TGF-{beta}1 stimulates monocyte chemoattractant protein-1 expression in mesangial cells through a phosphodiesterase isoenzyme 4-dependent process

Jingfei Cheng,1 Montserrat M. Diaz Encarnacion,1 Gina M. Warner,1 Catherine E. Gray,1 Karl A. Nath,2 and Joseph P. Grande1,2

1Department of Laboratory Medicine and Pathology, and 2Division of Nephrology and Hypertension, Mayo Clinic College of Medicine, Rochester, Minnesota

Submitted 1 April 2005 ; accepted in final form 26 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor (TGF)-{beta}1 are critical mediators of renal injury by promoting excessive inflammation and extracellular matrix deposition, thereby contributing to progressive renal disease. In renal disease models, MCP-1 stimulates the production of TGF-{beta}1. However, a potential role for TGF-{beta}1 in the regulation of MCP-1 production by mesangial cells (MCs) has not previously been evaluated. The objectives of this study were to define the role of TGF-{beta}1 in regulation of MCP-1 expression in cultured MCs and to define mechanisms through which rolipram (Rp), a phosphodiesterase isoenzyme 4 (PDE4) inhibitor with anti-inflammatory properties, alters MCP-1 expression. TGF-{beta}1 induced MCP-1 in a time- and dose-dependent manner without increasing transcription of the MCP-1 gene. TGF-{beta}1-mediated induction of MCP-1 occurred without activation of the NF-{kappa}B pathway. Rp blocked TGF-{beta}1-stimulated MCP-1 expression via a protein kinase A-dependent process, at least in part, by decreasing MCP-1 message stability. Rp exerted no effect on activation of the Smad pathway by TGF-{beta}1. TGF-{beta}1-mediated induction of MCP-1 required activation of ERK and p38, both of which were suppressed by a PDE4 inhibitor. TGF-{beta}1-stimulated reactive oxygen species (ROS) generation by MCs, and Rp inhibited ROS generation in TGF-{beta}1-stimulated MCs; in addition, both Rp and ROS scavengers blocked TGF-{beta}1-stimulated MCP-1 expression. We conclude that TGF-{beta}1 stimulates MCP-1 expression through pathways involving activation of ERK, p38, and ROS generation. Positive cross-talk between TGF-{beta}1 and MCP-1 signaling in MCs may underlie the development of progressive renal disease. Rp, by preventing TGF-{beta}1-stimulated MCP-1 production, may offer a therapeutic approach in retarding the progression of renal disease.

cAMP; inflammation; mitogen-activated protein kinase; reactive oxygen species


PROGRESSIVE RENAL DISEASE is driven by persistent inflammation and accumulation of extracellular matrix and commonly reflects the underlying role of transforming growth factor (TGF)-{beta}1, the single most important mediator of fibrosis (3, 5, 31, 35). A diverse array of stimuli upregulate TGF-{beta}1, including monocyte chemoattractant protein-1 (MCP-1). MCP-1 is a critical mediator of glomerular and interstitial inflammation during progressive renal disease by virtue of its potent chemotactic effects and its induction by a variety of growth factors and cytokines, all of which are relevant to progressive renal injury.

A particularly notable action of MCP-1 in its role in progressive disease is its inductive effect on TGF-{beta}1. For example, in a mesangial proliferative glomerulonephritis model, MCP-1 is rapidly induced after acute injury, promotes macrophage influx, and increases TGF-{beta}1 expression (53). MCP-1 stimulates TGF-{beta}1 production and collagen synthesis by rat lung fibroblasts, both in vitro and in vivo (13, 14). This induction of an archetypal fibrogenic cytokine (TGF-{beta}1) by a chemotactic one (MCP-1) thus sets the stage whereby inflammation culminates in progressive accumulation of extracellular matrix. Whether this interaction is bidirectional (that is, MCP-1 induces TGF-{beta}1 and TGF-{beta}1 in turn induces MCP-1) is an issue that is complex and unresolved (17, 32) and has not been examined to date in mesangial cells (MCs), with the latter cells critically contributing to progressive renal disease. Given the involvement of pathobiological events in MCs in progressive renal disease and the fact that MCs produce both TGF-{beta}1 and MCP-1 (often in exaggerated amounts in pathological states) and respond to these cytokines, in the present study, we examined the question whether TGF-{beta}1 induces MCP-1 in MCs. Such an effect may be of particular relevance to progressive glomerular disease because it provides a positive feedback relationship between TGF-{beta}1 and MCP-1 and thereby confers a self-perpetuating cycle of inflammation and fibrogenesis, with the latter representing a signature characteristic of progressive renal disease.

Signaling pathways initiated by TGF-{beta}1 and those that lead to the production of MCP-1 involve common intermediates. For example, TGF-{beta}1 rapidly activates the ERK and p38 signaling pathways, and this activation is necessary for TGF-{beta}1-mediated induction of extracellular matrix production by MCs (23). TGF-{beta}1 stimulates reactive oxygen species (ROS) generation (25, 30, 42, 56, 57). ROS in turn mediate TGF-{beta}1-stimulated matrix production in a variety of cells, including MCs (16, 36). Similarly, ROS have been identified as essential intermediates for cytokine-stimulated MCP-1 generation (4, 21, 34, 51).

Cytokine-stimulated MCP-1 generation is also influenced by cAMP, because cAMP analogs effectively block cytokine-stimulated MCP-1 expression (43, 50). Intracellular cyclic nucleotide levels can be regulated tightly in a cell type-specific manner with phosphodiesterase (PDE) inhibitors, which prevent the catabolism of cAMP, cGMP, or both (2, 10, 11, 26, 38). In this regard, we have previously demonstrated that cAMP hydrolysis in MCs is directed almost exclusively by PDE isoenzyme 3 (PDE3) and PDE isoenzyme 4 (PDE4) (6, 8, 40). Although both PDE3 and PDE4 inhibitors activate PKA and increase intracellular cAMP levels to a similar extent, only PDE3 inhibitors suppress growth factor-stimulated ERK activation and mitogenesis (6, 8, 40) and only PDE4 inhibitors suppress ROS generation by MCs (6, 8, 40).

On the basis of these considerations, in the present study, we sought to test the hypotheses that 1) TGF-{beta}1 stimulates the production of MCP-1 in MCs through a pathway involving activation of ERK or p38 and production of ROS as signaling intermediates and 2) PDE4 inhibitors are capable of blocking TGF-{beta}1-stimulated MCP-1 expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Primary antibodies for I{kappa}B-{beta}, Smad2/3, Smad4, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Amersham Biosciences (Piscataway, NJ). Phospho-Smad2 antibody was obtained from Cell Signaling Technology (Beverly, MA). Protein G magnetic beads were obtained from New England BioLabs (Beverly, MA). The ERK inhibitor U0126 was obtained from Promega (Madison, WI). The p38 inhibitor SB-202190 and the JNK inhibitor type II were obtained from Calbiochem (La Jolla, CA). The fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Molecular Probes (Eugene, OR). Other reagents, all of the highest purity grades, were purchased from standard suppliers.

Mesangial cell culture. A pSV3-Neo immortalized rat MC line (CRL-2573) was obtained from the American Type Culture Collection (Manassas, VA). Dulbecco's modified Eagle's medium (DMEM) containing 4,500 mg/l glucose and 15% fetal bovine serum (FBS) was used to culture CRL-2573. These transformed cells have been shown to exhibit characteristics similar to those of primary cultures of rat MCs. Parallel studies were conducted in MCs isolated from rat glomeruli to confirm that the cell line and primary cultures of MCs responded to TGF-{beta}1 in a similar fashion. Our handling of the rats conformed to institutional animal care guidelines established by the National Institutes of Health, and the study protocol was approved by the Mayo Clinic College of Medicine Institutional Animal Care and Use Committee (IACUC no. A17904 [GenBank] ). Primary MCs were obtained from 200-g male Sprague-Dawley rats using differential sieving as described previously (7, 19, 40). Briefly, rats were euthanized by intraperitoneal injection of pentobarbital sodium (100 mg/kg). The kidneys were excised, the renal capsule was removed, and the cortical tissue was minced and passed through a stainless steel sieve (200 µm pore size). The homogenate was sequentially sieved through nylon meshes of 390-, 250-, and 211-µm pore openings. The cortical suspension was then passed over a 60-µm sieve to collect glomeruli. The purity of glomerular preparations was evaluated using light microscopy. Preparations typically contained >90% glomeruli. Glomeruli were seeded on plastic tissue culture dishes and grown in complete Waymouth's medium [Waymouth's medium supplemented with 20% heat-inactivated FBS, 15 mM HEPES, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 1% ITS+ (insulin, transferrin, selenium, and bovine serum albumin)]. Fresh medium was added every 3 days. Cell outgrowths were characterized as MCs on the basis of positive immunohistochemical staining for vimentin and smooth muscle-specific actin and negative stains for cytokeratin, factor VIII-related antigen, and leukocyte common antigen (antibodies were obtained from Dako, Carpinteria, CA). MCs were passaged once per week after treatment with trypsin-ethylenediaminetetraacetic acid (EDTA; 0.25%; Sigma Chemical, St. Louis, MO). Cells used in experiments were from passages 515.

Northern blot analysis. MCs were plated in 10-cm dishes at 2 x 106 cells/dish in complete growth medium. Cells were rendered quiescent in withdrawal medium (growth medium with 0.5% FBS) for 24 h before the addition of vehicle (0.1% DMSO for both PDE inhibitors and MAPK inhibitors), PDE inhibitors, MAPK inhibitors, TGF-{beta}1, and other agents. Total cellular RNA was isolated using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA (20 µg/lane) was electrophoresed through a 1% agarose, 2.2 M formaldehyde denaturing gel and transferred to nylon membranes (Schleicher & Schuell, Keene, NH) as previously described (18, 20). Northern blot analysis was performed with a rat MCP-1 cDNA probe obtained as previously described (29) using reverse transcription-based polymerase chain reactions. Probes were labeled with [{alpha}-32P]dCTP by the random primer method, and blots were hybridized at 65°C overnight in a 0.5 M sodium phosphate buffer, pH 7.0, containing 1 mM EDTA, 7% sodium dodecyl sulfate (SDS), and 1% bovine serum albumin as described by Church and Gilbert (9). Autoradiograms were quantified using computer-assisted video densitometry. As a loading control, blots were reprobed for the housekeeping gene GAPDH as previously described (18).

Quantification of MCP-1 secretion using ELISA. Quiescent MCs were treated with PDE inhibitors or ROS scavengers for 30 min before addition of TGF-{beta}1 for 24 h. Control cells were treated with vehicle only. Conditioned medium was collected and centrifuged at 1,500 rpm for 5 min. Supernatants were stored at –70°C until assay. The concentration of MCP-1 was quantified using a rat-specific MCP-1 enzyme-linked immunosorbent assay (ELISA) kit (Biosource International, Camarillo, CA) according to the manufacturer's instructions.

Transfection studies. ERK and p38 activation were measured using the PathDetect in vivo signal transduction pathway trans-reporting system (Stratagene, La Jolla, CA). MCs were plated into 24-well culture plates at 8 x 104 cells/well in withdrawal medium. Twenty-four hours after being plated, cells were cotransfected with a firefly luciferase reporter vector (pFR-Luc), transactivator plasmids (pFA2-Elk1 for the ERK pathway and pFA2-CHOP for the p38 pathway), and a control Renilla luciferase reporter vector (phRG-TK) to control for transfection efficiency. For MCP-1 promoter activity studies, the MCP-1 promoter and flanking region was amplified from rat genomic DNA. Primers were based on GenBank accession no. AF079313 (forward primer sequence, 5'-TGTGAGAGCTGCTTGGCTGTAAC-3', nt 1,220–1,242; reverse primer sequence, 5'-TCTGGCTTCAGTGAGAGTTGGC-3', nt 3,635–3,614). The genomic PCR product was sequenced and subcloned into pGL3-Basic. MCs were cotransfected with the MCP-1 promoter construct (350 ng) and phRG-TK (20 ng). Control cells were cotransfected with pGL3-Basic and phRG-TK. NF-{kappa}B transcription studies were performed with slight modification as described by Trushin et al. (59). In brief, MCs were cotransfected with 350 ng of NF-{kappa}B-luc reporter plasmid and 5 ng of phRG-Basic (a control Renilla luciferase construct). Transfections were performed using FuGene 6 transfection reagent (Roche Molecular Biochemical, Indianapolis, IN) according to the manufacturer's instructions. Agonists were added 18 h after transfection. Cells were rinsed and lysed at various time points as described in RESULTS. Luciferase activity was assessed using the Dual-Luciferase reporter assay system (Promega, Madison, WI).

NF-{kappa}B DNA binding assay. Quiescent MCs were treated with TGF-{beta}1 or TNF-{alpha}, and nuclear extracts were prepared using Active Motif's nuclear extract kit (Carlsbad, CA). Activation of NF-{kappa}B subunit p65 was assessed colorimetrically using the TransAM NF-{kappa}B transcription factor assay kit (Active Motif) according to the manufacturer's instructions. Nuclear protein (20 µg/well) was used for the assay.

Western blot analysis. Vehicle, PDE inhibitor, and TGF-{beta}1-treated quiescent MCs were rinsed, harvested, and subjected to sonication (3 cycles of 10 s each, 8 µm amplitude) in 1x lysis buffer (Cell Signaling Technology, Beverly, MA). Homogenates were centrifuged at 10,000 g for 10 min at 4°C. Protein concentration of the supernatant was determined using the method described by Lowry (37). Equal amounts of lysate protein (~100 µg) were subjected to SDS-PAGE using the Criterion System (Bio-Rad Laboratories, Hercules, CA). Lysates were denatured for 5 min at 100°C in SDS loading buffer (Bio-Rad Laboratories). Electrophoresis was performed at a constant current (200 mA/gel), followed by transfer to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were blocked with 1x casein (Vector Laboratories, Burlingame, CA) in Tris-buffered saline (TBS) containing 0.1% Tween 20 and incubated with primary antibodies, followed by HRP-conjugated secondary antibodies. Blots were then visualized by exposing them to X-ray film using Amersham ECL Western blot analysis detection reagents (Amersham Biosciences, Piscataway, NJ). Blots were stripped and reprobed with a {beta}-actin antibody for equal loading.

Measurement of intracellular ROS generation. Quiescent MCs cultured in black, flat-bottomed, 96-well plates were loaded with the redox-sensitive dye H2DCFDA (5 µM) for 30 min at 37°C. Cells were then rinsed twice with phenol red-free RPMI (Invitrogen/Life Technologies, Carlsbad, CA) and treated with vehicle or PDE inhibitors for 15 min, followed by TGF-{beta}1 treatment for 90 min. H2DCFDA fluorescence was detected at excitation and emission wavelengths of 490 and 530 nm, respectively. ROS generation was measured using a microplate reader (Fusion Universal microplate analyzer; Packard Bioscience, Meriden, CT).

Apoptosis. Quiescent MCs were treated with vehicle, PDE inhibitors, ROS scavengers, and TGF-{beta}1. Caspase-3 activity was determined colorimetrically using the CaspACE assay system (Promega, Madison, WI). Caspase-8 activity was assessed using the Caspase-Glo8 assay system (Promega) according to the manufacturer's instructions.

Statistical analysis. Data presented are representative of at least three independent experiments performed in duplicate or in triplicate as indicated in the figure legends. Groups or pairwise comparisons were evaluated using Student's t-test, and P < 0.05 values were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TGF-{beta}1 induces MCP-1 mRNA expression in a time- and dose-dependent manner in MCs. Our initial studies determined whether TGF-{beta}1 influenced expression of MCP-1 in MCs. Steady-state MCP-1 mRNA levels were increased significantly after 2-h treatment with TGF-{beta}1, peak MCP-1 induction occurred at ~12 h, and the stimulatory effect of TGF-{beta}1 persisted through 48 h (Fig. 1A). TGF-{beta}1 increased MCP-1 mRNA in a dose-dependent fashion, and maximal induction was observed at concentrations ≥5 ng/ml TGF-{beta}1 (Fig. 1B).



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Fig. 1. Transforming growth factor (TGF)-{beta}1 induces monocyte chemoattractant protein (MCP)-1 mRNA expression in a time- and dose-dependent manner. A: mesangial cells (MCs) were treated with TGF-{beta}1 (10 ng/ml) for the indicated periods. B: MCs were exposed to the indicated doses of TGF-{beta}1 for 6 h. Total RNA was isolated and Northern blots were hybridized with cDNA probes specific for rat MCP-1 and for GAPDH, a housekeeping gene used to control for gel loading and transfer efficiency. Values represent means ± SE (n = 3). *P < 0.05 vs. control. Insets, blots of representative experiments.

 
PDE4 inhibitors block TGF-{beta}1-stimulated MCP-1 expression via a PKA-dependent process. In our previous studies, we demonstrated that cAMP hydrolysis in MCs is directed almost exclusively by PDE3 and PDE4. However, only PDE3 inhibitors, but not PDE4 inhibitors, are effective in blocking growth factor-stimulated mitogenesis of MCs (6). We therefore sought to determine whether PDE3 or PDE4 inhibitors had a differential effect on TGF-{beta}1-stimulated MCP-1 expression. Using Northern blot analysis, we found that the PDE4 inhibitor rolipram (Rp), but not the PDE3 inhibitor lixazinone, dose-dependently inhibited TGF-{beta}1-stimulated MCP-1 mRNA expression (Fig. 2, A and B). The structurally distinct PDE3 inhibitor cilostamide also had no effect on TGF-{beta}1-stimulated MCP-1 expression (Fig. 2C). The PKA inhibitor 14-22 amide reversed the inhibitory effect of Rp on MCP-1 expression (Fig. 2C). MCP-1 protein levels were quantified using a rat-specific MCP-1 ELISA kit. Both Rp and the structurally distinct PDE4 inhibitor Ro 20-1724 significantly blocked TGF-{beta}1-stimulated MCP-1 protein production; PDE3 inhibitors were without effect (Fig. 2D). These studies provide evidence that the observed suppressive effects on TGF-{beta}1-stimulated MCP-1 expression are due to inhibition of PDE4 activity and that the inhibitory effect of PDE4 inhibitors on TGF-{beta}1-stimulated MCP-1 expression is PKA dependent.



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Fig. 2. Phosphodiesterase isoenzyme 4 (PDE4) inhibitor rolipram (Rp) blocks TGF-{beta}1-stimulated MCP-1 expression in a dose-dependent, PKA-dependent fashion. A: dose response of Rp on TGF-{beta}1-stimulated MCP-1 expression. MCs were pretreated with vehicle (0.1% DMSO) or the indicated doses of Rp for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 6 h. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. B: dose response of lixazinone (Lix) on TGF-{beta}1-stimulated MCP-1 expression. MCs were pretreated with vehicle (0.1% DMSO) or the indicated doses of Lix for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 6 h. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. C: MCs were treated sequentially with the PKA inhibitor 14-22 amide (2 µM) and the PDE3 inhibitors Lix and cilostamide (CS) or the PDE4 inhibitor Rp (10 µM each) for 30 min and 1 h, respectively, before 10 ng/ml TGF-{beta}1 treatment for 6 h. Control cells were treated with vehicle (0.1% DMSO) only. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. D: MCs were treated with vehicle (0.1% DMSO) or PDE inhibitors (10 µM each) for 30 min before addition of TGF-{beta}1 (10 ng/ml) for 24 h. The concentration of MCP-1 secreted into the culture medium was quantified using a rat-specific MCP-1 ELISA kit. Values represent means ± SE (n = 3). #P < 0.05 vs. vehicle-treated levels. *P < 0.05 vs. TGF-{beta}1-treated levels. Insets, blots showing representative experiments.

 
TGF-{beta}1 does not significantly increase MCP-1 promoter activity. Transcription of the MCP-1 gene in response to diverse cytokines (e.g., LPS, TNF, IL-1) occurs through an NF-{kappa}B-dependent pathway. We prepared a chimeric MCP-1 promoter-luciferase construct containing NF-{kappa}B and activator protein-1 sequences essential for transcriptional activation of the MCP-1 gene (60). TGF-{beta}1 did not significantly increase transcriptional activity of the MCP-1 promoter-luciferase construct. However, as expected, transcriptional activity of the MCP-1 promoter construct was increased 2.3-fold by TNF-{alpha} (Fig. 3A). On the basis of these results, we conclude that TGF-{beta}1 induces MCP-1 through a mechanism that does not involve transcriptional activation of the MCP-1 gene.



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Fig. 3. Unlike TNF-{alpha}, TGF-{beta}1 does not increase MCP-1 promoter activity. NF-{kappa}B signaling pathway is not required for TGF-{beta}1-stimulated MCP-1 expression. A: MCs were cotransfected with a chimeric MCP-1 promoter-luciferase construct (350 ng) and a Renilla luciferase construct phRG-TK (20 ng). Control cells were cotransfected with pGL3-Basic and phRG-TK. Cells were then treated with TGF-{beta}1 or TNF-{alpha} (10 ng/ml each) for 24 h. Luciferase activity was assessed using the dual-luciferase reporter assay system (Promega, Madison, WI). Firefly/Renilla luciferase ratios for MCP-1-transfected cells were normalized to firefly/Renilla luciferase ratios from cells transfected with pGL3-Basic. Values represent means ± SE (n = 4). *P < 0.05 vs. control. B: MCs were treated with or without TGF-{beta}1 or TNF-{alpha} (10 ng/ml each) for 30 min. Nuclear proteins were extracted, and NF-{kappa}B subunit p65 activation was assessed colorimetrically using the TransAM NF-{kappa}B transcription factor assay kit (Active Motif) according to the manufacturer's instructions. Nuclear protein (20 µg/well) was used for p65 activity assay. Values represent means ± SE (n = 3). *P < 0.05 vs. control. C: MCs were cotransfected with 350 ng of NF-{kappa}B-luciferase reporter plasmid and 5 ng of phRG-Basic (a control Renilla luciferase construct) for 18 h before TGF-{beta}1 or TNF-{alpha} (10 ng/ml each) treatment for 2 and 6 h. Luciferase activity was assessed using the dual-luciferase reporter assay system. Values represent means ± SE (n = 3). *P < 0.05 vs. control.

 
NF-{kappa}B signaling pathway is not required for induction of MCP-1 by TGF-{beta}1. In many cell types, proinflammatory cytokines stimulate MCP-1 production through activation of the NF-{kappa}B pathway. Activation of the NF-{kappa}B pathway is associated with release and degradation of the inhibitory protein I{kappa}B from the p50/p65 complex. Using a p65 ELISA kit, we found that TGF-{beta}1 did not activate NF-{kappa}B. However, as expected, TNF-{alpha} strongly promoted NF-{kappa}B activation (+1,523%; P < 0.001) (Fig. 3B). In a complementary assay, TGF-{beta}1 did not stimulate transcriptional activity of a chimeric NF-{kappa}B-luciferase construct (59). However, as expected, TNF-{alpha} stimulated transcriptional activity of the NF-{kappa}B-luciferase construct (+2,695% at 2 h and +3,348% at 6 h) (Fig. 3C). Finally, TGF-{beta}1 had no effect on I{kappa}B degradation. On the other hand, TNF-{alpha} rapidly promoted I{kappa}B degradation as assessed by performing Western blot analysis (data not shown). On the basis of these considerations, we conclude that TGF-{beta}1 does not increase transcription of the MCP-1 gene significantly and that TGF-{beta}1-mediated induction of MCP-1 expression apparently does not involve activation of the NF-{kappa}B signaling pathway. These findings contrast with the well-recognized role of TNF-{alpha}, which increases transcription of the MCP-1 gene, promotes I{kappa}B degradation, and activates NF-{kappa}B.

PDE4 inhibitor Rp suppresses TGF-{beta}1-stimulated MCP-1 expression by decreasing message stability. Because TGF-{beta}1 does not increase transcription of the MCP-1 gene, we sought to determine whether TGF-{beta}1 promotes MCP-1 production by increasing MCP-1 mRNA stability and whether Rp decreases MCP-1 mRNA stability. The MCP-1 gene contains an A+T-rich region and an ATTA sequence that has been associated with unstable mRNA (45). The MCP-1 message half-life in untreated MCs was ~3 h (data not shown). After 6-h treatment with TGF-{beta}1, the MCP-1 message decayed slowly, with an estimated half-life of 9.5 h. However, Rp significantly reduced the MCP-1 half-life in TGF-{beta}1-treated cells to 5.5 h (Fig. 4). Lixazinone did not significantly alter MCP-1 message stability in TGF-{beta}1-treated MCs (data not shown). On the basis of these findings, TGF-{beta}1 increased steady-state MCP-1 mRNA levels at least in part through an increase in mRNA stability, whereas the PDE4 inhibitor Rp decreased MCP-1 message stability in TGF-{beta}1-treated MCs.



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Fig. 4. PDE4 inhibitor Rp suppresses TGF-{beta}1-stimulated MCP-1 expression by decreasing message stability. MCs were pretreated with vehicle (0.1% DMSO) or Rp (10 µM) for 30 min before addition of control and TGF-{beta}1 (10 ng/ml). After 6 h, cells were treated with actinomycin D (5 µg/ml) for 2 and 4 h. Total RNA was isolated, and Northern blots were probed with MCP-1 and GAPDH. MCs were treated with vehicle only before actinomycin D treatment ({bullet}), with TGF-{beta}1, before actinomycin D treatment ({circ}), or with Rp and TGF-{beta}1 before actinomycin D treatment ({blacktriangledown}). Values represent means ± SE (n = 3). #P < 0.05, significant difference in MCP-1-to-GAPDH ratios between vehicle and TGF-{beta}1-treated MCs. *P < 0.05, significant difference in MCP-1-to-GAPDH ratios between Rp-TGF-{beta}1- and TGF-{beta}1-treated MCs. Inset, blot showing a representative experiment.

 
Smad pathway is not required for the suppressive effect of PDE4 inhibitors on TGF-{beta}1-induced MCP-1 expression. It is well recognized that the Smad family of proteins are key intracellular mediators of TGF-{beta}1 signaling. We therefore sought to determine whether PDE4 inhibitors block TGF-{beta}1-stimulated MCP-1 expression through downregulation of the Smad signaling pathway. Smad2 phosphorylation was assessed using Western blot analysis with a phosphospecific antibody. We found that TGF-{beta}1 significantly increased Smad2 phosphorylation as expected; however, neither PDE3 nor PDE4 inhibitors altered TGF-{beta}1-stimulated Smad2 phosphorylation (Fig. 5A). We further immunoprecipitated PDE inhibitor and TGF-{beta}1-treated MC lysates with Smad2 antibody and probed the Western blots with Smad4 antibody. As expected, TGF-{beta}1 significantly increased Smad4 binding to Smad2. However, PDE inhibitors did not modulate Smad4 binding to Smad2 (Fig. 5B). These results indicate that PDE inhibitors do not alter TGF-{beta}1-initiated Smad signaling in MCs.



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Fig. 5. Smad pathway is not required for the suppressive effect of PDE4 inhibitors on TGF-{beta}1-stimulated MCP-1 expression. A: Rp has no effect on Smad2 activation by TGF-{beta}1. MCs were pretreated with vehicle (0.1% DMSO) or PDE inhibitors (10 µM each) for 15 min before TGF-{beta}1 (10 ng/ml) treatment for 1 h. Smad2 phosphorylation was assessed using Western blot analysis with a phosphospecific antibody. Blots were stripped and reprobed with a {beta}-actin antibody for equal loading. B: Rp did not alter Smad4 binding to Smad2/3. The same cell lysates were immunoprecipitated with Smad2 antibody, and Western blots were probed with Smad4 antibody. Blots were stripped and reprobed with a Smad2/3 antibody to reveal total Smad 2/3 levels. Data are representative of 3 experiments.

 
PDE4 inhibitor Rp blocks TGF-{beta}1-mediated induction of ERK and p38. TGF-{beta}1 induces the ERK and p38 pathways in MCs (23). Using Northern blot analysis, we found that the ERK inhibitor U0126 and the p38 inhibitor SB-202190 completely blocked TGF-{beta}1-stimulated MCP-1 expression, indicating that these pathways are necessary for TGF-{beta}1-stimulated MCP-1 expression. The JNK inhibitor only modestly blocked TGF-{beta}1-stimulated MCP-1 expression (Fig. 6A). The effect of PDE inhibitors on TGF-{beta}1-induced ERK and p38 activity was assessed using a transfection-based in vivo kinase assay. The PDE4 inhibitor Rp, but not the PDE3 inhibitor lixazinone, significantly suppressed TGF-{beta}1-induced ERK and p38 activity (Fig. 6B).



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Fig. 6. ERK and p38 pathways are necessary for TGF-{beta}1-stimulated MCP-1 expression. A: ERK and p38 inhibitors suppress TGF-{beta}1-stimulated MCP-1 mRNA expression. MCs were pretreated with vehicle (0.1% DMSO), the ERK inhibitor U0126 (25 µM), the p38 inhibitor SB-202190 (10 µM), or the JNK inhibitor (JNK II; 10 µM) for 1 h before TGF-{beta}1 (10 ng/ml) treatment for 6 h. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. Inset, blot showing a representative experiment. B: Rp blocks TGF-{beta}1-induced ERK and p38 activity. MCs were pretreated with vehicle (0.1% DMSO) or PDE inhibitors (10 µM each) for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 8 h. ERK and p38 activities were assessed using the PathDetect in vivo signal transduction pathway trans-reporting system (Stratagene, La Jolla, CA) as described in MATERIALS AND METHODS. Values represent means ± SE (n = 3). #P < 0.05 vs. vehicle-treated levels. *P < 0.05 vs. TGF-{beta}1-treated levels.

 
PDE4 inhibitor Rp blocks TGF-{beta}1-stimulated ROS generation. ROS are essential intermediates in many aspects of TGF-{beta}1 signaling (28, 30, 36). We have previously shown that PDE4 inhibitors suppress ROS generation by MCs (8). We therefore sought to determine whether ROS generation is necessary for TGF-{beta}1-stimulated MCP-1 expression and whether the PDE4 inhibitor Rp suppresses ROS generation in TGF-{beta}1-stimulated MCs. TGF-{beta}1 significantly induced hydrogen peroxide production as assessed using dichlorofluorescein fluorescence. Rp suppressed hydrogen peroxide generation by TGF-{beta}1, whereas lixazinone was without effect (Fig. 7A). Northern blot analysis indicated that ROS scavengers DMSO and N,N-dimethylthiourea (DMTU) significantly blocked TGF-{beta}1-stimulated MCP-1 expression (Fig. 7B). The suppressive effect of DMSO on TGF-{beta}1-stimulated MCP-1 expression was dose dependent. DMSO significantly blocked TGF-{beta}1-stimulated MCP-1 expression at doses ≥0.5%, whereas 0.1% DMSO (used as vehicle for control cells) was without effect (Fig. 7C). We further investigated the effect of 1% DMSO on MAPK pathways. A transfection-based in vivo kinase assay showed that DMSO significantly suppressed TGF-{beta}1-induced ERK and p38 activity (Fig. 7D). Using a rat-specific MCP-1 ELISA kit, we found that TGF-{beta}1 significantly stimulated MCP-1 protein expression as expected. DMSO at 1% concentration completely blocked the stimulatory effect of TGF-{beta}1 on MCP-1 protein expression. The ROS scavenger DMTU also significantly inhibited TGF-{beta}1-stimulated MCP-1 protein expression (Fig. 7E).



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Fig. 7. Reactive oxygen species (ROS) generation is involved in TGF-{beta}1-stimulated MCP-1 production, and Rp blocks TGF-{beta}1-stimulated ROS generation. A: Rp blocked TGF-{beta}1-stimulated ROS generation. MCs cultured in 96-well plates were loaded with the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; 5 µM) for 30 min at 37°C. Cells were then rinsed and treated with vehicle (0.1% DMSO) or PDE inhibitors (10 µM each) for 15 min, followed by TGF-{beta}1 (10 ng/ml) treatment for 90 min. H2DCFDA fluorescence was measured at excitation and emission wavelengths of 490 and 530 nm, respectively, using a microplate reader. B: ROS scavengers blocked TGF-{beta}1-stimulated MCP-1 mRNA expression. MCs were treated with vehicle (0.1% DMSO), 1% DMSO, or 10 mM N,N-dimethylthiourea (DMTU) for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 6 h. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. C: dose response of DMSO after TGF-{beta}1-stimulated MCP-1 expression. MCs were pretreated with vehicle (0.1% DMSO) or the indicated doses of DMSO for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 6 h. Total RNA was isolated, and Northern blots were probed for MCP-1 and GAPDH. D: DMSO blocked TGF-{beta}1-stimulated ERK and p38 activity. ERK and p38 activation were measured using the PathDetect in vivo signal transduction pathway trans-reporting system (Stratagene, La Jolla, CA) as described in MATERIALS AND METHODS. After 18-h transfection, MCs were pretreated with vehicle (0.1% DMSO) or 1% DMSO for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 8 h. E: ROS scavengers blocked TGF-{beta}1-stimulated MCP-1 protein expression. MCs cultured in 24-well plates were treated with vehicle (0.1% DMSO), 1% DMSO, or 10 mM DMTU for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 24 h. MCP-1 protein levels in culture medium were quantified using a rat-specific MCP-1 ELISA kit (BioSource International, Camarillo, CA) according to the manufacturer's instructions. Values represent means ± SE (n = 3). #P < 0.05 vs. vehicle-treated levels. *P < 0.05 vs. TGF-{beta}1-treated levels. Insets, blots showing representative experiments.

 
Both PDE4 inhibitors and ROS scavengers block TGF-{beta}1-stimulated apoptosis of MCs. In several cell lines, ROS are involved in TGF-{beta}1-stimulated apoptosis (24, 47). We therefore sought to determine whether PDE4 inhibitors could alter TGF-{beta}1-stimulated apoptosis in MCs. MCs were treated with PDE inhibitors, 1% DMSO, and TGF-{beta}1. TGF-{beta}1 significantly induced MC caspase-3 activity. Rp and DMSO significantly suppressed the apoptotic effect of TGF-{beta}1, whereas lixazinone had no effect (Fig. 8). A complementary assay for caspase-8 activity confirmed our findings described above (data not shown). Neither TGF-{beta}1 nor PDE3 or PDE4 inhibitors were cytotoxic to MCs as assessed by the lack of lactate dehydrogenase release into the culture medium after treatment (data not shown). On the basis of these considerations, we conclude that the inhibitory effect of PDE4 inhibitors on MCP-1 production is not due to decreased MC viability.



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Fig. 8. TGF-{beta}1 induced MC apoptosis, and PDE4 inhibitors and ROS scavengers blocked the effect of TGF-{beta}1 on MC apoptosis. MCs were pretreated with vehicle (0.1% DMSO), PDE inhibitors (10 µM each), or DMSO (1%) for 30 min before TGF-{beta}1 (10 ng/ml) treatment for 18 h. Caspase-3 activity was determined colorimetrically using the CaspACE assay system (Promega, Madison, WI) according to manufacturer's instructions. Values represent means ± SE (n = 3). #P < 0.05 vs. vehicle-treated levels. *P < 0.05 vs. TGF-{beta}1-treated levels.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
MCP-1, a member of the CC chemokine family, strongly recruits monocytes and subpopulations of T-cells during inflammation (44, 49, 52). Recent studies have indicated that MCP-1 may promote progressive renal disease by recruiting the TGF-{beta}1 pathway (13, 14, 53). However, the effect of TGF-{beta}1 on MCP-1 expression appears to be cell type specific. For example, TGF-{beta}1 suppresses MCP-1 expression by monocytes and/or macrophages (32, 58). This inhibition may serve to limit the activation of mononuclear cells after acute tissue injury. On the other hand, TGF-{beta}1 induces MCP-1 expression by fibroblasts (17, 55, 63) and, as reported here, glomerular MCs. There are no prior studies that have examined the effect of TGF-{beta}1 on the expression of MCP-1 in MCs. There was one prior study in which kidney cells were used, namely, proximal tubular epithelial cells, and that study demonstrated a suppressive effect of TGF-{beta}1 on MCP-1 (12). To the best of our knowledge, our present study is the first to examine the effect of TGF-{beta}1 on the expression of MCP-1 in MCs and to demonstrate the inductive effect of TGF-{beta}1 on MCP-1 in this cell type, one that critically contributes to progressive renal disease. Because MCP-1 induces TGF-{beta}1 expression in glomerular cells and fibroblasts (13, 14, 53), this cross-talk between MCP-1 and TGF-{beta}1 signaling may provide the basis for the persistent, dysregulated TGF-{beta}1 expression that is characteristic of many forms of progressive renal disease. On the basis of these considerations, agents that block TGF-{beta}1 induction of MCP-1 may be effective in preventing the progression of glomerular and/or interstitial renal disease.

Agonists of the cAMP-PKA pathway are potent anti-inflammatory agents, and nonhydrolyzable cAMP analogs suppress cytokine-stimulated MCP-1 production by MCs (43, 50). PDE inhibitors, which activate PKA by preventing the catabolism of cAMP, are used as selective and cell type-specific agonists of the cAMP-PKA pathway. In particular, PDE4 inhibitors are used to treat a number of inflammatory and autoimmune conditions in humans (15, 27, 39, 41). We have previously demonstrated that several structurally distinct PDE4 inhibitors are effective in blocking serum-opsonized zymosan-induced ROS generation by MCs (8). Because ROS are intermediates for some aspects of TGF-{beta}1 signaling (25, 30, 42, 57), we hypothesized that PDE4 inhibitors may be effective in blocking TGF-{beta}1-stimulated MCP-1 expression.

We found that TGF-{beta}1 induces MCP-1 mRNA and protein in a time- and dose-dependent manner. The PDE4 inhibitor Rp blocked TGF-{beta}1-stimulated MCP-1 mRNA expression. The PKA antagonist 14-22 amide reversed the inhibitory effect of Rp, indicating that Rp suppresses TGF-{beta}1-stimulated MCP-1 expression at least in part through activation of PKA. Although the PDE3 inhibitors lixazinone and cilostamide activate PKA to an extent similar to that of Rp, they did not significantly alter TGF-{beta}1-stimulated MCP-1 expression. TGF-{beta}1 did not significantly stimulate transcription of a chimeric MCP-1 promoter-luciferase construct containing NF-{kappa}B sequence elements, which are necessary for transcriptional activation by LPS (60). On the basis of these considerations, we conclude that TGF-{beta}1 does not induce MCP-1 by increasing transcription of the MCP-1 gene. Moreover, we found that TGF-{beta}1 treatment increased the half-life of MCP-1 from 3 h in untreated MCs to 9.5 h, while Rp decreased the MCP-1 message half-life in TGF-{beta}1-treated cells to 5.5 h. This suggests that TGF-{beta}1 stimulated MCP-1 expression, at least in part, by increasing message stability and that the inhibitory effect of Rp on TGF-{beta}1-stimulated MCP-1 expression may arise from decreased MCP-1 message stability. In support of our finding of a posttranscriptional mechanism for TGF-{beta}1-induced MCP-1 expression is the fact that the MCP-1 gene has an A+T-rich region and a copy of an ATTTA sequence that has been associated with unstable mRNA (45).

In many cell systems, proinflammatory cytokines such as TNF-{alpha} and IL-1 promote MCP-1 expression through the NF-{kappa}B pathway. NF-{kappa}B consists of dimers of the two subunits p50 and p65 (RelA) and exists in inactive form in cytoplasm associated with an inhibitory protein I{kappa}B (1). Activation of NF-{kappa}B results from the release of the inhibitory I{kappa}B subunit from the heterotrimeric complex, followed by nuclear translocation of the dimers to initiate transcription of target genes. We found that TGF-{beta}1 did not activate the NF-{kappa}B signaling pathway, as assessed by the inability of TGF-{beta}1 to promote degradation of I{kappa}B, to activate NF-{kappa}B subunit p65 and to increase transcriptional activity of the NF-{kappa}B promoter. As expected, TNF-{alpha}, which induces MCP-1 transcription through the NF-{kappa}B signaling pathway, was effective in promoting I{kappa}B degradation, p65 activation, and NF-{kappa}B promoter transcription.

It is well recognized that the Smad family of proteins are key intracellular mediators of TGF-{beta}1 signaling (61). As expected, we found that TGF-{beta}1 activated the Smad pathway as assessed by phosphorylation of Smad2 and the formation of heteromeric complexes containing Smad2/3 and Smad4. However, neither the PDE3 inhibitor lixazinone nor the PDE4 inhibitor Rp had any effect on Smad2 phosphorylation or on Smad4 binding to Smad2/3. These studies indicate that the inhibitory effect of the PDE4 inhibitor Rp on TGF-{beta}1-stimulated MCP-1 expression does not involve the Smad signaling pathway. Notably, these findings indicate that pathophysiologically relevant effects of TGF-{beta}1, namely upregulation of MCP-1, can be modulated through Smad-independent mechanisms.

In contrast to a lack of involvement of Smad proteins, our data demonstrate that a significant mechanism that accounts for the suppressive effect of PDE4 inhibitors on MCP-1 expression involves the MAPK signaling cascade. In MCs, TGF-{beta}1 activates the ERK, p38, and JNK signaling pathways (23, 48, 64), and cross-talk exists between the TGF-{beta}1-Smad signaling pathway and the MAPK signaling cascades (22, 33, 54, 62). Using pathway-specific MAPK inhibitors, we found that the ERK and p38 pathways are essential for TGF-{beta}1-stimulated MCP-1 expression. Moreover, we found that the PDE4 inhibitor Rp, but not the PDE3 inhibitor lixazinone, significantly blocked TGF-{beta}1-stimulated ERK and p38 activity in MCs. These studies indicate that the inhibitory effect of Rp on TGF-{beta}1-stimulated MCP-1 production may be related to inhibition of the ERK and/or p38 signaling pathways.

Recent studies have demonstrated that ROS are signaling intermediates for a number of pathways initiated by TGF-{beta}1, including apoptosis (47), IL-6 production (30), plasminogen activator inhibitor-1 upregulation (28), and matrix synthesis (36). We have previously shown that Rp inhibits serum-opsonized zymosan-stimulated ROS generation by MCs (8). Our present studies demonstrate that TGF-{beta}1 stimulated the production of hydrogen peroxide by MCs and that the PDE4 inhibitor Rp, but not the PDE3 inhibitor lixazinone, suppressed TGF-{beta}1-stimulated hydrogen peroxide production by MCs. The ROS scavengers DMSO and DMTU also inhibited TGF-{beta}1-stimulated MCP-1 expression. These studies indicate that Rp inhibits TGF-{beta}1-stimulated MCP-1 expression, at least in part, through inhibition of ROS generation by MCs. DMSO blocked TGF-{beta}1-stimulated ERK and p38 activity, further suggesting that ERK and p38 signaling pathways are involved in TGF-{beta}1-stimulated MCP-1 expression.

In summary, to the best of our knowledge, our present study provides the first demonstration that TGF-{beta}1 stimulates MCP-1 expression in MCs. Because available evidence in the literature attests to a stimulatory effect of MCP-1 on TGF-{beta}1, our present findings indicating a stimulatory effect of TGF-{beta}1 on MCP-1 uncover a potential positive feedback interaction between TGF-{beta}1 and MCP-1. Such an interaction sustains a recurrent cycle whereby an inflammatory cytokine (MCP-1) induces a fibrogenic one (TGF-{beta}1), which in turn supports heightened expression of MCP-1. Interrupting this feedback loop with the PDE4 inhibitor Rp thus provides an appealing strategy for retarding the progression of renal disease.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants R01 DK-16105 and R01 DK-55603 (to J. P. Grande) and by NIDDK Grant R01 DK-47060 (to K. A. Nath).


    ACKNOWLEDGMENTS
 
We thank Cherish Grabau for excellent secretarial assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. P. Grande, Mayo Clinic College of Medicine, 200 First St. SW, Stabile 7, Rochester, MN 55905 (e-mail: grande.joseph{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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