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From the Department of Medicine and Therapeutics, The Conway Institute for Biomolecular and Biomedical Research, University College Dublin, Dublin; and The Dublin Molecular Medicine Centre, Dublin, Ireland
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Abstract |
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PDGF is implicated in the progression of renal disease, being synthesized by infiltrating macrophages and platelets, as well as resident mesangial cells in response to multiple stimuli including endothelin, thrombin, and angiotensin II.3 PDGF bioactivity encompasses increased cellular proliferation,4 mesangial matrix expansion,5 and increased expression of the profibrotic cytokine TGF-ß1.6,7 The roles of both PDGF and TGF-ß1 in renal disease have been well characterized. However, current therapeutic interventions to regulate chronic renal inflammation are limited and PDGF has been proposed as a potential therapeutic target.8,9
There is growing evidence that lipoxins (LXs), endogenously produced eicosanoids, may have significant anti-inflammatory and proresolution bioactions. Biphasic lipid mediator production has been demonstrated in the context of an effective host defense. Initial proinflammatory mediator production is superseded by the production of anti-inflammatory, proresolution mediators including LXs, resolvins, and docosatrienes.10,11 LXs are well documented to inhibit neutrophil chemotaxis, adhesion, and transmigration12 and to stimulate monocyte chemotaxis.13 More recent evidence of their anti-inflammatory roles includes modulation of eosinophil activation.14 A role for LX promoting the resolution of inflammation has been demonstrated by stimulating phagocytic clearance of apoptotic neutrophils in vivo15 and in vitro.12 In the context of renal inflammation we have previously reported that human mesangial cells (hMC) express a G-protein-coupled receptor that binds LXA4, known as the ALXR.16 We have reported LXA4 inhibition of PDGF and epidermal growth factor (EGF)-induced hMC mitogenesis.4,6 These effects are mediated by modulation of receptor activation and inhibition of specific downstream signaling pathways, including the Akt/PKB pathway.6 Given the ability of LXA4 to inhibit the effects of PDGF receptor activation in hMCs, it was of further interest to elucidate the effects of LXA4 on the induction of PDGF-mediated gene expression changes. Here we report that PDGF stimulates the expression of multiple genes associated with matrix expansion and fibrosis, and that LXA4 modulates PDGF-induced gene expression. We report that soluble factors released by PDGF-stimulated hMCs can induce a profibrotic response in renal epithelia characterized by epithelial-to-mesenchymal transformation (EMT). The equivalent profibrotic response was prevented in supernatants from hMCs treated with LXA4 and PDGF. These data suggest that LXA4 may have distinct anti-fibrotic actions in human renal disease.
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Materials and Methods |
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LXA4 was obtained from Biomol (Plymouth Meeting, PA). Human recombinant PDGF-BB was acquired from Upstate Biotechnology, Milton Keynes, UK. Anti-thrombospondin and anti-fibronectin monoclonal antibodies were from Calbiochem (Nottingham, UK). Enzyme-linked immunosorbent assay (ELISA) kits for TGF-ß1 and matrix metalloproteinase (MMP)-1 were from R&D (Abingdon, Oxon, UK) and Amersham (Buckinghamshire, UK) respectively. Anti-
-smooth muscle actin monoclonal antibody was from Sigma-Aldrich (Tallaght, Dublin, Ireland) and anti-E-cadherin monoclonal antibody was obtained from BD Biosciences (Oxford, UK), alternatively, for immunoblotting anti-E-cadherin (U3254) from Sigma-Aldrich was used. Fluorescein isothiocyanate-conju-gated phalloidin was obtained from Molecular Probes (Eugene, OR).
Mesangial Cell Culture
hMCs were isolated from a nephrectomy sample obtained from the Mater Misercordiae University Hospital in accordance with institutional ethical guidelines. As previously described by Mitchell and colleagues,6
a sample of cortex was isolated and differentially sieved to extract the glomeruli, which were subsequently grown on collagen-coated plates. Cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml), which was selective for mesangial cell growth. These cells retained the phenotypic characteristics of hMCs, including stellate morphology, positive staining for vimentin and -smooth muscle actin, and negative staining for ZO-1 and occludin.17
In Vitro Model of EMT
Murine cortical tubular (MCT) cells were grown in Dulbecco’s modified Eagle’s medium-F12 Hams supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) and were stimulated in Dulbecco’s modified Eagle’s medium-F12 Hams supplemented with insulin-transferrin-selenium supplement (Sigma), L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and hydrocortisone (K1 media). Supernatant from hMCs treated with vehicle, LXA4 (1 nmol/L), PDGF (10 ng/ml), or LXA4 (15 minutes) pretreatment followed by PDGF were removed after 24 hours. MCT cells were stimulated with supernatants from pre-exposed hMCs, diluted in K1 media (Figure 1)
. To control for variability in mesangial cell number in PDGF versus vehicle-treated cells, supernatants were diluted accordingly. PDGF treatment was associated with a twofold increase in cell number as compared to vehicle, therefore conditioned media was diluted 1:3 with K1 media. Vehicle-treated supernatant was alternatively diluted 1:1 with K1 media.
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hMCs were serum restricted (RPMI 1640 supplemented with 0.2% fetal calf serum) for 48 hours before serum starving (RPMI 1640 supplemented with 0% fetal calf serum) for a further 1 hour before stimulating. Cells were subsequently treated with PDGF (10 ng/ml) ± LXA4 (1 nmol/L) in 0% fetal calf serum-RPMI 1640 for 24 hours. RNA from three independent experiments was pooled after isolation using lysis buffer, in accordance with the Qiagen minicolumn preparation (Qiagen, Valencia, CA). Complementary DNA (cDNA) synthesis, in vitro transcription, and microarray analysis were performed as we have previously reported.18 Briefly, cDNA was synthesized from total RNA using the Superscript Choice kit (Invitrogen, Carlsbad, CA). Biotin-labeled cRNA prepared from template cDNA was fragmented and hybridized to Affymetrix HGU133A arrays according to the Affymetrix protocol (Affymetrix, Santa Clara, CA). Arrays were then fluorescently labeled before scanning with a confocal scanner (Affymetrix). This process was repeated to obtain a duplicate chip from which data were compiled and analyzed.
Image files were obtained through Affymetrix GeneChip software (MAS5). Robust multichip analysis (RMA) was subsequently performed. RMA is a technique that analyzes directly from the Affymetrix microarray and is comprised of three steps; background adjustment, quantile normalization, and summarization. RMAexpress was used to make the data accessible to a Microsoft Windows operating system for further analysis, as per Sadlier and colleagues.19
The data from each microarray were collected and expression data for each condition were compared to control. Genes altered by PDGF, causing a 0.5 signal log ratio (SLR) or greater change, with respect to control on both microarrays (equivalent to a fold change in expression of 1.4 or greater) were termed significant and further analyzed for differential expression. Using unsupervised hierarchical cluster analysis as described in Eisen and colleagues20 a visual representation of genomic differential expression was attained. Furthermore, genes could be categorized using a web-based ontology program (Onto-Express).21 This program assigns genes a category based on current known biological function, however redundancy of genes between several categories may exist.
Promoter and Transcription Factor Analysis
Genes significantly altered by PDGF were analyzed using the web-based software Genomatix (Genomatix Software GmbH, Munich, Germany). Genes mapped to loci with experimentally verified promoter regions were further analyzed for common transcription factor binding sites.22 A random expectation value (re-value) was assigned to each transcription factor (the program assigns an expectation value for the number of transcription factor binding site matches per 1000 bp of random DNA sequence). The actual occurrence and random expectation of a given transcription factor were compared to confirm the presence of a binding site. Furthermore, we examined the binding of stimulating protein 1 (SP-1) to consensus SP-1 binding sites using the TransAM SP-1 kit (Active Motif, Rixensart, Belgium). hMCs were treated with PDGF (10 ng/ml) ± LXA4 (1 nmol/L) for 24 hours before extraction of nuclear lysate. Ten µg of nuclear lysate protein was added to each well and the assay was conducted according to the manufacturer’s protocol.
Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Quantitative real-time PCR was performed using TaqMan universal PCR master mix (P/N 4304437; Applied Biosystems, Weiterstadt, Germany) as per the manufacturer’s protocol. Samples were run in duplicate and were analyzed using the ABI Prism 7700 sequence detection system (Applied Biosystems). TaqMan PCR reactions were multiplexed for the gene of interest and VIC-labeled 18S rRNA, as an endogenous control (P/N 4310893E, Applied Biosystems). FAM-labeled forward primers, reverse primers, and TaqMan probes are listed below (Table 1)
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hMCs were treated as follows; cells were serum-starved for 24 hours before the addition of stimuli, they were then pretreated with LXA4 (1 nmol/L) or vehicle for 15 minutes before addition of PDGF (10 ng/ml). The treatment was for the indicated time periods (24 to 72 hours). Supernatants were retained and lysates were harvested in RIPA buffer (150 mmol/L NaCl, 50 mmol/L Tris, 5 mmol/L ethylenediamine tetraacetic acid, 10 mmol/L NaF, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate). The lysates were clarified by centrifugation at 10,621 x g for 10 minutes, the supernatant fraction was retained, and the protein concentration quantified using a Bradford protein assay. MCT cells were conditioned in K1 media, TGF-ß1 (10 ng/ml) and EGF (10 ng/ml) were added as a positive control for EMT. MCT cells were treated with pretreated hMC supernatant, the volume of supernatant from each condition was determined by hMC number. Cells were subsequently harvested as above and protein levels were determined by the Bradford assay. Specific protein levels were detected by immunoblotting lysates from hMC or MCT cell culture.
For Western blot analysis, 20 to 40 µg of hMC protein or 30 µg of MCT protein were loaded into each lane under reducing conditions on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) by electroblotting. Nonspecific antibody binding was reduced by blocking the membrane in 3% bovine serum albumin (Sigma, Dublin, Ireland) for 1 hour at room temperature. The antibody was then added at a concentration according to the manufacturer’s instructions and incubated on a rocking platform at 4°C, overnight. Membranes were subsequently incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature and visualized using enhanced chemiluminescence (Santa Cruz, Heidelberg, Germany) and X-ray film. To check for equal loading the membranes were stripped using sodium dodecyl sulfate, blocked and reprobed with anti-ß-actin monoclonal antibody (Sigma).
Quantitaion of TGF-ß1 Production
Quiescent hMCs were treated as indicated and at 72 hours the supernatant was retained and assayed for TGF-ß1 release by ELISA (R&D Systems) as per manufacturer’s protocol.
Quantitaion of MMP-1 Production
Quiescent hMCs were treated as above, at 6, 18, 24, 48 and 72 hours time-points the supernatant was retained and assayed for MMP-1 release by ELISA (Amersham Biosciences, Bucks, UK) as per the manufacturer’s protocol.
Immunofluorescent Microscopy
MCT cells were cultured on chamber slides (Nalge Nunc, Naperville, IL), and subsequently stimulated with pretreated hMC supernatant, as detailed above. Cells were then washed and fixed with 4% paraformaldehyde added directly to the wells. After washing in phosphate-buffered saline (PBS), cells were permeabilized in 0.5% Triton-X for 10 minutes, blocking agent (PBS containing 1% goat serum and 3% bovine serum albumin) was then added. Cells were kept on a rocking platform for 1 hour at room temperature. The primary antibody was subsequently added at concentrations according to the manufacturer’s instructions and incubated overnight on a rocking platform at 4°C. After washing, a fluorescein isothiocyanate-conjugated secondary antibody, Goat anti-mouse (Molecular Probes) was added at room temperature for 1 hour, after this, cells were washed in PBS containing 4,6-diamidino-2-phenylindole (DAPI) stain for nuclei visualization. Slides were then mounted with coverslips and analyzed using phase contrast (x40 magnification) and also using Axiovert 200 fluorescent microscopy DAPI and fluorescein isothiocyanate filters (Carl Zeiss, Jena, Germany).
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Results |
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Differential gene expression was examined using oligonucleotide microarrays (HGU133A), cDNA was extracted from hMCs exposed to 10 ng/ml PDGF for 24 hours with or without LXA4 pretreatment (15 minutes, 1 nmol/L). This procedure allowed whole genome analysis of hMCs in normal and growth factor-treated cells, identifying signatures of the conditions.20
Data obtained from each condition were normalized with respect to control (ie, vehicle-stimulated cells). An overview of the information acquired is represented in Figure 2A
, PDGF significantly altered (0.5 SLRs or greater) 4.16% of genes represented on the array (926 of 22,283 genes). Consistent with the modulation of PDGF signaling, pretreatment with LXA4 diminished the effects of PDGF. LXA4 pretreatment of the PDGF-stimulated response prevented significant PDGF responses in almost 40% of the 926 genes. Of the 582 genes increased significantly by PDGF, pretreatment of hMCs with LXA4 diminished this response to 353 genes stimulated by PDGF. Genes decreased by PDGF consisted of 344 transcripts, again LXA4 pretreatment diminished this to 205 genes. To gain more insight into the significance of these changes, genes are given functionality based on current known biological process using Onto-Express,21
a web-based ontological program (Figure 2B)
. Categories include embryogenesis, cell proliferation, fibrosis-related, immune response, and lipid metabolism. LXA4 decreased the number of PDGF-driven genes, with particular attention to cell proliferation (42% reduction) and fibrosis-related (49% reduction).
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. Genes significantly altered by PDGF (less than or equal to 0.5 SLR and greater than and equal to 0.5 SLR) were represented. Genome-wide data were clustered using statistical algorithms to arrange genes according to similarity in patterns of expression with co-expressed genes believed to have functional similarity.20
The primary cluster represented all genes significantly altered by PDGF, normalized with respect to control. Additional to this, each ontological category was clustered. The cell proliferation cluster displayed 65 transcripts, of these, 36 were significantly altered in the presence of LXA4, including various cyclins and p53-induced proteins. Of the 35 fibrosis transcripts, 18 were significantly altered by LXA4 pretreatment. Fibronectin, thrombospondin, and TGF-ß1 were among these. Immune response, embryogenesis, and lipid metabolism clusters also displayed altered transcript expression with LXA4 pretreatment.
Of particular interest to us was whether we could define specific transcription factors (TFs) activated in hMCs when exposed to PDGF and whether these might further be subdivided into LXA4-regulated and LXA4-independent genes. Analysis of gene promoter regions was conducted using Genomatix22
software. Genes significantly affected by PDGF were mapped to loci containing experimentally verified promoter regions. Subsequently, common TF binding regions were extracted using MatInspector software. Several ubiquitously expressed transcription factor-binding sites were found including cAMP response element binding site (CREB), early growth response factor (EGRF), and stimulating protein 1 (SP-1) (Figure 2D)
. SP-1 was found to be present on almost half of all PDGF-stimulated genes. Each TF is assigned a random-expectation (re) value based on the estimated number of binding sites for that TF per 1000 bp of sequence. For the SP-1 TF, the re-value was predicted at 1.7, however actual occurrence was averaged at 2.8. Subsequently, we analyzed SP-1 TF binding using a TransAM assay that measures SP-1 and posttranslationally modified SP-1. We found that PDGF significantly elevated levels of SP-1 (1.6-fold) and that this response was attenuated with LXA4 pretreatment (1.18-fold) (Figure 2E)
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Validation of Oligonucleotide Microarray Findings
A cohort of genes was selected for further analysis to validate gene expression changes identified by oligonucleotide microarray. In Figure 3a
quantitative RT-PCR data displayed PDGF-induced (10 ng/ml) increases in the expression of fibronectin and collagen type I 1 transcripts at 24 hours normalized with respect to 18S-ribosomal RNA (1.2- and 1.7-fold induction, respectively). Pretreatment with LXA4 (1 nmol/L) prevented PDGF-induced gene expression increases. PDGF also induced alterations in the protein levels of fibronectin, decorin, and thrombospondin (Figure 3b)
. Stimulation of mesangial cells with PDGF for 24 hours resulted in elevated levels of fibronectin and thrombospondin, a TGF-ß1 activator.23
Corresponding decreases were observed of the small proteoglycan decorin, a known inhibitor of the profibrotic cytokine TGF-ß1.24
These changes in protein expression were consistent with gene expression array findings and were altered with the pretreatment of hMCs with LXA4 (1 nmol/L).
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. Supernatant from stimulated cells was collected at the indicated time points and assayed using ELISA. TGF-ß1 levels were increased significantly by PDGF at 72 hours (2.1-fold change) with LXA4 pretreatment ablating this effect (1.1-fold change). Consistent with this, secreted MMP-1 levels were significantly enhanced in response to PDGF at 6, 18, and 24 hours, (1.2-, 1.5-, and 1.6-fold change, respectively) whereas LXA4 achieved significant diminution of the PDGF effect at 6 and 18 hours only (0.9- and 0.7-fold change). Epithelial-to-Mesenchymal Transformation of Proximal Tubular Epithelial Cells
It has been proposed that pleiotropic mediators released from the inflamed glomerulus play an important role in progressive renal disease by tubulointerstitial fibrosis.25,26
Tubulointerstitial fibrosis is frequently characterized by EMT. Consistent with this we observed that supernatants from hMCs treated with PDGF (10 ng/ml, 24 hours) induced a loss of E-cadherin expression concomitant with increased levels of -smooth muscle actin in MCT cells (Figure 4b)
. These phenotypic changes mirrored those seen with the prototypic inducers of EMT, TGF-ß1, and EGF (Figure 4a)
.27
MCT cells treated with cell supernatant from vehicle or LXA4-treated hMCs retained much of their epithelial phenotype. Remarkably, this observed change in MCT phenotype was less substantial after treatment with supernatant from PDGF-stimulated and LXA4-pretreated hMCs. Pretreatment with LXA4 diminished the effects of PDGF thereby impeding the indicated transformation. These changes were verified using Western blot analysis of MCT lysate (Figure 4c)
. PDGF-stimulated hMC supernatant caused decreases in E-cadherin and corresponding increases in -SMA levels. Again, supernatant from hMCs pretreated with LXA4 diminished this effect.
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. Consistent with this, supernatant from PDGF-stimulated (10 ng/ml, 24 hours) hMCs caused an increase in the expression of VEGF, CTGF, and fibronectin in MCT cells. Supernatant from hMCs pretreated with LXA4 (1 nmol/L, 15 minutes) and subsequently PDGF-stimulated, diminished these observed changes in gene expression (Figure 5
; B, C, and D), consistent with LXA4 modulation of PDGFR signals. Expression of all three genes was continuously decreased with LXA4-pretreated supernatant. Changes in epithelial cell gene expression induced by hMC supernatants were not due to residual PDGF in the media. This was verified by the addition of the tyrophostin PDGFR kinase inhibitor AG1296 (10 nmol/L) to hMC supernatant. Addition of the inhibitor did not significantly effect observed gene expression changes (data not shown). Furthermore, direct stimulation of the MCT cells with PDGF-BB did not induce significant expression of fibronectin, VEGF, or CTGF (Figure 5A)
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Discussion |
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LXs have been shown to exert potent anti-inflammatory actions in the early stages of GN mediated through inhibition of leukocyte infiltration and or adhesion30 and are proposed to act as endogenously produced proresolution agents in host defense and inflammation.31 As observed in intestinal enterocytes, expression of the LXA4 receptor was up-regulated in response to cytokines, furthermore addition of LXA4 or its stable, synthetic analogues inhibited interleukin-8 chemokine release, thereby modulating the initiation of inflammation.32 Previous data suggest that the anti-proliferative effects of LXA4 are mediated via intracellular mechanisms associated with modulation of growth factor receptor (PDGF and EGF) activation,4,6 and recruitment of specific SH-2 containing signaling molecules (Mitchell D, Gaffney A, Crean JK, Kinsella BT, Godson C, submitted). Here we demonstrate that LXA4 can counteract PDGF-induced gene expression. In agreement with other models in-vestigating LXA4-induced differential gene expression,18,33,34 we report that LXA4 modulated the expression of the NAB1 co-repressor, up-regulating expression by 0.3 SLRs. The stable synthetic analogue 15-epi-16-(para-fluorophenoxy)-lipoxin A (4)-methyl ester has been shown to exert remarkable renoprotection in a murine model of ischemia-reperfusion injury. Several of the genes differentially expressed in this model were also observed here, including interleukin-6, thrombospondin-1, metallothionein 1, transgelin, phosphoserine aminotransferase, and Enigma. LXA4 diminished the effects of PDGF-stimulated genes responsible for biological functions such as fibrosis, cell proliferation, immune response, lipid metabolism, and embryogenesis. Among the numerous genes identified as PDGF-responsive, those contributing to fibrosis-related changes in hMCs were clustered and examined further. We report that LXA4 modulates the PDGF-induced expression of several matrix-associated genes, including collagens, transforming growth factors, and fibronectin. Moreover, PDGF-increased expressions of matrix-associated proteins, thrombospondin, and fibronectin, were reduced by LXA4 to levels that were not significantly different to those of baseline. Such proteins contain multiple domains that bind proteoglycans, collagens, integrins, and cytokines, including TGF-ß1 and are therefore important in the control of cell viability, mitogenesis, and motility. Fibronectin is present basally within the mesangial matrix functioning in supporting mesangial cell viability,35 increases in fibronectin have been demonstrated in fibrotic disease processes.36 TSP is transiently expressed in matrix during development and repair23,37 and has previously been shown to respond to PDGF, TGF-ß1, and basic fibroblast growth factor,37 it is a known regulator of TGF-ß1 activity.23,38 We further verify that levels of decorin, a small leucine-rich proteoglycan that acts as an endogenous inhibitor of TGF-ß1,39 are decreased in response to PDGF and are recovered by LXA4 pretreatment. Overall, the up-regulation of both RNA and protein levels of the various fibrosis-associated genes, suggests an expansion of glomerular matrix and associated cytokines. The decreased expression of a negative regulator (decorin) and a corresponding increase in a positive regulator (thrombospondin) of TGF-ß1 demonstrates that PDGF-induced profibrotic activity in hMCs may be associated with increases in TGF-ß1. In contrast, LXA4 significantly reduced this PDGF-induced profibrotic activity observed in hMCs. These data further support the observed reduction in PDGF-induced secretion of TGF-ß1 by LXA4 at 24, 48,6 and 72 hours. The diverse changes in matrix-associated proteins occurring throughout time, relate to initial injury and long-term damage sustained and the ultimate healing process thereafter. Secreted MMP-1 (collagenase I) levels were significantly enhanced in response to PDGF at 6, 18, and 24 hours, whereas LXA4 achieved diminution of the PDGF effect only at 6 and 18 hours. However, consistent with this MMP-1 RNA levels in response to PDGF were not significantly altered by LXA4 at 24 hours.
The observed dynamics of extracellular matrix synthesis and degradation are consistent with various models of glomerular and tubulointerstitial renal disease. Adhikary and colleagues40 examined various extracellular matrix components in anti-glomerular basement membrane nephritis, mRNA for fibronectin and collagens I and IV were elevated in glomeruli from day 15, throughout the disease course. TIMP-1 and TGF-ß1 mRNA were also enhanced. A similar profile of extracellular matrix components and TGF-ß1 was observed in the cortex, increasing more gradually from day 15 to day 29 as tubular damage progressed.40
The reduction in secreted levels of both MMP-1 and TGF-ß1 in LXA4 pretreated cells may reflect reduced autokinase activity of the receptor tyrosine kinases (RTKs) and/or enhanced tyrosine phosphatase activity (Mitchell D, Gaffney A, Crean JK, Kinsella BT, Godson C, submitted). Significant evidence for the cross-talk between the LX G-protein-coupled receptor activated by LXA4 (ALXR) and the RTKs for both PDGF and EGF has been shown.4,6 In the context of LXA4 modulation of profibrotic changes, it is noteworthy that a role for the ALXR has recently been proposed in the anti-fibrotic treatment of a lung fibrosis model in mice.41 In these experiments LXA4 also significantly prevented enhanced proliferation of NIH3T3 fibroblasts and collagen expression by TGF-ß1. Furthermore Sodin-Semrl and colleagues42 described a role for LXA4 in regulating human synovial fibroblast activation, levels of MMP-1 and MMP-3 were diminished in response to LXA4 treatment of stimulated fibroblasts.
The effects of LXA4 on PDGFR activation are relatively specific, being restricted to recruitment to precise phosphotyrosine residues (Mitchell D, Gaffney A, Crean JK, Kinsella BT, Godson C, submitted). In this regard it was of interest to investigate whether LXA4 might modulate transcription factors downstream of specific signal transduction processes. It has been reported that LXA4 can mediate NF-
B-induced gene expression in intestinal epithelial cells.43
Using MatInspector, many ubiquitous TF binding sequences were observed in the promoters of PDGF-induced genes implicated in both matrix-associated and proliferative gene regulation. Verrecchia and colleagues44
showed that inhibition of the SP-1 TF prevented the expression of extracellular matrix genes in dermal fibroblasts. Although SP-1 is a ubiquitous TF, it has also been shown to respond to several growth factors, MAPK and glucose.45
Posttranslational modification of SP-1 may have a significant role in TF activation. In our study we measured the binding of nuclear SP-1 from hMCs treated with PDGF and PDGF pretreated with LXA4, we found that SP-1 was significantly increased in response to PDGF. LXA4 consistently diminished this effect.
The translation of a relatively modest glomerular injury to devastating tubulointerstitial fibrosis is increasingly appreciated. Such fibrosis can reflect on a combination of EMT of resident epithelia or progenitor cells, infiltration of circulating or proliferation of resident fibroblasts.46,47 Alterations in the mediators present in the interstitium and in the glomerular filtrate contribute to the development of tubulointerstitial fibrosis.26 Tubulointerstitial fibrosis encompasses loss of epithelium polarity, adherens junctions, tight junctions, desmosomes, and cytokeratin intermediate filaments to rearrange their F-actin stress fibers and express filopodia and lamellopodia.48 Epithelial cells gain plasticity during the remodeling process, promoting healing or scarring as a response to injury.49 Induction of EMT is associated with the expression of cytokines and the proteolytic degradation of epithelial basement membrane.50 Matrix metalloproteinases or membrane assembly inhibitors dismantle the membrane, whereas local expression of TGF-ß, EGF, IGF-II, or FGF-2 facilitates EMT.48,51,52 These cytokines contribute to elevated levels of matrix metalloproteinases and alter the cell proteome. Higgins and colleagues53 observed increases in the expression of collagen I and PAI-1, among others, in EMT using the in vivo model of unilateral ureteral obstruction and the in vitro model of stimulated murine proximal tubular (MCT) cultured cells. Signature markers for the disease were identified using oligonucleotide microarray.
We report here that PDGF-treated hMC supernatant caused a morphological change in renal tubular MCT cells, similar to that of TGF-ß1 and EGF. Loss of epithelial tight junction marker E-cadherin and gain of mesenchymal actin cytoskeleton marker -SMA in MCT cells treated with PDGF-stimulated hMC supernatant was observed. Interestingly, these effects were diminished by LXA4 pretreatment. LXA4 pretreatment could alter these effects perhaps by altering PDGF-induced receptor activation in the hMCs, changing the spectrum of soluble factors present in the ultrafiltrate. Furthermore, immunoblotting for E-cadherin and -SMA under the same conditions confirmed these findings. Previously, we have reported the inhibition of PDGF and EGF mediated PKB/Akt phosphorylation by LXA4.6
This pathway may be involved in the release of cytokines or mediators from hMCs, which we observe to cause morphological changes in MCT cells, a role for this pathway in fibrosis is well established. Stimulation of fibroblasts with endothelin-1 promotes enhanced contractile phenotype or activation, this is prevented by blockade of the PI3K/Akt pathway.54
In agreement with this Vittal and colleagues55
observed that inhibition of PKB/Akt in bleomycin-induced lung fibrosis markedly reduced accumulation of -smooth muscle actin-expressing myofibroblasts.
Supernatant from PDGF-stimulated hMCs stimulated an increase in RNA expression of VEGF, CTGF, and fibronectin. This effect was modulated by LXA4 pretreatment. To control for the possibility that residual PDGF in the media from pretreated hMCs might have induced profibrotic changes in the epithelial cells, these cells were treated with the PDGF-specific phosphotyrosine inhibitor, AG1296. However, in these cells changes in profibrotic gene expression persisted. Similarly, treatment of MCT cells with PDGF ligand did not induce profibrotic gene expression. Soluble factors released from hMCs in response to PDGF in our in vitro model may indicate the progression of renal disease seen in vivo.
Collectively these data demonstrate LXA4 as a potential anti-fibrotic agent, preventing growth factor-induced mesangial matrix production and the progression of renal disease, by alleviating the effect of hMC products on tubular cells. Further investigation into these bioactivities of LXA4 and its stable synthetic analogues will include examining effects on hMCs and tubular cells in an in vivo model of progressive renal disease.
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Acknowledgements |
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Footnotes |
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Supported by The Health Research Board Ireland, The Mater Foundation, The Wellcome Trust, The Government of Ireland Programe for Research in Third Level Institutes administered through the Higher Education Authority, and an Amgen Renal Research Bursary (to K.R.).
K.R. and B.M. contributed equally towards this manuscript.
Supplemental data (all Affymetrix data) are provided at www.ebi.ac.uk.
Accepted for publication May 26, 2005.
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 H.-J. Kim, B. C. Litzenburger, X. Cui, D. A. Delgado, B. C. Grabiner, X. Lin, M. T. Lewis, M. M. Gottardis, T. W. Wong, R. M. Attar, et al. Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-{kappa}B and Snail Mol. Cell. Biol., April 15, 2007; 27(8): 3165 - 3175. [Abstract] [Full Text] [PDF]
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 C. Bonnans, K. Fukunaga, M. A. Levy, and B. D. Levy Lipoxin A4 Regulates Bronchial Epithelial Cell Responses to Acid Injury Am. J. Pathol., April 1, 2006; 168(4): 1064 - 1072. [Abstract] [Full Text] [PDF]
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0.5 SLR and 
0.5 SLR) from the average of duplicate arrays. A: An overview of the numbers of differentially expressed genes. B: Changes in gene expression within major functional families using OntoExpress. C: Unsupervised hierarchical cluster analysis of the average of duplicate arrays and ontological clustering. D: Transcription factor consensus binding sequences on PDGF-altered genes, random-expectation values, and actual binding site occurrence are shown. E: SP-1 expression in hMC nuclear lysate after treatment with PDGF (10 ng/ml), LXA4 (1 nmol/L), vehicle, or LXA4 (15 minutes) followed by PDGF. Data are mean ± SEM of duplicate measurements from three independent experiments (*P < 0.05 compared to vehicle, #P < 0.05 compared to PDGF).
