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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-subunits and two transmembrane ß-subunits. Insulin binds to the {alpha}-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 40–60 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 kinase’s 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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo. 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. 1BGo, 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).

 
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. 2Go). 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.

 
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. 3AGo. Induction of MKP-1 mRNA by insulin was concentration-dependent, with a threshold response at 1 nM and a maximal response at 10–100 nM (Fig. 3BGo). 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. 2Go, 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).

 
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 4Go 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. 1Go. In addition to MKP-1, insulin also increased the expression of another protein with a molecular mass of 42–44 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.

 
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. 5Go, 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. 6AGo, 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. 6BGo) 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).

 
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. 1Go. The results of the densitometric scans are shown in Fig. 7AGo. 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.

 
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. 7BGo, 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. 7CGo(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. 7CGo(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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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). [{alpha}-32P]dCTP and [{gamma}-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 16–24 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 [{alpha}-32P] labeled rat MKP-1 cDNA and rat GAPDH cDNA. Hybridization was carried out in 50% deionized formamide, 5x SSC, 1x Denhardt’s, 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 6–16 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 [{gamma}-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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 MATERIALS AND METHODS
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