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Smad signaling cascade in renal mesangial cells upstream of mTOR
Cellular Signalling 21 (2009) 1806–1817
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR Bashier Osman, Anke Doller, El-Sayed Akool, Martin Holdener, Edith Hintermann, Josef Pfeilschifter, Wolfgang Eberhardt ⁎ Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
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Article history: Received 15 April 2009 Received in revised form 13 July 2009 Accepted 29 July 2009 Available online 7 August 2009 Keywords: FK506 binding protein p38 MAP kinase Rapamycin Renal fibrosis Smads TGFβ
a b s t r a c t The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ1 as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Excessive accumulation and deposition of glomerular and interstitial extracellular matrix (ECM) is a histological hallmark of many progressive glomerular diseases accompanied by glomerulosclerosis and interstitial fibrosis [1,2]. In the kidney the mesenchymal mesangial cells (MC) have been identified as the main producers of glomerular ECM and critically contribute to the structural and functional integrity of the glomerulus [3,4]. Fibrotic changes in the glomerulus do also account for a major complication of long-term immunosupressive therapy with calcineurin inhibitors (CNIs) such as ciclosporin A and tacrolimus [5]. An impaired ECM turnover in
Abbreviations: CNI, calcineurin inhibitor; CTGF, connective tissue growth factor; ECM, extracellular matrix; FKBP, FK506 binding protein; MC, mesangial cells; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; PAI-1, plasminogen activator inhibitor-1; SBE, Smad-binding element; siRNA, small interfering RNA. ⁎ Corresponding author. Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Tel.: +49 69 6301 6953; fax: +49 69 6301 7942. E-mail address: [email protected] (W. Eberhardt). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.07.016
addition to increased matrix protein synthesis is mainly attributable to a decrease in ECM degradation through increased synthesis of the intrinsic protease inhibitor plasminogen activator inhibitor-1 (PAI-1) [2]. Transforming growth factor β (TGFβ) upregulates both, matrix genes and matrix protease inhibitors and is one of the most prominent factors triggering the accumulation of ECM. Aberrant production of TGFβ critically contributes to the irreversible alteration of tissue architecture [6]. Besides upregulating collagens and the intrinsic inhibitors of matrix catabolizing proteases TGFβ can induce the expression of connective tissue growth factor (CTGF). CTGF is a member of the CCN (Cyr61/CTGF/Nov) family of matricellular proteins which besides TGFβ have emerged as further potent activators of fibrosis [6]. The critical role of TGFβ in fibrosis is convincingly demonstrated by the successful attenuation of ECM accumulation in different models of acute and chronic renal diseases following TGFβ inhibitory approaches [7,8]. Thereby, induction and activation of the TGFβ/Smad signaling cascade is critical for the initiation of fibrogenic cell responses. TGFβ signaling is initiated by the interaction of the type I and type II TGFβ receptors displaying intrinsic serine/threonine kinase activity. Subsequently, ligand binding causes a recruitment and phosphorylation of receptor-Smads (R-
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Smads), namely Smad 2, and Smad 3 proteins [9,10]. These activated R-Smads heterodimerize with the Co-Smad, mainly Smad 4, to build up a transcriptionally active complex which translocates to the nucleus where it modulates the expression of TGFβ target genes [10]. The macrocyclic lactone rapamycin (sirolimus) has firmly been established as an alternative immunosuppressant which is increasingly used to eliminate or at least lower CNI-induced side effects in the kidney [11]. However, despite this beneficial role rapamycin similar to CNIs has been shown to induce TGFβ expression [12]. Moreover, several reports have implicated a certain nephrotoxic potential of sirolimus especially when applied in combination with high doses of CNIs [12,13]. However, the mechanisms underlying these nephrotoxic actions are largely unknown. In pharmacological terms, rapamycin mainly acts through binding the immunophilin FK binding protein 12 (FKBP12), thereby inactivating the mammalian target of rapamycin (mTOR), a serine/threonine kinase which is critically involved in protein biosynthesis and cell cycle progression [14]. The primary targets of mTOR affected by rapamycin are the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the p70S6 kinase 1 (p70S6K) [14]. In this context, mTOR is a key sensor and integrator of different stimuli induced by nutrients and growth factors [15] acting downstream of the phosphatidylinositol 3-kinase (PI3Kinase)/AKT pathway [14]. Moreover, synthetic rapamycin derivatives are currently tested as potential anticancer agents in several preclinical and clinical studies [14]. Since similar to rapamycin, TGFβ-triggered inhibition of cell cycle regulators can impair cell cycle progression, a functional cross-talk between the mTOR and Smad signaling devices has been identified in several human cancer cells but also in nontransformed cells [16–18]. Based on our previous findings that CNIs via an activation of latent TGFβ initiate Smad-driven gene expression in renal mesangial cells [19], we aimed to elucidate whether rapamycin would interfere with TGFβ/Smad signaling and thereby modulate fibrogenic gene cascades. 2. Materials and methods 2.1. Materials Human recombinant TGFβ1 was purchased from Cell Concepts GmbH (Umkirch, Germany). A rabbit pan-specific TGFβ antibody and the Quantikine rat TGFβ1 immunoassay kit were purchased from R & D Systems (Wiesbaden-Nordenstadt, Germany). Rapamycin was from Axxora GmbH (Lörrach, Germany). The Alk-5 inhibitor, U0126, SP600125 and SB203580 were obtained from Calbiochem (Schwalbach, Germany). Diphenylene iodonium (DPI), N-acetyl cysteine (NAC), catalase, polyethylene glycol-superoxide dismutase (PEGSOD), xanthine oxidase, hypoxanthine, rotenone, insulin and phorbol 12-myristate 13-acetate (PMA) were derived from Sigma Aldrich (Taufkirchen, Germany). Antibodies specifically raised against phospho-Smad 2 (Ser 465/467), phospho-Smad 3 (Ser 423/425), total Smad-2, total Smad 3, Smad-2/Smad-3, phospho-p38 MAPK (Thr 180/ Tyr 182), p38 MAPK, mTOR, phospho-p70S6 kinase (Thr 389), phospho-4E-BP (Thr 37/46) and siRNA against mTOR were derived from Cell Signaling (Frankfurt am Main, Germany). Antibodies against CTGF, HDAC-1, PAI-1, Smad-4, TGFβ-RII, β-actin, as well as anti-rabbit and anti-mouse horseradish peroxidase-linked and anti-mouse Alexa 488-linked secondary antibodies, IgG and a predesigned siRNA against FKBP12 were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Customer siRNA against the TGFβ type II receptor was synthesized from Eurogentech (Seraing, Belgium). 5- (and -6) chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA), a cell-permeable indicator for ROS was from Molecular Probes (Karlsruhe, Germany) and 125I-labeled human recombinant TGFβ1 was purchased from Amersham Biosciences (Freiburg, Germany).
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2.2. Cell culture Rat glomerular MC were grown as described previously [20]. All cell culture media and supplements were purchased from LifeTechnologies (Karlsruhe, Germany). 2.3. Western blot analysis For detection of CTGF, FKBP12, mTOR, PAI-1, TGFβ-RII, phosphorylated and total Smads 2 and Smads 3, phosphorylated p38 and total p38, phosphorylated p70S6 kinase and phosphorylated 4E-BP1, whole cell lysates were prepared as described previously [21]. Total cell extracts containing 50 µg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE and Western blot analysis was performed by using standard procedures. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes before immunodetection. Nuclear extracts (20–50 µg) from MC were used for the assessment of the nuclear import of phosphorylated Smads as described previously [19]. 2.4. Indirect immunofluorescence microscopy Nuclear import of Smad 4 was analyzed by using indirect immunofluorescence microscopy as described previously [22] and images were analyzed using an inverse immunofluorescence microscope, BZ-7000 (Biozero, Keyence, Neu-Isenburg, Germany) equipped with a Zeiss Apo 20×/0.75 NA objective and images were analyzed by using the BZ-H1TL software from Biozero. 2.5. Small interfering (si)RNA Gene silencing was performed using small interfering (si)RNAs for human mTOR (Cell Signaling) and for FKBP12 (Santa Cruz Biotechnology). A siRNA against the TGFβ type II receptor was synthesized according to rat-specific sequence [23]. Subconfluent MC were transfected for 48 h with 25–50 nM of siRNA by using the Oligofectamine reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. 2.6. Determination of TGFβ1 antigen levels in conditioned media of MC The amount of TGFβ1 in cell culture supernatants was quantified by the Quantikine immunoassay kit from R&D Systems raised against rat TGFβ1. Confluent MC (1.0 to 1.5 × 106 cells) grown in six-well plates were preincubated in DMEM without FCS for 24 h before stimulation with or without rapamycin. 10–20 µl of conditioned media were directly transferred to the microtest strip wells of the ELISA plate. Omission of a prior acidification of samples allowed a specific detection of active TGFβ1. All further procedures were performed following the manufacturer's instructions. The absorbances at 450 nm were measured in a microtest plate spectrophotometer and antigen levels were determined by appropriate calibration curves using human TGFβ1 as a standard. 2.7. TGFβ neutralization experiments The impact of TGFβ on the rapamycin-induced cell responses was tested by the use of a neutralizing pan-specific TGFβ antibody (R & D Systems) and was applied as described previously [22]. 2.8. TGFβ binding competition assay Direct competition of rapamycin with recombinant TGFβ1 for binding to the TGFβ receptor was assessed by a TGFβ binding assay using 125I-labeled TGFβ1 [24]. Briefly, subconfluent MC were washed with cold PBS/EDTA (0.5 mM) before cells were trypsinized and
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resuspended in ice-cold binding buffer containing RPMI, 25 mM Hepes and 0.2% BSA. Aliquots of 340 µl (1 × 106 cells) were distributed into prelubricated polypropylene tubes and increasing concentrations of non-radiolabeled recombinant TGFβ1 or, alternatively, different amounts of rapamycin were added. The binding assay was initiated by addition of a 50 nM [125I] -TGFβ1 stock solution (final concentration: 80 pM) and reactions were incubated at 4 °C for 4 h. Finally, 150 µl aliquots of the binding assay were laid onto 150 µl cold silicon oil and unbound TGFβ1 subsequently removed by centrifugation (13,000 rpm). Finally, cell-bound radioactivity was measured in a γ-counter (Packard). 2.9. Reporter plasmids and transient transfection The transactivation of a Smad-driven reporter gene was measured by using the “pSBE4-Luc” reporter plasmid and “pMBE6-Luc” a corresponding luciferase vector which instead of the wild-type SBE contains three copies of a mutated SBE (GTTTATAC) [25]. Both vectors were kindly provided by Dr. Vogelstein (Johns Hopkins Oncology Center; Baltimore, Maryland, USA). A 1.0 kb promoter fragment from the PAI-1 gene (pGL-PAI-1) was kindly provided by Daniel Rifkin [26]. The pGL-CTGF plasmid which contains a 3.0 kb upstream promoter fragment of CTGF and which includes a functional SBE in a region between –173 and –166 [27] was a kind gift of R. Goldschmeding (Utrecht, The Netherlands). Transient transfections of rat MC were done by using the Effectene reagent (Quiagen) and measurements of luciferase activities were done as described previously [22]. 2.10. EMSA Preparation of nuclear extracts from rat MC and EMSA was performed as described previously [21]. For EMSA analysis a Smad consensus oligonucleotide encompassing a wild-type core Smadbinding element (SBE) (Santa Cruz Biotechnology) was used. DNAprotein complexes were separated from unbound oligonucleotide by electrophoresis through native 4.5% polyacrylamide gels and gels run in 0.5 x Tris-borate EDTA. Supershift analysis was done by preincubation of 1 µg supershift antibody to the binding reaction 30 min prior to the addition of the radioactively labeled oligonucleotides. 2.11. Detection of intracellular ROS production Measurement of intracellular formation of ROS was done by using the cell-permeable indicator CM-H2DCFDA. For ROS measurement, serum-starved MC were stimulated for 1 h and after having removed the stimulation media, cells were washed once with Hank's balanced salt solution (HBSS). Trypsinized cells were grounded by a short centrifugation (1000 rpm), and cell pellets were resuspended in CMH2DCFDA (5 µM in HBSS) and incubated in the dark at 37 °C for 30 min. Cells were washed and returned to HBSS media and ROS generation was
Fig. 2. Rapamycin activates TGFβ-inducible gene promoters. Subconfluent MC were transfected with 0.4 µg of either pSBE4-Luc (A, black bars) containing 4 tandem wildtype SBEs or alternatively, with pMBE6 (A, open bars) containing 6 point-mutated SBEs (A), or alternatively, with 0.4 µg of a 1.0 kb fragment of the pGL-PAI-1 promoter (B), or, alternatively with the same amount of a 3.0 kb fragment of the CTGF promoter (C). Transfection of the plasmids was supplemented by a cotransfection with 0.1 µg of RLCMV, coding for Renilla luciferase. After the transient transfection MC were treated for 16 h with either vehicle, or with the indicated concentrations of rapamycin before cell lysates were assayed for luciferase activities. The values for beetle luciferase were related to the values for Renilla luciferase and are depicted as relative light units (RLU). Data represent means ± S.D. (n = 6) of triplicate experiments ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 compared with vehicle.
subsequently determined by measuring the mean fluorescence intensity by flow cytometry on a FACS Calibur using the CellQuest Pro 5.2 software (BD Biosciences). Alternatively, to visualize ROS generation MC were grown on cover slips in 12-well plates before cells were treated in a similar way as described for FACS analysis. Culture dishes were
Fig. 1. Rapamycin activates the Smad signaling cascade in MC. (A) Quiescent MC were stimulated with either vehicle (-) or with rapamycin (50 ng/ml) for the indicated time periods before cells were lysed for Western blot analysis. Total protein (50 µg) was subjected to Western blot analysis and successively probed with an anti-phospho-Smad 2-specific antibody (p-Smad 2) and with an antibody raised against total Smad 2 (Smad 2). Data in the lower panel of (A) show a densitometric analysis of phospho-Smad 2 relative to total Smad 2 levels and represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 versus unstimulated control. (B) Dose-dependent nuclear import of phosphorylated R-Smads induced by rapamycin. MC were stimulated for 60 min with either vehicle (-) or the indicated concentrations of rapamycin before cells were fractionated for nuclear extracts. 30 µg of nuclear extracts were successively probed with a specific anti-phospho-Smad 2 and anti-phospho-Smad 3 antibody. Loading of equal amounts of nuclear extracts was ascertained by reprobing the blots with an anti-HDAC-1 antibody. Data in the lower panel of (B) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and are depicted as fold induction compared with nuclear phospho-Smad levels under unstimulated conditions. Data represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01, ⁎⁎⁎p ≤ 0.005 versus unstimulated control. (C, upper panel) Rapamycin-mediated entry of Smad 4 into the nucleus by indirect immunofluorescence. After simulation of cells with vehicle (control) or with the indicated concentration of rapamycin for 60 min, MC were fixed and stained with anti-Smad 4 and anti-mouse Alexa-488 antibodies, respectively before DAPI was added in order to counterstain cell nuclei (blue panel). (Lower panel) Densitometric analysis of nuclear FITC staining with data representing means ± S.D. (n = 3) ⁎⁎⁎p ≤ 0.005 versus unstimulated controls. (D) 5 µg of nuclear extracts from MC treated with either vehicle (-), or with rapamycin (+, 50 ng/ml), or, with TGFβ (+, 10 ng/ml) were subjected to EMSA (left panel) using 32P-labeled consensus SBE. (Right panel) Supershift analysis of rapamycin-induced SBE-bound complexes. For supershift analysis the indicated antibodies were added 30 min prior to the addition of the radiolabeled oligonucleotide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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transferred to an inverted immunofluorescence microscope (Zeiss) and images were analyzed by using the ImageJ software. 2.12. Statistical analysis Results are expressed as means ± S.D. Statistical analysis was performed using the Student's t-test and for multiple comparisons the ANOVA test for significance. The data are presented as relative induction compared with control conditions or compared with rapamycin-stimulated values. P ≤ 0.05, ≤ 0.01, and ≤ 0.001 were considered significant. 3. Results 3.1. Rapamycin activates the TGFβ Smad signaling cascade In a first attempt, we tested whether rapamycin can modulate the Smad signaling cascade and measured the phosphorylation state of the receptor (R) Smad indicative for an activation of the profibrotic Smad signaling cascade [10]. Stimulation of MC with rapamycin (50 ng/ml) induced a rapid phosphorylation which was maximal at 60 min and declined thereafter. This was not associated with the change in the total Smad 2 content (Fig. 1A). Since the phosphorylated R-Smads assemble with Smad 4 to build up a transcriptionally active complex, we next monitored for a nuclear import of these R-Smad proteins. As shown in Fig. 1B, stimulation of MC with rapamycin caused a dose-dependent increase in phosphorylated Smad 2/Smad 3 protein with a maximal increase in nuclear phospho-Smad 2/3 levels was observed with rapamycin concentrations of 10 ng/ml or higher (Fig. 1B). Activation of the TGFβ/Smad signaling cascade by rapamycin
is furthermore indicated by a significant increase in nuclear Smad 4 levels in MC stimulated for 60 min with rapamycin as monitored by indirect immunofluorescence microscopy (Fig. 1C). To test for functional effects of Smad phosphorylation by rapamycin, we monitored the DNA binding capacity of nuclear proteins to a radioactively labeled oligonucleotide bearing a consensus SBE by EMSA. As shown in Fig. 1D, treatment of MC with rapamycin caused a marked increase in DNA binding of a TGFβ inducible complex (Fig. 1D, left panel). By contrast, the constitutive binding of two faster migrating complexes was not affected by rapamycin treatment (Fig. 1D, left panel). The addition of antibodies raised against a common epitope of Smad 2 and Smad 3 (“anti-Smad 2/3”), or alternatively, of specific antibodies against Smad 3 (a.-Smad 3), or against phosphorylated Smad 2 (“a.-p.-Smad 2”) specifically impaired the DNA binding of the upper, rapamycin-inducible complex without affecting the constitutively bound complexes (Fig. 1D, right panel). Interestingly, only the addition of the anti-Smad 2/3 antibody, which recognizes a common epitope of Smad 2 and -3 caused a supershifted band (arrow in Fig. 1D, right panel) whereas all the other Smad antibodies applied reduced the DNA binding of the rapamycininduced complex without causing a real supershift which indicates that the binding of these antibodies somehow interferes with the DNA binding of corresponding Smad complexes (Fig. 1D, right panel). 3.2. SBE confers rapamycin induction of TGFβ inducible gene promoters Next, we tested whether rapamycin could also activate a TGFβ inducible control promoter bearing a tandem of four copies of SBE consensus motifs (pSBE4-Luc) [25]. Stimulation of MC with rapamycin for 12 h caused a significant increase in luciferase activity (Fig. 2A,
Fig. 3. Rapamycin induces expressions of PAI-1 and CTGF by an involvement of the TGFβ type II receptor. (A) Quiescent MC were stimulated with either vehicle (-), or with 50 ng/ml of rapamycin (+) for the indicated time periods before cells were harvested for total protein extracts. Total protein (50 µg) was subjected to Western blot analysis and successively probed with anti-PAI-1, anti-CTGF antibodies and with β-actin-specific antisera. Data in the lower panel of (A) show a densitometric analysis of PAI-1 and CTGF protein levels (filled bars) 8 h after the addition of rapamycin and represent means ± S.D. (n = 3) ⁎⁎p ≤ 0.01, ⁎⁎⁎p ≤ 0.005 versus unstimulated control (open bars). (B) Quiescent MC were transfected with either a control siRNA (control-siRNA) or with siRNA duplexes specific for rat TGFβ-RII (TGFβ-RII-siRNA). After transfection MC were serum-starved for 16 h before cells were stimulated with either vehicle (-), with the indicated amounts of rapamycin or, alternatively, with TGFβ (+, 10 ng/ml) for 8 h. Western blots were successively probed with antiTGFβ-RII, anti-PAI and anti-CTGF-specific antisera, respectively. (C) Activation of Smad 2 is abrogated after attenuation of the TGFβ-RII. MC were transfected as described in (B). After transfection cells were treated for 30 min with vehicle (-), or with different concentrations of rapamycin, or with TGFβ (+, 10 ng/ml) as indicated. Western blots show the total levels of TGFβ-RII, Smad 2 and phosphorylated Smad 2 (p-Smad 2). Data shown are representative of two independent experiments giving similar results.
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filled bars). By contrast, a reporter gene which instead of the wildtype SBE motifs contained a tandem of mutated SBE motifs (pMBE6Luc) was not induced by rapamycin (Fig. 2A, open bars) thus indicating that the promoter activation by rapamycin is mediated via SBEs. In order to prove activation of a native SBE containing promoter by rapamycin, we next tested whether rapamycin could activate a
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1.0 kb fragment of the PAI-1 gene promoter (pGL-PAI-1) which confers TGFβ responsiveness via a functional SBE and which is commonly used as a native control promoter of SBE-dependent transcription [26]. Rapamycin caused a dose-dependent induction in PAI-1 promoter activity with maximal effects at 100 ng/ml (Fig. 2B). As a further target gene which is induced upon activation of the TGFβ/Smad signaling cascade we tested a promoter fragment of the
Fig. 4. Activation of R-Smads depend on TGFβ and ALK-5 kinase activity. (A) Quiescent MC were pretreated for 60 min with vehicle (+ vehicle) or with either 20 µg of a pan-specific TGFβ antiserum (+ anti-TGFβ a.b.), 20 µg of control IgG (+IgG) or with 100 nM of ALK-5 inhibitor. Thereafter, MC were stimulated for 60 min with either vehicle (-) or with rapamycin (50 ng/ml) as indicated. 30 µg of nuclear extracts were subjected to SDS-PAGE and probed successively with an anti-phospho-Smad 2, anti-phospho-Smad 3 antibody and with an anti-HDAC-1-specific antibody. Data in the right panel of (A) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 versus unstimulated control; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.005 versus rapamycin-stimulated conditions. (B) Rapamycin induces rapid activation of latent TGFβ1. (Left panel) Quiescent MC were treated for 60 min with vehicle (control) or with rapamycin (Rapa, 50 ng/ml) before cell supernatants were assessed for active TGFβ1 by Quantikine ELISA as described in Section 2. Data represent means ± S.D. (n = 3), ⁎⁎ p ≤ 0.01 versus control. (Right panel) Activation of R-Smads by recombinant TGFβ1. Quiescent MC were stimulated for 30 min with either vehicle (-), or with the indicated concentrations of TGFβ1 before cells were lysed for nuclear extraction. 30 µg of nuclear extracts were subjected to SDS-PAGE and successively probed with an anti-phospho-Smad 2, anti-phospho-Smad 3 antibody and with an anti-HDAC-1-specific antibody. Data shown are representative of two independent experiments with similar results. (C) Rapamycin does not compete with TGFβ1 for receptor binding. Equal amounts of trypsinized MC were incubated with [125I]-TGFβ1 (80 pM), in the absence (-) or presence of the indicated concentrations of unlabeled TGFβ1 or rapamycin (Rapa) and receptor binding was allowed for 4 h. The cell-bound radioactivity was counted in a γ-counter and is blotted versus different TGFβ, or rapamycin concentrations. Data represent means ± S.D. (n = 3), #p ≤ 0.05, ##p ≤ 0.01 versus vehicle.
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Fig. 5. Rapamycin-mediated R-Smad and TGFβ activation depend on ROS. (A) Quiescent MC were treated for 60 min with vehicle (-), or rapamycin (Rapa, +, 50 ng/ml) in the absence or presence of either N-acetyl cysteine (NAC, 5 mM) or diphenylene iodonium (DPI, 10 µM) before nuclear extracts from cells were assessed for phospho-Smad 2 and phospho-Smad 3 contents. Loading of equal amounts of nuclear extracts was ascertained by assessment of nuclear HDAC-1 level. (Left panel) Data represent means ±S.D. (n= 3), *p≤0.05 versus unstimulated control; # p≤0.05 versus rapamycin-induced conditions. (B) MC were stimulated with either vehicle (-) or with rapamycin (Rapa, +, 50 ng/ml) in the absence or presence of either catalase (500 U/ ml), PEG-SOD (100 U/ml), allopurinol (100 µM), or rotenone (10 µM) for 60 min before nuclear extraction. Subsequently, the content of phosphorylated R-Smads in nuclear extracts (30 µg) was tested by Western blot analysis. The data in the lower panel of (B) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means± S.D. (n = 3) ⁎p ≤0.05, ⁎⁎p ≤0.01 versus unstimulated control; #p ≤0.05, versus the respective rapamycin-stimulated conditions. (C) Activation of latent TGFβ1 by rapamycin depends on ROS. Quiescent MC were treated for 60 min with vehicle (control), or with rapamycin (Rapa, 50 ng/ml) in the absence or presence of NAC (5 mM) before cell supernatants were assessed for active TGFβ1. Data are expressed as means± S.D. (n= 3), ⁎⁎p≤ 0.01 versus unstimulated control; ##p≤0.01 versus rapamycin-induced conditions. Fig. 6. Rapamycin induces ROS production and ROS activate R-Smads. MC were treated for 60 min with vehicle (control), rapamycin (Rapa, 50 ng/ml) in the presence or absence of NAC before radical formation was determined by dichlorofluorescein (DCF) formation. (A) Quiescent MC were grown on cover glass slides and incubated with NAC (5 mM) for 60 min before treated with rapamycin (Rapa, 50 ng/ml) for additional 60 min. Subsequently, cells were washed with HBSS and incubated in the dark for 30 min in 5 µM CM-H2DCFDA (in HBSS). ROS generation is visualized by inverse fluorescence microscopy and quantified by the Image software. Data represent means±S.D. (n=3), ⁎⁎p≤0.01 versus vehicle; ##p≤0.01 versus rapamycin-induced conditions. (B) Accumulation of intracellular ROS measured by FACS using the CM-H2DCFDA probe. Serum-starved MC were stimulated for 60 min with either vehicle (control), different concentrations of rapamycin as indicated, or, alternatively with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) used as a positive control, before cells were trypsinized and incubated with H2DCFDA. Fluorescence intensity was measured by using the CellQuest Pro 5.2 software (BD Biosciences) and data are representative of two independent experiments giving similar results. (C) Activation of R-Smads by the ROS generating xanthine oxidase system. Quiescent MC were treated for 60 min with vehicle (-), or with hypoxanthine (50 µM) plus the indicated concentrations of xanthine oxidase (HXXO). Nuclear extracts (30 µg) were subjected to SDS-PAGE and immunoblotted using the indicated antibodies. The data in the lower panel of (C) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means±S.D. (n=3) ⁎p≤0.05, ⁎⁎p≤0.01 versus unstimulated control.
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connective tissue growth factor (CTGF) gene, which similar to PAI-1, bears a functional SBE [27]. MC transiently transfected with pGLCTGF displayed a similar induction profile in luciferase activity as
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with pGL-PAI-1 (Fig. 2C). Collectively, these data demonstrate that rapamycin via increased Smad binding to SBEs activates TGFβinducible gene promoters.
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Fig. 7. Rapamycin-mediated R-Smad activation depends on p38 activity. (A) Quiescent MC were pretreated for 60 min with vehicle (+ vehicle), SB203580 (10 µM), SP600125 (10 µM), or U0126 (20 µM). Thereafter cells were left untreated (-) or stimulated for further 60 min with rapamycin (Rapa, 50 ng/ml). The data in the right panel show a densitometric analysis of the nuclear phospho-Smad 2 (filled bars) and phospho-Smad 3 (open bars) levels relative to nuclear HDAC-1 contents and represent means ± S.D. (n = 3) ⁎p ≤ 0.05 versus unstimulated control; #p ≤ 0.05, versus rapamycin-stimulated conditions. (B) Time-dependent activation of p38 by rapamycin. Quiescent MC were treated with vehicle (-), or with rapamycin (Rapa, 50 ng/ml) for the indicated time periods (left panel) or, alternatively, for 20 min either in the absence (+ vehicle) or, presence of NAC (5 mM), or ALK-5 inhibitor (100 nM) before cells were stimulated for further 30 min with rapamycin (right panel). Total protein extracts (50 µg) were subjected to Western blot analysis and successively probed with an anti-phospho-specific p38 antibody (p-p38) or, alternatively, with an antibody raised against total p38 (p38). Data shown are representative of two independent experiments giving similar results.
3.3. Rapamycin induces profibrotic gene expression via the TGFβ type II receptor Next, we tested whether the activation of SBE-driven promoters results in an upregulation of corresponding proteins. Stimulation of MC with rapamycin (50 ng/ml) caused a time-dependent increase in the steady-state level of PAI-1 and CTGF protein with a maximal increase observed at 8 h (Fig. 3A). To further test the possible involvement of the TGFβ signaling device in rapamycin-mediated upregulation of both genes we first applied siRNA specific for TGFβRII, which in contrast to TGFβ-RI exerts a direct ligand binding, and which in turn, completely prevented the stimulatory effects of rapamycin (Fig. 3B). Additionally, silencing of TGFβ-RII prevented rapamycin-induced Smad 2 phosphorylation thus indicating that the TGFβ type II receptor is indispensably involved in rapamycin-induced Smad activation (Fig. 3C). 3.4. Rapamycin activates R-Smads via TGFβ-RI kinase and extracellular TGFβ To further delineate the mechanism by which rapamycin activates R-Smads, we applied an activin receptor-like kinase (ALK)-5 inhibitor [28]. Incubation with 100 nM of the ALK-5 inhibitor decreased the rapamycin-induced Smad 2 and Smad 3 phosphorylation to a level which was beneath basal Smad phosphorylation (Fig. 4A). An involvement of extracellular TGFβ in the rapamycin-dependent response is furthermore shown by the effects of a neutralizing panspecific TGFβ antibody. Neutralization of TGFβ completely prevented the rapamycin-mediated increase in R-Smads phosphorylation but, in contrast to the effects caused by the ALK-5 inhibitor, it did not affect basal Smad 2/3 phosphorylation (Fig. 4A). To confirm the activation of
latent TGFβ by rapamycin, we measured the level of active TGFβ1 in conditioned media of rapamycin-treated MC by using a TGFβ1-specific ELISA without a prior acidification step. Treatment of MC with rapamycin for 60 min caused a significant increase in TGFβ1 levels (Fig. 4B, left panel), to amounts sufficient to induce R-Smads phosphorylation (Fig. 4B, right panel). Next, we tested whether rapamycin can directly bind to the TGFβ receptor and to this end we made use of a TGFβ binding assay using 125 I-labeled recombinant TGFβ1 [24]. Addition of unlabeled recombinant TGFβ dose-dependently competed with 125I TGFβ for receptor binding whereas rapamycin even at high doses did not compete thus indicating that rapamycin does not directly bind to the TGFβ-receptor (Fig. 4C). 3.5. Activation of R-Smads depends on ROS Based on our previous finding that CNIs induce Smad-2 activation via an increase in ROS production [19], we assessed whether rapamycin-induced R-Smad activation similarly would depend on ROS. To this end, MC were stimulated in the presence of the ROS scavenger N-acetyl cysteine (NAC, 5 mM), or alternatively, with DPI (10 µM), an inhibitor of NADPH oxidases. Whereas NAC caused an almost complete inhibition of rapamycin-induced R-Smad activation DPI had no significant effect on the levels of Smad 2 and -3 (Fig. 5A). We additionally tested further ROS inhibitors: catalase which decomposes H2O2, the membrane permeable superoxide dismutase PEG-SOD, allopurinol an inhibitor of the xanthine oxidase, and finally rotenone an inhibitor of the mitochondrial respiratory chain. Interestingly, only PEG-SOD caused a significant reduction in rapamycin-induced R-Smad 2 phosphorylation (Fig. 5B) thus
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indicating that mainly an increase in the intracellular superoxide generation is critical for Smad activation by rapamycin. Functionally, incubation with NAC almost completely blocked the rapamycintriggered release of active TGFβ1 (Fig. 5C).
depletion of FKBP12 thus indicating that Smad activation by rapamycin depends on the FKBP12 (Fig. 8B).
3.6. Rapamycin induces ROS formation in MC
Previously, we have shown that CNIs, at low nanomolar concentrations, can induce TGFβ-Smad signaling cascades in renal MC and thereby increase the expression of fibrogenic genes [19]. Here we provide a comprehensive analysis which demonstrates that most components of the TGFβ-Smad signaling cascade are similarly activated by rapamycin by a mechanism which is triggered by ROS. The immunosuppressive actions of rapamycin in most cases synergize with those of CNIs but, in contrast to CNIs, rapamycin inhibits lymphocyte proliferation via an IL-2 independent mechanism [32,33]. Regardless of this difference, an increase in TGFβ expression by both classes of immunosuppressants has been coincidently described by several reports [12,34]. Here, we demonstrate that rapamycin similar to CNIs induces a rapid activation of latent TGFβ which in turn leads to an activation of the profibrotic Smad signaling cascade. This cascade is activated already within minutes thus excluding the fact that stimulatory effects on fibrogenic gene expression are due to an increase in TGFβ gene expression. Our observations are in agreement with another report demonstrating activation of the bonemorphogenic protein 4 (BMP-4) by rapamycin in different prostate cancer cell lines [17]. All BMPs, in contrast to TGFβ, specifically induce ALK3 and -6 and thereby activate Smads 1, -5 and -8 [35–37]. A complex cross-talk between the TGFβ-Smad and rapamycin-sensitive signaling cascades has been implicated by several studies [15,16,38,39]. Importantly,
To further prove an increase in ROS formation by rapamycin, we next measured cellular radical formation by applying the cellpermeable ROS acceptor CM-H2-DCFDA which was either detected by immunofluorescence microscopy (Fig. 6A) or, alternatively, by FACS analysis (Fig. 6B). Independent of which approach we used, rapamycin caused a significant increase in ROS production (Fig. 6A, B) to an extent which was comparable to that evoked by the phorbol ester PMA (Fig. 6B). Importantly, preincubation with NAC almost completely abrogated the rapamycin-triggered ROS generation (Fig. 6A). Furthermore, applying the superoxide generating xanthine oxidase/hypoxanthine (HXXO) system caused a dosedependent and significant increase in Smad 2 and 3 phosphorylation (Fig. 6C). 3.7. Involvement of p38 MAPK in rapamycin-induced Smad activation Since an activation of several different MAPKs has been implicated in TGFβ signaling [29–31], we next investigated the impact of different MAPKs by using specific pharmacological inhibitors. We found that only preincubation with SB203580 (10 µM), an inhibitor of the p38 pathway, abrogated rapamycin-induced Smad 2 and Smad 3 phosphorylation whereas neither the JNK inhibitor SP600125 nor the ERK inhibitor U0126 had any suppressive effects on R-Smad activation by rapamycin (Fig. 7A). Correspondingly, we found a rapid and transient activation of the p38 MAPK by rapamycin with a peak in phospho p38 levels at 15 min after administration of rapamycin (Fig. 7B, left panel). Interestingly, the activation of p38 MAPK is prevented by both, the ALK-5 inhibitor and NAC thus indicating that the TGFβ-RI kinase and ROS generation are upstream of rapamycintriggered p38 MAPK activation (Fig. 7B, right panel).
4. Discussion
3.8. Rapamycin elicits Smad signaling independent of mTOR inhibition but requires FKBP12 In the next set of experiments, we asked whether the activation of TGFβ-Smad signaling by rapamycin is independent of its capacity to inhibit the serine/threonine kinase mammalian target of rapamycin (mTOR) and the downstream signaling which functionally results in the blockade of protein biosynthesis and cell cycle progression [32]. To this end we first tested whether rapamycin can block the insulininduced activation of the p70S6 kinase and the eukaryotic initiation factor 4E binding protein-1 (4E-BP1), two well-characterized mTOR effectors, using phosphorylation-specific antibodies. MC treated for 60 min with different concentrations of insulin, showed a dosedependent increase in p70S6 kinase phosphorylation (p-p70S6K) and, although to a lower extent, in 4E-BP1-phosphorylation (p-4E-BP1) when compared with vehicle-treated cells (Fig. 8A). Coincubation with rapamycin resulted in a total inhibition of insulin-induced p70S6 kinase phosphorylation and a clear reduction in the phospho 4E-BP levels which both signifies that in MC, rapamycin can negatively interfere with mTOR signaling. By contrast, the levels of phosphorylated p85S6 kinase levels (p-p85S6K) were not affected by rapamycin (Fig. 8A). Next, we tested for a possible involvement of FKBP12 the intrinsic binding protein of rapamycin. For this purpose we utilized small interference (si)RNA against FKBP12. MC transfected with siRNA against FKBP12 displayed a strong reduction in FKBP12 expression when compared with control siRNA transfected cells (Fig. 8B). Interestingly, phospho-Smad 2 levels were strongly impaired after
Fig. 8. Rapamycin-induced Smad activation depends on the FKBP12. (A) Rapamycin inhibits insulin-induced p70S6K and 4E-BP1 activation in rat MC. Quiescent MC were preincubated with rapamycin (Rapa, +, 50 ng/ml) for 30 min before stimulated with the indicated concentrations of insulin for 60 min before cells were harvested for total protein extraction. 30 µg of protein extract was subjected to SDS-PAGE and successively probed with an anti-phospho-p70S6K, anti-phospho-4E-BP1 and anti-β-actin antibody. (B) Rapamycin-induced Smad-2 activation depends on the FKBP12. MC were transfected with siRNA duplexes of mouse FKPB12 (FKBP12), or with a none-generelated control siRNA (control), or with the transfection reagent (mock). After transfection, MC were treated with either vehicle (-), or with rapamycin (Rapa, 50 ng/ml) for 60 min before cells were harvested for total protein extraction. To prove a knockdown of FKBP12, 30 μg οf protein extract was probed with anti-FKBP12 antibody by Western blot analysis. In parallel 30 µg of protein extracts were additionally probed with a phοsphο-Smad 2 and an antibody raised against total Smad 2. The data shown in the figure are representative of two independent experiments giving similar results.
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rapamycin through binding to its intracellular receptor, FKBP12 can reverse the inhibitory effects of FKBP12 on TGFβ type I receptor phosphorylation thus endowing a constitutive TGFβ signaling, resulting from the intimate tendency of both types of TGFβ receptors to interact with each other [38]. However, a similar mechanism seems not to be operative in MC, since depletion of FKBP12 by siRNA attenuation instead of elevating Smad signaling, completely prevented R-Smad activation by rapamycin (Fig. 8B). In contrast to reports on tumor cells [16,40], the rapamycin-induced TGFβ-Smad signaling observed in MC seems rather to be independent of mTOR signaling, since depletion of mTOR did not affect the rapamycin-induced activation of Smads (data not shown). Functionally, induction of the Smad-driven promotors of CTGF and PAI-1 by rapamycin strongly indicates that in renal MC the activation of TGFβ/Smad signaling by rapamycin is sufficient to elicit fibrogenic cell responses. In a further attempt to delineate the underlying mechanisms of how rapamycin induces Smad signaling we found a sequential activation of ROS and the p38 MAPK with both signals being indispensable for activation of R-Smads. ROS seem to act upstream of both, latent TGFβ activation and p38 activation as depicted in Fig. 9. Interestingly, in renal MC, rapamycin, similar to CNIs, activates p38 in a TGFβ receptor dependent manner [19] which is in contrast to the TGFβ receptor independent activation of different MAPKs by TGFβ reported by several studies [30,41]. Thus, our data suggest that rapamycin-triggered p38 MAPK acts through the TGFβ-receptor to induce the Smad signaling cascade (Fig. 9). Importantly, a critical role of ROS formation has also been proposed for cell responses induced by ciclosporine A [42] whereas
for rapamycin some reports have even described an inhibitory capacity of ROS [43]. We have observed that the rapid increase in ROS production and downstream Smad responses by rapamycin is diminished by a cell-permeable SOD mimetic (Fig 5B). Furthermore, the ineffectiveness of DPI, the xanthine oxidase inhibitor allopurinol and rotenone furthermore indicates that superoxide generation does not originate from one of the major cellular superoxide generating systems including NAD(P)H oxidase, xanthine oxidase or mitochondrial NAD(P)H dehydrogenases, respectively. Notably, in contrast to the ciclosporine-dependent increase in ROS production and subsequent Smad activation which seems independent from binding to ciclophilin [19], our results from siRNA experiments clearly demonstrate that FKBP12 is indispensable for rapamycin-mediated Smad activation (Fig. 8B). A challenging issue which needs to be addressed in the future is the identification of the intracellular ROS generator targeted by the rapamycin/FKBP12 complex. To the best of our knowledge our data for the first time demonstrate a rapid redoxsensitive increase in active TGFβ by rapamycin. Mechanistically, the release of TGFβ from the latent TGFβ binding protein (LTBP) complex can be achieved by proteolytic as well as by nonproteolytic events including the modulation by thrombospondin as well as different intergrins [44,45]. Importantly, the physiological activation of latent TGFβ is a target of redox-sensitive mechanisms. In this context, oxidation of specific amino acids in the latency conferring peptide can cause a conformational change in the latency-associated protein β (LAPβ) thus allowing a rapid release of TGFβ [46]. These results warrant further investigation in future studies to definitely unravel whether oxidation of LAPβ is involved in the rapamycin-triggered
Fig. 9. Schematic representation of profibrotic Smad signaling rapidly induced by rapamycin. Rapamycin via binding to the FKBP12 by a still unknown mechanism generates ROS which extracellularly induces a rapid release of latent TGFβ and subsequent TGFβ receptor triggered signaling. Thereby, the Smad 2/3 dependent pathway is activated through p38 and additional TGFβ-triggered signaling devices. The association of R-Smads with Co-Smad (Smad-4) leads to an active Smad transcription factor complex which specifically binds to a cognate SBE, present in the promoters of CTGF and PAI-1 genes. Abbreviations: CTGF, connective tissue growth factor; 4E-BP1, eukaryotic initiation factor binding protein; LAP, latency-associated protein; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; p70S6K, p70S6 kinase; ROS, reactive oxygen species; SBE, Smad-binding element; TGFβ, transforming growth factor β; PAI-1, plasminogen activator inhibitor-1.
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activation of latent TGFβ. A candidate protease which is commonly involved in the activation of latent TGFβ is matrix metalloproteinase2 which in MC similar to many other cell types, is constitutively expressed [47]. However, preliminary experiments with different MMP inhibitors clearly indicate, that MMPs are not involved in the activation of latent TGFβ by rapamycin (data not shown). In summary, we show that rapamycin induces a rapid activation of the fibrogenic Smad signaling cascade in mesangial cells via increased ROS generation and subsequent activation of latent TGFβ. Activation of Smad-triggered gene cascades furthermore highlights the complex repertoire of regulatory events triggered by the potent immunosuppressive drug rapamycin. Acknowledgements We thank R. Goldschmeding (Dept. of Pathology, University Medical Center, Utrecht, The Netherlands) for kindly providing the plasmid pGL-CTGF and Dr. Bert Vogelstein (The Johns Hopkins Oncology Center, Baltimore) for kindly providing the plasmids SBE4-Luc and MBE6-Luc. This work was supported by the German Research Foundation (DFG) grants, EB 257/3-1, PF 361/6-1, FOG 784, and SFB 815 and by the Excellence Cluster “Cardiopulmonary System (ECCPS)” EXC 147/1. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g
Rapamycin induces the TGFβ1/Smad signaling cascade in renal mesangial cells upstream of mTOR Bashier Osman, Anke Doller, El-Sayed Akool, Martin Holdener, Edith Hintermann, Josef Pfeilschifter, Wolfgang Eberhardt ⁎ Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany
a r t i c l e
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Article history: Received 15 April 2009 Received in revised form 13 July 2009 Accepted 29 July 2009 Available online 7 August 2009 Keywords: FK506 binding protein p38 MAP kinase Rapamycin Renal fibrosis Smads TGFβ
a b s t r a c t The mTOR kinase inhibitor rapamycin (sirolimus) is a drug with potent immunosuppressive and antiproliferative properties. We found that rapamycin induces the TGFβ/Smad signaling cascade in rat mesangial cells (MC) as depicted by the nuclear translocation of phospho-Smads 2, -3 and Smad-4, respectively. Concomitantly, rapamycin increases the nuclear DNA binding of receptor (R)- and co-Smad proteins to a cognate Smad-binding element (SBE) which in turn causes an increase in profibrotic gene expression as exemplified by the connective tissue growth factor (CTGF) and plasminogen activator inhibitor 1 (PAI-1). Using small interfering (si)RNA we demonstrate that Smad 2/3 activation by rapamycin depends on its endogenous receptor FK binding protein 12 (FKBP12). Mechanistically, Smad induction by rapamycin is initiated by an increase in active TGFβ1 as shown by ELISA and by the inhibitory effects of a neutralizing TGFβ antibody. Using an activin receptor-like kinase (ALK)-5 inhibitor and by siRNA against the TGFβ type II receptor (TGFβ-RII) we furthermore demonstrate a functional involvement of both types of TGFβ receptors. However, rapamycin did not compete with TGFβ for TGFβ-receptor binding as found in radioligand-binding assay. Besides SB203580, a specific inhibitor of the p38 MAPK, the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC) and a cell-permeable superoxide dismutase (SOD) mimetic strongly abrogated the stimulatory effects of rapamycin on Smad 2 and 3 phosphorylation. Furthermore, the rapid increase in dichlorofluorescein (DCF) formation implies that rapamycin mainly acts through ROS. In conclusion, activation of the profibrotic TGFβ/Smad signaling cascade accompanies the immunosuppressive and antiproliferative actions of rapamycin. © 2009 Elsevier Inc. All rights reserved.
1. Introduction Excessive accumulation and deposition of glomerular and interstitial extracellular matrix (ECM) is a histological hallmark of many progressive glomerular diseases accompanied by glomerulosclerosis and interstitial fibrosis [1,2]. In the kidney the mesenchymal mesangial cells (MC) have been identified as the main producers of glomerular ECM and critically contribute to the structural and functional integrity of the glomerulus [3,4]. Fibrotic changes in the glomerulus do also account for a major complication of long-term immunosupressive therapy with calcineurin inhibitors (CNIs) such as ciclosporin A and tacrolimus [5]. An impaired ECM turnover in
Abbreviations: CNI, calcineurin inhibitor; CTGF, connective tissue growth factor; ECM, extracellular matrix; FKBP, FK506 binding protein; MC, mesangial cells; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; PAI-1, plasminogen activator inhibitor-1; SBE, Smad-binding element; siRNA, small interfering RNA. ⁎ Corresponding author. Pharmazentrum Frankfurt/ZAFES, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Tel.: +49 69 6301 6953; fax: +49 69 6301 7942. E-mail address: [email protected] (W. Eberhardt). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.07.016
addition to increased matrix protein synthesis is mainly attributable to a decrease in ECM degradation through increased synthesis of the intrinsic protease inhibitor plasminogen activator inhibitor-1 (PAI-1) [2]. Transforming growth factor β (TGFβ) upregulates both, matrix genes and matrix protease inhibitors and is one of the most prominent factors triggering the accumulation of ECM. Aberrant production of TGFβ critically contributes to the irreversible alteration of tissue architecture [6]. Besides upregulating collagens and the intrinsic inhibitors of matrix catabolizing proteases TGFβ can induce the expression of connective tissue growth factor (CTGF). CTGF is a member of the CCN (Cyr61/CTGF/Nov) family of matricellular proteins which besides TGFβ have emerged as further potent activators of fibrosis [6]. The critical role of TGFβ in fibrosis is convincingly demonstrated by the successful attenuation of ECM accumulation in different models of acute and chronic renal diseases following TGFβ inhibitory approaches [7,8]. Thereby, induction and activation of the TGFβ/Smad signaling cascade is critical for the initiation of fibrogenic cell responses. TGFβ signaling is initiated by the interaction of the type I and type II TGFβ receptors displaying intrinsic serine/threonine kinase activity. Subsequently, ligand binding causes a recruitment and phosphorylation of receptor-Smads (R-
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Smads), namely Smad 2, and Smad 3 proteins [9,10]. These activated R-Smads heterodimerize with the Co-Smad, mainly Smad 4, to build up a transcriptionally active complex which translocates to the nucleus where it modulates the expression of TGFβ target genes [10]. The macrocyclic lactone rapamycin (sirolimus) has firmly been established as an alternative immunosuppressant which is increasingly used to eliminate or at least lower CNI-induced side effects in the kidney [11]. However, despite this beneficial role rapamycin similar to CNIs has been shown to induce TGFβ expression [12]. Moreover, several reports have implicated a certain nephrotoxic potential of sirolimus especially when applied in combination with high doses of CNIs [12,13]. However, the mechanisms underlying these nephrotoxic actions are largely unknown. In pharmacological terms, rapamycin mainly acts through binding the immunophilin FK binding protein 12 (FKBP12), thereby inactivating the mammalian target of rapamycin (mTOR), a serine/threonine kinase which is critically involved in protein biosynthesis and cell cycle progression [14]. The primary targets of mTOR affected by rapamycin are the eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the p70S6 kinase 1 (p70S6K) [14]. In this context, mTOR is a key sensor and integrator of different stimuli induced by nutrients and growth factors [15] acting downstream of the phosphatidylinositol 3-kinase (PI3Kinase)/AKT pathway [14]. Moreover, synthetic rapamycin derivatives are currently tested as potential anticancer agents in several preclinical and clinical studies [14]. Since similar to rapamycin, TGFβ-triggered inhibition of cell cycle regulators can impair cell cycle progression, a functional cross-talk between the mTOR and Smad signaling devices has been identified in several human cancer cells but also in nontransformed cells [16–18]. Based on our previous findings that CNIs via an activation of latent TGFβ initiate Smad-driven gene expression in renal mesangial cells [19], we aimed to elucidate whether rapamycin would interfere with TGFβ/Smad signaling and thereby modulate fibrogenic gene cascades. 2. Materials and methods 2.1. Materials Human recombinant TGFβ1 was purchased from Cell Concepts GmbH (Umkirch, Germany). A rabbit pan-specific TGFβ antibody and the Quantikine rat TGFβ1 immunoassay kit were purchased from R & D Systems (Wiesbaden-Nordenstadt, Germany). Rapamycin was from Axxora GmbH (Lörrach, Germany). The Alk-5 inhibitor, U0126, SP600125 and SB203580 were obtained from Calbiochem (Schwalbach, Germany). Diphenylene iodonium (DPI), N-acetyl cysteine (NAC), catalase, polyethylene glycol-superoxide dismutase (PEGSOD), xanthine oxidase, hypoxanthine, rotenone, insulin and phorbol 12-myristate 13-acetate (PMA) were derived from Sigma Aldrich (Taufkirchen, Germany). Antibodies specifically raised against phospho-Smad 2 (Ser 465/467), phospho-Smad 3 (Ser 423/425), total Smad-2, total Smad 3, Smad-2/Smad-3, phospho-p38 MAPK (Thr 180/ Tyr 182), p38 MAPK, mTOR, phospho-p70S6 kinase (Thr 389), phospho-4E-BP (Thr 37/46) and siRNA against mTOR were derived from Cell Signaling (Frankfurt am Main, Germany). Antibodies against CTGF, HDAC-1, PAI-1, Smad-4, TGFβ-RII, β-actin, as well as anti-rabbit and anti-mouse horseradish peroxidase-linked and anti-mouse Alexa 488-linked secondary antibodies, IgG and a predesigned siRNA against FKBP12 were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Customer siRNA against the TGFβ type II receptor was synthesized from Eurogentech (Seraing, Belgium). 5- (and -6) chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2-DCFDA), a cell-permeable indicator for ROS was from Molecular Probes (Karlsruhe, Germany) and 125I-labeled human recombinant TGFβ1 was purchased from Amersham Biosciences (Freiburg, Germany).
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2.2. Cell culture Rat glomerular MC were grown as described previously [20]. All cell culture media and supplements were purchased from LifeTechnologies (Karlsruhe, Germany). 2.3. Western blot analysis For detection of CTGF, FKBP12, mTOR, PAI-1, TGFβ-RII, phosphorylated and total Smads 2 and Smads 3, phosphorylated p38 and total p38, phosphorylated p70S6 kinase and phosphorylated 4E-BP1, whole cell lysates were prepared as described previously [21]. Total cell extracts containing 50 µg of protein were prepared in SDS sample buffer and subjected to SDS-PAGE and Western blot analysis was performed by using standard procedures. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes before immunodetection. Nuclear extracts (20–50 µg) from MC were used for the assessment of the nuclear import of phosphorylated Smads as described previously [19]. 2.4. Indirect immunofluorescence microscopy Nuclear import of Smad 4 was analyzed by using indirect immunofluorescence microscopy as described previously [22] and images were analyzed using an inverse immunofluorescence microscope, BZ-7000 (Biozero, Keyence, Neu-Isenburg, Germany) equipped with a Zeiss Apo 20×/0.75 NA objective and images were analyzed by using the BZ-H1TL software from Biozero. 2.5. Small interfering (si)RNA Gene silencing was performed using small interfering (si)RNAs for human mTOR (Cell Signaling) and for FKBP12 (Santa Cruz Biotechnology). A siRNA against the TGFβ type II receptor was synthesized according to rat-specific sequence [23]. Subconfluent MC were transfected for 48 h with 25–50 nM of siRNA by using the Oligofectamine reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. 2.6. Determination of TGFβ1 antigen levels in conditioned media of MC The amount of TGFβ1 in cell culture supernatants was quantified by the Quantikine immunoassay kit from R&D Systems raised against rat TGFβ1. Confluent MC (1.0 to 1.5 × 106 cells) grown in six-well plates were preincubated in DMEM without FCS for 24 h before stimulation with or without rapamycin. 10–20 µl of conditioned media were directly transferred to the microtest strip wells of the ELISA plate. Omission of a prior acidification of samples allowed a specific detection of active TGFβ1. All further procedures were performed following the manufacturer's instructions. The absorbances at 450 nm were measured in a microtest plate spectrophotometer and antigen levels were determined by appropriate calibration curves using human TGFβ1 as a standard. 2.7. TGFβ neutralization experiments The impact of TGFβ on the rapamycin-induced cell responses was tested by the use of a neutralizing pan-specific TGFβ antibody (R & D Systems) and was applied as described previously [22]. 2.8. TGFβ binding competition assay Direct competition of rapamycin with recombinant TGFβ1 for binding to the TGFβ receptor was assessed by a TGFβ binding assay using 125I-labeled TGFβ1 [24]. Briefly, subconfluent MC were washed with cold PBS/EDTA (0.5 mM) before cells were trypsinized and
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resuspended in ice-cold binding buffer containing RPMI, 25 mM Hepes and 0.2% BSA. Aliquots of 340 µl (1 × 106 cells) were distributed into prelubricated polypropylene tubes and increasing concentrations of non-radiolabeled recombinant TGFβ1 or, alternatively, different amounts of rapamycin were added. The binding assay was initiated by addition of a 50 nM [125I] -TGFβ1 stock solution (final concentration: 80 pM) and reactions were incubated at 4 °C for 4 h. Finally, 150 µl aliquots of the binding assay were laid onto 150 µl cold silicon oil and unbound TGFβ1 subsequently removed by centrifugation (13,000 rpm). Finally, cell-bound radioactivity was measured in a γ-counter (Packard). 2.9. Reporter plasmids and transient transfection The transactivation of a Smad-driven reporter gene was measured by using the “pSBE4-Luc” reporter plasmid and “pMBE6-Luc” a corresponding luciferase vector which instead of the wild-type SBE contains three copies of a mutated SBE (GTTTATAC) [25]. Both vectors were kindly provided by Dr. Vogelstein (Johns Hopkins Oncology Center; Baltimore, Maryland, USA). A 1.0 kb promoter fragment from the PAI-1 gene (pGL-PAI-1) was kindly provided by Daniel Rifkin [26]. The pGL-CTGF plasmid which contains a 3.0 kb upstream promoter fragment of CTGF and which includes a functional SBE in a region between –173 and –166 [27] was a kind gift of R. Goldschmeding (Utrecht, The Netherlands). Transient transfections of rat MC were done by using the Effectene reagent (Quiagen) and measurements of luciferase activities were done as described previously [22]. 2.10. EMSA Preparation of nuclear extracts from rat MC and EMSA was performed as described previously [21]. For EMSA analysis a Smad consensus oligonucleotide encompassing a wild-type core Smadbinding element (SBE) (Santa Cruz Biotechnology) was used. DNAprotein complexes were separated from unbound oligonucleotide by electrophoresis through native 4.5% polyacrylamide gels and gels run in 0.5 x Tris-borate EDTA. Supershift analysis was done by preincubation of 1 µg supershift antibody to the binding reaction 30 min prior to the addition of the radioactively labeled oligonucleotides. 2.11. Detection of intracellular ROS production Measurement of intracellular formation of ROS was done by using the cell-permeable indicator CM-H2DCFDA. For ROS measurement, serum-starved MC were stimulated for 1 h and after having removed the stimulation media, cells were washed once with Hank's balanced salt solution (HBSS). Trypsinized cells were grounded by a short centrifugation (1000 rpm), and cell pellets were resuspended in CMH2DCFDA (5 µM in HBSS) and incubated in the dark at 37 °C for 30 min. Cells were washed and returned to HBSS media and ROS generation was
Fig. 2. Rapamycin activates TGFβ-inducible gene promoters. Subconfluent MC were transfected with 0.4 µg of either pSBE4-Luc (A, black bars) containing 4 tandem wildtype SBEs or alternatively, with pMBE6 (A, open bars) containing 6 point-mutated SBEs (A), or alternatively, with 0.4 µg of a 1.0 kb fragment of the pGL-PAI-1 promoter (B), or, alternatively with the same amount of a 3.0 kb fragment of the CTGF promoter (C). Transfection of the plasmids was supplemented by a cotransfection with 0.1 µg of RLCMV, coding for Renilla luciferase. After the transient transfection MC were treated for 16 h with either vehicle, or with the indicated concentrations of rapamycin before cell lysates were assayed for luciferase activities. The values for beetle luciferase were related to the values for Renilla luciferase and are depicted as relative light units (RLU). Data represent means ± S.D. (n = 6) of triplicate experiments ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 compared with vehicle.
subsequently determined by measuring the mean fluorescence intensity by flow cytometry on a FACS Calibur using the CellQuest Pro 5.2 software (BD Biosciences). Alternatively, to visualize ROS generation MC were grown on cover slips in 12-well plates before cells were treated in a similar way as described for FACS analysis. Culture dishes were
Fig. 1. Rapamycin activates the Smad signaling cascade in MC. (A) Quiescent MC were stimulated with either vehicle (-) or with rapamycin (50 ng/ml) for the indicated time periods before cells were lysed for Western blot analysis. Total protein (50 µg) was subjected to Western blot analysis and successively probed with an anti-phospho-Smad 2-specific antibody (p-Smad 2) and with an antibody raised against total Smad 2 (Smad 2). Data in the lower panel of (A) show a densitometric analysis of phospho-Smad 2 relative to total Smad 2 levels and represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 versus unstimulated control. (B) Dose-dependent nuclear import of phosphorylated R-Smads induced by rapamycin. MC were stimulated for 60 min with either vehicle (-) or the indicated concentrations of rapamycin before cells were fractionated for nuclear extracts. 30 µg of nuclear extracts were successively probed with a specific anti-phospho-Smad 2 and anti-phospho-Smad 3 antibody. Loading of equal amounts of nuclear extracts was ascertained by reprobing the blots with an anti-HDAC-1 antibody. Data in the lower panel of (B) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and are depicted as fold induction compared with nuclear phospho-Smad levels under unstimulated conditions. Data represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01, ⁎⁎⁎p ≤ 0.005 versus unstimulated control. (C, upper panel) Rapamycin-mediated entry of Smad 4 into the nucleus by indirect immunofluorescence. After simulation of cells with vehicle (control) or with the indicated concentration of rapamycin for 60 min, MC were fixed and stained with anti-Smad 4 and anti-mouse Alexa-488 antibodies, respectively before DAPI was added in order to counterstain cell nuclei (blue panel). (Lower panel) Densitometric analysis of nuclear FITC staining with data representing means ± S.D. (n = 3) ⁎⁎⁎p ≤ 0.005 versus unstimulated controls. (D) 5 µg of nuclear extracts from MC treated with either vehicle (-), or with rapamycin (+, 50 ng/ml), or, with TGFβ (+, 10 ng/ml) were subjected to EMSA (left panel) using 32P-labeled consensus SBE. (Right panel) Supershift analysis of rapamycin-induced SBE-bound complexes. For supershift analysis the indicated antibodies were added 30 min prior to the addition of the radiolabeled oligonucleotide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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transferred to an inverted immunofluorescence microscope (Zeiss) and images were analyzed by using the ImageJ software. 2.12. Statistical analysis Results are expressed as means ± S.D. Statistical analysis was performed using the Student's t-test and for multiple comparisons the ANOVA test for significance. The data are presented as relative induction compared with control conditions or compared with rapamycin-stimulated values. P ≤ 0.05, ≤ 0.01, and ≤ 0.001 were considered significant. 3. Results 3.1. Rapamycin activates the TGFβ Smad signaling cascade In a first attempt, we tested whether rapamycin can modulate the Smad signaling cascade and measured the phosphorylation state of the receptor (R) Smad indicative for an activation of the profibrotic Smad signaling cascade [10]. Stimulation of MC with rapamycin (50 ng/ml) induced a rapid phosphorylation which was maximal at 60 min and declined thereafter. This was not associated with the change in the total Smad 2 content (Fig. 1A). Since the phosphorylated R-Smads assemble with Smad 4 to build up a transcriptionally active complex, we next monitored for a nuclear import of these R-Smad proteins. As shown in Fig. 1B, stimulation of MC with rapamycin caused a dose-dependent increase in phosphorylated Smad 2/Smad 3 protein with a maximal increase in nuclear phospho-Smad 2/3 levels was observed with rapamycin concentrations of 10 ng/ml or higher (Fig. 1B). Activation of the TGFβ/Smad signaling cascade by rapamycin
is furthermore indicated by a significant increase in nuclear Smad 4 levels in MC stimulated for 60 min with rapamycin as monitored by indirect immunofluorescence microscopy (Fig. 1C). To test for functional effects of Smad phosphorylation by rapamycin, we monitored the DNA binding capacity of nuclear proteins to a radioactively labeled oligonucleotide bearing a consensus SBE by EMSA. As shown in Fig. 1D, treatment of MC with rapamycin caused a marked increase in DNA binding of a TGFβ inducible complex (Fig. 1D, left panel). By contrast, the constitutive binding of two faster migrating complexes was not affected by rapamycin treatment (Fig. 1D, left panel). The addition of antibodies raised against a common epitope of Smad 2 and Smad 3 (“anti-Smad 2/3”), or alternatively, of specific antibodies against Smad 3 (a.-Smad 3), or against phosphorylated Smad 2 (“a.-p.-Smad 2”) specifically impaired the DNA binding of the upper, rapamycin-inducible complex without affecting the constitutively bound complexes (Fig. 1D, right panel). Interestingly, only the addition of the anti-Smad 2/3 antibody, which recognizes a common epitope of Smad 2 and -3 caused a supershifted band (arrow in Fig. 1D, right panel) whereas all the other Smad antibodies applied reduced the DNA binding of the rapamycininduced complex without causing a real supershift which indicates that the binding of these antibodies somehow interferes with the DNA binding of corresponding Smad complexes (Fig. 1D, right panel). 3.2. SBE confers rapamycin induction of TGFβ inducible gene promoters Next, we tested whether rapamycin could also activate a TGFβ inducible control promoter bearing a tandem of four copies of SBE consensus motifs (pSBE4-Luc) [25]. Stimulation of MC with rapamycin for 12 h caused a significant increase in luciferase activity (Fig. 2A,
Fig. 3. Rapamycin induces expressions of PAI-1 and CTGF by an involvement of the TGFβ type II receptor. (A) Quiescent MC were stimulated with either vehicle (-), or with 50 ng/ml of rapamycin (+) for the indicated time periods before cells were harvested for total protein extracts. Total protein (50 µg) was subjected to Western blot analysis and successively probed with anti-PAI-1, anti-CTGF antibodies and with β-actin-specific antisera. Data in the lower panel of (A) show a densitometric analysis of PAI-1 and CTGF protein levels (filled bars) 8 h after the addition of rapamycin and represent means ± S.D. (n = 3) ⁎⁎p ≤ 0.01, ⁎⁎⁎p ≤ 0.005 versus unstimulated control (open bars). (B) Quiescent MC were transfected with either a control siRNA (control-siRNA) or with siRNA duplexes specific for rat TGFβ-RII (TGFβ-RII-siRNA). After transfection MC were serum-starved for 16 h before cells were stimulated with either vehicle (-), with the indicated amounts of rapamycin or, alternatively, with TGFβ (+, 10 ng/ml) for 8 h. Western blots were successively probed with antiTGFβ-RII, anti-PAI and anti-CTGF-specific antisera, respectively. (C) Activation of Smad 2 is abrogated after attenuation of the TGFβ-RII. MC were transfected as described in (B). After transfection cells were treated for 30 min with vehicle (-), or with different concentrations of rapamycin, or with TGFβ (+, 10 ng/ml) as indicated. Western blots show the total levels of TGFβ-RII, Smad 2 and phosphorylated Smad 2 (p-Smad 2). Data shown are representative of two independent experiments giving similar results.
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filled bars). By contrast, a reporter gene which instead of the wildtype SBE motifs contained a tandem of mutated SBE motifs (pMBE6Luc) was not induced by rapamycin (Fig. 2A, open bars) thus indicating that the promoter activation by rapamycin is mediated via SBEs. In order to prove activation of a native SBE containing promoter by rapamycin, we next tested whether rapamycin could activate a
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1.0 kb fragment of the PAI-1 gene promoter (pGL-PAI-1) which confers TGFβ responsiveness via a functional SBE and which is commonly used as a native control promoter of SBE-dependent transcription [26]. Rapamycin caused a dose-dependent induction in PAI-1 promoter activity with maximal effects at 100 ng/ml (Fig. 2B). As a further target gene which is induced upon activation of the TGFβ/Smad signaling cascade we tested a promoter fragment of the
Fig. 4. Activation of R-Smads depend on TGFβ and ALK-5 kinase activity. (A) Quiescent MC were pretreated for 60 min with vehicle (+ vehicle) or with either 20 µg of a pan-specific TGFβ antiserum (+ anti-TGFβ a.b.), 20 µg of control IgG (+IgG) or with 100 nM of ALK-5 inhibitor. Thereafter, MC were stimulated for 60 min with either vehicle (-) or with rapamycin (50 ng/ml) as indicated. 30 µg of nuclear extracts were subjected to SDS-PAGE and probed successively with an anti-phospho-Smad 2, anti-phospho-Smad 3 antibody and with an anti-HDAC-1-specific antibody. Data in the right panel of (A) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means ± S.D. (n = 3) ⁎p ≤ 0.05, ⁎⁎p ≤ 0.01 versus unstimulated control; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.005 versus rapamycin-stimulated conditions. (B) Rapamycin induces rapid activation of latent TGFβ1. (Left panel) Quiescent MC were treated for 60 min with vehicle (control) or with rapamycin (Rapa, 50 ng/ml) before cell supernatants were assessed for active TGFβ1 by Quantikine ELISA as described in Section 2. Data represent means ± S.D. (n = 3), ⁎⁎ p ≤ 0.01 versus control. (Right panel) Activation of R-Smads by recombinant TGFβ1. Quiescent MC were stimulated for 30 min with either vehicle (-), or with the indicated concentrations of TGFβ1 before cells were lysed for nuclear extraction. 30 µg of nuclear extracts were subjected to SDS-PAGE and successively probed with an anti-phospho-Smad 2, anti-phospho-Smad 3 antibody and with an anti-HDAC-1-specific antibody. Data shown are representative of two independent experiments with similar results. (C) Rapamycin does not compete with TGFβ1 for receptor binding. Equal amounts of trypsinized MC were incubated with [125I]-TGFβ1 (80 pM), in the absence (-) or presence of the indicated concentrations of unlabeled TGFβ1 or rapamycin (Rapa) and receptor binding was allowed for 4 h. The cell-bound radioactivity was counted in a γ-counter and is blotted versus different TGFβ, or rapamycin concentrations. Data represent means ± S.D. (n = 3), #p ≤ 0.05, ##p ≤ 0.01 versus vehicle.
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Fig. 5. Rapamycin-mediated R-Smad and TGFβ activation depend on ROS. (A) Quiescent MC were treated for 60 min with vehicle (-), or rapamycin (Rapa, +, 50 ng/ml) in the absence or presence of either N-acetyl cysteine (NAC, 5 mM) or diphenylene iodonium (DPI, 10 µM) before nuclear extracts from cells were assessed for phospho-Smad 2 and phospho-Smad 3 contents. Loading of equal amounts of nuclear extracts was ascertained by assessment of nuclear HDAC-1 level. (Left panel) Data represent means ±S.D. (n= 3), *p≤0.05 versus unstimulated control; # p≤0.05 versus rapamycin-induced conditions. (B) MC were stimulated with either vehicle (-) or with rapamycin (Rapa, +, 50 ng/ml) in the absence or presence of either catalase (500 U/ ml), PEG-SOD (100 U/ml), allopurinol (100 µM), or rotenone (10 µM) for 60 min before nuclear extraction. Subsequently, the content of phosphorylated R-Smads in nuclear extracts (30 µg) was tested by Western blot analysis. The data in the lower panel of (B) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means± S.D. (n = 3) ⁎p ≤0.05, ⁎⁎p ≤0.01 versus unstimulated control; #p ≤0.05, versus the respective rapamycin-stimulated conditions. (C) Activation of latent TGFβ1 by rapamycin depends on ROS. Quiescent MC were treated for 60 min with vehicle (control), or with rapamycin (Rapa, 50 ng/ml) in the absence or presence of NAC (5 mM) before cell supernatants were assessed for active TGFβ1. Data are expressed as means± S.D. (n= 3), ⁎⁎p≤ 0.01 versus unstimulated control; ##p≤0.01 versus rapamycin-induced conditions. Fig. 6. Rapamycin induces ROS production and ROS activate R-Smads. MC were treated for 60 min with vehicle (control), rapamycin (Rapa, 50 ng/ml) in the presence or absence of NAC before radical formation was determined by dichlorofluorescein (DCF) formation. (A) Quiescent MC were grown on cover glass slides and incubated with NAC (5 mM) for 60 min before treated with rapamycin (Rapa, 50 ng/ml) for additional 60 min. Subsequently, cells were washed with HBSS and incubated in the dark for 30 min in 5 µM CM-H2DCFDA (in HBSS). ROS generation is visualized by inverse fluorescence microscopy and quantified by the Image software. Data represent means±S.D. (n=3), ⁎⁎p≤0.01 versus vehicle; ##p≤0.01 versus rapamycin-induced conditions. (B) Accumulation of intracellular ROS measured by FACS using the CM-H2DCFDA probe. Serum-starved MC were stimulated for 60 min with either vehicle (control), different concentrations of rapamycin as indicated, or, alternatively with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) used as a positive control, before cells were trypsinized and incubated with H2DCFDA. Fluorescence intensity was measured by using the CellQuest Pro 5.2 software (BD Biosciences) and data are representative of two independent experiments giving similar results. (C) Activation of R-Smads by the ROS generating xanthine oxidase system. Quiescent MC were treated for 60 min with vehicle (-), or with hypoxanthine (50 µM) plus the indicated concentrations of xanthine oxidase (HXXO). Nuclear extracts (30 µg) were subjected to SDS-PAGE and immunoblotted using the indicated antibodies. The data in the lower panel of (C) show a densitometric analysis of nuclear phospho-Smad 2 (filled bars), or phospho-Smad 3 (open bars) relative to nuclear HDAC-1 levels and represent means±S.D. (n=3) ⁎p≤0.05, ⁎⁎p≤0.01 versus unstimulated control.
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connective tissue growth factor (CTGF) gene, which similar to PAI-1, bears a functional SBE [27]. MC transiently transfected with pGLCTGF displayed a similar induction profile in luciferase activity as
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with pGL-PAI-1 (Fig. 2C). Collectively, these data demonstrate that rapamycin via increased Smad binding to SBEs activates TGFβinducible gene promoters.
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Fig. 7. Rapamycin-mediated R-Smad activation depends on p38 activity. (A) Quiescent MC were pretreated for 60 min with vehicle (+ vehicle), SB203580 (10 µM), SP600125 (10 µM), or U0126 (20 µM). Thereafter cells were left untreated (-) or stimulated for further 60 min with rapamycin (Rapa, 50 ng/ml). The data in the right panel show a densitometric analysis of the nuclear phospho-Smad 2 (filled bars) and phospho-Smad 3 (open bars) levels relative to nuclear HDAC-1 contents and represent means ± S.D. (n = 3) ⁎p ≤ 0.05 versus unstimulated control; #p ≤ 0.05, versus rapamycin-stimulated conditions. (B) Time-dependent activation of p38 by rapamycin. Quiescent MC were treated with vehicle (-), or with rapamycin (Rapa, 50 ng/ml) for the indicated time periods (left panel) or, alternatively, for 20 min either in the absence (+ vehicle) or, presence of NAC (5 mM), or ALK-5 inhibitor (100 nM) before cells were stimulated for further 30 min with rapamycin (right panel). Total protein extracts (50 µg) were subjected to Western blot analysis and successively probed with an anti-phospho-specific p38 antibody (p-p38) or, alternatively, with an antibody raised against total p38 (p38). Data shown are representative of two independent experiments giving similar results.
3.3. Rapamycin induces profibrotic gene expression via the TGFβ type II receptor Next, we tested whether the activation of SBE-driven promoters results in an upregulation of corresponding proteins. Stimulation of MC with rapamycin (50 ng/ml) caused a time-dependent increase in the steady-state level of PAI-1 and CTGF protein with a maximal increase observed at 8 h (Fig. 3A). To further test the possible involvement of the TGFβ signaling device in rapamycin-mediated upregulation of both genes we first applied siRNA specific for TGFβRII, which in contrast to TGFβ-RI exerts a direct ligand binding, and which in turn, completely prevented the stimulatory effects of rapamycin (Fig. 3B). Additionally, silencing of TGFβ-RII prevented rapamycin-induced Smad 2 phosphorylation thus indicating that the TGFβ type II receptor is indispensably involved in rapamycin-induced Smad activation (Fig. 3C). 3.4. Rapamycin activates R-Smads via TGFβ-RI kinase and extracellular TGFβ To further delineate the mechanism by which rapamycin activates R-Smads, we applied an activin receptor-like kinase (ALK)-5 inhibitor [28]. Incubation with 100 nM of the ALK-5 inhibitor decreased the rapamycin-induced Smad 2 and Smad 3 phosphorylation to a level which was beneath basal Smad phosphorylation (Fig. 4A). An involvement of extracellular TGFβ in the rapamycin-dependent response is furthermore shown by the effects of a neutralizing panspecific TGFβ antibody. Neutralization of TGFβ completely prevented the rapamycin-mediated increase in R-Smads phosphorylation but, in contrast to the effects caused by the ALK-5 inhibitor, it did not affect basal Smad 2/3 phosphorylation (Fig. 4A). To confirm the activation of
latent TGFβ by rapamycin, we measured the level of active TGFβ1 in conditioned media of rapamycin-treated MC by using a TGFβ1-specific ELISA without a prior acidification step. Treatment of MC with rapamycin for 60 min caused a significant increase in TGFβ1 levels (Fig. 4B, left panel), to amounts sufficient to induce R-Smads phosphorylation (Fig. 4B, right panel). Next, we tested whether rapamycin can directly bind to the TGFβ receptor and to this end we made use of a TGFβ binding assay using 125 I-labeled recombinant TGFβ1 [24]. Addition of unlabeled recombinant TGFβ dose-dependently competed with 125I TGFβ for receptor binding whereas rapamycin even at high doses did not compete thus indicating that rapamycin does not directly bind to the TGFβ-receptor (Fig. 4C). 3.5. Activation of R-Smads depends on ROS Based on our previous finding that CNIs induce Smad-2 activation via an increase in ROS production [19], we assessed whether rapamycin-induced R-Smad activation similarly would depend on ROS. To this end, MC were stimulated in the presence of the ROS scavenger N-acetyl cysteine (NAC, 5 mM), or alternatively, with DPI (10 µM), an inhibitor of NADPH oxidases. Whereas NAC caused an almost complete inhibition of rapamycin-induced R-Smad activation DPI had no significant effect on the levels of Smad 2 and -3 (Fig. 5A). We additionally tested further ROS inhibitors: catalase which decomposes H2O2, the membrane permeable superoxide dismutase PEG-SOD, allopurinol an inhibitor of the xanthine oxidase, and finally rotenone an inhibitor of the mitochondrial respiratory chain. Interestingly, only PEG-SOD caused a significant reduction in rapamycin-induced R-Smad 2 phosphorylation (Fig. 5B) thus
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indicating that mainly an increase in the intracellular superoxide generation is critical for Smad activation by rapamycin. Functionally, incubation with NAC almost completely blocked the rapamycintriggered release of active TGFβ1 (Fig. 5C).
depletion of FKBP12 thus indicating that Smad activation by rapamycin depends on the FKBP12 (Fig. 8B).
3.6. Rapamycin induces ROS formation in MC
Previously, we have shown that CNIs, at low nanomolar concentrations, can induce TGFβ-Smad signaling cascades in renal MC and thereby increase the expression of fibrogenic genes [19]. Here we provide a comprehensive analysis which demonstrates that most components of the TGFβ-Smad signaling cascade are similarly activated by rapamycin by a mechanism which is triggered by ROS. The immunosuppressive actions of rapamycin in most cases synergize with those of CNIs but, in contrast to CNIs, rapamycin inhibits lymphocyte proliferation via an IL-2 independent mechanism [32,33]. Regardless of this difference, an increase in TGFβ expression by both classes of immunosuppressants has been coincidently described by several reports [12,34]. Here, we demonstrate that rapamycin similar to CNIs induces a rapid activation of latent TGFβ which in turn leads to an activation of the profibrotic Smad signaling cascade. This cascade is activated already within minutes thus excluding the fact that stimulatory effects on fibrogenic gene expression are due to an increase in TGFβ gene expression. Our observations are in agreement with another report demonstrating activation of the bonemorphogenic protein 4 (BMP-4) by rapamycin in different prostate cancer cell lines [17]. All BMPs, in contrast to TGFβ, specifically induce ALK3 and -6 and thereby activate Smads 1, -5 and -8 [35–37]. A complex cross-talk between the TGFβ-Smad and rapamycin-sensitive signaling cascades has been implicated by several studies [15,16,38,39]. Importantly,
To further prove an increase in ROS formation by rapamycin, we next measured cellular radical formation by applying the cellpermeable ROS acceptor CM-H2-DCFDA which was either detected by immunofluorescence microscopy (Fig. 6A) or, alternatively, by FACS analysis (Fig. 6B). Independent of which approach we used, rapamycin caused a significant increase in ROS production (Fig. 6A, B) to an extent which was comparable to that evoked by the phorbol ester PMA (Fig. 6B). Importantly, preincubation with NAC almost completely abrogated the rapamycin-triggered ROS generation (Fig. 6A). Furthermore, applying the superoxide generating xanthine oxidase/hypoxanthine (HXXO) system caused a dosedependent and significant increase in Smad 2 and 3 phosphorylation (Fig. 6C). 3.7. Involvement of p38 MAPK in rapamycin-induced Smad activation Since an activation of several different MAPKs has been implicated in TGFβ signaling [29–31], we next investigated the impact of different MAPKs by using specific pharmacological inhibitors. We found that only preincubation with SB203580 (10 µM), an inhibitor of the p38 pathway, abrogated rapamycin-induced Smad 2 and Smad 3 phosphorylation whereas neither the JNK inhibitor SP600125 nor the ERK inhibitor U0126 had any suppressive effects on R-Smad activation by rapamycin (Fig. 7A). Correspondingly, we found a rapid and transient activation of the p38 MAPK by rapamycin with a peak in phospho p38 levels at 15 min after administration of rapamycin (Fig. 7B, left panel). Interestingly, the activation of p38 MAPK is prevented by both, the ALK-5 inhibitor and NAC thus indicating that the TGFβ-RI kinase and ROS generation are upstream of rapamycintriggered p38 MAPK activation (Fig. 7B, right panel).
4. Discussion
3.8. Rapamycin elicits Smad signaling independent of mTOR inhibition but requires FKBP12 In the next set of experiments, we asked whether the activation of TGFβ-Smad signaling by rapamycin is independent of its capacity to inhibit the serine/threonine kinase mammalian target of rapamycin (mTOR) and the downstream signaling which functionally results in the blockade of protein biosynthesis and cell cycle progression [32]. To this end we first tested whether rapamycin can block the insulininduced activation of the p70S6 kinase and the eukaryotic initiation factor 4E binding protein-1 (4E-BP1), two well-characterized mTOR effectors, using phosphorylation-specific antibodies. MC treated for 60 min with different concentrations of insulin, showed a dosedependent increase in p70S6 kinase phosphorylation (p-p70S6K) and, although to a lower extent, in 4E-BP1-phosphorylation (p-4E-BP1) when compared with vehicle-treated cells (Fig. 8A). Coincubation with rapamycin resulted in a total inhibition of insulin-induced p70S6 kinase phosphorylation and a clear reduction in the phospho 4E-BP levels which both signifies that in MC, rapamycin can negatively interfere with mTOR signaling. By contrast, the levels of phosphorylated p85S6 kinase levels (p-p85S6K) were not affected by rapamycin (Fig. 8A). Next, we tested for a possible involvement of FKBP12 the intrinsic binding protein of rapamycin. For this purpose we utilized small interference (si)RNA against FKBP12. MC transfected with siRNA against FKBP12 displayed a strong reduction in FKBP12 expression when compared with control siRNA transfected cells (Fig. 8B). Interestingly, phospho-Smad 2 levels were strongly impaired after
Fig. 8. Rapamycin-induced Smad activation depends on the FKBP12. (A) Rapamycin inhibits insulin-induced p70S6K and 4E-BP1 activation in rat MC. Quiescent MC were preincubated with rapamycin (Rapa, +, 50 ng/ml) for 30 min before stimulated with the indicated concentrations of insulin for 60 min before cells were harvested for total protein extraction. 30 µg of protein extract was subjected to SDS-PAGE and successively probed with an anti-phospho-p70S6K, anti-phospho-4E-BP1 and anti-β-actin antibody. (B) Rapamycin-induced Smad-2 activation depends on the FKBP12. MC were transfected with siRNA duplexes of mouse FKPB12 (FKBP12), or with a none-generelated control siRNA (control), or with the transfection reagent (mock). After transfection, MC were treated with either vehicle (-), or with rapamycin (Rapa, 50 ng/ml) for 60 min before cells were harvested for total protein extraction. To prove a knockdown of FKBP12, 30 μg οf protein extract was probed with anti-FKBP12 antibody by Western blot analysis. In parallel 30 µg of protein extracts were additionally probed with a phοsphο-Smad 2 and an antibody raised against total Smad 2. The data shown in the figure are representative of two independent experiments giving similar results.
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rapamycin through binding to its intracellular receptor, FKBP12 can reverse the inhibitory effects of FKBP12 on TGFβ type I receptor phosphorylation thus endowing a constitutive TGFβ signaling, resulting from the intimate tendency of both types of TGFβ receptors to interact with each other [38]. However, a similar mechanism seems not to be operative in MC, since depletion of FKBP12 by siRNA attenuation instead of elevating Smad signaling, completely prevented R-Smad activation by rapamycin (Fig. 8B). In contrast to reports on tumor cells [16,40], the rapamycin-induced TGFβ-Smad signaling observed in MC seems rather to be independent of mTOR signaling, since depletion of mTOR did not affect the rapamycin-induced activation of Smads (data not shown). Functionally, induction of the Smad-driven promotors of CTGF and PAI-1 by rapamycin strongly indicates that in renal MC the activation of TGFβ/Smad signaling by rapamycin is sufficient to elicit fibrogenic cell responses. In a further attempt to delineate the underlying mechanisms of how rapamycin induces Smad signaling we found a sequential activation of ROS and the p38 MAPK with both signals being indispensable for activation of R-Smads. ROS seem to act upstream of both, latent TGFβ activation and p38 activation as depicted in Fig. 9. Interestingly, in renal MC, rapamycin, similar to CNIs, activates p38 in a TGFβ receptor dependent manner [19] which is in contrast to the TGFβ receptor independent activation of different MAPKs by TGFβ reported by several studies [30,41]. Thus, our data suggest that rapamycin-triggered p38 MAPK acts through the TGFβ-receptor to induce the Smad signaling cascade (Fig. 9). Importantly, a critical role of ROS formation has also been proposed for cell responses induced by ciclosporine A [42] whereas
for rapamycin some reports have even described an inhibitory capacity of ROS [43]. We have observed that the rapid increase in ROS production and downstream Smad responses by rapamycin is diminished by a cell-permeable SOD mimetic (Fig 5B). Furthermore, the ineffectiveness of DPI, the xanthine oxidase inhibitor allopurinol and rotenone furthermore indicates that superoxide generation does not originate from one of the major cellular superoxide generating systems including NAD(P)H oxidase, xanthine oxidase or mitochondrial NAD(P)H dehydrogenases, respectively. Notably, in contrast to the ciclosporine-dependent increase in ROS production and subsequent Smad activation which seems independent from binding to ciclophilin [19], our results from siRNA experiments clearly demonstrate that FKBP12 is indispensable for rapamycin-mediated Smad activation (Fig. 8B). A challenging issue which needs to be addressed in the future is the identification of the intracellular ROS generator targeted by the rapamycin/FKBP12 complex. To the best of our knowledge our data for the first time demonstrate a rapid redoxsensitive increase in active TGFβ by rapamycin. Mechanistically, the release of TGFβ from the latent TGFβ binding protein (LTBP) complex can be achieved by proteolytic as well as by nonproteolytic events including the modulation by thrombospondin as well as different intergrins [44,45]. Importantly, the physiological activation of latent TGFβ is a target of redox-sensitive mechanisms. In this context, oxidation of specific amino acids in the latency conferring peptide can cause a conformational change in the latency-associated protein β (LAPβ) thus allowing a rapid release of TGFβ [46]. These results warrant further investigation in future studies to definitely unravel whether oxidation of LAPβ is involved in the rapamycin-triggered
Fig. 9. Schematic representation of profibrotic Smad signaling rapidly induced by rapamycin. Rapamycin via binding to the FKBP12 by a still unknown mechanism generates ROS which extracellularly induces a rapid release of latent TGFβ and subsequent TGFβ receptor triggered signaling. Thereby, the Smad 2/3 dependent pathway is activated through p38 and additional TGFβ-triggered signaling devices. The association of R-Smads with Co-Smad (Smad-4) leads to an active Smad transcription factor complex which specifically binds to a cognate SBE, present in the promoters of CTGF and PAI-1 genes. Abbreviations: CTGF, connective tissue growth factor; 4E-BP1, eukaryotic initiation factor binding protein; LAP, latency-associated protein; MMP, matrix metalloproteinase; mTOR, mammalian target of rapamycin; p70S6K, p70S6 kinase; ROS, reactive oxygen species; SBE, Smad-binding element; TGFβ, transforming growth factor β; PAI-1, plasminogen activator inhibitor-1.
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activation of latent TGFβ. A candidate protease which is commonly involved in the activation of latent TGFβ is matrix metalloproteinase2 which in MC similar to many other cell types, is constitutively expressed [47]. However, preliminary experiments with different MMP inhibitors clearly indicate, that MMPs are not involved in the activation of latent TGFβ by rapamycin (data not shown). In summary, we show that rapamycin induces a rapid activation of the fibrogenic Smad signaling cascade in mesangial cells via increased ROS generation and subsequent activation of latent TGFβ. Activation of Smad-triggered gene cascades furthermore highlights the complex repertoire of regulatory events triggered by the potent immunosuppressive drug rapamycin. Acknowledgements We thank R. Goldschmeding (Dept. of Pathology, University Medical Center, Utrecht, The Netherlands) for kindly providing the plasmid pGL-CTGF and Dr. Bert Vogelstein (The Johns Hopkins Oncology Center, Baltimore) for kindly providing the plasmids SBE4-Luc and MBE6-Luc. This work was supported by the German Research Foundation (DFG) grants, EB 257/3-1, PF 361/6-1, FOG 784, and SFB 815 and by the Excellence Cluster “Cardiopulmonary System (ECCPS)” EXC 147/1. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]
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