Synergistic induction of monocyte chemoattractant protein-1 by integrins and platelet-derived growth factor via focal adhesion kinase in mesangial cells

Kaori Kanegae1, Masahito Tamura2, Narutoshi Kabashima2, Ryota Serino1, Masaki Tokunaga2, Shigeru Oikawa1 and Yasuhide Nakashima1

1 Second Department of Internal Medicine and 2 Kidney Center, University of Occupational and Environmental Health University Hospital, Kitakyushu, Japan

Correspondence and offprint requests to: Associate Professor Masahito Tamura, MD, PhD, Kidney Center, University of Occupational and Environmental Health University Hospital, 1-1 Iseigaoka, Yahatanishi, Kitakyushu 807-8555, Japan. Email: mtamura{at}med.uoeh-u.ac.jp



   Abstract
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Background. Growth factors, extracellular matrix and its receptor integrins are upregulated in various glomerular diseases. We investigated the mechanism of collaboration between integrins and platelet-derived growth factor (PDGF) in focal adhesion kinase (FAK)- and extracellular signal-related kinase (ERK)1/2-mediated signal pathways that lead to monocyte chemoattractant protein (MCP)-1 expression in cultured rat mesangial cells (MCs).

Methods. Serum-starved MCs were plated on fibronectin- or polylysine-coated plates with or without PDGF, and examined for phosphorylation of ERK1/2, mitogen-activated protein or ERK kinase (MEK)1/2 and FAK by western blotting, and for expression of MCP-1 mRNA and protein by reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The effects of dominant-negative FAK on MCP-1 expression were examined.

Results. Cell adhesion to fibronectin increased phosphorylation of FAK, MEK1/2 and ERK1/2, and induced MCP-1 mRNA and protein expression. PDGF increased phosphorylation of FAK, MEK1/2 and ERK1/2 even without cell adhesion to fibronectin, and induced MCP-1 mRNA and protein expression. PDGF with integrin activation by fibronectin synergistically increased phosphorylation of FAK, MEK1/2 and ERK1/2, and expression of MCP-1 mRNA and protein. Dominant-negative FAK attenuated fibronectin enhancement of PDGF-induced ERK1/2 phosphorylation and MCP-1 expression, indicating involvement of FAK in this signalling.

Conclusions. Our results suggest the cooperative role of integrin and PDGF receptor in activation of the ERK pathway possibly via FAK in MCs. The synergistic activation of integrin and PDGF signalling may play an important role in the progression of glomerular diseases through the induction of MCP-1.

Keywords: cell adhesion; integrin; extracellular matrix; growth factor; gene expression; glomerulonephritis



   Introduction
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 Abstract
 Introduction
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The pathological features of many forms of progressive glomerulonephritis (GN) are proliferation of mesangial cells and abnormal accumulation of mesangial extracellular matrix (ECM). The pathological mesangial ECM remodelling results from a complex process that involves increased expression of growth factors and chemokines. Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine family and has been reported to have a high degree of specificity as a chemotactic factor for monocytes/macrophages, and plays an important role in the progression of inflammatory processes including renal diseases [1].

Recent studies have elucidated the mechanisms that regulate MCP-1 expression. Inflammatory cytokines and growth factors, such as platelet-derived growth factor (PDGF) and interleukin-1, can trigger the expression of MCP-1 through activation of several signalling pathways such as extracellular signal-related kinase (ERK)1/2 [2], p38 mitogen-activated protein (MAP) kinase [3], protein kinase C [4], activator protein (AP)-1 [5] and nuclear factor (NF)-{kappa}B [6]. We have reported that integrin-mediated activation of intracellular signalling molecules also induces MCP-1 expression in mesangial cells [7], suggesting that abnormal remodelling of ECM observed in many types of glomerular diseases can upregulate MCP-1 expression.

Previous studies elucidated the mechanisms responsible for mediation and conversion of mechanical stress and cell adhesion on ECM into intracellular biochemical signals. Integrins, which are transmembrane receptors for ECM composed of {alpha} and ß subunits, are considered potential receptors of mechanical stress. Recently, we reported that mechanical stress activates p38 MAPK and induces cellular hypertrophy through the integrin–focal adhesion kinase (FAK)–Src–Ras pathway [8]. We also reported that activation of integrins by cell adhesion to ECM proteins induces MCP-1 expression in mesangial cells through activation of FAK [7], indicating that integrin activation by mechanical stress and ligand binding can trigger specific activation of intracellular signalling. Integrins and their cytoplasmic tails are involved in forming large complexes of cytoskeletal proteins and signalling molecules upon cell adhesion to ECM called focal adhesions that include FAK and other signal transduction molecules, such as PDGF receptor and ERK1/2 [9], suggesting the collaboration between integrins and growth factor receptors. Thus, many of the well-known signalling pathways identified previously for growth factors and chemokines could be activated by integrins, and lead to the induction of expression of specific genes.

PDGF is upregulated in many types of human and experimental GN and has been implicated as one of the growth factors that mediate and modulate the biological processes that occur during renal tissue injury. Increased expression of integrins as well as quantitative and constitutional changes in ECM components such as fibronectin and glomerular hypertension are also observed in many types of progressive GN, suggesting that activation of integrins by ECM proteins and mechanical strain, and enhanced expression of PDGF may contribute to the progression of GN through collaborative activation of intracellular signalling pathways. Based on this background, the present study was designed to determine the consequence of collaboration between integrins and PDGF involving the ERK1/2–MAP kinase pathway. The results showed that integrin activation of FAK plays a pivotal role in PDGF-mediated induction of MCP-1 in mesangial cells. Stimulation of mesangial cells by PDGF and cell adhesion to integrin ligand matrix synergistically increased phosphorylation of MEK1/2 and ERK1/2 and induced MCP-1 expression via activation of FAK. These results suggest that collaboration between integrin and PDGF signalling may contribute to the progression of glomerular diseases through the induction of MCP-1.



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 Subjects and methods
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Materials
Human fibronectin, poly-L-lysine and PDGF-BB were purchased from Sigma (St Louis, MO). Petri dishes were coated by incubating with 10 µg/ml fibronectin or poly-L-lysine in phosphate-buffered saline (PBS) for 1 h at 37°C, washed three times with PBS, and used for each assay. Mono-clonal antibody for FAK was obtained from Transduction Laboratories (Lexington, KY). Polyclonal antibody against Y397-phosphorylated FAK was from BioSource International (Camarillo, CA). Monoclonal antibody against phospho-ERK1/2 and polyclonal antibodies against ERK1/2, phospho-MEK1/2 and MEK1/2 were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Cell culture
Glomerular mesangial cells from male Wistar rats were isolated by the differential sieving method and identified by their positive immunostaining for vimentin and smooth muscle-specific actin, and negative staining for cytokeratins, factor VIII-related antigen and leukocyte common antigen (antibodies from Dako Corp., Carpinteria, CA) as described previously [10]. Mesangial cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and amphotericin B at 37°C in a 5% CO2 incubator. To synchronize the cells in quiescence, mesangial cells were maintained in DMEM containing 0.5% fetal calf serum for 24 h prior to the experiments. Mesangial cells were used between passages 5 and 15.

Western blotting
Serum-starved mesangial cells were detached from the dishes by trypsin/EDTA digestion followed by inactivation of trypsin by incubation with 1 mg/ml soybean trypsin inhibitor. Cells were then plated on 10 µg/ml fibronectin- or polylysine-coated dishes in 5% bovine serum albumin-containing DMEM with or without 10 ng/ml PDGF. Phosphorylation of FAK, MEK1/2 and ERK1/2 was analysed by western blotting. Briefly, mesangial cells were solubilized in RIPA buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride] and a protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN). Homogenates were clarified by centrifugation at 15 000 g for 15 min at 4°C, and equal amounts of total cell lysates (30 µg of protein) were analysed by western blotting using anti-phospho-FAK (1:2000), total FAK (1:1000), phospho-ERK1/2 (1:2000), total ERK1/2 (1:1000), phospho-MEK1/2 (1:1000) or total MEK1/2 (1:1000) antibodies. Blots were visualized by the enhanced chemiluminescence reaction (Amersham Life Science, Arlington Heights, IL). Phosphorylation levels of FAK, ERK1/2 and MEK1/2 were determined as ratios of phosphorylated FAK, ERK1/2 or MEK1/2 against total FAK, ERK1/2 or MEK1/2 levels, respectively.

Reverse transcription–polymerase chain reaction (RT–PCR) for MCP-1
RT–PCR for MCP-1 was performed as described [2,7]. Briefly, mRNA was extracted with the QuickPrep mRNA purification kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). The first-strand cDNA was synthesized from 25 ng of poly(A)+ RNA in 50 mM Tris–HCl buffer (pH 8.3) containing 200 ng of random hexamers, 3 mM MgCl2, 400 U of murine Moloney leukaemia virus reverse transcriptase, 500 µM dNTP, 15 mM dithiothreitol and 75 mM KCl in a final volume of 15 µl for 1 h at 37°C using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech). Expression of MCP-1 and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNAs was determined by RT–PCR using specific primers for MCP-1 sense, 5'-TATGCAGGTCTCTGTCACGC-3', antisense, 5'-AAGTGTTGAACCAGGATTCACA-3'; GAPDH sense, 5'-TCCCTCAAGATTGTCAGCAA-3', antisense, 5'-AGATCCACAACGGATACATT-3' [2,7]. PCR for MCP-1 and GAPDH was performed for 24 cycles under the following conditions: 1 min at 95°C, 45 s at 60°C and 45 s at 72°C. Primer sets for MCP-1 and GAPDH generated 595 and 308 bp products, respectively. The samples were subjected to agarose gel electrophoresis and stained with ethidium bromide to visualize DNA bands, followed by scanning densitometry (FB1200S; Canon, Tokyo, Japan). To confirm the accuracy of the mRNA quantity amplified by RT–PCR, PCR for MCP-1 and GAPDH was performed by changing PCR cycles or by incubating serially increasing amounts of mesangial cDNA synthesized from 25 ng of mRNA as described previously by our group [2,7], which revealed a dose- and cycle-dependent increase in the PCR product (data not shown). We therefore amplified 25 ng of each mRNA for 24 cycles for MCP-1 and GAPDH in the following experiments.

Enzyme-linked immunosorbent assay (ELISA) for MCP-1
MCP-1 protein concentrations in the culture supernatants of mesangial cells were measured by using an ELISA kit (Cosmo Bio, Tokyo, Japan) following the method recommended by the manufacturer as described previously [2]. We plated 1 x 105 cells for each well and stimulated cells by fibronectin or PDGF for 6 h to induce maximal expression of MCP-1 protein [7]. The culture supernatants (50 µl) were placed into flat-bottomed 96-well ELISA plates coated with goat anti-rat MCP-1 serum together with biotinylated anti-MCP-1 solution and incubated for 30 min at room temperature. Stabilized chromogen (100 µl) was added to each well and incubated for an additional 30 min. After adding stop solution (100 µl), the absorbance at 450 nm was analysed by using a plate reader (model 450, BioRad, Hercules, CA). The minimum sensitivity of ELISA for MCP-1 was 8.0 pg/ml.

Plasmids and transfection assay
A human focal adhesion targeting domain (FAT) of FAK expression plasmid vsv-FAT was kindly provided by Dr Kenneth M. Yamada (National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD). Puromycin-resistant plasmid pHA262pur was obtained from Dr Heinte Riele (Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, The Netherlands). vsv-FAT (8 µg) was transfected into mesangial cells using the cationic liposome- (LipoTAXI® Mammalian Transfection Kit, Stratagene, La Jolla, CA) mediated transfection method together with pHA262pur (1 µg) [2]. To increase the expression of transfected genes, 5 mM sodium butyrate was added to the culture medium for 24 h after transfection [11]. Cells expressing vsv-FAT were selected as described previously [2]. Briefly, at 24 h after transfection, the cells were maintained for 2 days in a culture medium containing 1 µg/ml puromycin, and cultured for an additional 24 h in the regular culture medium containing 0.5% serum without puromycin. Expression of vsv-FAT plasmid was confirmed by western blotting using anti-vsv antibody.

Statistical analysis
Data were expressed as the mean±SD. Differences between groups were examined for statistical significance using analysis of variance (ANOVA). A P-value of <0.05 denoted the presence of a statistically significant difference.



   Results
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 Abstract
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 Subjects and methods
 Results
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 References
 
Effects of cell adhesion to fibronectin and PDGF-BB on phosphorylation of FAK, MEK1/2 and ERK1/2
First, we examined the dynamics of the effects of cell adhesion to the matrix on the intracellular signal pathways. We previously reported that cell spreading on fibronectin was significantly increased when cells were spread on >5 µg/ml fibronectin although few cells were spread on <2.5 µg/ml fibronectin in cultured rat mesangial cells [7]. Therefore, we used 10 µg/ml fibronectin in the following assays. Cell adhesion to fibronectin increased phosphorylation levels of Y397-FAK, a key tyrosine phosphorylation site for full activation of FAK [12], in a time-dependent manner (Figure 1A). Phosphorylation levels of FAK rapidly increased from 15 min, with a peak noted at 60–120 min. Similarly, cell adhesion to fibronectin increased phosphorylation of not only FAK but also MEK1/2 and its downstream ERK1/2 in a time-dependent manner, with a peak noted at similar time points as seen in FAK (Figure 1B and C).



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Fig. 1. Activation of FAK, MEK1/2 and ERK1/2 by fibronectin. Serum-starved mesangial cells were plated on 10 µg/ml fibronectin-coated dishes for the indicated times in 5% bovine serum albumin containing serum-free DMEM, and were analysed by western blotting using anti-phospho Y397 FAK, anti-phospho MEK1/2 or anti-phospho ERK1/2 antibodies, respectively. Relative FAK, MEK1/2 and ERK1/2 phosphorylation levels were determined by densitometric analysis as the relative ratio of phospho-FAK, MEK1/2 or ERK1/2 to total FAK, MEK1/2 or ERK1/2, respectively. The value at time 0 was set as 100% and the data were reported as percentages of the amount at time 0. Values represent the mean±SD of four independent experiments. *P<0.05 and **P<0.01 vs time 0.

 
Next, we examined the effects of PDGF on phosphorylation levels of FAK, MEK1/2 and ERK1/2. PDGF rapidly increased the phosphorylation levels of ERK1/2 and MEK1/2, with a peak noted at 10 min (Figure 2B and C). Interestingly, although the effect of PDGF on the phosphorylation level of FAK was weaker than that of fibronectin, PDGF rapidly increased the phosphorylation levels of FAK as well as MEK1/2 and ERK1/2 even without cell adhesion to ECM (Figure 2A), suggesting a cross-talk between cell adhesion-mediated signal pathways and the growth factor-mediated MAPK pathway.



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Fig. 2. Activation of FAK, MEK1/2 and ERK1/2 by PDGF. Serum-starved mesangial cells plated on Petri dishes coated with 10 µg/ml polylysine were incubated with 10 ng/ml PDGF-BB for the indicated times in 5% bovine serum albumin containing serum-free DMEM, and were analysed by western blotting using anti-phospho-Y397 FAK, anti-phospho-MEK1/2 or anti-phospho-ERK1/2 antibodies, respectively. Values represent the mean±SD of four independent experiments. Data are expressed as percentages of the amount in cells at time 0. *P<0.05 and **P<0.01 vs time 0.

 
Synergism of integrin- and PDGF-mediated intracellular signalling
Because both fibronectin and PDGF could independently stimulate FAK, MEK1/2 and ERK1/2, we examined the collaborative effects of fibronectin and PDGF on these signalling molecules. Serum-starved mesangial cells were plated on either fibronectin- or polylysine-coated plates followed by stimulation with or without PDGF. Cell adhesion to fibronectin resulted in an increase in phosphorylation levels of FAK, in contrast to cell adhesion to polylysine, which mediates integrin-independent cell adhesion (Figure 3, lanes 1 and 3). Simultaneous stimulation of cells with PDGF and fibronectin dramatically increased FAK phosphorylation (Figure 3, lane 4), in contrast to cells stimulated with either PDGF or fibronectin (Figure 3, lanes 2 and 3). The increase in FAK phosphorylation in cells stimulated with both PDGF and fibronectin was more than the sum of the increases in cells stimulated with either PDGF or fibronectin. Thus, PDGF and fibronectin synergistically increased FAK phosphorylation.



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Fig. 3. Effects of fibronectin and PDGF on FAK phosphorylation. (A) Serum-starved mesangial cells were plated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes for 50 min followed by incubation with or without 10 ng/ml PDGF-BB for an additional 10 min. Phosphorylation levels of FAK were assayed by western blotting using anti-phospho- and total FAK antibodies. (B) Relative phosphorylation levels of FAK were quantified with normalization of phospho-FAK levels by total FAK levels as determined by densitometric scanning of transblot bands. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 and {dagger}P<0.005.

 
We also examined the collaborative effects of PDGF and fibronectin in the phosphorylation of MEK1/2 and ERK1/2. Phosphorylation levels of MEK1/2 and ERK1/2 were increased after stimulation with either PDGF or fibronectin (Figures 4 and 5). Similarly, simultaneous stimulation of cells with PDGF and fibronectin synergistically increased the phosphorylation levels of MEK1/2 and ERK1/2.



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Fig. 4. Effects of fibronectin and PDGF on MEK1/2 phosphorylation. (A) Serum-starved mesangial cells were plated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes with or without 10 ng/ml PDGF-BB for 10 min. Phosphorylation levels of MEK1/2 were assayed by western blotting using anti-phospho- and total MEK1/2 antibodies. (B) Relative phosphorylation levels of MEK1/2 were quantified with normalization of phospho-MEK1/2 levels by total MEK1/2 levels as determined by densitometric scanning of transblot bands. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 and {dagger}P<0.005.

 


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Fig. 5. Effects of fibronectin and PDGF on ERK1/2 phosphorylation. (A) Serum-starved mesangial cells were plated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes with or without 10 ng/ml PDGF-BB for 10 min. Phosphorylation levels of ERK1/2 were assayed by western blotting using anti-phospho- and total ERK1/2 antibodies. (B) Relative phosphorylation levels of ERK1/2 were quantified with normalization of phospho-ERK1/2 levels by total ERK1/2 levels as determined by densitometric scanning of transblot bands. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 and {dagger}P<0.005.

 
Synergistic induction of MCP-1 by PDGF and fibronectin
We reported previously that PDGF-induced activation of the MEK/ERK signal pathway and ECM-induced integrin/FAK pathway can independently increase MCP-1 expression in mesangial cells [2,7]. Therefore, we next examined whether PDGF and fibronectin have collaborative effects on MCP-1 expression as well as the effects on FAK, MEK1/2 and ERK1/2. Cell adhesion to fibronectin induced MCP-1 mRNA expression in a time-dependent manner (Figure 6A). Maximal induction of MCP-1 mRNA was noted at 3 h, consistent with our previous report [7]. PDGF as well as fibronectin induced MCP-1 mRNA expression (Figure 6B, lane 2). The expression level of MCP-1 mRNA was synergistically increased after simultaneous stimulation of cells with PDGF and fibronectin (Figure 6B, lane 4). We also examined the expression levels of MCP-1 protein. Both PDGF and fibronectin independently increased MCP-1 protein levels in culture supernatants (Figure 7, lanes 2 and 3). Similar synergism in MCP-1 protein expression was observed when cells were stimulated with PDGF and fibronectin (Figure 7, lane 4).



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Fig. 6. Effects of fibronectin and PDGF on MCP-1 mRNA expression. (A) Serum-starved mesangial cells were plated on 10 µg/ml fibronectin-coated dishes for the indicated times. Expression of MCP-1 and GAPDH mRNA was analysed by RT–PCR. (B) Serum-starved mesangial cells were plated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes with or without 10 ng/ml PDGF-BB for 3 h. Expression of MCP-1 and GAPDH mRNA was analysed by RT–PCR. Relative expression levels of MCP-1 mRNA were expressed as the ratio against cells without fibronectin and PDGF. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 and {dagger}P<0.005.

 


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Fig. 7. Effects of fibronectin and PDGF on MCP-1 protein expression. Serum-starved mesangial cells were plated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes with or without 10 ng/ml PDGF-BB for 6 h. Levels of MCP-1 protein in the culture medium were analysed by ELISA. Data were expressed as the ratio against cells without fibronectin and PDGF. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 and {dagger}P<0.005.

 
Role of FAK in PDGF- and fibronectin-induced MCP-1 expression
We further investigated the role of FAK in PDGF- and fibronectin-induced MCP-1 expression to explore the molecular interaction in PDGF- and integrin-mediated signalling, because FAK is a key molecule in integrin-mediated signal transduction that leads to gene expression [12]. We co-transfected vsv-FAT, a dominant-negative truncation of FAK, with a plasmid containing the puromycin resistance gene, and selected transfectants over 2 days using puromycin, as described previously [7]. This puromycin selection procedure routinely yielded ~90% pure populations of transfectants according to fluorescence analyses using vsv markers. After selection by puromycin, the selected cells were analysed further for ERK1/2 phosphorylation and MCP-1 expression. Again, ERK1/2 phosphorylation and MCP-1 mRNA expression were synergistically increased after simultaneous stimulation by both PDGF and fibronectin in non-transfected cells (Figure 8A and B). However, in the cells expressing vsv-FAT, such increases in ERK phosphorylation and MCP-1 induction by fibronectin stimulation were completely reduced to the levels seen on the cells plated on polylysine.



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Fig. 8. Effects of dominant-negative FAK on fibronectin- and PDGF-induced ERK1/2 activation and MCP-1 expression. (A) Mesangial cells transfected with or without vsv-FAT plasmid were incubated in DMEM without serum for 24 h and replated on either 10 µg/ml fibronectin- or 10 µg/ml polylysine-coated dishes for 50 min followed by incubation with or without 10 ng/ml PDGF-BB for an additional 10 min. Phosphorylation of ERK1/2 was assayed by western blotting using anti-phospho- and total ERK1/2 antibodies. (B) Mesangial cells were transfected with or without vsv-FAT plasmid, and the expression level of MCP-1 mRNA was analysed by RT–PCR. The relative expression levels of MCP-1 mRNA were expressed as the ratio against cells without vsv-FAT, fibronectin and PDGF. Data represent the mean±SD of four independent experiments. *P<0.05, **P<0.01 vs cells without vsv-FAT, fibronectin and PDGF.

 


   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Several lines of evidence have recently implicated enhanced expression of growth factors and ECM proteins in the pathogenesis of various glomerular diseases. However, the relationship of the intracellular signalling pathways triggered by growth factors and integrins has not been well elucidated. The major findings of the present study were: (i) fibronectin increased phosphorylation of MEK1/2 and its downstream ERK1/2 as well as FAK even without PDGF; (ii) PDGF increased tyrosine phosphorylation of FAK as well as MEK1/2 and ERK1/2 even without cell adhesion; (iii) fibronectin and PDGF synergistically enhanced tyrosine phosphorylation of FAK, MEK1/2 and ERK1/2; (iv) synergism was also observed between fibronectin and PDGF for inducing MCP-1; and (v) a dominant-negative form of FAK diminished the synergistic effects of fibronectin and PDGF on ERK1/2 phosphorylation and MCP-1 expression. These results indicate a cross-talk between integrin and PDGF signalling possibly via phosphorylation of FAK, and suggest that activation of the MEK/ERK pathway by PDGF is partly dependent on integrin-mediated cell adhesion in mesangial cells. It has been reported that the interaction of integrins and angiotensin II results in synergistic activation of FAK, MEK and ERK in vascular smooth muscle cells [13]; however, this study demonstrates for the first time that simultaneous stimulation of mesangial cells by integrins and PDGF can synergistically induce MCP-1 expression. These findings suggest the importance of abnormal ECM accumulation in modulating the pattern of synthesis of a chemokine by glomerular mesangial cells.

Integrins are multifunctional molecules. They regulate a variety of biological processes, such as organization of the actin-based cytoskeleton and cell adhesion and migration, by functioning as receptors for ECM molecules. In addition, integrin interaction with ECM ligands also triggers transmembrane effects on the localization of signalling molecules as well as cytoskeletal proteins into adhesion sites [9]. Thus, although integrins lack intrinsic enzymatic activity, they activate signal transduction involving FAK and MAP kinase pathways such as ERK [12,14]. These signal pathways activated by integrins eventually lead to regulation of gene expression, such as immediate early genes and inflammatory cytokines [7]. In the present study, in cooperation with integrins, PDGF produced a marked activation of the MEK/ERK pathway followed by induction of MCP-1. Although the precise mechanism(s) of such synergism remains to be elucidated, it has been reported that integrins can collaborate or synergize functionally with growth factors in a variety of biological processes such as cell growth and differentiation [14]. Activation of intracellular signalling triggered by integrins depends on the molecular hierarchies of cytoskeletal and signalling molecules, which are regulated by integrin aggregation, occupancy and tyrosine phosphorylation [9]. Although simple integrin aggregation induces non-synergistic activation of the ERK class of MAP kinase, growth factors such as epidermal growth factor (EGF), PDGF and basic fibroblast growth factor (bFGF) produce maximal activation of MAP kinase if the integrins are both aggregated and occupied by ligands in human fibroblasts [15].

FAK is a non-receptor tyrosine kinase that is widely expressed in adherent cells and plays a key role in integrin-mediated intracellular signalling. Cell adhesion to fibronectin triggers both integrin receptor aggregation and occupancy, and causes full tyrosine phosphorylation of FAK [15]. The mechanism that links integrins to the MAP kinase pathway remains controversial. Shlaepfer et al. [16,17] postulated that the Grb2–Sos complex previously implicated in EGF receptor function also accumulates in focal adhesion sites upon integrin activation by binding to phosphorylated Y925 in FAK, and thereby induces ERK phosphorylation via Ras activation in 3T3 fibroblasts and human 293 cells. Giancotti et al. [18] reported that ligation of {alpha}6ß4 integrin in human keratinocytes causes tyrosine phosphorylation of Shc, which binds to tyrosine-phosphorylated Y397-FAK, and recruits Grb2 followed by activation of Ras and stimulation of the MAP kinases ERK and JNK. In addition to the involvement of FAK in integrin-mediated MAP kinase activation, Juliano et al. [19] reported the existence of at least two distinct integrin signalling pathways that include the FAK-independent pathway in 3T3 fibroblasts. In the present study, cell adhesion to fibronectin, but not to non-specific substrate polylysine, induced tyrosine phosphorylation of Y397 in FAK, which is a key tyrosine phosphorylation site for full activation of FAK including phosphorylation of Y925 [14]. Furthermore, dominant-negative FAK diminished fibronectin-induced ERK activation and MCP-1 expression. These results indicate that activation of MEK and ERK and expression of MCP-1 are specific to integrin-mediated cell adhesion, and suggest that such synergistic effects depend on at least FAK activation.

The MAP kinase pathway is recognized as a regulator of cell growth, differentiation and gene activation pathways, and can be independently stimulated by integrins and PDGF [2,7]. Activation of ERK members of the MAP kinase pathway by integrins is induced after their juxtamembrane accumulation, and the time course was reported to differ from stimulation by the growth factor PDGF in being more gradual and persistent, and associated with cell spreading [20], consistent with our results that fibronectin increased phosphorylation levels of MEK and ERK from 15 min and reached a peak level at 60–120 min, while PDGF rapidly and transiently increased phosphorylation of these molecules, with a peak noted at 10 min.

Several signalling pathways are involved in the regulation of MCP-1 expression, such as FAK, MAP kinases, protein kinase C and NF-{kappa}B, which can also be activated by integrins [14]. In the present study, fibronectin- and PDGF-induced MCP-1 expression was accompanied by activation of FAK and MEK/ERK. We have reported that integrin- or PDGF-induced MCP-1 expression was completely inhibited by downregulation of FAK or MEK, respectively, suggesting the critical roles of FAK and MEK in integrin- and PDGF-mediated induction of MCP-1 [2,7]. MCP-1 is implicated in glomerular injury in various human and experimental forms of GN, diabetic nephropathy and hypertensive kidney by functioning as a major chemotactic factor for monocytes/macrophages. These findings suggest that enhanced expression of growth factors such as PDGF and ECM proteins might have a pivotal role in the progression of glomerular diseases by inducing MCP-1.

In conclusion, we have demonstrated in the present study that stimulation by the growth factor PDGF and the ECM protein fibronectin can synergistically activate intracellular signalling pathways including MEK, ERK and FAK accompanied by MCP-1 expression in mesangial cells. Although the precise mechanism that explains the synergism remains to be elucidated, FAK seems to be located at the crossroads of PDGF- and integrin-mediated signal pathways. Because MCP-1 plays an important role in the progression of many glomerular diseases by attracting monocytes/macrophages and inducing certain intracellular signalling pathways that lead to expression of various cytokines, these findings suggest that expansion of the ECM together with enhanced expression of growth factors might be involved in the progression of glomerular diseases by manipulating the number of infiltrating monocytes and stimulation of cytokine gene expression through the induction of MCP-1 expression.



   Acknowledgments
 
This work was supported in part by JSPS.KAKENHI (to M.T., no. 15590872). Part of this manuscript was presented in an abstract form at the 37th Congress of the American Society of Nephrology, 2004.

Conflict of interest statement. None declared.



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 4. 4.05
Accepted in revised form: 10. 6.05





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