexpression,
the levels of MKP-1 and GAPDH transcript expression were measured by
dot blot analysis in transfected cells at various times after
transfection. The mRNA levels were quantitated and normalized as in
Fig. 1
. The results of the densitometric scans are shown in Fig. 7A
. There was a time-dependent increase
in MKP-1 mRNA expression in transfected cells. Maximal expression was
observed 48 h after transfection (30-fold over basal), and
thereafter the expression declined. This result indicates that MKP-1
can be overexpressed in Hirc B cells.

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Figure 7. MKP-1 Expression Inhibits Insulin-Stimulated MAP
Kinase Activity and MAP Kinase-Sensitive Gene Transcription
A, MKP-1 mRNA levels in Hirc B cells at various times after
transfection. Hirc B cells were transfected with the expression plasmid
pSG5 MKP-1 by the Ca3(PO4)-mediated
transfection procedure. At various times after transfection, Hirc B
cells were harvested and total RNA was prepared as described in
Materials and Methods. MKP-1 mRNA levels were examined
by dot blot analysis using a 32P-labeled MKP-1 cDNA probe.
The autoradiograms were quantitated by scanning densitometry, and the
data were normalized against GAPDH mRNA. The results are expressed
relative to the amount of MKP-1 mRNA present in untransfected cells.
This figure provides the mean ± SE for four independent
experiments. B, MAP kinase activity in control and MKP-1- transfected
Hirc B cells under basal and insulin-stimulated conditions. Forty eight
hours after transfection, Hirc B cells were incubated at 37 C in the
absence and presence of 100 nM insulin for 10 min. MAP
kinase activity was then measured in cell extracts as described in
Materials and Methods. This figure provides the mean ±
SE for three experiments. *, P < 0.001
in comparison with all other groups. C, Effects of MKP-1 on the (i) SRE
and (ii) CRE-mediated expression of a ß-galactosidase reporter gene.
Hirc B cells were transfected with either 5XSRE/Z or 5XCRE/Z along with
either pSG5 or pSG5-MKP-1. Forty eight hours after transfection, Hirc B
cells were incubated at 37 C in the absence and presence of 100
nM insulin or 1 mM each of 8-Br-cAMP and IBMX
for 24 h and cell extracts were prepared as described in
Materials and Methods. ß-Galactosidase activity was
measured in cell extracts using a ß-galactosidase assay system kit.
The results are expressed relative to the ß-galactosidase activity
observed under basal conditions in cells cotransfected with 5XSRE/Z or
5XCRE/Z and pSG5 vector. This figure provides the mean ±
SE for three experiments. **, P <
0.001 in comparison with all other groups.
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To determine the consequence of MKP-1 overexpression, MAP kinase
activity was measured in control and transfected Hirc B cells under
basal and insulin- stimulated conditions. As shown in Fig. 7B
, incubation of untransfected Hirc cells with 100 nM insulin
for 10 min resulted in an 8.0-fold increase in MAP kinase activity over
basal levels (lane 2 vs. 1). Similar observations have been
reported in Hirc B cells by Pang et al. (42). Overexpression
of MKP-1 inhibited both basal (20% as compared with untransfected
cells, lane 3 vs. 1) and insulin-stimulated (50% as
compared with untransfected cells, lane 4 vs. 2) MAP kinase
activity. However, insulin-stimulated MAP kinase activity was
significantly reduced in MKP-1 overexpressing cells (P
< 0.001). When compared with untransfected cells, transfection of Hirc
B cells with the pSG5 vector alone altered neither basal nor
insulin-stimulated MAP kinase activity (not shown). Furthermore,
lysates from control and MKP-1 transfected cells had equivalent
insulin-stimulated MEK activity (not shown), suggesting that MKP-1 does
not inhibit insulin-stimulated events upstream of MAP kinase.
MAP kinase is cytoplasmic in quiescent cells. Upon growth factor
stimulation, cytosolic MAP kinase is activated and translocated rapidly
to the nucleus (43, 44, 45, 46). Once in the nucleus, MAP kinase phosphorylates
and activates various transcription factors, including c-Myc, ATF2, and
p62TCF (47). Since MKP-1 is a nuclear phosphatase (26), it
is likely that the enzyme dephosphorylates the translocated MAP kinase
within the nucleus, thus inactivating it. To examine the effects of
MKP-1 expression on an insulin- stimulated nuclear event linked to MAP
kinase activation, we measured the transcriptional activity of the
c-fos serum response element (SRE) containing promoter,
under basal and insulin-stimulated conditions in control and MKP-1
overexpressing cells. Upon phosphorylation by activated MAP kinase, the
transcription factor p62TCF binds to the SRE and activates
transcription through the SRE promoter (43, 44). Thus, the activation
of the SRE containing promoter acts as a read out of the activation of
MAP kinase.
To determine the transcriptional activity of the SRE containing
promoter, we transfected the reporter plasmid 5XSRE/Z into Hirc B cells
and measured ß-galactosidase activity in a transient assay. 5XSRE/Z
contains five copies of the SRE, placed adjacent to a basic promoter
element and linked to the ß-galactosidase gene (48). As shown in Fig. 7C
(i), insulin increased ß-galactosidase activity (2.69-fold over
control, lane 2 vs. 1) in Hirc B cells transfected with
5XSRE/Z DNA. There was very little ß-galactosidase activity in
untransfected cells under basal and insulin-stimulated conditions, and
the enzyme activity was not affected by cotransfection with control
pSG5 vector (not shown). Cotransfection of the expression plasmid
pSG5-MKP-1 inhibited both basal (0.75-fold control, lane 3
vs. 1) and insulin-stimulated (1.39-fold over control, lane
4 vs. 2) ß-galactosidase activities. Insulin-stimulated
ß-galactosidase activity was significantly reduced in
MKP-1-overexpressing cells (P < 0.001).
It is interesting to note that MKP-1 does not act nonspecifically to
inhibit all promoter activation, as demonstrated by similar studies
performed in the absence and presence of 8-Br-cAMP and
isobutylmethylxanthine (IBMX) in Hirc B cells cotransfected with MKP-1
and 5XCRE/Z DNAs. The plasmid 5XCRE/Z contains five copies of the cAMP
response element (CRE) placed adjacent to a Rous sarcoma virus promoter
and linked to the ß-galactosidase gene (49). Upon phosphorylation by
protein kinase A, the transcription factor CREB binds to the CRE and
can activate transcription through the CRE promoter (50). As shown in
Fig. 7C
(ii), 8-Br-cAMP and IBMX increased ß-galactosidase activity
(2.34-fold over control, lane 2 vs. 1) in Hirc B cells
transfected with 5XCRE/Z DNA. However, there was no significant
alteration either in basal (lane 3 vs. 1) or cAMP-induced
(lane 4 vs. 2) ß-galactosidase activity by cotransfection
of pSG5-MKP-1. These results suggest that the overexpression of MKP-1
specifically inhibits MAP kinase, preventing SRE-dependent
transcription. PAC-1 and HVH2, other members of the dual specificity
phosphatase family, have also been shown to inhibit MAP kinase-induced
transcription of an SRE-containing promoter (19, 22).
In summary, insulin induces a time- and concentration-dependent
increase in MKP-1 mRNA and protein expression in cells overexpressing
the human insulin receptor. We previously reported similar observations
with another tyrosine phosphatase, PTPase 1B, in L6 myotubes (38). The
time course for the insulin-mediated induction of MKP-1 mRNA expression
correlates with the time course for the inactivation of
insulin-stimulated MAP kinases. Insulin-stimulated receptor tyrosine
kinase activity is essential for the induction of the MKP-1 message.
The overexpression of MKP-1 significantly inhibits insulin-stimulated
MAP kinase activity and MAP kinase-dependent gene expression. This
study demonstrates that MKP-1 is regulated by insulin. MKP-1 is, in
turn, part of the insulin-mediated signal transduction pathway
regulating MAP kinase activity. MKP-1 is, to our knowledge, the third
tyrosine phosphatase to be identified as a negative regulator of
insulin signaling. PTPase 1B and leukocyte antigen-related phosphatase
have been previously shown to attenuate insulin action (51, 52, 53).
The expression and activity of MKP-1 are increased in response to
insulin. Insulin-stimulated MKP-1 dephosphorylates threonine and
tyrosine residues of activated MAP kinase, inhibiting its activity.
Therefore, the insulin signal, which leads to MAP kinase activation,
also leads to the induction of the specific MAP kinase attenuator,
MKP-1. Continuous MAP kinase activity during inappropriate phases of
insulin signaling may be prevented in this manner. However, the
mechanism of insulin-induced MKP-1 expression is not yet clear. Since
MKP-1 is principally regulated at the transcriptional level, it is
possible that insulin-stimulated MAP kinase activity could increase the
expression of MKP-1. However, fibroblast transformation by oncogenes
such as v-ras, v-raf or constitutively active forms of MEK all elicit
constitutive MAP kinase activity (54, 55, 56), and there is no report so
far that these oncogenes activate MKP-1 activity. If the activation of
MAP kinase by insulin is the signal that activates MKP-1, we would
expect that constitutive activation of MAP kinase by oncogenic ras
would activate MKP-1. Hence, MAP kinase activation alone is probably
insufficient for or uninvolved with MKP-1 induction. Recently, it has
also been shown that the expression of MKP-1 is induced by activation
of the MEKK-SEK-SAPK but not by stimulation of the
Raf-MEK-MAP-signaling pathway (57). Thus, in addition to inducing the
MAP kinase-signaling pathway, insulin may activate the SAPK signaling
pathway to increase the expression of MKP-1, which in turn regulates
MAP kinase activity. Further studies are warranted to explore this
possibility. Work on the regulation of this dual specificity
phosphatase will generate vital information to further elucidate
insulin- mediated signal transduction pathways utilizing MAP
kinase.
Though the insulin dependent activation of MAP kinase has offered the
tantalizing suggestion that MAP kinase activity plays a role in insulin
signaling, previous data have failed to establish the physiological
relevance of MAP kinase activity within the insulin-signaling matrix.
Our present data suggest an essential role for MAP kinase in
insulin-induced gene expression. A similar conclusion has also
been independently reached by another laboratory (58). Our results also
indicate that MKP-1 may be a critical component of the insulin signal
transduction pathway and that defects in MKP-1 expression and/or
activity may have profound effects on cellular responsivity to
insulin.
 |
MATERIALS AND METHODS
|
---|
Materials
DMEM-F12, FCS, gentamycin, and glutamax were obtained from Life
Technologies, Inc. (Gaithersburg, MD). Insulin was kindly provided by
Eli Lilly and company (Indianapolis, IN). Methotrexate and forskolin
were purchased from Calbiochem (San Diego, CA). MKP-1 and MAP kinase
antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA)
and Upstate Biotechnology, Inc. (Lake Placid, NY), respectively.
Tween-20, protein molecular weight standards, acrylamide, and TEMED
were purchased from Bio-Rad (Hercules, CA). Nonfat dry milk was from
Nestle Foods Co.(Glendale, CA). Anti-rabbit IgG and enhanced
chemiluminescence (ECL) detection system were obtained from Amersham
Life Science (Arlington Heights, IL). Nitrocellulose membrane was from
Schleicher & Schuell (Keene, NH). Eukaryotic expression vector-pSG5 was
obtained from Stratagene (La Jolla, CA). [
-32P]dCTP
and [
-32P]ATP were purchased from Dupont, NEN (Boston,
MA). RNASTAT-60 was purchased from Tel-Test-"B", Inc. (Friendswood,
Texas). ß-Galactosidase enzyme assay system was obtained from Promega
(Madison, WI). All other reagents were purchased from Sigma (St. Louis,
MO) and were the highest quality available.
Cell Lines
The cell lines used in this study were described previously (39, 59). Briefly, Hirc B and AK1018 are rat fibroblast cell lines
expressing normal human insulin receptors (Hirc B) and human insulin
receptors with lysine 1018 replaced by alanine (AK1018), respectively.
Both of these cell lines were propagated in DMEM-F12 containing 10%
FCS, 50 µg/ml gentamycin, 2 mM glutamax, and 500
nM methotrexate.
RNA Isolation
Approximately 90% confluent Hirc B or AK1018 cells were serum
starved for 1624 h in medium with 0.1% FCS. After serum starvation
cells were incubated in the absence and presence of 100 nM
insulin for various periods of time at 37 C. Thereafter, total RNA was
isolated using RNASTAT-60 as previously described (60).
Northern Blot Analysis
Northern blot analysis was carried out as previously described
(61). Briefly, 50 µg of total RNA were electrophoresed through a
1.2% agarose, 2.2 M formaldehyde gel and stained with
ethidium bromide. The RNA was visualized and photographed by UV
transillumination to ensure that the total RNA was intact and to allow
the calculation of molecular weight, based on the migration of 28 and
18 S ribosomal subunits. RNA was transferred to nitrocellulose filters
in 20 x sodium chloride/ sodium citrate (SSC) (3 M
NaCl, 0.3 M sodium citrate, pH 7.0). The blots were
hybridized sequentially to [
-32P] labeled rat MKP-1
cDNA and rat GAPDH cDNA. Hybridization was carried out in 50%
deionized formamide, 5x SSC, 1x Denhardts, 0.1 mg/ml heat-denatured
sheared salmon sperm DNA, and 1 x 106 cpm/ml
32P-labeled cDNA probe at 42 C for 16 h. The blots
were washed three times (5 min each) at room temperature in 2x
SSC/0.1% SDS and three times (15 min each) at 55 C in 0.1x SSC/0.1%
SDS. The filters were air-dried and exposed to XAR-5 film for 616 h.
The relative mRNA levels were determined by laser scanning densitometry
of the autoradiographs using Adobe photoshop V 2.5.1 (Adobe Systems,
Inc., San Jose, CA) and NIH Image (NIH, Bethesda, MD). Probes were
stripped from the filters by washing the blots in distilled water at 95
C for 15 min.
Dot Blot Analysis
In most of the experiments described, total RNA was analyzed by
dot hybridization (61). All RNA samples were analyzed at two or three
serial 2-fold dilutions containing 20 and 10 or 20, 10, and 5 µg of
total RNA. The autoradiograms were scanned with a densitometer-scanner
using Adobe photoshop V 2.5.1 and NIH Image. Only those readings that
increased co-linearly with increasing total RNA concentrations were
used for calculations. To obtain such linearity for samples in the same
experiment having very different signal intensities, it was sometimes
necessary to scan several autoradiograms with variable amounts of
exposure.
Immunoblot Analysis
Ninety percent confluent Hirc B cells were serum starved and
exposed to insulin as described above in RNA Isolation.
Cells were then harvested in cell lysis buffer (50 mM
Tris-HCl, pH 7.3, 150 mM NaCl, 1% Triton X-100, 5
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1
mM dithiothreitol, 1 mM sodium vanadate, 25
mM NaF, 10 mM sodium pyrophosphate, 25
mM ß-glycerophosphate, and 50 U/ml aprotonin) and
centrifuged at 12,000 x g for 15 min at 4 C. The
supernatants containing 100 µg of protein were subjected to 12.5%
SDS-PAGE and transferred onto nitrocellulose membranes. The membranes
were probed with anti-MKP-1 antibody (diluted at 1:200). A horseradish
peroxidase-conjugated secondary antibody (1:1000) was used to allow
detection of immunoreactive bands using enhanced chemiluminescence.
Transient Transfection
Hirc B cells were grown as described above to 80% confluence,
then transfected with and without 7 µg of the mammalian expression
vector pSG5 containing rat MKP-1 cDNA (pSG5-MKP-1) by the
Ca3(PO4)2-mediated transfection
procedure (62). One day after transfection, cells were exposed to 10%
glycerol for 3 min, and then washed three times with PBS. The cells
were incubated in fresh medium for various periods of time and then
harvested for analysis of MKP-1 mRNA levels as described above.
MAP Kinase Assay
Untransfected or transfected Hirc B cells were serum starved for
16 h in medium with 0.1% FCS, then incubated in the absence and
presence of 100 nM insulin for 10 min at 37 C. The cells
were harvested in 100 µl of cell lysis buffer as described above and
centrifuged at 12,000 x g for 15 min at 4 C. The
supernatants containing 200 µg of protein were incubated with 2 µl
of anti-MAP kinase antibody (1 µg/µl) for 1 h at room
temperature. The immunoprecipitates were recovered by incubating
with 20 µl of 50% protein G-Sepharose for 1 h at room
temperature, centrifuging, and washing three times with cell lysis
buffer, then once with a kinase buffer without ATP (18 mM
HEPES, pH 7.5, and 10 mM magnesium acetate). The
immunoprecipitates were incubated with 20 µg myelin basic protein in
40 µl kinase buffer (36) containing 2 µCi
[
-32P]ATP for 10 min at 30 C. Ten microliters of the
reaction mixture were spotted on a P-81 filter paper (Whatman Inc.,
Clifton, NJ), and the filter was washed with 180 mM
phosphoric acid five times for 5 min each, followed by one wash with
95% ethanol. The paper was dried, and the radioactivity bound on the
filter paper was counted in a scintillation counter. Results are
expressed in terms of counts per min released per µg protein.
Gene Expression
Hirc B cells were transfected with 1 µg of either 5XSRE/Z or
5XCRE/Z plasmid along with 6 µg of either pSG5, or construct
expressing MKP-1 using calcium phosphate precipitation as described
previously. Thirty two hours later, cells were serum starved for
16 h in medium with 0.1% FCS. After serum starvation, cells were
incubated in the absence and presence of 100 nM insulin or
1 mM each of 8-Br-cAMP and IBMX for 24 h at 37 C. Cell
extracts were prepared from the various treatment groups and assayed
for ß-galactosidase activity using a ß-galactosidase enzyme assay
system kit (Promega, Madison, WI).
Statistical Analysis
Statistical analysis was performed using ANOVA.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. Jerrold M. Olefsky for providing rat 1 fibroblasts
overexpressing human wild type or mutant insulin receptors. We also
thank Drs. Nikki J. Holbrook for providing the rat MKP-1 cDNA and Dave
Rose for the 5XSRE/Z and 5XCRE/Z constructs.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jyotirmoy Kusari, Department of Physiology, SL 39, School of Medicine, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699.
This work was supported by NIH Grant DK-46490 and by start-up funds
from Tulane University (to J.K.).
Received for publication March 19, 1997.
Accepted for publication June 24, 1997.
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