Hormones, Growth Factors, and Cell Signaling

Mechanisms of Mitogen-Activated Protein Kinase Activation in Experimental Diabetes

MIDORI AWAZU, KENJI ISHIKURA, MARIKO HIDA and MAKIKO HOSHIYA
JASN April 1999, 10 (4) 738-745;

Abstract

Abstract. Various growth factors and vasoactive substances are implicated in the pathogenesis of renal growth seen in early diabetes mellitus (DM). Mitogen-activated protein kinase (MAPK) is an important mediator of these extracellular stimuli. Protein kinase C (PKC), an enzyme known to be stimulated in DM, also activates MAPK. Thus, MAPK activity was examined in glomeruli from streptozotocin-induced DM rats. MAPK activity, measured as myelin basic protein kinase, was elevated by approximately 50% in DM versus controls (CON). Increased protein contents of p42mapk and p44mapk, as well as increased tyrosine phosphorylation and mobility shift of p42mapk, were also observed in DM. Tyrosine dephosphorylation of pp42mapk, on the other hand, assessed by incubating glomerular membrane with or without sodium orthovanadate (vanadate), was significantly diminished in DM. Protein expression of MAPK phosphatase-1 (MKP-1), a dual specificity phosphatase that inactivates MAPK, was approximately 60% of CON. Reduction in MKP-1 was reproduced in cultured mesangial cells grown under high glucose (30 mM; HG). The suppression of MKP-1 was PKC-dependent since incubation of HG cells with phorbol 12-myristate 13-acetate for 24 h abolished it. Furthermore, calcium ionophore A23187 reversed the suppression, suggesting that blunted Ca2+ signalling, characteristic of HG cells secondary to PKC stimulation, may be the cause. These results demonstrate that glomerular MAPK is activated in DM by multiple mechanisms i.e., increases in protein contents, increased phosphorylation, and decreased dephosphorylation of the enzyme due to suppression of MKP-1. These alterations may have an implication in the pathogenesis of diabetic nephropathy.

Diabetic kidney is characterized by early hypertrophy of both glomerular and tubuloepithelial structure (1,2). The mechanisms for these processes are not fully elucidated but include the elaboration of various growth factors and vasoactive substances such as transforming growth factor-β, angiotensin II, endothelin, thromboxane A2, insulin-like growth factor-1, fibroblast growth factor, and platelet-derived growth factor 3,4). Mitogen-activated protein kinase (MAPK), also known as extracellular signal-regulated protein kinase (ERK), is a crucial intermediary signaling molecule in the signal transduction cascade of these substances.

Activation of protein kinase C (PKC), due to de novo synthesis of diacylglycerol induced by high glucose, is another key feature of diabetes mellitus (DM) (5). It is considered to play an important role in the pathogenesis of diabetic nephropathy. A recent study demonstrated that orally administered PKC inhibitor ameliorated the development of diabetic nephropathy in rats (6). PKC is known to activate MAPK through activation of Raf-1 (7).

MAPK is a serine/threonine kinase found ubiquitously expressed in tissues. Two isoforms of MAPK, p44mapk and p42mapk, also called ERK1 and ERK2, are known to exist. MAPK is activated by phosphorylation on both tyrosine and threonine and inactivated by dephosphorylation of either residue (8,9). The direct upstream activator of MAPK is a dual-specificity protein kinase, MAPK kinase or MEK (10). MEK is in turn activated by Raf-1 (11). The phosphorylation state of MAPK is determined by phosphatases as well as kinases. Thus, recently cloned dual-specificity protein tyrosine phosphatases draw particular attention as a potential regulator of MAPK cascade (12). These enzymes are capable of removing both phosphotyrosine and phosphothreonine from target proteins. MAPK phosphatase-1 (MKP-1) is a member of such phosphatases, which has been shown to dephosphorylate MAPK in vitro and in vivo (13,14). MKP-1 exhibits substrate specificity toward MAPK and is located in the nucleus (15). Since phosphorylated MAPK migrates to the nucleus where it activates several transcription factors and becomes inactivated (16), the localization of MKP-1 strongly suggests its role as a physiologic inactivator of MAPK. MKP-1 has been demonstrated to be expressed in cultured mesangial cells and regulated by multiple signaling molecules (17,18). Little is known, however, on the precise role of MKP-1 in vivo especially in pathophysiologic settings.

In the present study, we examined whether the glomerular MAPK activity is altered during the early phase of diabetic kidney growth. Furthermore, we sought to determine the mechanisms for the stimulation with a special focus on the dephosphorylation of the enzyme.

Materials and Methods

Materials

Bovine myelin basic protein (MBP), protein kinase inhibitor, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, 1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA), A23187, phorbol 12-myristate 13-acetate (PMA), D-mannitol, and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). Arginine vasopressin (AVP) was from Peptide Institute (Osaka, Japan). Streptozotocin was from Wako Pure Chemical Industries (Osaka, Japan). Monoclonal anti-phosphotyrosine antibody (PY20-alkaline phosphatase conjugate) was from Transduction Laboratories (Lexington, KY). Anti-rat MAPK R2 (Erkl-CT, rabbit polyclonal IgG) was from Upstate Biotechnology (Lake Placid, NY). Anti-MKP-1 antibody (V-15, rabbit polyclonal) was from Santa Cruz Biotechnology (Santa Cruz, CA). IgGsorb was from The Enzyme Center (Malden, MA). γ-[32P] ATP was from Amersham (Tokyo, Japan).

Induction of Diabetes

Male Sprague Dawley rats weighing 180 g were injected with 60 mg/kg streptozotocin (60 mg/ml in 0.01 M citric acid, 0.09% saline, pH 4) by tail vein. Two rats were sacrificed 1 wk later, and the remaining six, 3 wk after the injection. Rats matched for age and weight at the time of streptozotocin administration served as controls (CON). At death, blood was drawn from the abdominal aorta for determination of nonfasting plasma glucose by the glucose oxidase method.

Isolation of Glomeruli

Glomeruli were isolated from rat kidneys using a method previously reported (19). In brief, rats were anesthetized, and the kidneys were removed immediately and placed in ice-cold phosphate-buffered saline. All the following procedures were performed at 4°C. The cortices were dissected, minced, and homogenized. The resulting suspension was passed through three consecutive sieves (stainless steel ASTM #140, #80, #230). The sieved specimens were then collected, centrifuged 3 min at 2000 rpm, and the supernatant was discarded. This washing was repeated one more time, and one-half of glomeruli were suspended in lysis buffer containing 20 mM Hepes, pH 7.2, 1% Triton X-100, 10% glycerol, 20 mM sodium fluoride, 1 mM sodium orthovanadate (vanadate), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Glomeruli were homogenized by Dounce (200 strokes to achieve microscopic verification of complete disruption). Insoluble material was removed by centrifugation (10,500 × g, 10 min). Cleared lysates were used for MAPK assay and immunoblotting. The remaining half of glomeruli were resuspended in membrane buffer (0.32 M sucrose, 10 mM Tris, pH 7.5, 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetra-acetic acid, 1 mM ethylenediamine tetra-acetic acid, homogenized, and centrifuged for 30 min. The particulate pellet was resuspended in membrane preparation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM PMSF, bacitracin 20 μg/ml, 1% NP-40) and used for dephosphorylation assay. Protein content in cell lysates was determined by DC protein assay (Bio-Rad Laboratories, Tokyo, Japan).

Mesangial Cell Culture

Rat mesangial cells were isolated and cultured using the method described elsewhere (20). Cells were grown in regular RPMI 1640 (glucose 9.2 mM) or high glucose medium (glucose 30 mM) containing 17% fetal bovine serum 100 μ/ml penicillin, 100 μg/ml streptomycin, and 250 μg/L fungizone. Glucose concentration of 9.2 mM was used to simulate the blood glucose levels in control rats. In control experiments, mannitol was added to 9.2 mM medium to bring total osmolality to values equivalent to 30 mM glucose. Media were replaced every day. Experiments were performed after 5 d at approximately 80% confluence. Unless indicated otherwise, cells were fed 16 to 18 h before the experiment. For experiments examining the effects of AVP on MKP-1 expression, cells were serum-deprived for 24 h with appropriate concentrations of glucose media. Cells were washed with cold phosphate-buffered saline and lysed in lysis buffer. Insoluble material was removed by centrifugation (10,500 × g, 10 min). Cleared lysates were used for immunoblotting. Cells from passages 9 through 13 were used. The cultures were maintained at 37°C in a humidified atmosphere of 95% O2/5% CO2.

MAPK Assay

MAPK activity was measured by [32P]ATP incorporation into MBP as previously described (21). Assays were performed at 25°C for 15 min in a final volume of 40 μl containing 25 mM Tris-Cl, pH 7.4, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM ethyleneglycol-bis (β-aminoethyl ether)-N,N′-tetra-acetic acid, 1 μM protein kinase inhibitor, 50 μM ATP, 2 μCi γ-[32P] ATP, 20 μg MBP, and 10 μl of samples. The reactions were terminated by the addition of 10 μl of Laemmli's buffer, and phosphorylated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After autoradiography to visualize phosphorylation of MBP, the radioactivity was determined by liquid scintillation counter.

Immunoblot Analysis

Cleared lysates were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA). Nonspecific binding sites were blocked in TBS buffer (10 mM Tris-Cl, pH 7.4, 0.15 M NaCl) containing 5% BSA overnight at 4°C or for 1 h at 25°C. Antibodies were added to TBS with 5% BSA in saturating titers and incubated with mixing for 2 h at 25°C. Blots were washed twice, then developed (anti-phosphotyrosine antibody). For analyses with MAPK and MKP-1 antibody, blots were further incubated with goat anti-rabbit Ig/alkaline phosphatase conjugate for 1 h with mixing and then washed. Membranes were washed once in substrate buffer (0.1 M Tris-Cl, pH 9.5, 0.1 M NaCl, and 5 mM MgCl2), then developed by the addition of fresh substrate (20 mg of nitroblue tetrazolium in 60 ml of substrate buffer, mixed immediately before blot exposure with 10 mg of 5-bromo-4-chloro-3-indolyl phosphate in 200 μl of N,N′ dimethylformamide). Alternatively bound antibodies were detected using the enhanced chemiluminescence Western blotting system from Amersham (Tokyo, Japan). Blots were scanned and quantitatively analyzed using NIH Image.

Dephosphorylation Assay

Glomerular membranes (60 to 70 μg) were incubated in dephosphorylation buffer (250 mM Hepes, pH 7.2, 50 mM dithiothreitol, 25 mM ethylenediaminetetra-acetic acid) for 30 min at 25°C with or without 1 mM vanadate. The reactions were terminated by the addition of Laemmli's buffer, and phosphorylated proteins were separated by SDS-PAGE. Immunoblotting with anti-phosphotyrosine and anti-MAPK antibody was performed as described above.

Statistical Analyses

The results are expressed as mean ± SEM. Comparisons were made by ANOVA and unpaired t test as appropriate. Statistical significance was determined as P < 0.05.

Results

Animal Data

Because there was no difference in all parameters studied between rats injected with streptozotocin 1 and 3 wk before the experiment, the data were pooled. All streptozotocin-treated rats were diabetic with a mean plasma glucose of 490 ± 58 mg/dl (n = 8, P < 0.05 versus CON) (Table 1). Kidney weight corrected by body weight was significantly greater in DM than control rats.

View this table:
Table 1.

Characteristics of study animals

MAPK Activity, Protein Content, and Tyrosine Phosphorylation in Glomeruli from Diabetic and Control Rats

Glomerular MAPK activity, measured as MBP kinase, was increased in DM (151 ± 10% versus CON, P < 0.05, n = 6) (Figure 1, A and B). To examine the mechanisms for MAPK activation, glomerular protein contents of two isoforms of MAPK were assessed by immunoblot analysis. Both p42mapk and p44mapk were more abundant in DM than CON (166 ± 21 and 137 ± 10% versus CON, respectively, both P < 0.05, n = 4) (Figure 1C). Furthermore, a slow-migrating band of p42mapk that corresponds to the phosphorylated active enzyme was observed in DM, whereas it was hardly detectable in CON. Immunoblot analysis with anti-phosphotyrosine also showed that tyrosine phosphorylation of p42mapk was significantly enhanced in DM (151 ± 11% versus CON, P < 0.05, n = 4) (Figure 1D). Tyrosine phosphorylation of p44mapk was not detected either in DM or CON. These results indicate that activation of MAPK in DM was accompanied by increased protein contents and tyrosine phosphorylation of the enzyme.

Figure 1.
Figure 1.

Activation of mitogen-activated protein kinase (MAPK) in glomeruli from diabetes mellitus (DM). (A) Stimulation of MAPK activity in glomeruli from DM. MAPK activity was measured as myelin basic protein (MBP) kinase activity in glomerular lysates as described in Materials and Methods. Values are expressed as percentage of control (CON), mean ± SEM, n = 6. *P < 0.05 versus CON. (B) A representative autoradiogram of phosphorylated MBP. (C) Increases in MAPK proteins in glomeruli from DM. Equal amounts of glomerular lysates were loaded and immunoblotted with anti-MAPK antibody. A representative immunoblot. Quantitative analysis is shown below (n = 4). (D) Increases in tyrosine phosphorylation of p42mapk in glomeruli from DM. Equal amounts of glomerular lysates were loaded and immunoblotted with anti-phosphotyrosine antibody (anti-PTyr). To indicate the position of pp42mapk, 3T3 cell lysate blotted with anti-MAPK antibody is shown as a positive control (n = 4).

Dephosphorylation of pp42mapk in Glomerular Membranes from Diabetic and Control Rats

Since the activity of MAPK is regulated by the balance of phosphorylation and dephosphorylation, we examined whether MAPK dephosphorylating activity is altered in DM. Among several serine/threonine and tyrosine phosphatases suggested to dephosphorylate MAPK, MKP-1, a dual specificity phosphatase, has been shown to inactivate MAPK in vivo (13). Since MKP-1 is located in the nucleus, we used glomerular particulate fractions for the assay. As shown in Figure 2A, tyrosine phosphorylated p42mapk (pp42mapk) was present in the particulate fractions of both CON and DM (top panel). Similarly to whole cell lysates, MAPK protein contents were slightly increased in particulate fractions of DM compared with CON (Figure 2A, bottom panel). In CON, pp42mapk was completely tyrosine-dephosphorylated after 30 min of incubation in dephosphorylation buffer. Vanadate, an MKP-1 inhibitor, inhibited the dephosphorylation of pp42mapk. In striking contrast, there was no difference in pp42mapk in DM in the presence or absence of vanadate, suggesting an absence of vanadate-sensitive tyrosine phosphatase activity toward pp42mapk.

Figure 2.
Figure 2.

Reduced tyrosine dephosphorylation of MAPK in DM. (A) Dephosphorylation of pp42mapk in glomerular membranes from DM. Glomerular membranes were incubated in dephosphorylation buffer with or without 1 mM vanadate. After stopping reactions by addition of Laemmli's buffer, immunoblot analyses with anti-phosphotyrosine (anti-PTyr) and anti-MAPK were performed. To indicate the position of pp42mapk, 3T3 cell lysate blotted with anti-MAPK antibody is shown as a positive control (top panel, far right). The blots were reprobed with anti-MAPK (bottom panel). In control membranes, bands for p44 and p42mapk (faint bands below p44) are visible. In DM, bands for p44 and p42mapk are fused. Results are representative of three experiments. (B) Reduced glomerular MAPK phosphatase-1 (MKP-1) expression in DM. Equal amounts of glomerular lysates were loaded and immunoblotted with anti-MKP-1 antibody. A representative immunoblot.

Reduction in MKP-1 Protein Expression in Glomeruli from Diabetic Rats

Next we assessed the glomerular protein content of MKP-1 in DM and CON. Immunoblot analysis with anti-MKP-1 antibody revealed MKP-1 of 39 kD (Figure 2B). In DM, MKP-1 protein was decreased to 63 ± 9% of CON (P < 0.05, n = 5).

Reduction in MKP-1 Protein Expression in Mesangial Cells Grown under High Glucose Conditions

To explore the mechanisms for the reduction in MKP-1 in diabetic glomeruli, we examined MKP-1 expression in cultured mesangial cells grown under high glucose conditions (30 mM; HG). We examined the MKP-1 expression in serumreplete cells, since diabetic glomeruli are exposed to various growth factors. As shown in Figure 3A, MKP-1 protein in HG cells was 56 ± 2% of cells grown under control medium (9.2 mM glucose, P < 0.05, n = 5). The effect of HG was not due to an osmotic effect, since mannitol with the same osmolality as HG had no significant effect on MKP-1 expression (97 ± 3% versus control, NS, n = 2) (Figure 3A). PKC has been shown to be activated in HG cells due to de novo synthesis of diacylglycerol (22). Thus, we tested whether reduced MKP-1 in HG was due to the increase in PKC activity. Downregulation of PKC by treating cells with 100 nM PMA for 24 h completely abolished the reduction in MKP-1 in HG cells (Figure 3B). Recent reports have demonstrated that intracellular Ca2+ signaling is important for the induction of MKP-1 (23,24). Since Ca2+ signaling has been shown to be blunted in cells grown under high glucose due to the activation of PKC (25,26), we examined whether this could be the mechanism for the reduction in MKP-1. HG or control cells were exposed to intracellular Ca2+ chelator BAPTA (10 μM) or the Ca2+ ionophore A23187 (3 μM) for 4 h. As shown in Figure 3C, BAPTA decreased MKP-1 expression in control cells to the same level as HG, whereas no further decrease was observed in HG cells. A23187, on the other hand, increased MKP-1 in HG with no effect on CON. These results suggest that the suppression of MKP-1 in HG cells was caused by the impaired Ca2+ signaling due to PKC activation. To further confirm the roles of Ca2+ and PKC, MKP-1 induction by AVP was examined. Previous studies demonstrated that Ca2+ elevations in response to vasoactive substances such as AVP was suppressed in HG environment (25,26). Thus, one would expect MKP-1 induction by AVP to be reduced. For these experiments, cells were serum-deprived for 24 h to remove stimuli for MKP-1 induction. In contrast to the serum-replete cells, MKP-1 expression was slightly but significantly increased in HG environment (139 ± 10%, P < 0.05, n = 3) (Figure 3D). This finding could be expected since PKC is a known activator of MKP-1. In control cells, incubation with 100 nM AVP for 2 h increased MKP-1 protein by 38 ± 11% (P < 0.05, n = 3). In contrast, MKP-1 did not increase but decreased by 36 ± 4% (P < 0.05 versus HG baseline, and NS versus control baseline, n = 3) in HG cells. To test whether the blunted response in HG was mediated by PKC, the effect of PKC activation on AVP-induced MKP-1 expression was examined. As shown in Figure 3E, incubation with 100 nM PMA for 2 h increased MKP-1 protein by 47 ± 5% (P < 0.05, n = 3). AVP (100 nM) for 2 h significantly increased MKP-1 protein by 28 ± 3%, whereas pretreatment with PMA 3 min before the addition of AVP significantly inhibited MKP-1 induction (97 ± 5% of controls, NS, n = 3). These findings, together with the experiments using BAPTA and A23187, demonstrate that the suppressed MKP-1 in serum-replete HG cells is due to the reduced Ca2+ elevations secondary to PKC activation.

Figure 3.
Figure 3.

Mechanisms of reduced MKP-1 in mesangial cells grown under high glucose (HG). (A) Reduced MKP-1 expression in mesangial cells grown under high glucose. Mesangial cells were grown under 30 mM (HG) or 9.2 mM (CON) glucose supplemented with 17% fetal bovine serum (FBS) for 5 d. Cells grown under mannitol served as an osmotic control. Sixteen to eighteen hours after the last medium exchange, cells were lysed. Equal amounts of cell lysates were loaded and immunoblotted with anti-MKP-1 antibody. A representative immunoblot. (B) Effect of protein kinase C downregulation on MKP-1 expression in mesangial cells grown under high glucose. Mesangial cells grown similarly to A were treated with 100 nM phorbol 12-myristate 13-acetate for 24 h. Quantitative analysis is shown below. Values are expressed as percentage of CON, mean ± SEM, n = 3. *P < 0.05 versus CON. (C) Effects of 1,2-bis-(2-aminophenoxy)-ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) and A23187 on MKP-1 protein in mesangial cells grown under high glucose. Mesangial cells grown similarly to A were treated with 10 μM BAPTA or 3 μM A23187 for 4 h (n = 3). (D) Effects of arginine vasopressin (AVP) on MKP-1 protein in mesangial cells grown under high glucose. Mesangial cells grown similarly to A were serum-deprived for 24 h before the experiments. Cells were then treated with 100 nM AVP for 2 h (n = 3). (E) Effects of PMA on AVP-mediated increase in MKP-1 protein. Mesangial cells grown under 9.2 mM glucose supplemented with 17% FBS for 5 d were serum-deprived for 24 h before the experiments. Cells were then treated with 100 nM PMA, 100 nM AVP, or both for 2 h (n = 3).

Discussion

The findings presented here demonstrate that MAPK activity is stimulated in glomeruli isolated from streptozotocin-induced diabetic rats. Recently, Haneda and coworkers reported increases in MAPK activity in glomeruli from this diabetic model (27). They also demonstrated an increase in MEK activity in DM, which is consistent with the increased tyrosine phosphorylation and electrophoretic mobility shift of p42mapk shown in the present study. On the other hand, increased protein expression of MAPK and changes in electrophoretic mobility of MAPK observed by us were not found by the same authors. The reason for this discrepancy is unclear since animal species and duration of diabetic state were not different from the present study. It has been reported that stimulation with platelet-derived growth factor (PDGF) increases de novo synthesis of MAPK as well as MEK protein in mesangial cells. It is considered to be one of the mechanisms for potent and sustained activation of MAPK by PDGF (28). PDGF mRNA expression has been reported to be elevated in glomeruli from streptozotocin-induced diabetic rats (4). Thus, the increased MAPK protein demonstrated in the present study could be due to the upregulation of PDGF.

Protein phosphorylation is determined by a dynamic balance between protein kinases and protein phosphatases. In this regard, we have demonstrated decreased tyrosine phosphatase activity toward MAPK as another mechanism for the stimulation of MAPK in diabetic glomeruli. Since phosphorylation on both threonine and tyrosine residue is required for MAPK activity, phosphatases specific for phosphoserine/threonine, phosphotyrosine, or recently identified dual-specificity phosphatases may be relevant for its inactivation. MKP-1, an immediate early gene product, also known as 3CH134, CL100, or erp, is one of the dual-specificity phosphatases that may play an important role in the regulation of MAPK activity. The kinetics of serum-stimulated expression of MKP-1 was correlated with the inactivation of MAPK (13). Overexpression of MKP-1 has been demonstrated to inhibit cellular proliferation (15). Moreover, introduction of MKP-1 antisense oligonucleotide has been shown to prolong activation of MAPK (29). MKP-1 is induced by extracellular stimuli, including growth factors, phorbol esters, hydrogen peroxide, angiotensin II, cAMP, and cGMP-raising agents (14,17,29,30,31). Since growth factors and phorbol esters that activate MAPK cascade are known to induce MKP-1, the reduced protein expression of MKP-1 in diabetic glomeruli was puzzling. To clarify the mechanisms, MKP-1 expression was examined in mesangial cells cultured under high glucose conditions. Cells grown in medium containing 30 mM glucose and serum showed suppressed MKP-1 protein levels compared with cells grown under 9.2 mM glucose. PKC has been shown to be activated in mesangial cells cultured under high glucose due to de novo synthesis of diacylglycerol (22). Downregulation of PKC by incubating HG cells with PMA for 24 h completely abolished the MKP-1 suppression, suggesting that the observed phenomenon was PKC-dependent. Although PKC has been shown to induce MKP-1 expression, recent studies by Scimeca et al. and others have demonstrated that intracellular Ca2+ signalling is essential for the induction of the enzyme (23,24). Thus, Ca2+ chelation blocked the induction of MKP-1 expression to all stimuli including PMA in different cell types. Furthermore, increasing intracellular Ca2+ concentration with Ca2+ ionophore A23187 was sufficient to induce MKP-1. Of particular interest, high glucose has been shown to inhibit intracellular Ca2+ signalling in response to vasoactive substances in mesangial cells (25,26). PKC inhibitors or 24-h exposure to PMA completely reversed the effects of high glucose, indicating PKC involvement. Thus, we tested whether the suppression of MKP-1 in HG was due to the blunted Ca2+ signalling secondary to PKC activation. In support of our hypothesis, Ca2+ ionophore completely reversed the MKP-1 suppression in HG cells. Moreover, intracellular chelator BAPTA decreased MKP-1 expression in control cells to the same level as HG cells. To confirm the roles of Ca2+ and PKC, induction of MKP-1 by vasoactive agent AVP was examined in HG and control cells. These experiments were performed in serumdepleted cells to remove stimuli for MKP-1 induction. In contrast to serum-replete cells, HG caused a slight but significant increase in MKP-1 protein. This was not surprising in view of the fact that PKC is an inducer of MKP-1. The AVP-mediated MKP-1 induction, however, was suppressed in HG cells. These effects of HG were mimicked by PMA. Thus, while PMA alone induces MKP-1, it blunts the AVP-induced expression in MKP-1. These results demonstrate that the suppressed MKP-1 expression in glomeruli and serum-replete mesangial cells under the diabetic state may be due to the impaired Ca2+ signaling secondary to PKC activation.

Other phosphatases such as serine/threonine phosphatase and tyrosine phosphatase are also able to inactivate MAPK, and therefore may be involved in the reduced MAPK inactivation in diabetic glomeruli. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase, largely located in the cytoplasm, and is responsible for inactivation of cytosolic MAPK by the dephosphorylation of threonine residue (32,33). The reduced MAPK dephosphorylation in diabetic glomeruli shown in the present study, however, is unlikely to be due to PP2A since its activity is inhibited by okadaic acid but not by vanadate. On the other hand, vanadate-sensitive protein tyrosine phosphatases are known to inactivate MAPK both in vitro and in vitro and therefore may be responsible for our observation (32,33,34). We also cannot exclude the possibility that other dual-specificity phosphatases such as PAC1 and B23 demonstrated to be present in mesangial cells contribute to the reduced inactivation of MAPK in diabetic glomeruli (18). Nevertheless, this study provides evidence supporting a role for MKP-1 in glomerular MAPK activation in DM.

In summary, MAPK activity is stimulated in diabetic glomeruli by multiple mechanisms i.e., increased protein contents, increased tyrosine phosphorylation, and decreased inactivation of the enzyme. In view of the pivotal role of MAPK in regulating cellular events including growth, these alterations may contribute to the development of glomerular hypertrophy seen in early diabetes. Furthermore, increased MAPK activity could be a potential therapeutic target for diabetic nephropathy. Inhibition of PKC, another enzyme known to be activated in DM, has been shown to ameliorate the progression of diabetic complications including nephropathy (6). It is generally considered that targeting the downstream signaling cascade shared by many growth factors is more effective than targeting a single growth factor. In a pathophysiologic condition such as diabetic nephropathy, in which multiple factors are implicated in the pathogenesis, including growth factors, vasoactive substances, and increased PKC secondary to high glucose, inhibition of MAPK, a common downstream signaling molecule, may prove to be more effective in the prevention and treatment of the disease.

Acknowledgments

Acknowledgment

This study was supported by Keio Gijuku Developmental Fund, Keio University Medical Science Fund, a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (10670757), and Pharmacia-Upjohn Fund for Growth and Development Research.

Footnotes

  • American Society of Nephrology

References

  1. ↵
    Mauer SM, Steffes MW, Ellis EN, Sutherland DE, Brown DM, Goetz FC: Structural-functional relationships in diabetic nephropathy. J Clin Invest 74: 1143-1155,1984
  2. ↵
    Seyer-Hansen K, Hausen J, Gundersen HJ: Renal hypertrophy in experimental diabetes: A morphometric study. Diabetologia18 : 501-505,1980
    OpenUrlPubMed
  3. ↵
    Mogyorósi A, Ziyadeh FN: Update on pathogenesis, markers and management of diabetic nephropathy. Curr Opin Nephrol Hypertens 5:243 -253, 1996
    OpenUrlPubMed
  4. ↵
    Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, Tomino Y, Koide H: mRNA expression of growth factors in glomeruli from diabetic rats.Diabetes 42:450 -456, 1993
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Craven PA, DeRubertis FR: Protein kinase C is activated in glomeruli from streptozotocin diabetic rats: Possible mediation by glucose.J Clin Invest 83:1667 -1675, 1989
  6. ↵
    Ishii H, Jirousek MR, Koya D, Takagi C, Xia P, Clermont A, Bursell S-E, Kern TS, Ballas LM, Heath WF, Stramm LE, Feener EP, King GL: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC β inhibitor.Science 272:728 -731, 1996
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Marquardt B, Frith D, Stabel S: Signalling from TPA to MAP kinase requires protein kinase C, raf, and MEK: Reconstitution of the signalling pathway in vitro. Oncogene 9:3213 -3218, 1994
    OpenUrlPubMed
  8. ↵
    Cobb MH, Boulton TG, Robbins DJ: Extracellular signal-regulated kinases: ERKs in progress. Cell Regul 2:965 -978, 1991
    OpenUrlPubMed
  9. ↵
    Anderson NG, Maller JL, Tonks NK, Sturgill TW: Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651 -653, 1990
    OpenUrlCrossRefPubMed
  10. ↵
    Payne DM, Rossomando AJ, Martino P, Erickson AK, Her J-H, Shabanowitz J, Hunt DF, Weber MJ, Sturgill TW: Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J 10:885 -892, 1991
    OpenUrlPubMed
  11. ↵
    Kyriakis JM, App H, Zhang XF, Banerjee P, Brautigan DL, Rapp UR, Avruch J: Raf-1 activates MAP kinase-kinase. Nature358 : 417-421,1992
    OpenUrlCrossRefPubMed
  12. ↵
    Guan KL, Broyles SS, Dixon JE: A Tyr/Ser protein phosphatase encoded by vaccinia virus. Nature 350:359 -362, 1991
    OpenUrlCrossRefPubMed
  13. ↵
    Sun H, Charles CH, Lau LF, Tonks NK: MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell 75:487 -493, 1993
    OpenUrlCrossRefPubMed
  14. ↵
    Charles CH, Sun H, Lau LF, Tonks NK: The growth factor-inducible immediate-early gene 3CH134 encodes a protein-tyrosine-phosphatase.Proc Natl Acad Sci USA 90:5272 -5296, 1993
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Brondello JM, McKenzie FR, Sun H, Tonks NK, Pouysségur J: Constitutive MAP kinase phosphatase (MKP-1) expression blocks G1 specific gene transcription and S-phase entry in fibro-blasts. Oncogene10 : 1895-1904,1995
    OpenUrlPubMed
  16. ↵
    Davis RJ: The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553 -14556, 1993
    OpenUrlFREE Full Text
  17. ↵
    Sugimoto T, Haneda M, Togawa M, Isono M, Shikano T, Araki S, Nakagawa T, Kashiwagi A, Guan K-L, Kikkawa R: Atrial natriuretic peptide induces the expression of MKP-1, a mitogenactivated protein kinase phosphatase, in glomerular mesangial cells. J Biol Chem271 : 544-547,1996
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Bokemeyer D, Sorokin A, Dunn MJ: Differential regulation of the dual-specificity protein-tyrosine phosphatases CL100, B23, and PAC1 in mesangial cells. J Am Soc Nephrol 8:40 -50, 1997
    OpenUrlAbstract
  19. ↵
    Awazu M, Yared A, Swift LL, Hoover RL, Ichikawa I: Dietary fatty acid modulates glomerular atrial natriuretic peptide receptor. Kidney Int 42: 265-271,1992
    OpenUrlPubMed
  20. ↵
    Harper PA, Robinson JM, Hoover RL, Wright TC, Karnovsky MJ: Improved methods for culturing rat glomerular cells. Kidney Int26 : 875-880,1984
    OpenUrlPubMed
  21. ↵
    Sugimoto T, Kikkawa R, Haneda M, Shigeta Y: Atrial natriuretic peptide inhibits endothelin-1-induced activation of mitogen-activated protein kinase in cultured rat mesangial cells. Biochem Biophys Res Commun 195: 72-78,1993
    OpenUrlCrossRefPubMed
  22. ↵
    Ayo SH, Radnik R, Garoni JA, Troyer DA, Kreisberg JI: High glucose increases diacylglycerol mass and activates protein kinase C in mesangial cell cultures. Am J Physiol 261:F571 -F577, 1991
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Scimeca J-C, Servant MJ, Dyer J-O, Meloche S: Essential role of calcium in the regulation of MAP kinases phosphatase-1 expression.Oncogene 15:717 -725, 1997
    OpenUrlCrossRefPubMed
  24. ↵
    Cook SJ, Beltman J, Cadwallader KA, McMahon M, McCormick F: Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells. J Biol Chem272 : 13309-13319,1997
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Menè P, Pugliese G, Pricci F, Di Mario U, Cinotti GA, Pugliese F: High glucose inhibits cytosolic calcium signaling in cultured rat mesangial cells. Kidney Int43 : 585-591,1993
    OpenUrlCrossRefPubMed
  26. ↵
    Menè P, Pugliese G, Pricci F, Di Mario U, Cinotti GA, Pugliese F: High glucose level inhibits capacitative Ca2+ influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism. Diabetologia 40:521 -527, 1997
    OpenUrlCrossRefPubMed
  27. ↵
    Haneda M, Araki S, Togawa M, Sugimoto T, Isono M, Kikkawa R: Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions.Diabetes 46:847 -853, 1997
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Huwiler A, Fabbro D, Pfeilschifter J: Platelet-derived growth factor stimulates de-novo synthesis of mitogen-activated protein kinase in renal mesangial cells. Eur J Biochem227 : 209-213,1995
    OpenUrlPubMed
  29. ↵
    Duff JL, Monia BP, Berk BC: Mitogen-activated protein (MAP) kinase is regulated by the MAP kinase phosphatase (MKP-1) in vascular smooth muscle cells. J Biol Chem 270:7161 -7166, 1995
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Charles CH, Abler AS, Lau LF: cDNA sequence of a growth factor-inducible immediate early gene and characterization of its encoded protein. Oncogene 7:187 -190, 1992
    OpenUrlPubMed
  31. ↵
    Keyse SM, Emslie EA: Oxidative stress and heat shock induce a human gene encoding a protein-tyrosine phosphatase. Nature359 : 644-647,1992
    OpenUrlCrossRefPubMed
  32. ↵
    Alessi DR, Gomez N, Moorhead G, Lewis T, Keyse SM, Cohen P: Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol 5: 283-295,1995
    OpenUrlCrossRefPubMed
  33. ↵
    Anderson NG, Maller JL, Tonks NK, Sturgill TW: Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 343:651 -653, 1990
  34. ↵
    Zhao Z, Tan Z, Wright JH, Diltz CD, Shen SH, Krebs EG, Fischer EH: Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation. J Biol Chem 270:11765 -11769, 1995
    OpenUrlAbstract/FREE Full Text
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Mechanisms of Mitogen-Activated Protein Kinase Activation in Experimental Diabetes
MIDORI AWAZU, KENJI ISHIKURA, MARIKO HIDA, MAKIKO HOSHIYA
JASN Apr 1999, 10 (4) 738-745;
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