点击显示 收起
【摘要】 Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.
【关键词】 protein kinase A mitogenactivated protein kinase cyclin E p Raf
MANY GLOMERULAR DISEASES ARE characterized by excessive proliferation of mesangial cells (MC) ( 1 ). A wide variety of growth factors and cytokines are produced by parenchymal cells and circulating inflammatory cells in response to glomerular injury ( 2 ). After injury, signals for growth factor-directed mitogenesis are transduced through the Ras-Raf-MEK-ERK signaling cascade ( 75, 82 ). In MC and some other cell types, activation of the ERK signaling pathway and mitogenesis in response to growth factors may be inhibited through "negative cross talk" with the cAMP-PKA pathway ( 22, 43, 67, 69, 85, 98 ). For example, nonhydrolizable analogs of cAMP or stimuli of adenylate cyclase markedly inhibit proliferation of vascular smooth muscle cells (VSMC) ( 43, 78 ), fibroblasts ( 22, 98 ), adipocytes ( 85 ), and MC ( 69 ).
In some cell types, the antiproliferative effects of cAMP have been associated with protein kinase A-mediated phosphorylation of the Raf isoform Raf-1 on Ser 43 or other serine residues, including S233 and S259. Phosphorylation of Raf-1 is associated with reduced Raf-1 kinase activity, decreased interaction of Raf-1 with its upstream effector Ras, and decreased ERK activity. Although previous studies demonstrated that cAMP inhibits MC mitogenesis, the site(s) on Raf-1 phosphorylated by PKA in MC has not been previously identified.
Recent studies have indicated that phosphorylation of Raf-1 by PKA or suppression of ERK activity may not be necessary for cAMP-mediated inhibition of proliferation, at least in some cell types. In NIH 3T3 cells expressing Raf-1 with S43, S233, and S259 substituted with alanine residues to prevent their phosphorylation, cAMP still inhibited mitogenesis without suppressing Raf-1 or ERK activity ( 30 ). In CCL39 cells, cAMP inhibits mitogenesis without suppressing ERK activation. These studies indicate that pathways other than Raf-1 that regulate cell growth are targeted by PKA. Depending on cell type, potential targets of cAMP-PKA signaling have included cyclin D, cyclin E, cyclin A, and the cell cycle inhbitors p21 and p27 ( 25, 37, 38, 44, 47, 50, 51, 56 ). It is not known whether suppression of ERK activity is necessary for cAMP agonists to inhibit proliferation or whether cAMP agonists alter expression of cell cycle-regulatory proteins in MC.
However, there is a great deal of evidence indicating that the cAMP-PKA pathway interacts with the ERK pathway in a cell type-specific manner. For example, in some cell types that express B-Raf as the predominant Raf isoform, cAMP promotes ERK activation and stimulates mitogenesis ( 31, 35, 79 ). In some ( 31, 35, 79 ) but not all cell types ( 11 ), cAMP promotes mitogenesis through PKA-mediated phosphorylation of Rap-1, which increases B-Raf kinase activity. Whereas Raf-1 has a ubiquitous tissue distribution, B-Raf is highly expressed in cell types that proliferate in response to cAMP, including neuronal tissues and testis ( 92 ). Based on these considerations, some investigators postulated that B-Raf is a primary determinant of the cellular proliferative response to cAMP ( 36 ). No previous studies have determined whether B-Raf is expressed in MC.
Although cAMP agonists are potent inhibitors of MC proliferation ( 69 ), therapeutic efficacy of these agents is limited by a number of untoward systemic side effects. Recent studies have indicated that phosphodiesterase (PDE) inhibitors, which prevent the catabolism of cAMP and thereby lead to PKA activation, may have therapeutic efficacy as selective and cell type-specific agonists of the cAMP-PKA pathway. The PDE superfamily is large and complex. At present, at least 12 families of PDEs have been described, which catalyze the hydrolysis of cAMP, cGMP, or both ( 7, 20, 48, 65, 89, 90 ). PDED family members are distinguished by primary structure, modes of regulation, and capacity for inhibition by specific PDE inhibitors ( 7, 8, 28 ). In most cells, the capacity to hydrolyze cAMP by PDE far exceeds the capacity for cAMP synthesis ( 24 ). The activity of PDE isozymes is tightly regulated ( 24, 65, 81 ). A small change in PDE isozyme activity can have a profound effect on cAMP signaling without large changes in total intracellular cAMP levels ( 7, 51, 94 ). Recent evidence suggests that PDE isozymes are capable of compartmentalizing intracellular cAMP-mediated responses within a cell ( 15, 27, 32, 33, 64, 65, 83 ).
We previously showed that cAMP hydrolysis in MC is almost exclusively directed by members of the PDE3 and PDE4 family ( 69 ). PDE3 family members have a high affinity and specificity for cAMP hydrolysis. A number of agents are potent and specific inhibitors of PDE3 activity, which include lixazinone, cilostamide, cilostazol, and others ( 3, 8, 28, 74 ). There are two subfamilies of PDE3-PDE3A, which is expressed in cardiac myocytes, VSMC, oocytes, and other tissues, and PDE3B, which is expressed in adipocytes, hepatocytes, and pancreatic cells ( 18, 55, 80, 87 ). Recent studies have demonstrated the presence of additional isoforms generated through alternative transcriptional start sites and posttranscriptional processing ( 18, 97 ).
There are at least 18 isoforms of PDE4 encoded by 4 distinct genes (PDE4A, PDE4B, PDE4C, and PDE4D) ( 12, 93 ). These isoforms are expressed in a cell-specific fashion and show distinct activities, distribution, and regulation. PDE4 activity is specifically inhibited by a number of pharmacological agents, including rolipram, RO-1724, and denbufylline ( 28, 69, 74 ). These agents have been employed as anti-inflammatory agents in a number of experimental and human studies ( 9, 40, 84 ). We previously demonstrated that PDE4 inhibitors suppress reactive oxygen species (ROS) generation by MC ( 15 ). However, expression of specific PDE3 or PDE4 isoforms in MC has not previously been determined. These studies are necessary to provide the basis for future studies to define potential mechanisms whereby PDE inhibitors regulate MC mitogenesis and response to inflammatory stimuli.
Although we previously demonstrated that PDE3 but not PDE4 inhibitors suppress MC mitogenesis and PDE4 but not PDE3 inhibitors suppress MC ROS generation, potential mechanisms underlying the differential effect of PDE3 and PDE4 inhibitors in regulation of mitogenesis have not been established in MC or any other cell type. The primary objectives of this study were 1 ) to identify specific PDE3 and PDE4 isoforms present in MC; 2 ) to determine whether MC express both Raf-1 and B-Raf isoforms; 3 ) to determine whether PDE3 and PDE4 inhibitors differentially regulate Raf-1 or B-Raf kinase activity; 4 ) to determine whether ERK inhibition is mechanistically related to the antiproliferative effects of PDE3 inhibitors; and 5 ) to define cell cycle targets of PDE3 vs. PDE4 inhibitors in MC. These studies provide further support for the notion that PDE inhibitors may be employed as therapeutic agents to regulate distinct functions in MC.
MATERIALS AND METHODS
Materials. [ 3 H]thymidine was purchased from DuPont/New England Nuclear Research Products (Boston, MA). Primary antibodies for p21, p27, cyclin D, cyclin E, cyclin A, cdk-2, cdk-4, Raf-1, B-Raf, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-Raf-1 (Ser43) antibody was obtained from BioSource International (Camarillo, CA). Phospho-Raf-1 (S259) and phospho-Raf-1 (S338) antibodies were obtained from Cell Signaling Technology (Beverly, MA). Protein G Plus agarose was obtained from Santa Cruz Biotechnology. Histone H1 was obtained from Calbiochem (La Jolla, CA). MEK-1 (FL) was obtained from Santa Cruz Biotechnology. Other reagents and supplies were obtained through standard commercial suppliers.
MC preparation and culture. Handling of rats conformed to the institutional animal care guidelines established by The National Institute of Health. MC cultures were obtained from 200-g male Sprague-Dawley rats by differential sieving, as described previously ( 13, 41, 69 ). Briefly, rats were anesthetized by intraperitoneal injection of a 50:50 mixture containing 20 mg/ml xylazine and 100 mg/ml ketamine. 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 by light microscopy. Preparations 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 fetal bovine serum, 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 MC by positive immunohistochemical staining for vimentin, smooth muscle-specific actin, and negative stains for cytokeratin, factor VIII-related antigen, and leukocyte-common antigen (antibodies from Dako, Carpinteria, CA). MC were passed once a week following treatment with trypsin-EDTA (0.02%; Sigma, St. Louis, MO). Cells used in experiments were from passages 5 - 15.
PCR analysis for PDE profiles. Total RNA was isolated from rat MC using the RNeasy Total RNA Isolation Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Reverse-transcription and PCR amplification were performed using the Gene-Amp System (PerkinElmer, Branchburg, NJ). PCR analysis of PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, and PDE4D was performed essentially as previously described ( 57a ), with the addition of a DNase I treatment (RNase-free DNase I; Roche Molecular Chemicals, Indianapolis, IN) to eliminate residual genomic DNA. All PCR products were sequenced in both orientations.
Transfection studies. PKA and ERK activation was measured using the PathDetect In Vivo Signal Transduction Pathway trans -Reporting System (Stratagene, La Jolla, CA). Constitutively active Ras and Raf vector set, Dominant Negative Ras and Raf Vector Set (Clontech Laboratories, Palo Alto, CA) were used to investigate the effect of Ras and Raf overexpression on ERK activation in MC. MC were plated into 24-well culture dishes at 8 x 10 4 cells/well in complete Waymouth's medium. Twenty-four hours after being plated, cells were cotransfected with a firefly luciferase reporter vector (pTRE-Luc), a control Renilla luciferase reporter vector, and transactivator plasmids, e.g., pFA2-CREB for the PKA pathway and pFA2-Elk1 for the ERK pathway. Transfections were performed using FuGENE 6 Transfection Reagent (Roche Molecular Biochemical), according to the manufacturer's instructions. Eighteen hours after transfection, lixazinone, or rolipram (10 µM each), and epidermal growth factor (EGF; 20 ng/ml) were added. Control cells received vehicle only. 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).
Assay for cAMP. MC were incubated with PDE inhibitors (10 µM) in 24-well culture dishes for 60 min. Supernatants were removed and the reactions were terminated with 5% TCA (final concentration). The TCA was extracted with water-saturated ether, and the cAMP content was measured using RIA as previously described ( 16, 99 ).
Measurement of [ 3 H]thymidine incorporation. MC were plated into 24-well culture dishes at 5 x 10 4 cells/well and grown for 24-48 h in complete Waymouth's medium. Cells were rendered quiescent for 24 h in Waymouth's medium containing 0.5% fetal calf serum and then treated with PDE inhibitors (10 µM each). Control cells were treated with an equal volume of vehicle. In some experiments, EGF (20 ng/ml) was added 30 min after treatment with PDE inhibitors. After 20 h, cells were treated with methyl-[ 3 H]thymidine (1 µCi/ml) and incubated for an additional 4 h. Cells were washed three times with 10% TCA and once with water and lysed by the addition of 0.2 N NaOH. Radioactivity was determined by liquid scintillation counting. Incorporation of [ 3 H]thymidine was used as a measure of the rate of mitogenic synthesis of DNA.
Western blot analysis. MC were treated with PDE inhibitors, as described above. After incubation, MC were rinsed, harvested, and subjected to sonication (3 cycles of 10 s each, 8-µm amplitude) in 1 x lysis buffer (Cell Signaling Technology). The homogenates were centrifuged at 10,000 g for 10 min at 4°C. Protein concentration of the supernatant was determined by the method of Lowry ( 62 ). Equal amounts of lysate proteins ( 100 µg) were subjected to SDS-PAGE in the PROTEAN II minigel system or the Criterion System (Bio-Rad Laboratories, Hercules, CA). Lysates were denatured for 5 min at 100°C in SDS-loading buffer according to Laemmli ( 60 ). Electrophoresis was performed at a constant current (200 mA/gel), followed by transfer to polyvinylidene difluoride membranes (Bio-Rad Laboratories). The membranes were blocked with 5% nonfat dry milk in TBS containing 0.5% Tween 20 and incubated with primary antibodies followed by HRP-conjugated secondary antibodies. The blots were then visualized by exposure to X-ray film using the Phototype-HRP Western Detection System (Cell Signaling Technology).
In vitro kinase assays for ERK activity. A p44/42 MAP kinase Assay Kit (Cell Signaling Technology) was used to measure ERK activity, according to the manufacturer's instructions. Briefly, after treatment with PDE inhibitors (10 µM), MC were rinsed, harvested, and sonicated four times for 5 s each in 1 x lysis buffer plus 1 mM PMSF. Samples were microcentrifuged for 10 min at 4°C, and protein concentration of the supernatants was determined, as described above. Two hundred microliters of cell lysate containing 200 µg of total protein were added to 15 µl of resuspended immobilized phospho-p44/42 MAP kinase (Thr202/Tyr204) monoclonal antibody and incubated with gentle rocking overnight at 4°C. After samples were microcentrifuged for 30 s at 4°C, pellets were washed twice with 1 x lysis buffer and twice with 1 x kinase buffer. The washed pellets were suspended in 50 µl of 1 x kinase buffer supplemented with 200 µM ATP and 2 µg Elk-1 fusion protein and incubated for 30 min at 30°C. Reactions were terminated with 25 µl of 3 x SDS sample buffer. Samples were boiled for 5 min, vortexed, microcentrifuged for 2 min, and then loaded (30 µl) on SDS-PAGE gels (12%). Samples were analyzed by Western blotting, as described above.
Kinase assays for cyclin D and cyclin E activity. After treatment with PDE inhibitors (10 µM), cells were lysed in 1 x lysis buffer, and protein concentration was determined, as described above. Equal amounts of lysate protein (200 µg) were immunoprecipitated with antibodies specific for cyclin D and cyclin E. The immune complexes were collected with protein G plus agarose and washed twice with 1 x lysis buffer. Complexes were resuspended and washed twice with kinase buffer (50 mM Tris·HCl, pH 7.4, 10 mM MgCl 2, 1 mM DTT). Complexes were then resuspended in 50 µl of kinase buffer containing 2 µg of histone H1 200 µM ATP, and 10 µCi [ - 32 P]ATP (3,000 Ci/mM), and incubated at 30°C for 30 min. After incubation, 25 µl of 3 x SDS loading buffer were added, and the samples were boiled and electrophoresed on a SDS-PAGE gel. The gels were dried, and incorporation of 32 P was visualized by autoradiography and quantitated with a Kodak image analysis system.
Raf-1 and B-Raf kinase assay. Cells were treated with PDE inhibitors for 15 min, lysed, and immunoprecipitated with 5 µg of anti-Raf-1 and anti-B-Raf antibodies (Santa Cruz Biotechnology) overnight. The Raf-1 and B-Raf kinase activity in immunoprecipitates was measured in vitro using Raf-1 and B-Raf Kinase Cascade Assay Kits (Upstate Biotechnology, Lake Placid, NY), according to the manufacturer's instructions. Briefly, after 30-min incubation at 30°C in the presence of Mg 2+, MEK-1 and MAP kinase2/Erk2, MAP kinase2/Erk2 substrate myelin basic protein (MBP), and [ - 32 P]ATP were added and incubated at 30°C for 10 min. Twenty-five microliters of each reaction were spotted onto P81 phosphocellulose squares, and the squares were washed three times with 0.75% phosphoric acid, once with acetone. The level of 32 P incorporation into MBP was determined by liquid scintillation counting.
Caspase 3 assay. Caspase 3 activity of control and PDE inhibitor (10 µM)-treated MC was determined fluorometrically using the CaspACE Assay System (Promega), according to the manufacturer's instructions.
Statistical analysis. Data presented are representative of at lease three independent experiments performed in duplicate or triplicate, as indicated in the figure legends. Groups or pairwise comparisons were evaluated by the Student's t -test; P values of <0.05 were considered statistically significant.
RESULTS
Multiple PDE3 and PDE4 isoforms are present in MC. In our previous studies, we determined that cAMP hydrolysis in MC was directed almost exclusively by PDE3 and PDE4. However, the isoforms of PDE3 and PDE4 expressed in MC have not been previously characterized. Expression of PDE3 and PDE4 isoforms in RNA isolated from MC was determined by RT-PCR, using rat isoform-specific PDE3 and PDE4 primers ( 83 ). MC express both PDE3 isoforms (3A and 3B), and all four PDE4 genes (4A, 4B, 4C, 4D) ( Fig. 1 ). The multitude of cAMP-PDE isoforms expressed by MC may provide the basis for functional compartmentalization of cAMP signaling ( 15 ).
Fig. 1. Phosphodiesterase (PDE) profile in rat mesangial cells (RMC). cDNA fragments of PDE3A (508 bp), PDE3B (499 bp), PDE4A (233 bp), PDE4B (787 bp), PDE4C (539 bp), and PDE4D (262 bp) were amplified from mesangial cell (MC) cDNA, as described in MATERIALS AND METHODS.
Although both PDE3 and PDE4 inhibitors activate PKA and increase intracellular cAMP levels, only PDE3 inhibitors suppress MC mitogenesis. In MC, cAMP-PDE activity attributable to PDE4 is two times higher than cAMP-PDE activity attributable to PDE3 ( 69 ). We therefore sought to determine whether there were any differences in the ability of PDE3 or PDE4 inhibitors to activate PKA or increase intracellular cAMP levels. A transfection-based PKA assay was employed to determine the role of PDE3 and PDE4 inhibitors in PKA activation. Both lixazinone (a PDE3 inhibitor) and rolipram (a PDE4 inhibitor) activated PKA to a similar extent. PKA activation by PDE3 and PDE4 inhibitors was 20% that observed in MC treated with the potent adenylate cyclase agonist forskolin (FSK) and the nonselective PDE inhibitor IBMX ( Fig. 2 A ). Both lixazinone and rolipram modestly increased cAMP levels to a similar extent (+70 and +83%, respectively; Fig. 2 B ). As expected, FSK markedly increased cAMP content (+1,800%; Fig. 2 B ). Although both lixazinone and rolipram activated PKA and modestly increased intracellular cAMP content, only the PDE3 inhibitor lixazinone suppressed basal (-38%; Fig. 2 C ) and EGF-stimulated (-57%; see Fig. 7 B ) DNA synthesis, as assessed by [ 3 H]thymidine incorporation. Rolipram had no significant effect on basal or EGF-stimulated DNA synthesis ( Fig. 2 C and see Fig. 7 B ). Differences in thymidine incorporation were paralleled by similar differences in cell number, as assessed by serial cell counts 48-72 h after administration of PDE inhibitors (data not shown). This finding is in accord with our previous observations that several structurally distinct PDE3 inhibitors, but not PDE4 inhibitors, suppress MC mitogenesis ( 69 ). Potential mechanisms underlying this differential effect of PDE3 and PDE4 inhibitors on MC mitogenesis have not been established. We therefore sought to test the hypothesis that PDE3 and PDE4 inhibitors differentially regulate the Ras-Raf-MEK-ERK signaling pathway.
Fig. 2. Effect of PDE inhibitors on PKA activation, intracellular cAMP content, and mitogenesis. A : PKA activation. MC were treated with 10 µM lixazinone (Lix), 10 µM rolipram (Rp), or 10 µM forskolin (FSK) plus 10 µM IBMX for 4 h. PKA activation was assessed by a transfection-based in vivo kinase assay, as described in MATERIALS AND METHODS. B : cAMP content. MC were treated with 10 µM Lix, 10 µM Rp, or 10 µM FSK for 60 min. cAMP content was measured using RIA. C : MC mitogenesis. MC were treated with 10 µM Lix or 10 µM Rp for 20 h, and [ 3 H]thymidine incorporation was measured using liquid scintillation counting. Values represent means ± SE ( n = 3). * P < 0.05 vs. control.
Fig. 7. Overexpression of upstream effectors blocks the inhibitory effect of PDE3 inhibitors on ERK activation and MC mitogenesis. A : overexpression of constitutively active Ras and Raf blocks the inhibitory effect of PDE3 inhibitors on ERK activation. ERK activation was assessed by a transfection-based in vivo kinase assay. MC were cotransfected with Elk1 and constitutively active Ras (c.a.Ras) and Raf (c.a.Raf) or dominant negative Ras (d.n.Ras) and Raf (d.n.Raf) constructs for 18 h. Control (Ctrl) group was cotransfected with Elk1 and pBluescript (pBS). Cells were treated with 10 µM Lix or 10 µM Rp 60 min before EGF (20 ng/ml) treatment for 8 h. B : overexpression of constitutively active MEK-1 reverses the suppressive effect of PDE3 inhibitors on MC mitogenesis. MC were pretreated with Lix or Rp (10 µM each) for 30 min before EGF (20 ng/ml) treatment for 20 h. [ 3 H]thymidine uptake was assessed by liquid scintillation counting. Values represent means ± SE ( n = 3). # P < 0.05 vs. basal. * P < 0.05 vs. EGF-treated control.
MC express both Raf-1 and B-Raf. It is widely recognized that cAMP suppresses mitogenesis of some cell types, whereas it stimulates the proliferation of others ( 91 ). In many cell types, cAMP activates PKA, which phosphorylates Raf-1 on a number of serine residues, leading to downregulation of the Raf-MEK-ERK signaling cascade. However, cAMP activates the ERK pathway and stimulates mitogenesis in other cell types in which B-Raf rather than Raf-1 is the major Raf isoform expressed ( 29 ). Based on previous reports that low levels of B-Raf protein have been detected in kidney ( 5 ), we sought to determine whether both Raf-1 and B-Raf isoforms were expressed in MC. By Western blot analysis, we found that MC express high levels of B-Raf as well as Raf-1. Treatment of MC with either lixazinone or rolipram did not alter expression of B-Raf or Raf-1 ( Fig. 3 ). These studies indicate that the proliferative vs. antiproliferative effects of cAMP in various cell types are not merely dictated by expression of B-Raf rather than Raf-1.
Fig. 3. MC express both B-Raf and Raf-1; PDE inhibitors do not alter the levels of B-Raf and Raf-1 protein expression. MC were treated with 10 µM Lix or 10 µM Rp for 8 h. B-Raf and Raf-1 levels were assessed by Western blot analysis.
PDE3 and PDE4 inhibitors differentially phosphorylate Raf-1 in MC. In many cell types, cAMP blocks cell growth by phosphorylating Raf-1 on serine 43 and other serine residues. We sought to determine whether lixazinone and rolipram differentially phosphorylated Raf-1. MC were treated with lixazinone, rolipram, or FSK and IBMX for 10 min before addition of EGF. Phosphorylation of Raf-1 on different sites was assessed by Western blotting, using phospho-specific antibodies. After lixazinone treatment, Raf-1 was rapidly phosphorylated on serine 43 and serine 259. Raf-1 phosphorylation was stimulated 10 min after lixazinone administration and persisted 5 min through 1 h after EGF treatment. Rolipram had no effect on Raf-1 phosphorylation. It has been shown that when African green monkey kidney (COS) cells are growth factor stimulated, phosphorylation of serine 338 is required for Raf-1 activation ( 26 ). After EGF treatment, Raf-1 is recruited to the plasma membrane by activated Ras and is phosphorylated on serine 338 ( 30, 68 ). However, our data show that lixazinone, but not rolipram, inhibited Raf-1 phosphorylation on serine 338 ( Fig. 4 ).
Fig. 4. Effect of PDE inhibitors on Raf-1 phosphorylation in MC. MC were pretreated with 10 µM Lix, 10 µM Rp, or 10 µM FSK plus IBMX as positive control for 10 min, and then epidermal growth factor (EGF) was added for 5 and 60 min. Raf-1 phosphorylation was assessed by Western blot analysis using phospho-Raf-1-specific antibodies to S43 ( top ), S259 ( middle ), and S338 ( bottom ). Values represent means ± SE ( n = 3). # P < 0.05 vs. untreated cells. * P < 0.05 vs. EGF-treated cells.
PDE3 and PDE4 inhibitors differentially regulate Raf-1 kinase activity: both PDE3 and PDE4 inhibitors suppress B-Raf kinase activity. Raf-1 or B-Raf was immunoprecipitated from extracts prepared from MC treated with lixazinone or rolipram, and Raf-1 and B-Raf kinase activity was assessed using Raf-1 or B-Raf Kinase Cascade Assay Kits, as described in MATERIALS AND METHODS. Lixazinone treatment reduced Raf-1 kinase activity by 32%, whereas rolipram had no significant effect on Raf-1 kinase activity ( Fig. 5 ). Both lixazinone and rolipram suppressed B-Raf kinase activity. However, lixazinone inhibited B-Raf activity to a greater extent (44 and 31%, respectively) ( Fig. 5 ).
Fig. 5. PDE3 and PDE4 inhibitors differentially regulate Raf-1 kinase activity, whereas both PDE3 and PDE4 inhibitors suppress B-Raf kinase activity. MC were treated with 10 µM Lix or 10 µM Rp for 15 min. Raf-1 and B-Raf kinase activity was assessed in vitro using Raf-1 and B-Raf Kinase Cascade Assay Kits as described in MATERIALS AND METHODS. Values represent means ± SE ( n = 3). * P < 0.05 vs. control.
PDE3 inhibitors suppress mitogenesis through inhibition of the Ras-Raf-MEK-ERK signaling pathway. The effect of PDE3 or PDE4 inhibitors on the ERK signaling pathway was assessed by an in vitro kinase assay, as described in MATERIALS AND METHODS. Lixazinone significantly suppressed basal ERK activation. The inhibitory effect of lixazinone on ERK activation was observed after 15 min. ERK activation was suppressed by 38% after 1-h treatment with lixazinone and returned to baseline levels after 8 h. Rolipram had no effect on ERK activity ( Fig. 6 A ). Addition of EGF to quiescent MC produced a sixfold increase in ERK activity. One-hour treatment with lixazinone significantly suppressed ERK activation (-68%). Rolipram had no significant effect on EGF-stimulated ERK activation ( Fig. 6 B ).
Fig. 6. PDE3, but not PDE4, inhibitors suppress ERK activation. Basal and EGF (20 ng/ml)-stimulated ERK activity was assessed by in vitro kinase assay. Values represent means ± SE ( n = 3). A : effect of PDE inhibitors on basal ERK activity. MC were treated with 10 µM Lix or 10 µM Rp for the indicated times. * P < 0.05 vs. control (C). B : effect of PDE inhibitors on EGF-stimulated ERK activity. MC were treated with 10 µM Lix or 10 µM Rp 15 min before EGF (20 ng/ml) treatment for 60 min. # P < 0.05 vs. basal activity. * P < 0.05 vs. EGF-treated control. Insets : blots of representative experiments.
To determine the mechanisms by which PDE3 inhibitors suppress MC mitogenesis, MC were cotransfected with Elk-1 and constitutively active or dominant negative Ras or Raf constructs or cotransfected with Elk-1 and pBluescript as control. In control cells, lixazinone, but not rolipram, inhibited EGF-stimulated ERK activity. Constitutively active Ras or Raf plasmids significantly increased EGF-stimulated ERK activity (by 61 and 51%, respectively). Both the constitutively active Ras and Raf plasmids abrogated the inhibitory effect of lixazinone on ERK activity. The dominant negative Ras or Raf-1 plasmids significantly inhibited EGF-stimulated ERK activity (by 95 and 52%, respectively). Lixazinone had no effect on the low level of ERK activity observed in MC cotransfected with a dominant negative Ras plasmid. Lixazinone suppressed EGF-stimulated ERK activity in MC cotransfected with a dominant negative Raf plasmid, possibly reflecting an effect of lixazinone on endogenous wild-type Raf-1 ( Fig. 7 A ).
In complementary studies, MC were transfected with either a constitutively active MEK-1 construct or pBluescript as control and were treated with PDE inhibitors and EGF. [ 3 H]thymidine uptake was assessed. Lixazinone significantly inhibited EGF-stimulated DNA synthesis in control plasmid-transfected cells, whereas the constitutively active MEK-1 plasmid blocked the inhibitory effect of lixazinone on MC mitogenesis ( Fig. 7 B ). These data suggest that inhibition of the Ras-Raf-MEK-ERK signaling pathway is mechanistically linked to the inhibitory effect of lixazinone on MC mitogenesis.
PDE3 and PDE4 inhibitors differentially modulate cell cycle-regulatory proteins. MC were treated with lixazinone or rolipram. Cyclin and cdk levels were determined by Western blot analysis; activity of cyclin D and cyclin E was assessed by histone H1 kinase assay. Lixazinone significantly suppressed cyclin D expression and activity at 8 h ( Fig. 8, A and B ). Although lixazinone did not suppress cyclin E expression (data not shown), cyclin E kinase activity was inhibited after 1 h of lixazinone treatment and the inhibitory effect persisted for up to 18 h. Rolipram alone reduced cyclin E kinase activity by 23% at 8 h; however, lixazinone inhibited cyclin E kinase activity to a greater extent ( P < 0.01; Fig. 9 ). After 8 h of treatment, lixazinone inhibited cyclin A expression (by 27%); rolipram was without effect ( Fig. 10 ). Neither lixazinone nor rolipram significantly altered expression of cdk2 and cdk4 (data not shown).
Fig. 8. Effect of PDE inhibitors on cyclin D expression and activity in MC. MC were treated with 10 µM Lix or 10 µM Rp for 1 and 8 h. A : PDE3, but not PDE4, inhibitors suppress cyclin D expression in MC. Cyclin D levels were assessed by Western blot analysis. B : PDE3, but not PDE4, inhibitors suppress cyclin D activity in MC. Cyclin D activity was measured by histone H1 kinase assay. Values represent means ± SE ( n = 3). * P < 0.05 vs. control. Insets : blots of representative experiments.
Fig. 9. PDE3, but not PDE4, inhibitors decrease cyclin E activation. MC were treated with 10 µM Lix or 10 µM Rp for the indicated times. Cyclin E activity was assessed by histone H1 kinase assay. Values represent means ± SE ( n = 3). * P < 0.05 vs. control. Insets : blots of representative experiments.
Fig. 10. Effect of PDE inhibitors on cyclin A expression in MC. MC were treated with 10 µM Lix or 10 µM Rp for 8 h. Cyclin A levels were assessed by Western blot analysis. Values represent means ± SE ( n = 3). * P < 0.05 vs. control. Inset : blot of a representative experiment.
PDE3 and PDE4 inhibitors differentially regulate the cell cycle-inhibitory proteins p21 and p27. The antiproliferative effect of lixazinone was associated with induction of the cell cycle inhibitor p21 after 8 h of treatment (+90%). Rolipram inhibited p21 expression after 18 h (-31%) but had no significant effect on p21 levels at any other time point ( Fig. 11 A ). Lixazinone had no significant effect on p27 expression. Rolipram modestly increased p27 levels after 8-h treatment (+30%; Fig. 11 B ).
Fig. 11. Effect of PDE inhibitors on cell cycle-inhibitory proteins. MC were treated with 10 µM Lix or 10 µM Rp for the indicated times. p21 And p27 levels were measured by Western blot analysis. A : effect of PDE inhibitors on p21 expression. B : effect of PDE inhibitors on p27 expression. Values represent means ± SE ( n = 3). * P < 0.05 vs. control. Insets : blots of representative experiments.
Low-dose FSK eliminates compartmentalization of the antiproliferative effects of PDE3 and PDE4 inhibitors. Although lixazinone and rolipram activate PKA and increase cAMP levels to a similar extent, only lixazinone inhibits mitogenesis, through blocking the Ras-Raf-MEK-ERK signaling pathway and reducing cyclin D and cyclin E kinase activity. We sought to test the hypothesis that rolipram could inhibit MC proliferation when administered with a low dose of the adenylate cyclase agonist FSK. MC were treated with 30 nM FSK with lixazinone or rolipram (1 µM). At those low doses, neither FSK nor rolipram alone had an inhibitory effect on MC proliferation. However, combined treatment of MC with 30 nM forskolin and 1 µM rolipram significantly inhibited MC proliferation by 30%. One micromolar lixazinone alone or with 30 nM FSK inhibited MC mitogenesis by 39 and 53%, respectively ( Fig. 12 ). These studies provide further evidence for functional compartmentalization of cAMP pools differentially regulated by PDE3 and PDE4.
Fig. 12. Low-dose FSK eliminates compartmentalization of the antiproliferative effect of PDE3 and PDE4 inhibitors. MC were treated with FSK (30 nM) and Lix or Rp (1 µM) for 18 h before measuring [ 3 H]thymidine uptake. Values represent means ± SE ( n = 3). * P < 0.05 vs. control.
PDE3 and PDE4 inhibitors do not induce apoptosis in MC. Apoptosis was assessed in MC treated with lixazinone or rolipram. Neither lixazinone nor rolipram had any significant effect on caspase 3 activity. As a positive control, caspase 3 activity was significantly increased by treatment of MC with 20 ng/ml TNF plus 10 µg/ml cycloheximide (3 independent experiments, data not shown).
DISCUSSION
These studies provide evidence that ERK signaling in MC is differentially regulated by functionally compartmentalized intracellular pools of cAMP directed by PDE3 and PDE4. We previously showed that cAMP is hydrolyzed in MC almost exclusively through the actions of PDE3 and PDE4 ( 69 ). Although total cAMP hydrolytic activity attributable to PDE4 is approximately twice that of cAMP hydrolytic activity attributable to PDE3, both PDE3 and PDE4 inhibitors activate PKA and elevate intracellular cAMP levels to a similar extent. Only PDE3, but not PDE4, inhibitors are effective in suppressing MC mitogenesis. This differential effect of PDE inhibitors on MC mitogenesis is associated with differential phosphorylation of Raf-1, decreased Raf-1 kinase activity, decreased ERK activity, and modulation of cell cycle proteins. Expression of constitutively active Ras or Raf constructs abrogates the inhibitory effect of PDE3 inhibitors on ERK activity, whereas overexpression of MEK-1 eliminates the suppressive effect of PDE3 inhibitors on MC mitogenesis. These studies provide evidence that the antiproliferative effect of PDE3 inhibitors was mechanistically linked to suppression of the Ras-Raf-MEK-ERK signaling pathway.
The distribution of PDE3 and PDE4 isoforms in MC has not been previously characterized in detail. We found that MC express both PDE3 isoforms (PDE3A and PDE3B) and express all four gene products comprising the PDE4 family (PDE4A, PDE4B, PDE4C, and PDE4D) ( 93 ). PDE3A is widely expressed in arterial tissues, platelets, and cardiac tissue ( 40, 97 ). Recent studies showed that alternative transcriptional and posttranscriptional processing of the PDE3A gene is responsible for the generation of at least three isoforms that differ in the number of membrane association domains, protein kinase A or protein kinase B phosphorylation sites ( 18, 97 ). PDE3B is primarily expressed in adipocytes, hepatocytes, and pancreatic cells ( 80 ). Both PDE3A and PDE3B are expressed in cultured arterial smooth muscle cells ( 76 ). The PDE4 family is the largest PDE family characterized to date, with at least 18 isoforms ( 10, 21, 48, 49, 93 ). Recent studies have defined important functions for specific PDE4 isoforms. For example, PDE4D knockout mice have impaired growth and fertility ( 54 ). The airways of mice deficient in the PDE4D gene are refractory to muscarinic cholinergic stimulation ( 46, 71 ). These animals still exhibit pulmonary inflammation, indicating that other PDE4 isoforms such as PDE4A or PDE4B regulate this process ( 39, 66 ). Along these lines, PDE4B has been shown to be essential for TNF synthesis in response to LPS stimulation ( 53 ). The PDE4D5 isoform, but not other PDE4D isoforms, interacts with the RACK1 signaling scaffold protein ( 100 ), whereas the PDE4D3 isoform contains a unique NH 2 -terminal domain that allows interactions with specific SH3 proteins ( 6 ).
Using a functional assay, we previously demonstrated that most cAMP PDE activity in MC resides within the cytosol ( 69 ). However, additional studies are needed to determine which PDE3 isoform is responsible for inhibition of mitogenesis and which PDE4 isoform is responsible for inhibition of ROS generation in MC and to determine whether these activities reside within the cytosol or are localized to the plasma membrane or other cellular compartments.
It has been postulated that the relative expression of B-Raf vs. Raf-1 may dictate whether cAMP stimulates or inhibits proliferation ( 91 ). Raf-1 has a ubiquitous tissue distribution, whereas B-Raf is expressed primarily in neuronal cells and testis ( 92 ). We found that MC expressed both Raf-1 and B-Raf. The PDE3 inhibitor lixazinone inhibited Raf-1 kinase activity, whereas the PDE4 inhibitor rolipram was without significant effect. However, both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. It is likely that the effect of cAMP on B-Raf kinase activity depends on cell culture conditions and/or cell type. For example, cAMP stimulates B-Raf and ERK activity in melanocytes ( 11 ) and PC12 pheochromocytoma cells cultured in the presence of serum ( 34 ). However, cAMP inhibits Raf-1 and B-Raf activity in serum-starved PC12 cells ( 34, 77, 96 ) or PC12 cells treated with EGF, nerve growth factor, or platelet-derived growth factor ( 96 ). In differentiating promyelocytic HL-60 leukemia cells, cAMP inhibits B-Raf activity but activates ERK ( 17 ). In Rat-1 fibroblasts (which lack B-Raf), cAMP inhibits Raf-1 activity, leading to suppression of ERK activity and inhibition of mitogenesis. However, expression of B-Raf renders the cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of ERK and mitogenesis, suggesting that the relative levels of B-Raf and Raf-1 may determine whether cells proliferate or are growth inhibited by cAMP ( 34 ). The ability of B-Raf to activate MAPK may be related to its ability to complex with 14-3-3 proteins ( 79 ). In cell types inhibited by cAMP, approximately fivefold less 14-3-3 protein was associated with B-Raf than was observed in cell types in which cAMP stimulated MAPK ( 79 ). Further studies are needed to address this issue in MC.
In cell types that are growth inhibited by cAMP, it is thought that inhibition of mitogenesis is associated with PKA-mediated phosphorylation of Raf-1 on serine 43 and other serine residues, including serine 259 and serine 621 ( 22, 30, 45, 85, 98 ). We therefore sought to determine whether the differential effect of PDE3 and PDE4 inhibitors on MC mitogenesis was associated with a differential effect on Raf-1 phosphorylation. We found that the PDE3 inhibitor lixazinone promoted rapid phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338. The PDE4 inhibitor rolipram had no significant effect on phosphorylation of Raf-1. In at least some cell types, phosphorylation of Raf-1 on serine 43 by PKA inhibits binding of Raf-1 to Ras-GTP ( 19, 30, 98 ) and suppresses Raf-1 kinase activity. PKA phosphorylates Raf-1 at other serine residues, including serine 259 and serine 621 ( 30, 73 ). However, elevated cAMP does not stimulate phosphorylation of serine 621 or suppress activity of the catalytic domain of Raf-1 in vivo, indicating that this may be an in vitro artifact ( 88 ). Recent studies showed that growth factor-stimulated mitogenesis is associated with phosphorylation of Raf-1 at serine 338 ( 26 ). This phosphorylation apparently promotes membrane localization of Raf-1 and subsequent activation of ERK signaling ( 26, 68 ). We found that lixazinone, but not rolipram, decreased phosphorylation of Raf-1 at serine 338 and suppressed Raf-1 kinase activity. Further studies are needed to determine whether lixazinone-mediated decreases in Raf-1 kinase activity are due to decreased phosphorylation of Raf-1 at serine 338.
We found that the inhibitory effect of lixazinone on ERK activity was abrogated when MC were transfected with a constitutively active Ras or Raf plasmid. Furthermore, the antiproliferative effect of lixazinone was reversed when MC were transfected with a constitutively active MEK-1 construct. These studies underscore the importance of the ERK pathway in regulation of MC mitogenesis and indicate that strong activation of the Ras-Raf-MEK-ERK pathway can stimulate cAMP-independent proliferation.
Although these studies demonstrate a critical role of cAMP in regulation of the Ras-Raf-MEK-ERK pathway, recent studies have suggested that cAMP may regulate mitogenesis through other pathways. In some cell systems, inhibition of ERK by cAMP does not appear to explain the growth-inhibitory effects of cAMP ( 23, 43, 70 ). In rat smooth muscle cells, cAMP at concentrations that strongly inhibit DNA synthesis does not inhibit ERK activation ( 42 ). In MC, addition of cAMP at a time when transient growth factor activation of ERK has declined to basal levels still results in potent inhibition of DNA synthesis. The efficiency whereby cAMP elevation inhibits smooth muscle cell DNA synthesis is nearly identical when cAMP is added before growth factor stimulation or 6-12 h later ( 58 ). The same phenomenon has been observed in other cell types ( 70 ). Furthermore, recent studies showed that ERK is activated normally in cells derived from Raf-1 knockout animals ( 50, 72 ). These observations indicate the presence of Raf-1-independent pathways for ERK activation, at least in some circumstances.
Based on these considerations, we sought to define other potential cell cycle targets that may be subject to differential regulation by PDE3 and PDE4 inhibitors. We found that the antiproliferative effects of PDE3 inhibitors were associated with reduced cyclin D levels, cyclin E kinase activity, and decreased cyclin A levels. In mammalian cells, G1 to S transition is governed by the cyclin-cyclin-dependent kinase (cdk) complexes, in which the cdk acts as the catalytic subunit and the cyclin acts as the regulatory subunit. Expression of cyclin D increases in early-mid part of G1 to form complexes with cdk4 and cdk6 ( 57, 63, 86 ). In the mid-part of G1, cyclin E associates with cdk2 to form an active complex. In contrast to cyclin D, cyclin E expression is not generally induced by growth factors. However, activity of the cyclin E-cdk2 complex is markedly increased in mid-G1 following growth factor stimulation, and inhibition of cdk2 activity prevents cells from entering the S phase of the cell cycle ( 86 ). In the S phase, cdk2 complexes with cyclin A ( 86 ).
Activity of cyclin-cdk complexes is also regulated by cdk inhibitor proteins that bind to and inhibit cdks. In smooth muscle cells ( 38, 59, 95 ) and other cell types ( 4, 56 ), cAMP suppresses induction of cyclin D and cyclin A and upregulates the cdk inhibitor protein p27. In smooth muscle cells, p27 levels are induced and levels of p27 associated with cdk2 are also increased ( 58 ), resulting in an abrogation of both cdk2 and cdk4 activities ( 38 ). We found that PDE3 inhibitors increase expression of the cdk inhibitor protein p21, whereas PDE4 inhibitors had no effect on p21 expression. These studies indicate that PDE3 inhibitors suppress MC mitogenesis, at least in part, through upregulation of the cdk inhibitor p21.
We previously showed that MC possess functionally compartmentalized pools of cAMP regulated by distinct PDE isoforms. A pool of cAMP directed by PDE3 inhibits mitogenesis, whereas a pool of cAMP directed by PDE4 suppresses ROS generation by MC ( 15 ). In our current studies, we demonstrate that PDE3 inhibitors promote phosphorylation of Raf-1 on serine 43 and serine 259 and inhibit phosphorylation of Raf-1 on serine 338, and PDE4 inhibitors were without effect. PDE3, but not PDE4, inhibitors suppress Raf-1 kinase activity and ERK activation, indicating MAPK signaling is regulated by functionally compartmentalized pools of cAMP. The compartmentalization can be overcome by increasing intracellular cAMP levels with the adenylate cyclase agonist FSK. The inhibitory effect of PDE3 inhibitors on ERK activity is abolished when constitutively active Ras or Raf constructs are expressed and the suppressive effects of PDE3 inhibitors on MC mitogenesis are reversed when constitutively active MEK-1 is expressed in MC. These studies provide evidence that, at least in MC, PDE3 inhibitors act by suppressing ERK activation. Other targets of cAMP signaling directed by PDE3 include cyclins D, E, and A and the cell cycle inhibitor p21. Additional studies are needed to determine whether the observed effects of PDE3 inhibitors on cell cycle protein expression and activity are mechanistically related to suppression of ERK activity. Although nonselective PDE inhibitors such as IBMX have antiproliferative effects in MC and other cell types, a variety of systemic side effects limits their clinical use. In contrast, isoform-specific PDE inhibitors have been used to treat a variety of inflammatory and autoimmune conditions. PDE inhibitors may provide useful therapeutic targets for modulating ERK activation in response to acute or chronic renal injury and may thereby arrest progression of a variety of chronic renal diseases.
GRANTS
This work is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-16105 and DK-55603.
ACKNOWLEDGMENTS
We thank C. Grabau for excellent secretarial assistance.
【参考文献】
Abboud HE. Growth factors and the mesangium. J Am Soc Nephrol 2: S185-S189, 1992.
Abboud HE. Growth factors in glomerulonephritis. Kidney Int 43: 252-267, 1993.
Alvarez R, Banerjee GL, Bruno JJ, Jones GL, Littschwager K, Strosberg AM, and Venuti MC. A potent and selective inhibitor of cyclic AMP phosphodiesterase with potential cardiotonic and antithrombotic properties. Mol Pharmacol 29: 554-560, 1986.
Barlat I, Henglein B, Plet A, Lamb N, Fernandez A, McKenzie F, Pouyssegur J, Vie A, and Blanchard JM. TGF- 1 and cAMP attenuate cyclin A gene transcription via a cAMP responsive element through independent pathways. Oncogene 11: 1309-1318, 1995.
Barnier JV, Papin C, Eychene A, Lecoq O, and Calothy G. The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. J Biol Chem 270: 23381-23389, 1995.
Beard MB, O'Connell JC, Bolger GB, and Houslay MD. The unique N-terminal domain of the cAMP phosphodiesterase PDE4D4 allows for interaction with specific SH3 domains. FEBS Lett 460: 173-177, 1999.
Beavo J. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75: 725-748, 1995.
Beavo JA and Reifsnyder DH. Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends Pharmacol Sci 11: 150-155, 1990.
Bielekova B, Lincoln A, McFarland H, and Martin R. Therapeutic potential of phosphodiesterase-4 and -3 inhibitors in Th1-mediated autoimmune diseases. J Immunol 164: 1117-1124, 2000.
Bolger G, Michaeli T, Martins T, St John T, Steiner B, Rodgers L, Riggs M, Wigler M, and Ferguson K. A family of human phosphodiesterases homologous to the dunce learning and memory gene product of Drosophila melanogaster are potential targets for antidepressant drugs. Mol Cell Biol 13: 6558-6571, 1993.
Busca R, Abbe P, Mantoux F, Aberdam E, Peyssonnaux C, Eychene A, Ortonne JP, and Ballotti R. Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes. EMBO J 19: 2900-2910, 2000.
Chen CN, Denome S, and Davis RL. Molecular analysis of cDNA clones and the corresponding genomic coding sequences of the Drosophila dunce+ gene, the structural gene for cAMP phosphodiesterase. Proc Natl Acad Sci USA 83: 9313-9317, 1986.
Cheng J, Yusufi AN, Thompson MA, Chini EN, and Grande JP. Nicotinic acid adenine dinucleotide phosphate: a new Ca 2+ releasing agent in kidney. J Am Soc Nephrol 12: 54-60, 2001.
Chini C, Grande J, Chini E, and Dousa T. Compartmentalization of cAMP signaling in mesangial cells by phosphodiesterase isozymes PDE3 and PDE4. Regulation of superoxidation and mitogenesis. J Biol Chem 272: 9854-9859, 1997.
Chini CCS, Chini EN, Williams JM, Matousovic K, and Dousa TP. Formation of reactive oxygen metabolites in glomeruli is suppressed by inhibition of cAMP phosphodiesterase isozyme type IV. Kidney Int 46: 28-36, 1994.
Cho YJ, Kim JY, Jeong SW, Lee SB, and Kim ON. Cyclic AMP induces activation of extracellular signal-regulated kinases in HL-60 cells: role in cAMP-induced differentiation. Leuk Res 27: 51-56, 2003.
Choi YH, Ekholm D, Krall J, Ahmad F, Degerman E, Manganiello VC, and Movsesian MA. Identification of a novel isoform of the cyclic-nucleotide phosphodiesterase PDE3A expressed in vascular smooth muscle myocytes. Biochem J 353: 41-50, 2001.
Chuang E, Barnard D, Hettich L, Zhang XF, Avruch J, and Marshall MS. Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. Mol Cell Biol 14: 5318-5325, 1994.
Conti M and Jin SL. The molecular biology of cyclic nucleotide phosphodiesterases. Prog Nucleic Acid Res Mol Biol 63: 1-38, 1999.
Conti M, Richter W, Mehats C, Livera G, Park JY, and Jin C. Cyclic AMP-specific PDE4 phosphodiesterases as critical components of cyclic AMP signaling. J Biol Chem 278: 5493-5496, 2003.
Cook S and McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 262: 1069-1072, 1993.
Cospedal R, Lobo M, and Zachary I. Differential regulation of extracellular signal-regulated protein kinases (ERKs) 1 and 2 by cAMP and dissociation of ERK inhibition from anti-mitogenic effects in rabbit vascular smooth muscle cells. Biochem J 342: 407-414, 1999.
D'Angelo G, Lee H, and Weiner RI. cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation. J Cell Biochem 67: 353-366, 1997. <a href="/cgi/external_ref?access_num=10.1002/(SICI)1097-4644(19971201)67:3
Depoortere F, Pirson I, Bartek J, Dumont JE, and Roger PP. Transforming growth factor 1 selectively inhibits the cyclic AMP- dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27 kip1. Mol Biol Cell 11: 1061-1076, 2000.
Diaz B, Barnard D, Filson A, MacDonald S, King A, and Marshall M. Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling. Mol Cell Biol 17: 4509-4516, 1997.
Dodge KL, Khouangsathiene S, Kapiloff MS, Mouton R, Hill EV, Houslay MD, Langeberg LK, and Scott JD. mAKAP assembles a protein kinase A/PDE4 phosphodiesterase cAMP signaling module. EMBO J 20: 1921-1930, 2001.
Dousa T. Cyclic-3',5'-nucleotide phosphodiesterase isozymes in cell biology and pathobiology of the kidney. Kidney Int 55: 29-62, 1999.
Dugan LL, Kim JS, Zhang Y, Bart RD, Sun Y, Holtzman DM, and Gutmann DH. Differential effects of cAMP in neurons and astrocytes. Role of B-raf. J Biol Chem 274: 25842-25848, 1999.
Dumaz N, Light Y, and Marais R. Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms. Mol Cell Biol 22: 3717-3728, 2002.
Dumont JE, Jauniaux JC, and Roger PP. The cyclic AMP-mediated stimulation of cell proliferation. Trends Biochem Sci 14: 67-71, 1989.
Elks ML and Manganiello VC. Antilipolytic action of insulin: role of cAMP phosphodiesterase activation. Endocrinology 116: 2119-2121, 1985.
Elks ML and Manganiello VC. Selective effects of phosphodiesterase inhibitors on different phosphodiesterases, adenosine 3',5'-monophosphate metabolism, and lipolysis in 3T3-L1 adipocytes. Endocrinology 115: 1262-1268, 1984.
Erhardt P, Troppmair J, Rapp R, and Cooper G. Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells. Mol Cell Biol 15: 5524-5530, 1995.
Frodin M, Peraldi P, and Van Obberghen E. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells. J Biol Chem 269: 6207-6214, 1994.
Fujita T, Meguro T, Fukuyama R, Nakamuta H, and Koida M. New signaling pathway for parathyroid hormone and cyclic AMP action on extracellular-regulated kinase and cell proliferation in bone cells. Checkpoint of modulation by cyclic AMP. J Biol Chem 277: 22191-22200, 2002.
Fukumoto S, Hosoi M, Koyama H, Yamakawa K, Nishizawa Y, and Morii H. Inhibitors of cAMP-phosphosphodiesterase types III and IV suppress the proliferation of human vascular smooth uscle cells at G1/S phase by inhibiting cdk2 activity. Circulation 96: 503-I, 1997.
Fukumoto S, Koyama H, Hosoi M, Yamakawa K, Tanaka S, Morii H, and Nishizawa Y. Distinct role of cAMP and cGMP in the cell cycle control of vascular smooth muscle cells: cGMP delays cell cycle transition through suppression of cyclin D1 and cyclin-dependent kinase 4 activation. Circ Res 85: 985-991, 1999.
Giembycz M. PDE4D-deficient mice knock the breath out of asthma. Trends Pharmacol Sci 21: 291-292, 2000.
Giembycz MA. Cilomilast: a second generation phosphodiesterase 4 inhibitor for asthma and chronic obstructive pulmonary disease. Expert Opin Investig Drugs 10: 1361-1379, 2001.
Grande JP, Jones ML, Swenson CL, Killen PD, and Warren JS. Lipopolysaccharide induces monocyte chemoattractant protein production by rat mesangial cells. J Lab Clin Med 124: 112-117, 1994.
Graves LM, Bornfeldt KE, Argast GM, Krebs EG, Kong X, Lin TA, and Lawrence JC Jr. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc Natl Acad Sci USA 92: 7222-7226, 1995.
Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, and Krebs EG. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci USA 90: 10300-10304, 1993.
Gubler U and Hoffman BJ. A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269, 1983.
Hafner S, Adler HS, Mischak H, Janosch P, Heidecker G, Wolfman A, Pippig S, Lohse M, Ueffing M, and Kolch W. Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol 14: 6696-6703, 1994.
Hansen G, Jin S, Umetsu DT, and Conti M. Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D. Proc Natl Acad Sci USA 97: 6751-6756, 2000.
Hayashi S, Morishita R, Matsushita H, Nakagami H, Taniyama Y, Nakamura T, Aoki M, Yamamoto K, Higaki J, and Ogihara T. Cyclic AMP inhibited proliferation of human aortic vascular smooth muscle cells, accompanied by induction of p53 and p21. Hypertension 35: 237-243, 2000.
Houslay MD. PDE4 cAMP-specific phosphodiesterases. Prog Nucleic Acid Res Mol Biol 69: 249-315, 2001.
Houslay MD, Sullivan M, and Bolger GB. The multienzyme PDE4 cyclic adenosine monophosphate-specific phosphodiesterase family: intracellular targeting, regulation, and selective inhibition by compounds exerting anti-inflammatory and antidepressant actions. Adv Pharmacol 44: 225-342, 1998.
Huser M, Luckett J, Chiloeches A, Mercer K, Iwobi M, Giblett S, Sun XM, Brown J, Marais R, and Pritchard C. MEK kinase activity is not necessary for Raf-1 function. EMBO J 20: 1940-1951, 2001.
Inoue Y, Toga K, Sudo T, Tachibana K, Tochizawa S, Kimura Y, Yoshida Y, and Hidaka H. Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model. Br J Pharmacol 130: 231-241, 2000.
Jin SL and Conti M. Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF- responses. Proc Natl Acad Sci USA 99: 7628-7633, 2002.
Jin SL, Richard FJ, Kuo WP, D'Ercole AJ, and Conti M. Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice. Proc Natl Acad Sci USA 96: 11998-12003, 1999.
Kasuya J, Liang SJ, Goko H, Park SH, Kato K, Xu ZD, Hockman S, Manganiello VC, and Fujita-Yamaguchi Y. Cardiac type cGMP-inhibited phosphodiesterase (PDE3A) gene structure: similarity and difference to adipocyte type PDE3B gene. Biochem Biophys Res Commun 268: 827-834, 2000.
Kato JY, Matsuoka M, Polyak K, Massague J, and Sherr CJ. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell 79: 487-496, 1994.
Kitagawa M, Higashi H, Jung H, Suzuki-Takahashi I, Ikeda M, Tamai K, Kato J, Segawa K, Yoshida E, Nishimura S, and Taya Y. The consensus motif for phosphorylation by cyclin D1-cdk4 is different from that for phosphorylation by cyclin A/E-cdk2. EMBO J 15: 7060-7069, 1996.
Kostic MM, Erdogan S, Rena G, Borchert G, Hoch B, Bartel S, Scotland G, Huston E, Houslay MD, and Krause EG. Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditioned rat heart. J Mol Cell Cardiol 29: 3135-3146, 1997.
Koyama H, Bornfeldt KE, Fukumoto S, and Nishizawa Y. Molecular pathways of cyclic nucleotide-induced inhibition of arterial smooth muscle cell proliferation. J Cell Physiol 186: 1-10, 2001. <a href="/cgi/external_ref?access_num=10.1002/1097-4652(200101)186:1
Kronemann N, Nockher W, Busse R, and Schini-Kerth V. Growth-inhibitory effect of cyclic GMP- and cyclic AMP-dependent vasodilators on rat vascular smooth muscle cells: effect on cell cycle and cyclin expression. Br J Pharmacol 126: 349-357, 1999.
Laemmli U. Cleavage of structural proteins during assembly of the head of bacteriophage T 4. Nature 227: 680-685, 1970.
Liu H and Maurice DH. Expression of cyclic GMP-inhibited phosphodiesterases 3A and 3B (PDE3A and PDE3B) in rat tissues: differential subcellular localization and regulated expression by cyclic AMP. Br J Pharmacol 125: 1501-1510, 1998.
Lowry O, Rosebrough A, Farr A, and Randall R. Protein measurement with folin phenol reagent. J Biol Chem 193: 265-275, 1951.
Lundberg A and Weinberg R. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol Cell Biol 18: 753-761, 1998.
MacKenzie SJ and Houslay MD. Action of rolipram on specific PDE4 cAMP phosphodiesterase isoforms and on the phosphorylation of cAMP-response-element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase in U937 monocytic cells. Biochem J 347: 571-578, 2000.
Manganiello V and Elks M. Regulation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes: the role of particulate phosphodiestease in the antilipolytic action of insulin. In: Mechanisms of Insulin Action, edited by Belfrage P, Donner J, and Stralfors P. Amsterdam: Elsevier Science, 1986, p. 147-166.
Manning CD, Burman M, Christensen SB, Cieslinski LB, Essayan DM, Grous M, Torphy TJ, and Barnette MS. Suppression of human inflammatory cell function by subtype-selective PDE4 inhibitors correlates with inhibition of PDE4A and PDE4B. Br J Pharmacol 128: 1393-1398, 1999.
Marx J. Two major signal pathways linked. Science 262: 988-990, 1993.
Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, and Marais R. Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18: 2137-2148, 1999.
Matousovic K, Grande JP, Chini CCS, Chini EN, and Dousa TP. Inhibitors of cyclic nucleotide phosphodiesterase isozymes type-III and type-IV suppress mitogenesis of rat mesangial cells. J Clin Invest 96: 401-410, 1995.
McKenzie FR and Pouyssegur J. cAMP-mediated growth inhibition in fibroblasts is not mediated via mitogen-activated protein (MAP) kinase (ERK) inhibition. cAMP-dependent protein kinase induces a temporal shift in growth factor-stimulated MAP kinases. J Biol Chem 271: 13476-13483, 1996.
Mehats C, Jin SL, Wahlstrom J, Law E, Umetsu DT, and Conti M. PDE4D plays a critical role in the control of airway smooth muscle contraction. FASEB J 17: 1831-1841, 2003.
Mikula M, Schreiber M, Husak Z, Kucerova L, Ruth J, Wieser R, Zatloukal K, Beug H, Wagner EF, and Baccarini M. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J 20: 1952-1962, 2001.
Mischak H, Seitz T, Janosch P, Eulitz M, Steen H, Schellerer M, Philipp A, and Kolch W. Negative regulation of Raf-1 by phosphorylation of serine 621. Mol Cell Biol 16: 5409-5418, 1996.
Nicholson CD, Challiss RAJ, and Shahid M. Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci 12: 19-27, 1991.
Nishida E and Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci 18: 128-131, 1993.
Palmer D and Maurice DH. Dual expression and differential regulation of phosphodiesterase 3A and phosphodiesterase 3B in human vascular smooth muscle: implications for phosphodiesterase 3 inhibition in human cardiovascular tissues. Mol Pharmacol 58: 247-252, 2000.
Peraldi P, Frodin M, Barnier JV, Calleja V, Scimeca JC, Filloux C, Calothy G, and Van Obberghen E. Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP. FEBS Lett 357: 290-296, 1995.
Plevin R, Malarkey K, Aidulis D, McLees A, and Gould GW. Cyclic AMP inhibitors inhibits PDGF-stimulated mitogen-activated protein kinase activity in rat aortic smooth muscle cells via inactivation of c-Raf-1 kinase and induction of MAP kinase phosphatase-1. Cell Signal 9: 323-328, 1997.
Qiu W, Zhuang S, von Lintig FC, Boss GR, and Pilz RB. Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins. J Biol Chem 275: 31921-31929, 2000.
Reinhardt RR, Chin E, Zhou J, Taira M, Murata T, Manganiello VC, and Bondy CA. Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases. J Clin Invest 95: 1528-1538, 1995.
Rybalkin SD and Bornfeldt KE. Cyclic nucleotide phosphodiesterases and human arterial smooth muscle cell proliferation. Thromb Haemost 82: 424-434, 1999.
Sadowski HB, Shuai K, Darnell JE Jr, and Gilman MZ. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science 261: 1739-1744, 1993.
Scotland G, Beard M, Erdogan S, Huston E, McCallum F, MacKenzie S, Peden A, Pooley L, Rena N, Ross A, Yarwood S, and Houslay M. Intracellular compartmentalization of PDE4 cyclic AMP-specific phosphodiesterases. Methods 14: 65-79, 1998.
Sekut L, Yarnall D, Stimpson SA, Noel LS, Bateman-Fite R, Clark RL, Brackeen MF, Menius JA Jr, and Connolly KM. Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation. Clin Exp Immunol 100: 126-132, 1995.
Sevetson BR, Kong X, and Lawrence JC. Increasing cAMP attenuates activation of mitogen activated protein kinase. Proc Natl Acad Sci USA 90: 10305-10309, 1993.
Sherr CJ. Mammalian G1 cyclins. Cell 73: 1059-1065, 1993.
Shitsukawa K, Andersen CB, Richard FJ, Horner AK, Wiersma A, van Duin M, and Conti M. Cloning and characterization of the cyclic guanosine monophosphate-inhibited phosphodiesterase PDE3A expressed in mouse oocyte. Biol Reprod 65: 188-196, 2001.
Sidovar MF, Kozlowski P, Lee JW, Collins MA, He Y, and Graves LM. Phosphorylation of serine 43 is not required for inhibition of c-Raf kinase by the cAMP-dependent protein kinase. J Biol Chem 275: 28688-28694, 2000.
Soderling SH and Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 12: 174-179, 2000.
Sonnenburg WK and Beavo JA. Cyclic GMP and regulation of cyclic nucleotide hydrolysis. Adv Pharmacol 26: 87-114, 1994.
Stork PJ and Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12: 258-266, 2002.
Storm S, Cleveland J, and Rapp U. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene 5: 345-351, 1990.
Swinnen JV, Joseph DR, and Conti M. Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes. Proc Natl Acad Sci USA 86: 5325-5329, 1989.
Takahashi S, Oida K, Fujiwara R, Maeda H, Hayashi S, Takai H, Tamai T, Nakai T, and Miyabo S. Effect of cilostazol, a cyclic AMP phosphodiesterase inhibitor, on the proliferation of rat aortic smooth muscle cells in culture. J Cardiovasc Pharmacol 20: 900-906, 1992.
Vadiveloo PK, Filonzi EL, Stanton HR, and Hamilton JA. G1 phase arrest of human smooth muscle cells by heparin, IL-4 and cAMP is linked to repression of cyclin D1 and cdk2. Atherosclerosis 133: 61-69, 1997.
Vaillancourt RR, Gardner AM, and Johnson GL. B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP. Mol Cell Biol 14: 6522-6530, 1994.
Wechsler J, Choi YH, Krall J, Ahmad F, Manganiello VC, and Movsesian MA. Isoforms of cyclic nucleotide phosphodiesterase PDE3A in cardiac myocytes. J Biol Chem 277: 38072-38078, 2002.
Wu J, Dent P, Jelinek T, Wolfman A, Weber M, and Sturgill T. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262: 1065-1069, 1993.
Yamaki M, McIntyre S, Rassier ME, Schwartz JH, and Dousa TP. Cyclic 3',5'-nucleotide diesterases in dynamics of cAMP and cGMP in rat collecting duct cells. Am J Physiol Renal Fluid Electrolyte Physiol 262: F957-F964, 1992.
Yarwood SJ, Steele MR, Scotland G, Houslay MD, and Bolger GB. The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 274: 14909-14917, 1999.
作者单位：Renal Pathophysiology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905