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【摘要】 In a separate study, we identified PGE 2 as a potent inhibitor of TGF- 1 -induced epithelial-mesenchymal transition (EMT) in cultured Madin-Darby canine kidney (MDCK) cells (Zhang A, Wang M-H, Dong Z, and Yang T. Am J Physiol Renal Physiol 291: F1323-F1331, 2006). This finding prompted us to examine the roles of other prostanoids: PGD 2, PGF 2, PGI 2, and thromboxane A 2 (TXA 2 ). Treatment with 10 ng/ml TGF- 1 for 3 days induced EMT as reflected by conversion to the spindle-like morphology, loss of E-cadherin, and activation of -smooth muscle actin ( -SMA). Treatment with PGD 2 remarkably preserved the epithelial-like morphology, restored the expression of E-cadherin, and abolished the activation of -SMA. In contrast, PGF 2, carbocyclic thromboxane A 2, PGI 2 and its stable analog beraprost were without an effect. MDCK cells expressed DP 1 and DP 2 receptors; however, the effect of PGD 2 was neither prevented by DP 1 antagonist BW-A868C or DP 2 antagonist BAY-u3405 nor was mimicked by DP 1 agonist BW-245C. cAMP-elevating agents forskolin and 8-Br-cAMP blocked EMT. However, cAMP blockers H89 and Rp-cAMP failed to block the effect of PGD 2. PGD 2 did not seem to act via its metabolites as 15-deoxy-Delta( 12, 14 )-prostaglandin J 2 (15d-PGJ 2 ) levels in the medium following incubation with 3 µM PGD 2 were well below the values predicted from the cross activity of the assay. Exposure to TGF- 1 induced a threefold increase in reactive oxygen species production that was completely abolished by PGD 2. We conclude that 1 ) PGD 2, but not PGI 2, PGF 2, and TXA 2 inhibit EMT, 2 ) PGD 2 inhibits EMT independently of DP 1 and DP 2 receptors, and 3 ) PGD 2 exhibits antioxidant property which may, in part, account for the antifibrotic action of this PG.
【关键词】 reactive oxygen species transforming growth factor MadinDarby canine kidney cells
EPITHELIAL - MESENCHYMAL TRANSITION (EMT) is a complex process by which polarized epithelial cells are converted into motile, myofibroblast-like cells ( 12, 45 ). The phenotypic conversion involves loss of epithelial polarity and E-cadherein, disruption of tubular basement membrane, acquisition of spindle-like morphology, de novo synthesis of -smooth muscle actin ( -SMA), and production of matrix proteins ( 22 ). EMT plays a central role in development and carcinogenesis ( 45 ). It is recently established that EMT is critically involved in organ fibrosis including the kidney. EMT is a major event in the pathogenesis of renal tubulointerstitial fibrosis (TIF) which is a common pathway responsible for progression of chronic renal disease into end-stage renal failure ( 9, 10, 37, 44 ). A landmark study by Iwano et al. ( 19 ) using mice with genetically tagged proximal tubular cells demonstrates that 36% of renal fibroblasts originate from the epithelium in a TIF model.
Prostaglandins (PGs) are a group of autacoids derived from arachidonic acid through the cyclooxygenase (COX) pathway by constitutive COX-1 and inducible COX-2. COX, the so-called PGH 2 synthase, converts arachidonic acid to PGH 2, which is further metabolized by individual PG synthases to five primary bioactive prostanoids, PGE 2, PGI 2, PGD 2, PGF 2, and thromboxane A 2 (TXA 2 ) ( 3, 41, 42 ). These prostanoids typically act in an autocrine and/or paracrine fashion via specific transmembrane G protein-coupled receptors designated EP (for E-prostanoid receptor), IP, DP, FP, and TP, respectively ( 4, 6, 28 ). Prostanoids are abundantly produced in the kidney and have been recognized as important homeostatic regulators of a wide spectrum of renal function as evidenced by the deleterious renal side effects of COX inhibitors (NSAIDs) and the severe renal phenotype in COX-2 knockout mice. We recently demonstrated that PGE 2 almost completely reversed TGF- 1 -induced EMT in cultured MDCK cells, partially mediating the antifibrotic effect of hepatocyte growth factor (HGF) ( 52 ). To complement this observation, the present study was undertaken to examine the roles of other prostanoids, including PGD 2, PGF 2, PGI 2, and TXA 2 in TGF- 1 -induced EMT in the same cell culture model.
MATERIALS AND METHODS
Reagents and antibodies. Recombinant human TGF- 1 was purchased from Calbiochem (Cambridge, MA). cAMP-elevating regents, 8-Br-cAMP, forskolin, 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), Rp-cAMP, N -[2-( p -bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), mouse monoclonal anti- -SMA (catalog number: A5228), and rat monoclonal anti-E-cadherin (catalog number: U3254) antibodies were purchased from Sigma (St. Louis, MO). PGD 2, carbocyclic thromboxane A 2 (CTA 2; a stable analog of thromboxane A 2 ), PGF 2, PGI 2 and beraprost, 15d-PGJ 2, rabbit polyclonal anti-DP1 (catalog number: 101640), anti-DP 2 (catalog number: 10007002), and anti-murine COX-2 (catalog number: 160106) antiserum were from Cayman (Ann Arbor, MI). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG (catalog number: sc-2011), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (catalog number: sc-2005), and anti-rabbit IgG (catalog number: sc-2004) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture. MDCK cells were cultured in DMEM, containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (GIBCO, Burlington, ON) at 37°C under 5% CO 2 in a humidifiedincubator. Cells were grown to 80% confluence, serum-starved for 24 h, and then treated with vehicle or human recombinant TGF- 1 in the presence or absence of prostanoids or their analogs for the appropriate periods of time. Microscopic examination was performed during each experiment to assess the morphological changes before sample analysis.
Immunoblotting. MDCK cells were lysed and subsequently sonicated in PBS containing 1% Triton X-100, 250 µM PMSF, 2 mM EDTA, and 5 mM DTT (pH 7.5). Protein concentration was determined by Coomassie reagent. Thirty micrograms of protein from whole cell lysates were denatured in boiling water for 10 min, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with mouse anti- -SMA monoclonal antibody, or rabbit anti-DP 1 or DP 2 polyclonal antibodies at a dilution of 1:1,000. After being washed with TBS, blots were incubated with a goat anti-HRP-conjugated secondary antibody (1:1,000 dilution) and visualized with ECL kits (Amersham).
Immunofluorescence microscopy for E-cadherin and -SMA. Cells were grown on coverslips and stimulated with TGF- 1 in the presence or absence of prostaglandins. The medium was removed, and the cell layer was rinsed with PBS. Cells were fixed and permeabilized with acetone-methanol for 10 min at -20°C, then were rehydrated with PBS, blocked with 5% BSA in PBS for 1 h. Coverslips were sequentially incubated with rat monoclonal anti-E-cadherin (1:400 dilution) or mouse monoclonal anti- -SMA (1:200 dilution) and FITC-labeled goat anti-rat or mouse antibodies (1:200 dilution) each for 60 min at room temperature. Cells were then visualized and photographed by fluorescence microscopy at x 400 magnification. Negative controls were performed using nonimmune serum or IgG instead of first antibodies.
cAMP assay. Serum-starved MDCK cells grown in six-well plates were pretreated with 10 µM IBMX for 30 min and then treated with 2 µM PGD 2 in the presence or absence of DP 1 antagonist, BW-A868C, or DP 2 antagonist, BAY-u3405, or treated with DP 1 agonist BW-245C. After treatment, medium was removed and the cells were washed with PBS. Immediately after being washed, 0.3 ml of 0.1 M HCl was added. After 20-min incubation, the cells were scraped and transferred into a centrifuge tube and spun for 10 min at 1,000 g to pellet the cell debris. The cAMP enzyme-linked immunosorbent assay was performed according to manufacturer?s instructions (Cayman Chemicals). The results were normalized with protein concentration.
15d-PGJ 2 enzyme immunoassay. 15d-PGJ 2 in the culture media was measured with an enzyme immunoassay kit from Assay Designs (Ann Arbor, MI). The assay was performed according to the manufacturer?s instruction. Briefly, 100 µl of the medium, along with a serial dilution of 15d-PGJ 2 standard samples, were mixed with appropriate amounts of alkaline phosphatase-conjugated 15d-PGJ 2 and 15d-PGJ 2 antiserum, and incubated at room temperature for 2 h. After the wells were emptied and rinsed with wash buffer, 200 µl of substrate solution were added. The optical density was read at 405 nm.
DCFDA fluorescence measurement of ROS. The fluorogenic substrate 2',7'-dichlorofluorescein diacetate (DCFDA) is a cell-permeable dye that is oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by H 2 O 2 and can therefore be used to monitor intracellular generation of ROS. For measurement of ROS, cells were grown onto glass cover slides. When the cells reached confluence, they were washed twice with PBS and incubated for 30 min with 50 µM DCFDA and PGD 2. Then the cells were treated by TGF- 1 for 30 min. At the end of the incubation period, the cells were again washed twice with PBS and imaged by confocal laser microscopy. To quantitate ROS levels, cells were seeded to 96-well plates and were treated as abovementioned. Relative fluorescence was measured by a fluorescence plate reader (FLUOstar OPTIMA) at excitation and emission wavelengths of 485 and 528 nm, respectively, three times at 90-s intervals. Relative fluorescence units (RFU) was expressed as fold increase over untreated cells.
Caspase activity assays. Serum-starved MDCK cells grown in six-well plates were treated with TGF- 1 in the presence or absence of PGD 2. After treatment, the cells were washed with PBS and collected in CasPASE lysis buffer. The caspase-3/7/10 assay was performed by using Cas-PASE-3/7/10 assay kit (GenoTechnology, St. Louis, MO) according to the manufacturer?s instructions. Caspase activity was monitored as optical absorbance at 405 nm in a time-dependent manner.
Statistical analysis. One-way ANOVA was performed to test for statistical significance of differences among the values observed in each treatment group followed by a Bonferroni posttest. A P value <0.05 was considered significant. Values shown represent means ± SE.
RESULTS
Effects of various prostanoids on TGF- 1 -induced EMT. In an earlier study, we showed that PGE 2 had a remarkable inhibitory effect on TGF- 1 -induced EMT in cultured MDCK cells. This prompted us to examine the effects of other prostanoids: PGD 2, PGF 2, PGI 2, and TXA 2. To evaluate EMT, we used three independent parameters: cell morphology, the level of E-cadherin, and expression of -SMA, assessed by phase-contrast microscopy, immunostaining, and immunoblotting, respectively. We first monitored the morphological changes in MDCK cells treated by TGF- 1 alone or in combination with individual prostanoids or their analogs. Under basal state, MDCK cells exhibited typical epithelial-like morphology ( Fig. 1 A ), whereas TGF- 1 treatment (10 ng/ml) for 3 days induced a complete conversion to spindle-like morphology ( Fig. 1 B ). Strikingly, the TGF- 1 -induced morphological changes were completely prevented by treatment with 2 µM PGD 2 ( Fig. 1 C ). In contrast, CTA 2 ( Fig. 1 D ), PGF 2 ( Fig. 1 E ), PGI 2 ( Fig. 1 F ), and the stable PGI 2 analog beraprost ( Fig. 1 G ) had no obvious effect on EMT.
Fig. 1. Effects of various prostanoids on the morphologic changes in Madin-Darby canine kidney (MDCK) cells. The cells were grown in 6-well plates until 80% confluence and then treated for 3 days with vehicle ( A ), TGF- 1 alone (10 ng/ml; B ), TGF- 1 plus PGD 2 (2 µM; C ), TGF- 1 plus CTA 2 (5 µM; D ), TGF- 1 plus PGF 2 (2 µM; E ), TGF- 1 plus PGI 2 (2 µM; F ), or TGF- 1 plus beraprost (1 µM; G ) for 3 days. Photographs were taken using a Nikon microscope (phase contrast). Shown are representatives of 2-3 independent experiments.
E-cadherin, a classic epithelial cell marker, is a membrane-bound protein involved in cell-cell interactions in intact renal tubular epithelial cells. We then performed immunocytochemical analyses to monitor changes in E-cadherin protein expression. As expected, E-cadherin was expressed exclusively in the basolateral membrane of MDCK cells in basal state ( Fig. 2 A ). In contrast, incubation with 10 ng/ml TGF- 1 for 3 days dramatically reduced E-cadherin expression ( Fig. 2 B ). The reduction of E-cadherin was completely prevented in the presence of 2 µM PGD 2 ( Fig. 2 C ), but not in CTA 2 ( Fig. 2 D ), PGF 2 ( Fig. 2 E ), or PGI 2 ( Fig. 1 F ), or beraprost ( Fig. 2 G ).
Fig. 2. Effects of various prostanoids on E-cadherin expression in MDCK cells. The cells were grown in 6-well plates until 80% confluence and then treated for 3 days with vehicle ( A ), TGF- 1 alone (10 ng/ml; B ), TGF- 1 plus PGD 2 (2 µM; C ), TGF- 1 plus CTA 2 (5 µM; D ), TGF- 1 plus PGF 2 (2 µM; E ), TGF- 1 plus PGI 2 (2 µM; F ), or TGF- 1 plus beraprost (1 µM; G ). Immunofluorescence was performed using rat monoclonal anti-E-cadherin and FITC-labeled goat anti-rat antibody. Cells were visualized and photographed by fluorescence microscopy at x 400 magnification. Shown are representatives of 2-3 independent experiments.
-SMA is an actin isoform specific to myofibroblasts and its expression undergoes characteristic changes during EMT. The effect of PGD 2 on EMT was further analyzed by immunoblotting analysis of -SMA. -SMA protein expression in MDCK cells was detected at low level in basal state and was remarkably induced by a 3-day treatment with TGF- 1. PGD 2 in the dose range of 0.1-2 µM inhibited -SMA expression in a dose-dependent manner with a noticeable effect at 0.25 µM and a maximal effect at 2 µM ( Fig. 3 ). Given the selective and striking effects of PGD 2 on EMT, we conducted a series of following experiments to determine the underlying mechanism for PGD 2 actions in cultured MDCK cells.
Fig. 3. Effects of PGD 2 on TGF- 1 -induced -smooth muscle actin (SMA) protein expression in MDCK cells. The cells were grown on 6-well plates until 80% confluence and then treated with vehicle or TGF- 1 in the presence or absence of PGD 2 at the indicated concentrations (0.1-2.0 µM). Top : representative immunoblots. Bottom : densitometric analysis of -SMA expression; n = 3 in each group. * P < 0.01 vs. TGF- 1 -treated cells.
The EMT induced by TGF- 1 appeared to be associated with reduced cell proliferation rate. Therefore, we tested whether TGF- 1 (10 ng/ml) in the presence or absence of PGD 2 (2 µM) induced apoptosis in MDCK cells. Caspase 3/7/10 activities, detected by using the Cas-PASE-3/7/10 assay kit, were not significantly affected by TGF- 1 regardless of the presence or absence of PGD 2 (control: 1.00 ± 0.24; TGF- 1 alone: 1.13 ± 0.15; TGF- 1 + PGD 2 : 1.01 ± 0.2, n = 5 in each group, P 0.05). The values represent fold changes over controls.
Examination of DP receptors involved. The biological effects of PGD 2 are transduced by D prostanoid receptor (DP) 1 and DP 2, two G protein-coupled receptors. To determine the mechanisms involved in the PGD 2 inhibition of EMT, we first examined expression of the two PGD 2 receptors using immunoblotting. As shown in Fig. 4 A, DP 1 receptor was detected as a single 43-kDa protein. DP 2 receptor protein was detected as a double band; the smaller molecular weight band of 39 kDa was of predicted size while the identity of the slightly larger molecular weight band ( 50 kDa) is unclear. Cayman?s DP 2 receptor polyclonal antibody detects both unglycosylated and glycosylated protein from human ranging from 35-40 to 55-70 kDa, as reported by Nagata et al. ( 27 ). This raises a possibility that the 50-kDa band detected in MDCK cells might be the glycosylation product. To address the functional aspect of the DP receptors, we determined effects of PGD 2 on intracellular cAMP, along with the use of DP receptor agonists and antagonists to discriminate the types of DP receptors. PGD 2 at 2 µM significantly elevated intracellular cAMP levels that were blocked by DP 1 antagonist BW-A868C (10 µM), but not DP 2 antagonist BAY-u3405 (10 µM), suggesting involvement of the DP 1 receptor. Indeed, like PGD 2, the selective DP 1 agonist BW-245C (10 µM) was able to elevate intracellular cAMP ( Fig. 4 B ), thereby confirming the presence of a functional DP 1 receptor in MDCK cells.
Fig. 4. Existence of DP 1 and DP 2 receptors in MDCK cells. A : immunoblotting of DP 1 and DP 2 receptors. Three wells of MDCK cells ( n = 3 for each DP receptor subtype) were grown to confluence and serum-starved for 24 h, DP 1 and DP 2 receptors were detected by immunoblotting using rabbit anti-DP 1 and anti-DP 2 antibodies. B : measurement of intracellular cAMP. MDCK cells were grown to confluence and serum-starved for 24 h and then pretreated with 10 µM IBMX for 30 min, followed by incubation with serum-free media alone or 2 µM PGD 2 in the presence or absence of BW-A868C or BAY-u3405, or treated by DP 1 agonist BW-245C. Cells were harvested in 0.1 M HCl and supernatants were assayed for cAMP levels by ELISA. Results were normalized by protein concentration. Values represent means ± SE, n = 6 in each group. * P < 0.01 vs. control. P < 0.01 vs. PGD 2 -treated cells. P < 0.01 vs. PGD 2 plus BW-A868C treated.
To investigate the involvement of DP receptors in the PGD 2 inhibition of EMT, we used respective DP receptor antagonists and agonists to assess the roles of individual DP receptors. Unexpectedly, neither the DP 1 antagonist BW-A868C (10 µM) nor DP 2 antagonist BAY-u3405 (10 µM) was able to block the PGD 2 effect as assessed by morphological changes ( Fig. 5, D and E ), E-cadherin expression ( Fig. 5, J and K ), and -SMA expression ( Fig. 6, D, E, and G ). Unlike PGD 2, DP 1 agonist BW-245C (10 µM) did not affect TGF- 1 -induced EMT as shown in Fig. 5 F for morphology, Fig. 5 L for E-cadherin immunostaining, and Fig. 6 F for -SMA immunostaining and Fig. 6 G for -SMA immunoblotting.
Fig. 5. Effects of DP receptor antagonists or agonists on the morphologic changes and E-cadherin expression in MDCK cells. The cells were grown in 6-well plates until 80% confluence and then treated with vehicle ( A, G ), TGF- 1 alone (10 ng/ml; B, H ), TGF- 1 plus PGD 2 (2 µM; C, I ), TGF- 1 plus PGD 2 and BW-A868C (10 µM; D, J ), TGF- 1 plus PGD 2 and BAY-u3407 (10 µM; E, K ), or TGF- 1 plus BW-245C (10 µM; F, L ) for 3 days. A - F : morphologic changes. Photographs were taken using a Nikon microscope (phase contrast). G - L : E-cadherin expression. Immunofluorescence was performed using rat monoclonal anti-E-cadherin and FITC-labeled goat anti-rat antibody. Cells were visualized and photographed by fluorescence microscopy at x 400 magnification.
Fig. 6. Effects of DP receptor antagonists or agonists on TGF- 1 -induced -SMA protein expression in MDCK cells. A - F : cells were grown on 6-well plates until 80% confluence and then treated with vehicle ( A ), TGF- 1 (10 ng/ml; B ), or TGF- 1 with PGD 2 (2 µM; C ) in the presence or absence of BAY-u3407 (10 µM; D ), BW-A868C (10 µM; E ), and BW-245C (10 µM; F ). Immunofluorescence was performed using mouse monoclonal anti- -SMA and FITC-labeled goat anti-mouse antibody. Cells were visualized and photographed by fluorescence microscopy at x 400 magnification. G : cells were treated as aforementioned and -SMA expression was detected by immunoblotting. Top : representative immunoblots. Bottom : densitometric analysis of -SMA expression; n = 3 for each group. * P < 0.01 vs. TGF- 1 -stimulated cells.
Role of cAMP. cAMP pathway has been shown to antagonize the TGF- 1 -elicited signaling ( 21 ). Thus we examined whether the cAMP pathway affected TGF- 1 -induced EMT and whether this mechanism was applicable to the PGD 2 action. We examined the effect of cAMP on EMT. MDCK cells were preincubated with cell membrane-permeable cAMP analog 8-Br-cAMP (50 µM), or an adenylyl cyclase activator, forskolin (50 µM), for 30 min, and then treated with TGF- 1 for 3 days. As seen in Fig. 7, both 8-Br-cAMP and forskolin completely blocked TGF- 1 -induced EMT as assessed by morphological changes ( Fig. 7, C and D ) and -SMA expression ( Fig. 7 E ). Both Rp-cAMP and PKA inhibitor H89 blocked the effect of forskolin on the inhibition of EMT induced by TGF- 1, indicating that cAMP was involved in the inhibition of forskolin on EMT ( Fig. 8 ). We next used Rp-cAMP and H89 to block the cAMP activity and determined their impact on the PGD 2 effect. As shown in Fig. 9, neither Rp-cAMP (200 µM) nor H89 (10 µM) was able to block the PGD 2 effect as assessed by morphological changes and -SMA expression.
Fig. 7. Effect of cAMP-elevating agents on TGF- 1 -induced EMT. A - D : morphologic changes. The cells were grown in 6-well plates until 80% confluence and then treated with vehicle ( A ), TGF- 1 alone (10 ng/ml; B ), TGF- 1 plus 50 µM 8-Br-cAMP (50 µM; C ), or TGF- 1 plus forskolin (50 µM; D ) for 3 days. Photographs were taken using a Nikon microscope (phase contrast). E : cells were treated as aforementioned and -SMA expression was detected by Western blotting. Top : representative immunoblots. Bottom : densitometric analysis of -SMA expression; n = 3 for each group. * P < 0.01 vs. TGF- 1 -stimulated cells.
Fig. 8. Effects of cAMP blockade on the forskolin inhibition of epithelial-mesenchymal transition (EMT). A - E : morphological changes. The cells were grown in 6-well plates until 80% confluence and then treated with vehicle ( A ), TGF- 1 alone ( B ), TGF- 1 plus 50 µM forskolin ( C ), TGF- 1 plus 50 µM forskolin and 200 µM Rp-cAMP ( D ), or TGF- 1 plus 50 µM forskolin and 10 µM H89 ( E ) for 3 days. Photographs were taken using a Nikon microscope (phase contrast). F : -SMA expression. MDCK cells were treated as aforementioned, and -SMA expression was determined by immunoblotting. Top : representative immunoblots. Bottom : densitometric analysis of -SMA expression; n = 3 for each group. * P < 0.01 vs. TGF- 1 -stimulated cells.
Fig. 9. Effects of cAMP blockade on the PGD 2 inhibition of EMT. A - E : morphologic changes. The cells were grown in 6-well plates until 80% confluence and then treated with vehicle ( A ), TGF- 1 alone ( B ), TGF- 1 plus 2 µM PGD 2 ( C ), TGF- 1 plus 2 µM PGD 2 and 200 µM Rp-cAMP ( D ), or TGF- 1 plus 2 µM PGD 2 and 10 µM H89 ( E ) for 3 days. Photographs were taken using a Nikon microscope (phase contrast). F : -SMA expression. MDCK cells were treated as aforementioned, and -SMA expression was determined by immunoblotting. Top : representative immunoblots. Bottom : densitometric analysis of -SMA expression; n = 3 for each group. * P < 0.01 vs. TGF- 1 -stimulated cells.
Measurement of production of 15d-PGJ 2 from PGD 2. To address the possibility that PGD 2 may act via its end metabolite 15d-PGJ 2, we determined the levels of 15d-PGJ 2 in the medium from PGD 2 -treated MDCK cells. MDCK cells were treated with 3 µM PGD 2 for 48 and 72 h, and the medium was subjected to 15d-PGJ 2 EIA. Our data showed that the concentrations of 15d-PGJ 2 in the medium at both 48 and 72 h following the addition of 3 µM PGD 2 were 0.12 ± 0.02 µM ( n = 3 in each group). The actual concentration of 15d-PGJ 2 was well below the values predicted from the cross activity of 15d-PGJ 2 antibody to PGD 2 (5% cross-reactivity equivalent to 0.16 µM). Moreover, 15d-PGJ 2 at the dose of 0.1 µM had no effect on EMT in cultured MDCK cells (data not shown).
Effect of PGD 2 on ROS production. In light of the recent reports on an important role of ROS in mediating EMT ( 33, 38 ), we examined the possibility that PGD 2 may act via inhibition of ROS production. We found that cellular ROS levels at 30 min after TGF- 1 treatment were threefold higher than the basal value ( Fig. 10 ). This increase was completely blocked by treatment with 2 µM PGD 2 ( Fig. 10 ).
Fig. 10. Effects of PGD 2 on the cellular ROS generation. Confluent MDCK cells in chamber slides were pretreated with 2 µM PGD 2 for 30 min and then exposed to TGF- 1 for further 30 min in the presence of DCFDA. A : control. B : 10 ng/ml TGF- 1. C : 10 ng/ml TGF- 1 plus 2 µM PGD 2. D : quantitation of DCF fluorescence. Confluent MDCK cells in 96-well plates were pretreated with 2 µM PGD 2 for 30 min and then stimulated by 10 ng/ml TGF- 1 for further 30 min in the presence of DCF. Fluorescence was quantified using FLUOstar OPTIMA. Values represent means ± SE, n = 8. * P < 0.01 vs. control. P < 0.01 vs. TGF- 1 group.
DISCUSSION
EMT has emerged as a critical event in the pathogenesis of TIF. Knowledge concerning the regulatory mechanism of EMT may lead to the development of effective therapies to halt the progression of ESRD. The identification of a limited number of negative regulators of EMT, namely, HGF ( 23, 24 ) and bone morphogenic protein 7 ( 51 ), has already shed light on the therapeutic interventions of the disease process. In an earlier study, we identified PGE 2 as a potent inhibitor of EMT, suggesting that eicosanoids may represent a novel class of EMT regulators ( 52 ). Inspired by this finding, we moved forward to examining the effects of other prostanoids on EMT in the same cell culture model. Our results showed that PGD 2 remarkably blocked EMT but PGF 2, PGI 2, and TXA 2 had no effect. The PGD 2 effect appeared independent of DP 1 and DP 2 receptors. PGD 2 exhibited an antioxidant property that was likely relevant to the antifibrotic action of this PG.
As expected, exposure of MDCK cells to TGF- 1 for 3 days induced a complete conversion of the epithelial cells to myofibroblasts as evidenced by acquisition of spindle-like morphology, loss of E-cadherin, and activation of -SMA. Additionaly, the TGF- 1 -induced EMT in MDCK cells appeared to be associated with reduced cell number. In a general agreement with this observation, reduced cell number has been noticed during EMT in primary proximal tubular cells and HK-2 cells ( 40 ) and in mouse mammary epithelial cells ( 33 ). We performed the caspase-3/7/10 assay but did not find evidence for apoptosis. Whether there is a cause-effect relationship between reduced cell number and EMT is unclear but certainly warrants further investigation.
Among various prostanoids tested, PGD 2 was highly efficient in inhibiting TGF- 1 -induced EMT. In contrast, PGD 2 treatment induced a full restoration of epithelial morphology and E-cadherin expression and a complete abolishment of -SMA stimulation. This effect was observed with PGD 2 at the range of 0.25 to 2 µM, which is likely to be within the range of physiological concentrations of PGD 2 in the renal tissues. Tissue concentration of PGD 2 in the rat kidney was detected at 200 ng/g tissue weight which was even higher than that of PGE 2 ( 46 ). This raises a possibility that PGD 2 may function as an endogenous inhibitor of EMT in the kidney.
PGD 2 elicits its effects through two G protein-coupled receptors (GPCRs), the DP 1 and the recently discovered chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2, also named DP 2 receptor). The DP 1 and DP 2 receptors are coupled to G s and G I, respectively, mediating divergent effects; DP 2 is primarily responsible for the proinflammatory action of PGD 2 in allergic inflammation ( 43 ), whereas DP 1 activation decreases inflammation ( 1, 13, 16 ). To determine the DP receptors involved, we examined effects of the DP 1 antagonist BW-A868C and the DP 2 antagonist BAY-u3405 on PGD 2 -induced inhibitory effects on EMT. Unexpectedly, neither BW-A868C nor BAY-u3405 was able to block the effect of PGD 2. In line with this finding, the DP 1 agonist BW-245C did not affect TGF- 1 -induced EMT. We also provided evidence that the lack of responses to the DP receptor agonist and antagonist was not due to the absence of the DP receptors in MDCK cells. Taken together, these findings may suggest the existence of additional DP receptors responsible for PGD 2 -dependent regulation of EMT.
Recently, Lin and colleagues ( 21 ) demonstrated that PKA activators, db-cAMP and forskolin, all efficiently inhibited TGF- 1 -induced, Smad3/4-dependent connective tissue growth factor (CTGF) expression. Smad3/4 activation is the common mediator of TGF- 1 -induced EMT. Thus it is possible that cAMP may exhibit an inhibitory effect on EMT. Our data showed that 8-Br-cAMP and forskolin completely blocked TGF- 1 -induced EMT as assessed by morphologic change and -SMA expression. Moreover, both Rp-cAMP and PKA inhibitor H89 blocked the inhibitory effect of forskolin on EMT, indicating a counterregulatory role of cAMP pathway during EMT. Although PGD 2 stimulated production of intracellular cAMP, blockade of endogenous cAMP activity had no effect of PGD 2 -induced inhibition of EMT, nearly ruling out the involvement of cAMP. Despite this negative aspect of the finding, to our knowledge, the counterregulatory role of cAMP in EMT has not been reported. It is possible that the cAMP pathway may be utilized by other antifibrotic factors with more effective cAMP-elevating capability. More importantly, this finding lends support to the use of cAMP-elevating agents for treatment of chronic renal disease.
PGD 2 can be degraded nonenzymatically to biologically active J-series cyclopentone PGs, especially the end product of 15d-PGJ 2 which is considered as a naturally occurring endogenous ligand of peroxisome proliferator-activated receptor (PPAR ) ( 14, 20 ). 15d-PGJ 2 and other synthetic PPAR ligands are reported to exert anti-inflammatory and antifibrotic effects in a wide variety of tissues including the kidney ( 7, 15, 36 ). There is a possibility that PGD 2 may act through its metabolite 15d-PGJ 2 in renal cells. To rule out this possibility, we determined the levels of 15d-PGJ 2 in the medium of MDCK cells 48 and 72 h after adding 3 µM PGD 2. The enzyme immunoassay detected a substantially low medium concentration of 15d-PGJ 2 that can be fully attributed to the cross activity of 15d-PGJ 2 antibody to PGD 2. This finding substantiates the notion that PGD 2 may act via its receptors rather than its metabolite 15d-PGJ 2.
Whatever DP receptors are involved, we provide evidence for antioxidant activity of PGD 2 that likely accounts for the antifibrotic effect of this PG. ROS have an established role in the pathogenesis of chronic renal disease. Two recent studies have made a landmark observation that ROS mediate EMT in both cancer cells and renal proximal tubule cells ( 33, 38 ). We found that TGF- 1 at 10 ng/ml induced a threefold increase in DCF-sensitive cellular ROS that was completely blocked by treatment with 2 µM PGD 2. This finding suggests that ROS serve as a target of PGD 2 during EMT. To our knowledge, this is the first report on the antioxidant property of PGD 2. A previous report on antioxidant capability of PGI 2 in cultured aortic smooth muscle cells provides an important insight into the atheroprotection of PGI 2 ( 11 ).
PGD 2 is formed from the product of COX activity, PGH 2, by two distinct PGD synthases, the lipocalin-type synthase ( L -PGDS) and the glutathione-dependent hematopoietic PGD synthase (H-PGDS). PGD 2 is the major arachidonic acid product in mast cells, playing a critical role in allergic inflammation. Other pharmacological activities of PGD 2 include vasodilation and bronchoconstriction, inhibition of platelet aggregation, sleep induction, hypothermia, and reduction of intraocular pressure. In contrast, there is scant information concerning PGD 2 actions in the kidney. The demonstration of the antifibrotic and antioxidant properties of PGD 2 in cultured renal epithelial cells indicates potential beneficial effects of this PG on renal injury. In line with this notion, systemic administration of PGD 2 improves renal blood flow and function in dogs ( 35 ). In Dahl salt-sensitive rats susceptible to hypertension-induced kidney injuries, tissue concentration of PGD 2 in the outer medulla was significantly lower than those of salt-resistant rats ( 46 ). A large number of studies report a creatinine-like increase in plasma L -PGDS in chronic renal disease ( 18, 25, 32 ). More importantly, urinary L -PGDS excretion markedly increases in the early stage of kidney injury such as diabetic nephropathy, and urinary L -PGDS is considered as a useful predictor of the forthcoming renal injury ( 47, 48 ). There is debate, however, as to whether the increase in L -PGDS levels is a consequence of reduced glomerular filtration or due to de novo synthesis as a compensatory mechanism. The latter is supported by recent genetic evidence that L -PGDS knockout mice develop glomerular hypertrophy, tubular damage, and renal fibrosis ( 34 ). It seems likely that in response to kidney injury the PGDS-PGD 2 pathway becomes activated, functioning as a renoprotective mechanism. In line with this notion, NSAIDs are well known to be associated with various types of nephrotoxicities, including interstitial nephritis ( 2, 49 ) and COX-2-deficient mice develop severe renal pathologies and progressive renal failure ( 8, 26 ). Whether COX activity in these situations is related to regulation of EMT is unknown but certainly represents an interesting area for future studies. Contrary to this hypothesis, however, it has been shown that selective COX-2 inhibitors exhibit renoprotective effects in animal models of 5/6 nephropathy ( 39 ) and diabetic and hypertensive nephropathy ( 5 ). The reason for the discrepancy is unclear but appears, in part, to be related to a distinct heterogeneity of coxibs. In this regard, Hermann et al. ( 17 ) recently compared the effects of two selective COX-2 inhibitors, celecoxib and rofecoxib, and a nonselective COX inhibitor, diclofenac, on renal morphology and function in salt-sensitive hypertension and demonstrated distinct renal effects of the two coxibs, celecoxib and rofecoxib, with the former showing protective effects while the latter showing detrimental effects. This finding raises a concern that some of renal effects of the coxibs may not be entirely related to the blockade of COX-2 activity.
It is somewhat surprising that PGI 2 did not exhibit any effect on TGF- 1 -induced EMT despite increasing reports on the role of PGI 2 in renal fibrotic response ( 29 ). In cultured mesangial cells, the PGI 2 analog cicaprost reduces fibronectin levels by 40% and induces a threefold increase in the level of matrix metalloproteinase-2, a key enzyme in matrix degradation ( 30, 31 ). In a general agreement with this observation, prostacyclin synthase knockout mice develop severe renal fibrosis ( 50 ). We speculate that different mechanisms may underlie the beneficial effects of various types of prostanoids.
In summary, the present study complements an earlier observation on PGE 2 inhibition of EMT in cultured MDCK cells ( 52 ) by examining the roles of other prostanoids: PGD 2, PGF 2, PGI 2, and TXA 2. Like PGE 2, PGD 2 completely reversed TGF- 1 -induced EMT while PGF 2, PGI 2, and CTA 2 were without an effect. The mechanism of the PGD 2 effect is unclear but appears to be independent of DP 1 or DP 2 receptors and may be related to its antioxidant property. The new information will help in an understanding of the role and mechanism of prostanoid-dependent pathway in chronic renal disease and will also shed light on the use of prostanoids for therapeutic interventions of the devastating disease.
GRANTS
This work was supported by National Institutes of Health Grants RO-1-HL-079453, RO-1-DK-066592, R21-DK-069460, and KO-1-DK-064981 (to T. Yang).
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作者单位：1 Division of Nephrology, University of Utah and Veterans Affairs Medical Center, Salt Lake City, Utah; and 2 Department of Cellular Biology and Anatomy, Medical College of Georgia; and 3 Medical Research Service, Veterans Affairs Medical Center, Augusta, Georgia