NDT Advance Access published online on June 21, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn333
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The mitogen-activated protein kinase Erk5 mediates human mesangial cell activation
1 Instituto Reina Sofía de Investigación Nefrológica, Departamento de Fisiología y Farmacología, Universidad de Salamanca 2 Red de Investigación en Enfermedades Renales (RedinRen), Instituto Carlos III de Investigación, Ministerio de Sanidad y Consumo 3 Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca 4 Servicio de Urología, Hospital Universitario de Salamanca, Salamanca, Spain
Correspondence and offprint requests to: Alicia Rodríguez-Barbero, Departamento de Fisiología y Farmacología, Universidad de Salamanca, Campus Miguel de Unamuno, Edificio Departamental, 37007 Salamanca, Spain. Tel: +34-923-294472; Fax: +34-923-294669; E-mail: barberoa{at}usal.es
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Abstract |
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Top
Abstract
IntroductionBackground. Mesangial activation occurs in many forms of renal disease that progress to renal failure. Mitogen-activated protein kinases (MAPKs) are important mediators involved in the intracellular network of interacting proteins that transduce extracellular stimuli to intracellular responses. The extracellular signal-regulated kinases 5 (Erk5) MAPK pathway has been involved in regulating several cellular responses. Thus, we examined the expression of Erk5 in human renal tissue and the function of Erk5 in cultured human mesangial cells.
Methods. Erk5 was visualized in human renal tissue by immunohistochemistry and in mesangial cells by immunofluorescence microscopy using the anti-Erk5 C-terminus antibody. Erk5 expression and activation, and collagen I expression were determined by western blot. To generate a dominant-negative form of the Erk5 in human mesangial cells, an EcoRI fragment from wild-type pCEFL-HA-Erk5 was subcloned into the EcoRI site of pCDNA3. Cell proliferation was analysed by an MTT-based assay. Cell contraction was analysed by studying the changes in the planar cell surface area.
Results. Erk5 was expressed in the kidney, mainly localized at the glomerular mesangium. In cultured human mesangial cells, Erk5 was activated by foetal calf serum (FCS), high glucose, endothelin-1, platelet-activating factor (PAF), epidermal growth factor (EGF) and transforming growth factor beta-1 (TGF-β1). The expression of a dominant-negative form of Erk5 in human mesangial cells resulted in a significant decrease in proliferation, EGF-induced cell contraction and TGF-β1-induced collagen I expression.
Conclusions. These results suggest that Erk5 is involved in agonist-induced mesangial cell contraction, proliferation and ECM accumulation and point to a multifunctional role of Erk5 in the pathophysiology of glomerular mesangial cells.
Keywords: cell contraction; cell proliferation; Erk5 MAPK; extracellular matrix; mesangial cells
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Introduction |
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Glomerular mesangial cells have a central role in maintaining the structure and function of the glomerular capillary tuft. Mesangial cell activation, characterized by cell contraction, proliferation and extracellular matrix (ECM) synthesis, occurs in many forms of renal disease that progress to renal failure [1,2]. Regardless of the type of glomerular injury, the altered mesangial cell function plays a central role in the pathogenesis of progressive glomerulopathy [3]. Thus, the knowledge of the cellular and molecular pathways responsible for pathological glomerular alterations may help to elucidate the pathogenesis of glomerular diseases. Studies on the mechanisms involved in renal pathophysiology have pointed to mitogen-activated protein kinases (MAPKs) as important intermediaries of glomerular diseases. Thus, glomerular MAPKs are activated in experimental glomerular diseases, such as hypertension [4,5], diabetes mellitus [6] and glomerulonephritis [7].
MAPKs are important mediators involved in the transduction of extracellular stimuli to intracellular responses [8]. Three classical families of MAPKs with different substrate specificities have been described, and include the extracellular signal-regulated kinases (Erk)-1 and -2, c-Jun NH2-terminal kinase (JNK) and the p38 MAPK [9]. Each subfamily may be regulated via different signal transduction pathways and modulate specific cell functions [10]. Erk1/2 are activated primarily in response to proliferative stimuli [11,12], whereas the other MAPKs are activated primarily in response to inflammatory and stressful stimuli, including oxidant and osmotic stresses [13–15]. All three classical MAPKs, Erk1/2, JNK and p38, are expressed in the kidney and have been detected in various renal cells [16].
Another more recently discovered MAPK, the extracellular signal-regulated kinase 5 (Erk5)/Big MAP Kinase 1 (BMK1), has been involved in the regulation of cell proliferation, apoptosis and responses to physical stimuli [17,18]. Erk5 was identified as a MAPK family member with a large COOH-terminal and a unique loop-12 sequence that shares the TEY activation motif with Erk1/2 but is activated by MAPK/Erk kinase 5 (MEK5) [18]. Erk5 contains an 
400-amino-acid C-terminal extension containing a transcriptional activation domain and a region that can phosphorylate the myocyte enhancer factor-2 (MEF2) family of transcription factors leading to the up-regulation of the immediate early response gene c-jun [19,20]. Erk5 was reported to be activated by physical stress [13,19], serum and certain growth factors such as epidermal growth factor (EGF) [21] in several cells. Currently, the role of Erk5 in mesangial cell activation is not well known. Therefore, we assessed whether Erk5 may be activated in human mesangial cells after treatment with several agonists involved in renal damage.
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Materials and methods |
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Reagents
Culture media and foetal calf serum (FCS) were purchased from Invitrogen (Paisley, UK). Protein A-sepharose was from Amersham-Pharmacia (Piscataway, NJ, USA). EGF was from Becton Dickinson (Bedford, MA, USA) and TGF-β1 was from R&D Systems (Minneapolis, MN, USA). Other generic chemicals were purchased from Sigma, Roche or Merck. The anti-haemaglutinin antibody and the MTT reagent were from Roche (Indianapolis, IN, USA). The goat anti-Erk5 C-20 and the mouse anti-phosphotyrosine antibodies were from Santa Cruz (Santa Cruz, CA, USA), as was the horseradish peroxidase conjugate (HRP) anti-goat immunoglobulin-G. A GST-EGFR fusion protein that included the 301 COOH-terminal residues of the human EGFR was used to generate polyclonal anti-EGFR antibodies (R03) as previously described [22]. The Cy3-conjugated secondary antibody was from Jackson Immunoresearch (West Grove, PA, USA). The mouse anti-alpha-tubulin was from Oncogene (San Diego, CA, USA), and the rabbit anti-collagen I was from Chemicon (Temecula, CA, USA). HRP anti-rabbit and anti-mouse immunoglobulin-G was from Bio-Rad Laboratories (Cambridge, MA, USA.). The anti-Erk5 C-terminus antibody, raised in rabbits against residues 791–805 of Erk5, and the anti-pErk5 antibody have been previously described [21].
Human tissue and cell culture
Renal tissue was obtained from patients undergoing nephrectomy because of circumscribed renal tumours. Kidney tissue was distant from the neoplasm and macroscopically normal. Within 30 min after nephrectomy, samples of cortex from the normal renal pole were obtained for cell culture or immunohistochemistry. Biopsies were fixed in 4% formaldehyde and embedded in paraffin.
Primary cultures of human mesangial cells were obtained from glomeruli isolated by differential sieving and grown in the RPMI 1640 medium supplemented with 10% FCS as previously described [23,24]. PT67 packaging cells (Clontech Laboratories, Mountain View, CA, USA) were grown in DMEM with 10% FBS, 50 U/ml penicillin and 50 µg/ml streptomycin. Cells were cultured at 37°C in a humidified atmosphere in the presence of 5% CO2 and 95% air.
Plasmids
HA-Erk5 was subcloned into the pCEFL mammalian expression vector. This is a modified version of the pCDNA3 vector that includes the elongation factor 1 promoter that controls the expression of an N-terminal HA tag after which the Erk5 cDNA is located [25]. To generate a dominant-negative form of Erk5, an EcoRI fragment from wild-type pCEFL-HA-Erk5 was subcloned into the EcoRI site of pCDNA3. Site-directed mutagenesis of the region containing the activating TEY microdomain (TEY to AEF) was performed using standard procedures [26]. For retrovirus production, HA-Erk5AEF was subcloned into the pLZR-IRES-GFP vector [21].
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Retrovirus production and infection |
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For stable production of retroviruses, PT67 packaging cells (Clontech) were transfected with the retroviral plasmid pLZR-HA-Erk5AEF or the empty vector (pLZR-IRES-GFP). After transfection, cells were plated in selection medium containing hygromycin (300 µg/ml, Calbiochem, La Jolla, CA, USA) and resistant clones selected for their ability to infect MCF7 cells, as we previously described [21]. Infection products were routinely analysed by SDS–PAGE and autoradiography.
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Immunohistochemistry and immunofluorescence microscopy |
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Fixed kidney blocks embedded in paraffin were incubated with a specific antibody raised against peptides corresponding to the C-terminus of Erk5 (residues 719–805), followed by a peroxidase label polymer amplified detection system (Envision System; Dako, Glostrup, Denmark). The chromogen used was 3,3'-diaminobencidine, and the tissues were counterstained with haematoxylin.
Immunofluorescence microscopy was performed essentially as we previously described [24]. Briefly, mesangial cells were fixed and permeabilized, and then incubated with the anti-Erk5 C-terminus antibody. Erk5 binding was detected using the Cy3-conjugated antibody. Coverslips were examined using a Leica DMRXA microscope (Wetzlar, Germany).
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Immunoprecipitation and western blot analysis |
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Renal tissues and cells were lysed on an ice-cold lysis buffer, and proteins were determined as previously described [24]. For immunoprecipitation, equal amounts of total protein extracts were incubated with the indicated antibodies along with protein A-sepharose. Protein samples were separated by SDS-PAGE and membranes blocked before incubation with the primary antibodies. The anti-alpha-tubulin antibody was used to confirm loading of a comparable amount of protein in each lane. After incubation with HRP-conjugated secondary antibodies, bands were visualized by a luminol-based detection system with p-iodophenol enhancement [21]. Densitometry was analysed using the NIH Scion Image software (Scion, Frederick, MD, USA).
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Cell proliferation measurements |
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Cell proliferation was analysed at Days 0 and 5 of treatment with 10 nM EGF or 50 pM TGF-β1 by an MTT-based assay (Roche, Penzberg, Germany) following the commercial instructions. The absorbance was measured at 595 nM and transformed into a number of cells by using a curve that correlated the absorbance and number of human mesangial cells (HMC). The results are presented as mean ± SEM of three experiments performed with duplicates.
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Analysis of cell contraction |
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Cell contraction was analysed by studying the changes in the planar cell surface area (PCSA) [23]. Cells were observed under phase contrast with an inverted PFX photomicroscope (magnification 40x; Nikon, Tokyo, Japan). The images were captured by a Hitachi KP-113 solid-state television camera (Hitachi Denshi, Tokyo, Japan). Photographs were taken under various experimental conditions and at different time periods. The results are presented as the mean ± SEM of at least three experiments. PCSA was determined by computer-aided planimetric techniques using the NIH Scion Image software (Scion, Frederick, MD, USA).
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Statistical analyses |
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Numerical data are presented as mean ± SEM and were analysed by Student's t-test or two-way ANOVA with the SPSS 12.0 software (SPSS, Chicago, IL, USA). Significance was assigned at P < 0.01.
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Results |
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Erk5 expression in human kidney and mesangial cells
Erk5 expression in human kidney was analysed by western blot as shown in Figure 1A. Immunohistochemistry analysis revealed that Erk5 was mainly localized at the endothelium of some vessels and in the glomerular mesangium (Figure 1B). To extend the above findings, we examined Erk5 expression and activation in primary cultured HMC. In growth-arrested cells, foetal calf serum (FCS) induced a clear change in the electrophoretic mobility of Erk5, indicative of its dual phosphorylation and activation. Furthermore, Erk5 phosphorylation, assessed with an anti-phospho-Erk5 antibody, was also increased (Figure 1C). Immunofluorescence microscopy analysis revealed that under resting conditions Erk5 was diffusely distributed in the cytosol, whereas in cells growing with FCS, Erk5 was observed mainly in the nucleus (Figure 1D).
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Renal damage-related agonists induce Erk5 activation in HMC
We examined Erk5 activation by western blot analysis after incubation with several agonists involved in mesangial pathophysiology. Cells were exposed to endothelin-1 (10 nM), platelet-activating factor (PAF) (10 nM), high glucose (35 mM) or angiotensin II (1 µM). Erk5 was clearly activated by endothelin-1 (Figure 2A), PAF (Figure 2B) and high glucose (Figure 2C), whereas angiotensin II moderately activated Erk5 (Figure 2D). High glucose and endothelin-1 induced a rapid activation of Erk5 that increased at 5 min and persisted for 20 min (Figure 2A,C). PAF-induced Erk5 activation was slower, peaked at 20 min and then declined rapidly (Figure 2B). The activation of Erk5 by angiotensin II was weak and peaked at about 15 min (Figure 2D).
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Transforming growth factor beta-1 (TGF-β1) is a critical factor in kidney diseases such as glomerulosclerosis [28] and mesangioproliferative glomerulonephritis [29]. Because some of the effects of angiotensin II, endothelin-1 and glucose on the kidney have been found to be mediated by TGF-β [30], the action of TGF-β1 was tested for its potential effect on Erk5 activation in HMC. Treatment with TGF-β1 (50 pM) resulted in a significant activation of Erk5 (Figure 2E). The TGF-β1-induced Erk5 activation peaked at 15 min and then declined rapidly to peak again at 24 h, decreasing by 48 h (Figure 2E).
We also determine Erk5 activation after 15 min of treatment, by using a specific anti-phospho-Erk5 antibody [21]. TGF-β1, endothelin-1, PAF and high glucose phosphorylated visibly Erk5 whereas the phosphorylation of Erk5 by angiotensin II was moderate (Figure 2F).
EGF induces Erk5 activation in HMC
Previous studies reported that EGF is involved in Erk5 activation [21] and that EGF receptors (EGFR) are expressed by mesangial cells [27]. Thus, we tested the action of EGF on Erk5 activation in HMC. First, the activation characteristics of the EGFR were analysed by immunoprecipitation with the R03 rabbit polyclonal anti-EGFR antibody followed by western blotting with an anti-phosphotyrosine antibody. Serum-starved cells were treated for various time periods and with different concentrations of EGF. The effect of EGF on EGFR activation was found to be time and dose dependent. EGFR was activated at concentrations from 0.1 to 10 nM (Figure 2G). The effect of EGF (10 nM) on EGFR activation was detectable at 5 min of treatment, continued by 60 min and decreased progressively by 2 h (Figure 2H). In parallel, the action of EGF on Erk5 activation was tested. In the examined concentration range (0.1–10 nM EGF), Erk5 activation increased in a dose-dependent manner. The maximal effects occurred at concentrations between 1 and 10 nM (Figure 2G). The effect of EGF (10 nM) on Erk5 activation was detectable at 5 min of treatment, reached a maximum between 10 and 30 min and decreased progressively by 45 min (Figure 2H). Furthermore, we determined Erk5 activation by using a specific anti-phospho-Erk5 antibody [21]. Treatment with EGF (10 nM) during 10 min resulted in a significantly increased phosphorylation of Erk5 (Figure 2I). To investigate whether EGF affects the subcellular distribution of Erk5, the location of Erk5 was analysed by immunofluorescence microscopy. EGF induced a substantial decrease in the cytosolic staining of Erk5 that was accompanied by its accumulation into the nucleus (Figure 2J).
Role of Erk5 in mesangial proliferation
Many forms of renal disease that progress to renal failure are characterized by mesangial proliferation [31,32]. Since Erk5 was activated by TGF-β1 and EGF and both have been implicated in cell growth [33,34], we investigated the potential effects of EGF and TGF-β1 on mesangial proliferation. Cells were treated with 10 nM EGF or 50 pM TGF-β1, and the proliferation rate was assessed after 5 days. When cells were incubated without FCS, neither untreated, nor EGF- or TGF-β1-treated cells were able to proliferate (data not shown). Incubation with FCS and EGF increased the number of cells. Incubation with FCS and TGF-β1 decreased the number of cells (Figure 3A). To investigate the role of Erk5 in mesangial proliferation, we expressed, using retrovirus infection, a HA-tagged form of Erk5 (HA-Erk5AEF) that has been reported to act in a dominant-negative fashion [21,33]. In parallel, control cells were infected with retroviruses containing the empty vector. The expression of HA-Erk5AEF was assessed by western blotting using an anti-HA antibody (Figure 3B). Expression of dominant-negative (dn)Erk5 (HMC-Erk5AEF) resulted in a significant decrease in proliferation of cells treated with FCS, EGF or TGF-β1 compared to the effect of these agonists in cells expressing the empty vector (HMC) (Figure 3A). The phosphorylation of Erk5 in HMC-Erk5AEF was significantly lower in EGF- or TGF-β1-treated cells compared to the effect of these agonists in cells expressing the empty vector (HMC) (Figure 3B).
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Role of Erk5 in mesangial cell contraction
Because of their position surrounding glomerular capillaries, glomerular mesangial cells seem to play a major role in the regulation of the glomerular filtration rate [1,2]. The percentage of change in PCSA with respect to time zero (100%) was evaluated every 10 min during 1 h. Under control conditions there were no significant changes in PCSA (Figure 4A). Addition of 10 nM EGF resulted in a progressive PCSA reduction that was maximal at 60 min (89 ± 2%), and significantly different from control cells (Figure 4A). Cells treated with 50 pM TGF-β1 showed a decrease in PCSA to only 95 ± 2%, which was not significantly different to control conditions (data not shown). Expression of dnErk5 resulted in a significant inhibition in EGF-induced mesangial PCSA reduction compared with control cells at 60 min of treatment (Figure 4B). Western analysis using an anti-HA antibody demonstrated expression of HA-Erk5AEF in infected cells (data not shown). Control cells were infected in parallel with retroviruses coding for the empty vector.
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Role of Erk5 in collagen I expression
Collagen I is a component of renal ECM that increases in glomerular fibrosis. As Figure 5 shows, 50 pM TGF-β1 increases collagen I expression in HMC. Expression of dnErk5 (HMC-Erk5AEF) resulted in a significant decrease in collagen I after 24 h of TGF-β1 treatment compared with control cells (Figure 5).
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-tubulin. The figure shows a representative experiment of three independent experiments performed. |
Discussion |
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The present study demonstrates the expression of Erk5 in human kidney and in primary cultured HMC, as well as its activation by agonists involved in renal damage. As Erk5 was originally shown to be activated by serum [19,33], we assessed the effect of serum on Erk5 activation in HMC. Erk5 was localized in the cytosol of resting cells and shifted to a nuclear localization in cells treated with serum as a result of its activation as already shown in other cell types [19,21,35].
Several stimuli with a major role in renal diseases such as angiotensin II and endothelin-1 have been demonstrated to activate Erk5 in vascular smooth muscle cells [13,36]. It was also reported that glucose activated Erk5 in endothelial and mesangial cells [37,38]. However, it is not yet documented whether Erk5 is activated by other agonists in HMC. The present data demonstrate that Erk5 was moderately activated by angiotensin II whereas high glucose, endothelin-1 and PAF rapidly and significantly activated Erk5 in HMC. These results are consistent with those of Suzaki et al. [38] reporting that glucose caused Erk5 activation in rat mesangial cells. Therefore, Erk5 could play a role in the pathophysiology of glomerular diseases in which angiotensin II, endothelin-1, glucose or PAF are involved. In this regard, the synthesis and liberation of vasoconstrictors such as PAF, angiotensin II and endothelin-1 has been related to the nephrotoxicity induced by cyclosporine, cisplatin and gentamicin [39].
Some evidence supports the possibility that some of the effects of angiotensin II, endothelin-1, glucose or PAF are mediated by cytokines such as EGF and TGF-β1. Matsubara et al. [40] demonstrated in cardiac fibroblasts the involvement of EGFR in angiotensin II-induced synthesis of fibronectin and TGF-β. Similarly, Hua et al. [41] showed that endothelin-1 activates Erk1/2 in rat mesangial cells predominantly through a pathway involving EGFR trans-activation. In the present study, we demonstrate that HMC respond to EGF and TGF-β1 by inducing Erk5 activation in a concentration- and time-dependent manner. Since EGF and TGF-β1 have been demonstrated to play a major role in chronic glomerular diseases, EGF- and TGF-β1-induced Erk5 activation may mediate some of the effects of these substances on glomerular pathology.
In the adult kidney, mesangial cells are largely quiescent [1]. However, glomerular injury can alter the phenotype of mesangial cells, resulting in hypertrophy, hyperplasia, cellular contraction and/or expansion of the mesangial matrix. Mesangial proliferation is a predominant pathological feature of many forms of glomerular diseases, such as IgA nephropathy, lupus nephropathy and diabetic nephropathy and frequently precedes the development of glomerulosclerosis [32]. Erk5 MAPK pathway has a critical role for S-phase entry in the cell cycle, and also mediates proliferation in response to EGFR [33]. Thus, we investigated the involvement of Erk5 activation in mesangial proliferation induced by EGF [27]. In our system, EGF increases whereas TGF-β1 reduces FCS-induced mesangial cell growth, as it has been referred in other cell types [42]. Infection with a dnErk5 form reduced FCS-induced cell proliferation, inhibited EGF-induced cell proliferation and increased TGF-β1-induced inhibition in cell proliferation. These results suggest that Erk5 plays a role in the regulation of mesangial proliferation. The pro-fibrotic effects of TGF-β are recognized to be a key factor in the glomerulosclerosis and interstitial fibrosis that characterizes chronic progressive renal disease. TGF-β1 causes ECM accumulation by enhancing glomerular mesangial production of collagen and fibronectin, suppressing the expression of ECM degrading proteases and increasing the synthesis of ECM protease inhibitors [43–46]. Our results demonstrate that a dnErk5 reduced TGF-β1-induced collagen synthesis, thus suggesting that Erk5 activation plays a role in the regulation of ECM synthesis by mesangial cells. In addition, Erk5 has been demonstrated to be activated in the glomeruli of diabetic rats where a marked mesangial cell proliferation and ECM accumulation also take place [38].
Another purpose of the present study was to assess the role of Erk5 in mesangial cell contraction. Mesangial cells are subjected to multiple forms of mechanical strain [47]. Furthermore, their morphological position juxtaposed to the vascular compartment renders them susceptible to a number of vasoactive substances [48–50]. Glomeruli and mesangial cells contracted in response to vasoconstrictor agonists [23,51–53]. Our data demonstrate that transfection of a dominant-negative form of Erk5 blunted the decrease in PCSA induced by EGF in mesangial cells. This finding supports the possibility that Erk5 could be involved in EGF-induced mesangial contraction. The finding that cells expressing a dominant-negative form of Erk5 were resistant to EGF-induced cell contraction opens the possibility that Erk5 may have functions that are not dependent on the regulation of gene expression. However, we cannot exclude that the Erk5 influences the expression of proteins that affect mesangial cell contraction.
As already mentioned, Erk5 is required for growth-factor-induced cell proliferation and cell cycle progression [33]. During growth-factor-induced cell stimulation, Erk5 activates the serum and glucocorticoid inducible kinase 1 (SGK1) [54]. Erk5 activates SGK by phosphorylation at serine 78, and this Erk5-mediated phosphorylation event is necessary for the activation of SGK and for cell proliferation induced by EGF [55]. Interestingly, SGK1 is heavily expressed in fibrosing tissue, including the kidney [56], and TGF-β1 has been shown to enhance its transcription [57]. In addition, it has been demonstrated that SGK1 activation mediates fibronectin deposition in HMC exposed to high glucose environment [58]. Closely related to this observation, Suzaki et al. [38] have demonstrated that blocking Erk5 activation by transfection of a dnMEK5, which is an upstream regulator of Erk5, abolished the Erk5-mediated rat mesangial cell proliferation stimulated by high glucose.
In conclusion, our results suggest that Erk5 is involved in agonist-induced mesangial cell contraction, proliferation and ECM accumulation and point to a multifunctional role of Erk5 in the pathophysiology of glomerular mesangial cells.
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Acknowledgments |
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We thank Dr Miguel Arévalo, Department of Anatomy, University of Salamanca, for his help with immunohistochemistry. These studies have been supported by grants from Junta de Castilla y León (SA089/02) to JML-N, DGCYT (BFU2004–0028/BFI) to JML-N, Instituto de Salud Carlos III (RETIC RD06/0016 to JML-N and 01/1060 to AP).
Conflict of interest statement. F.D. is a fellow from Junta de Castilla y León and S.V. is a fellow from Ministerio de Educación y Ciencia. The results presented in this paper have not been published previously in whole or part, except in abstract format. A part of these results have been published as an Abstract in Nephrology Dialysis and Transplantation (18 Suppl 4: 26, 2003).
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Accepted in revised form: 21. 5.08
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