Published online before print February 7, 2008, doi: 10.1161/CIRCRESAHA.107.167395
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© 2008 American Heart Association, Inc.
UltraRapid Communication |
Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1
From the Centre for Vascular Research (N.Y.T., V.C.M., M.M.K., F.S.S., X.L., R.P., R.G.F., L.M.K.), School of Medical Sciences, University of New South Wales, Sydney; Department of Immunology (M.C.B.), Monash University, Clayton; and Australian Proteome Analysis Facility (M.P.M.), Macquarie University, Sydney, Australia.
Correspondence to Levon M. Khachigian, PhD, DSc, Centre for Vascular Research, Department of Pathology, University of New South Wales, Sydney NSW 2052, Australia. E-mail L.Khachigian{at}unsw.edu.au
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Abstract |
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Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein–DNA interactions, protein–protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-
. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC- and have generated novel anti-peptide antibodies recognizing the PKC-–phosphorylated form of Sp1. Angiotensin II, which activates PKC- phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-–phospho-Sp1 axis and angiotensin II–inducible gene expression.
Key Words: Sp1 phosphorylation • PKC- • PDGF-D • transcription
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Introduction |
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Sp1 regulates gene expression via multiple mechanisms including direct protein–DNA interaction, protein–protein interactions with other transcription factors and kinases, chromatin remodeling, and maintenance of methylation-free CpG island.1 It is a phosphoprotein with a molecular mass of 100 to 110 kDa and is extensively glycosylated with O-linked sugars.2,3 Growing evidence indicates that the phosphorylation state of Sp1 influences its transcriptional activity. For example, Sp1 phosphorylation has been linked with shear stress induction of the tissue factor promoter,4 hepatocyte growth factor-inducible vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) transcription,5 epidermal growth factor–inducible apolipoprotein A-I expression,6 and T-cell receptor–inducible interleukin-21 receptor gene expression.7 Protein kinases able to phosphorylate Sp1 include extracellular signal-regulated kinase,8,9 MEK1,10 casein kinase II,11 HIV type 1 Tat,12 and DNA-dependent protein kinase.3
We have shown previously that Sp1 phosphorylation and expression of the extrinsic apoptotic ligand, FasL are processes critically dependent on the integrity of the atypical protein kinase C, PKC-, as demonstrated by the blockade of both of these processes with dominant-negative (kinase dead) PKC-.13,14 Furthermore, we and others have shown that the transcriptional regulation of some members of the platelet-derived growth factor (PDGF)/VEGF family, such as PDGF B-chain14 and VEGF/VPF,5,15,16 are dependent on PKC-–Sp1 phosphorylation. PKC-–induced Sp1 phosphorylation–dependent transcription also regulates the luteinizing hormone receptor gene.17 PKC-– and Sp1-dependent transcription are activated by pharmacological agents such as nogalamycin (NOG) and camptothecin (CAM).14 For example, dominant negative (kinase dead) PKC- blocks NOG-inducible Sp1 phosphorylation14 and inhibits CAM-inducible Sp1-dependent gene transcription.13 The PKC-–Sp1 phosphorylation axis may thus be a general regulatory pathway.
PKC- is a member of the Ser/Thr PKC family that regulates signal transduction pathways that control apoptosis, proliferation, ion channel function, and extracellular secretion.18 It is an atypical PKC with the general consensus phosphorylation site of Ser/Thr-X-Arg/Lys (where X represents any amino acid). On activation, it translocates to the nucleus19 and is phosphorylated on Thr410, a modification that stimulates kinase activity in vitro and in cells.20 PKC- phosphorylation sites have been defined in some proteins, such as Ser333 in the human parainfluenza virus type III phosphoprotein.21 Studies by Pal et al investigating VEGF transcription revealed the region in Sp1 phosphorylated by PKC-.15 Recombinant and cellular Sp1 physically interact with PKC- but not PKC-
, -β, or -
.15 Further studies by this group demonstrated that PKC- binds to and phosphorylates the zinc finger (ZNF) domain of Sp1 but fails to interact with other regions of the protein.15 ZNFs are relatively small zinc-binding protein motifs commonly found in certain transcription factors, such as Egr-1.22–24 Identification of the actual residues phosphorylated in the ZNF region of Sp1 by PKC- would shed valuable light on the mechanisms involved in Sp1 activation and on the regulation of PKC-–dependent phospho-Sp1–dependent gene expression.
Angiotensin II (Ang II), the primary effector of the renin–angiotensin system, is a multifunctional hormone that plays an important role in vascular function and maintenance and has been linked to PKC activation in smooth muscle cells (SMCs).22,23 Ang II is generally regarded to be a proatherogenic factor through its regulation of signaling cascades that lead to vascular SMC growth and migration, endothelial function, production and release of proinflammatory molecules, and modification of the extracellular matrix.25 PDGFs, potent mitogens and chemoattractants for cells of mesenchymal origin, are a family of growth factors that are under the transcriptional control of Ang II.25–27 Furthermore, the promoter regions of PDGF-A,26 -B,27 -C,28 and more recently PDGF-D29 have been shown to bind Sp1 under basal and/or stimulatory conditions. Whether the Ang II–PKC-–Sp1 phosphorylation axis plays a role in regulating members of the PDGF family is not presently known, nor is the identity of the specific residue(s) in Sp1 modified by PKC- regulating PDGF ligand transcription. A better understanding of the regulatory mechanisms controlling PDGF expression would provide important clues on novel interventional approaches that may be used to interfere with the positive regulatory role PDGFs play in a variety of vascular pathologies, including atherosclerosis and restenosis.
Phospho-specific antibodies targeting a given regulatory factor that ignore the inactive (unphosphorylated) form are extremely valuable as research and diagnostic tools. We have demonstrated that active PKC-(pThr410) is expressed in vascular SMCs in human atherosclerotic lesions.30 Phospho-specific antibodies recognizing the PKC- modified form of Sp1 would help establish the existence of this regulatory pathway in other pathological settings. These antibodies could also be used to provide information on protein–DNA interactions that are dependent on the phosphorylation of Sp1. In this study, we have identified specific amino acids in Sp1 modified by PKC-, developed phospho-specific Sp1 antibodies, and used these to define mechanisms controlling Ang II–inducible Sp1-dependent gene expression.
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Materials and Methods |
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Cell Culture
WKY12-22 SMCs (generous gift of Dr Stephen M. Schwartz, University of Washington, Seattle) and human SMCs (HSMCs) (obtained from Cell Applications Inc) were maintained in Waymouth medium (Sigma), pH 7.4 at 37°C in a humidified atmosphere of 5% CO2 in medium supplemented with penicillin 10 U/mL, streptomycin 10 µg/mL, 1 mmol/L L-glutamine, and 10% fetal bovine serum (Invitrogen). The medium was changed to serum-free for 6 hours before transfection or 24 hours before the addition of reagents.
Transient Transfection and Sp1 Plasmid Mutations
Wild-type cytomegalovirus (CMV)-Sp1 vector was obtained from Dr Robert Tjian (Howard Hughes Medical Institute, University of California, Los Angeles). CMV-gutless vector was made by cutting CMV-Sp1 with XhoI to remove the Sp1 cDNA.29 Mutant forms of Sp1 were created using the QuikChange Site-Directed Mutagenesis kit (Stratagene) according to the instructions of the manufacturer. Transient transfections were performed at 60% confluence with indicated constructs using FuGENE6 transfection agent (Roche Applied Science). After 24 hours, the transfected cells were either assayed for luciferase activity or incubated with camptothecin (CAM) (1 µg/mL) (Sigma Aldrich) or Ang II (10–7 mol/L) (Calbiochem) for another 24 hours. As a control for luciferase assays, 1 µg of pRL (Renilla luciferase)-TK was used in experiments, and luciferase activity was quantified with the dual luciferase assay system (Promega). Firefly luciferase was normalized to Renilla activity.
Peptide and Phosphopeptide Synthesis
Peptides were synthesized by Auspep Pty Ltd (Melbourne) and purified by high-performance liquid chromatography to >75%. The peptides were resuspended in distilled water and stored at –20°C.
In Vitro Kinase Reactions
Fifty to 200 µg of peptide or 100 ng of recombinant Sp1 was incubated with 2.5 µCi of 32P-ATP and 100 µg of human recombinant PKC- (Sigma) in PKC- reaction buffer (10 mmol/L NaCl, 400 µmol/L Hepes, 50 µmol/L dithiothreitol [DTT], 8 µmol/L EDTA, 20 µmol/L EGTA, 25% glycerol, 0.05% Triton X-100) or PKC-
reaction buffer (30 mmol/L Tris-HCl, pH 7.5, 0.01% Triton X-100, 10.73 µmol/L β-mercaptoethanol, 200 µmol/L phenylmethylsulfonyl fluoride, 0.2 ng/mL leupeptin, 400 µmol/L EGTA, 9.75 mmol/L MgCl2) in a final volume of 40 µL. The samples were incubated at 30°C for 30 minutes and then spun in Millipore Microcon YM-30 for 12 minutes (30-kDa cutoff). The filtrate was applied to phosphocellulose paper (Amersham MAP kinase kit) and allowed to incubate at 22°C for 5 minutes. Filters were washed 3 times with 1 mL of 1% acetic acid, then twice with 1 mL of H2O. Filter papers were placed into scintillation vials before assessment of β radioactivity. A fragment containing the ZNF region of Sp1 (amino acids 432 to 701) was cloned into pQE-82L vector containing His6 tag coding sequence. When wild-type Sp1 was produced, this plasmid was used as a template to produce ZNFmThr668/Ser670/Thr681 by site-directed mutagenesis (Stratagene). Proteins were expressed using 1 mmol/L isopropyl β-D-thiogalactoside and purified using Ni-NTA system from Qiagen.
Mass Spectrometry
The in vitro kinase reaction was carried out as described above in a total volume of 30 µL; however, 3.33 mmol/L cold ATP was substituted in place of 32P-ATP. The samples were spun in Millipore Microcon YM-30 for 12 minutes, and the filtrate was used for mass spectrometric analysis. Two microliters of sample were diluted with 18 µL of 30% acetonitrile/0.1% formic acid and briefly centrifuged at 14 000g. Five microliters of the supernatant was loaded into a PicoTip Emitter (New Objective, Woburn, Mass) and analyzed by static nanoelectrospray mass spectrometry using an Applied Biosystems 4000 Q-Trap. The presence of phosphorylated peptide was detected by conducting neutral loss scans to detect the loss of +98 m/z or +49 m/z. The peptide sequence responsible for producing the diagnostic phospho-ion was verified by tandem mass spectrometry (MS/MS).
Generation of Antibodies Against Sp1 Phosphopeptides and Dot Immunoblot Analysis
Sp1 peptides, GKRF(pT)R(pS)DELQR(pT indicates phosphorylated Thr and pS indicates phosphorylated Ser), GKRFTRSDELQR, HKRTH(pT)GEKKF, and HKRTHTGEKKF (Table 1), all containing an N-terminal cysteine (C), were synthesized by Chiron Mimotopes (Melbourne, Australia) and supplied at >90% to 95% purity. Anti–phospho-Sp1 antibodies were raised by immunizing NZW rabbits with phosphorylated peptides conjugated to keyhole limpet hemocyanin (Sigma). The anti-peptide antibody was affinity-purified with the immunizing peptide conjugated to BSA coupled on a 1:1 column of Affi-Gel 10/15 (Bio-Rad). The antibody was absorbed with the relevant nonphosphorylated peptide conjugated to BSA, also coupled on a 1:1 column of Affi-Gel 10/15 (Bio-Rad). Specificity of the anti-Sp1 antibodies was verified by dot blots against peptides conjugated to albumin (1:10 wt/wt). Antibody reactivity was visualized using horseradish peroxidase–conjugated goat anti-rabbit IgG and enhanced chemiluminescence kit (Amersham Biosciences). All procedures involving immunization were performed in accordance with institutional guidelines.
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Preparation of Nuclear Extracts
WKY12-22 cells treated with or without 10 µmol/L NOG (Sigma) or 1 µg/mL CAM for 6 hours or growth-quiescent HSMCs treated with or without 10–7 mol/L Ang II (Calbiochem) were washed twice and scraped in 10 mL of cold PBS, pH 7.4. The cells were centrifuged at 500g for 10 minutes at 4°C, and the pellet was resuspended in 100 µL of cold Buffer A (10 mmol/L Hepes–NaOH, pH 8.0, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 20 mmol/L sucrose, 0.5% Nonidet P-40) and incubated on ice for 5 minutes. The suspension was centrifuged at 18 000g for 40 seconds, and the pellet of nuclei was lysed with 20 µL of cold Buffer C (20 mmol/L Hepes–NaOH, pH 8, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 1 mmol/L DTT) by gently mixing for 20 minutes at 4°C. After recentrifugation at 18 000g for 1 minute, 20 µL of supernatant was combined with 20 µL of cold Buffer D (20 mmol/L HEPES-NaOH, pH 8, 100 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 1 mmol/L DTT) and was stored at –80°C until use. Buffers contained protease inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 4 µg/mL aprotinin, and 10 µg/mL leupeptin).
Electrophoretic Mobility-Shift Analysis
Oligodeoxynucleotides were synthesized by Sigma Genosys and annealed before radiolabeling with 
-32P-ATP (Perkin Elmer) using T4 polynucleotide kinase (New England Biolabs) and purified using Illustra Microspin G25 columns (GE Healthcare). Binding reactions were carried out in volume of 20 µL as described.26 Samples were resolved by electrophoresis on 6% nondenaturing polyacrylamide gel and vacuum dried at 80°C for 30 minutes before visualization by autoradiography.
Western Blot Analysis
WKY12-22 cells were treated with or without 10 µmol/L NOG (Sigma) or 1 µg/mL CAM, and growth-quiescent HSMCs were treated with or without 10–7 mol/L Ang II for the indicated times. Nuclear extracts were prepared as described above. Cytosolic extracts were prepared by washing and scraping cells in PBS, transferring to precooled tubes, and centrifuging at 500g for 15 minutes (4°C). The pellet was resuspended in 100 µL of Buffer A. Samples were placed on ice for 5 minutes, spun at 18 000g for 40 seconds, the supernatant (cytosolic extracts) was collected and stored at –80°C. Samples were prepared by adding 6 µL of 4x SDS protein loading sample buffer (0.5 mol/L Tris-HCl, pH 6.8, 50% glycerol, 20% SDS 0.05%, bromophenol blue), 2 µL of 0.5 mol/L DTT, and 10 µg nuclear extract protein and then boiled for 5 minutes at 100°C. Proteins were resolved by 8% SDS-PAGE and transferred onto Immobilon-P transfer membranes (Millipore). Membranes were blocked overnight in PBS containing 5% skim milk and 0.05% Tween-20. PKC-(pThr410), p668/670-Sp1, and p681-Sp1 were detected using rabbit polyclonal antibodies, followed by an horseradish peroxidase–linked secondary antibody and Western Blotting Luminol Reagent (Santa Cruz Biotechnology). For Western blot analysis of wild-type and mutant Sp1ZNF, in vitro kinase reactions using unlabeled ATP to produce "cold" protein. Samples were run on 12% SDS-PAGE gels and probed with pSer, pThr, and pTyr (Zymed). β-Actin antibodies and endothelin (ET)-1 were purchased from Sigma, PKC- antibodies were obtained from Santa Cruz Biotechnology, and PKC- antibodies were purchased from BD Bioscience.
Immunoprecipitation Analysis
Total protein was isolated from WKY12-22 SMCs treated with CAM using radioimmunoprecipitation assay (RIPA) buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% SDS, 1% Triton X-100, and protease inhibitors (1% aprotinin, 2 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 0.5 mmol/L dithiothreitol). Samples were incubated with prewashed protein G–Sepharose beads for 1 hour. Samples were centrifuged, and the supernatant was incubated with rabbit polyclonal Sp1 antibodies (Santa Cruz Biotechnology) overnight at 4°C with gentle shaking. Prewashed G-Sepharose beads were further incubated with lysate/antibody mixture for 2 hours. Beads were washed twice in 200 mmol/L NaCl/1x RIPA, followed by a final wash in 1x RIPA. Proteins were resolved by 8% SDS-PAGE and subsequent Western blot analysis for rabbit polyclonal anti-pSer, pThr, pTyr (Zymed), and Sp1 (Santa Cruz Biotechnology). Alternatively, cytosolic extracts from SMCs treated with Ang II (10 to 7 mol/L for 1 hour) preincubated with losartan or PD123319 (1 µmol/L for 1 hour) were first immunoprecipitated with rabbit polyclonal PKC-(pThr410) or PKC- antibodies by Western blot analysis.
Chromatin Immunoprecipitation Analysis
Human SMCs grown in 100-mm Petri dishes at 80% to 90% confluence were washed with cold PBS, pH 7.4, before formaldehyde fixation and chromatin immunoprecipitation (ChIP) as previously described31 with the indicated antibody. PCR was performed in 1 mmol/L MgCl2, 0.1 mmol/L dNTPs, 0.1 µmol/L primers and 1U Platinum Taq Polymerase (Invitrogen). Amplification conditions were as follows: 94°C for 2 minutes; 40 cycles of 94°C for 30 seconds, 54°C for 10 seconds and 72°C for 30 seconds; with another extension time of 4 minutes. The region of the PDGF-D promoter (390 bp) spanning Sp1 site –472/–469 was amplified with primers ChipPDGFDF2 primer 5'-CAT CAG TCT CGA CCT TTT CTC-3' and ChipPDGFDR2 primer 5'-GCT CAG GAA ACA AAC TCG C-3'.
Real-Time PCR
Growth-quiescent HSMCs were treated with or without 10–7 mol/L Ang II for the indicated times, with prior incubation with myristoylated PKC- pseudosubstrate inhibitor (Calbiochem) for 1 hour. Total RNA was prepared using TRIzol (Life Technologies Inc) according to the instructions of the manufacturer. cDNA was generated using 5 µg of total RNA, 0.5 µg/µL Oligo (dT)15 primer (Sigma), 1 mmol/L dNTP mix (Roche), and dH2O to 20 µL. The reaction was heated to 65°C for 5 minutes and immediately chilled on ice. First Strand Buffer (Invitrogen), 0.1 mol/L DTT (Sigma), and 40 U/µL RNAsin (Promega) were added to the sample mixture and heated for 2 minutes at 42°C, followed by the addition of 1 µL of Superscript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42°C for 50 minutes, and then at 70°C for 15 minutes to heat-inactivate the enzyme. The cDNA samples were stored at –20°C. Real-time quantitative PCR was performed using a Corbett RotorGene 3000 Detection System in a final volume of 10 µL containing 1 µL of cDNA, 5 µL of SYBR Green Master Mix (Applied Biosystems), and 0.5 µmol/L primers (Sigma) in DNAse-free water at the following PCR conditions: 50°C for 2 minutes; 95°C for 10 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 45 seconds, and 72°C for 20 seconds. Primer sequences for human PDGF-D were (forward) 5'-CGG TAT CGA GGC AGG TCA TAC-3' and (reverse) 5'-ACG CTT GGC ATC ATC ATT GAG-3'; and for human GAPDH were (forward) 5'-CCT TCA TTG ACC TCA ACT ACA TGG-3' and (reverse) 5'-GCT CCT GGA AGA TGG TGA TG-3'. Amplicon size was verified on 2% agarose/Tris/boric acid/EDTA gels.
Animal Models and Immunohistochemical Analysis
Carotid permanent ligation32 and balloon catheter injury studies33 were performed with Sprague–Dawley rats as previously described. Atherosclerotic carotid artery specimens were obtained by endarterectomy from St Vincent’s Hospital, Sydney. Immunohistochemistry was performed on paraffin sections of formalin-fixed tissue as previously described.34 Briefly, before staining, deparaffinized sections were boiled in citrate buffer, pH 6.0, to retrieve antigenicity and treated with 3% hydrogen peroxidase to block the endogenous peroxidase activities. The standard avidin–biotin complex immunoperoxidase technique was used.35 After washing in 50 mmol/L Tris-NaCl, pH 7.6, sections were incubated with primary antibodies (2 µg/mL for Sp1 and p676/686) for 60 minutes at 22°C. Sections were washed in 50 mmol/L Tris-NaCl, pH 7.6, and incubated with appropriate secondary antibody for 20 minutes and finally with avidin–biotin complex (Elite Vector PK-6100) for 30 minutes. The product of the immunohistochemical reaction was visualized by treatment with 3,3'-diaminobenzidine solution for 2 minutes, which produced brown coloration. Sections were counterstained with Mayer’s hematoxylin. For negative controls, the primary antibody was omitted, or the sections were treated with immunoglobulin fraction of appropriate nonimmune serum as substitute for the primary antibody.
Statistics
The data are expressed as the means±SE of the mean and analyzed using Student’s t test or ANOVA, where a P<0.05 was considered significant. Each in vitro experiment was performed independently multiple times, each time in duplicate or triplicate. Statistical analysis was performed on the means of 3 independent experiments. Rat injury experiments were performed in groups of 6.
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Results |
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PKC-
Phosphorylates Sp1 on Ser and Thr ResiduesWe used CAM14 as a model agonist of PKC-
–dependent Sp1 phosphorylation. Western blot analysis using antibodies recognizing the active form of PKC-(pThr410) revealed that CAM induces PKC- phosphorylation in vascular SMCs within 30 minutes and that PKC- remains phosphorylated for up to 3 hours (Figure 1A). Immunoprecipitation of Sp1 and subsequent immunoblotting determined that CAM-stimulated phosphorylation on Ser and Thr residues, but not on Tyr residues, in Sp1 (Figure 1B).
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Identification of Putative PKC- Phosphorylation Sites in Sp1 ZNF Domain
Inspection of the ZNF domain of Sp1 (residues 628 to 708) (Table 2) revealed the existence of 5 Ser (Ser641, Ser661, Ser670, Ser698, Ser702) and 7 Thr residues (Thr640, Thr651, Thr659, Thr668, Thr679, Thr681, Thr707). Computer analysis using NetPhos 2.0 Server software (http://www.cbs.dtu.dk/services/NetPhos) determined that Ser641, Thr668, Ser670, and Thr681 were the best scoring candidates for phosphorylation; however, software prediction does not ensure phosphorylation and requires experimental validation. Therefore, to explore the specific roles of Ser641, Thr668 and Ser670, and Thr681 as substrates of PKC- phosphorylation, we performed kinase reactions using human recombinant PKC- and 32P-ATP. In control experiments, we demonstrate that purified PKC- stimulates incorporation of 32P-ATP into full-length recombinant Sp1 (Figure 2A) and that the kinase is itself phosphorylated (Figure 2A). PKC- phosphorylated synthetic peptides 665/674 (containing Thr668 and Ser670) and 677/686 (containing Thr681) but did not affect 32P incorporation into peptide 637/646 (containing Ser641) (Figure 2B, top left). PKC- phosphorylation of peptide 677/686, unlike 637/646, was dose-dependent (Figure 2B, bottom graphs). PKC- also phosphorylated a 24-aa synthetic peptide, 663/686, spanning all 3 candidate residues (Figure 2C). 32P-incorporation, however, was dramatically reduced with peptide m663/686, bearing Ala mutations in Thr668, Ser670, and Thr681 (Figure 2C). In contrast, neither wild-type nor mutant peptide was phosphorylated by PKC- (Figure 2B, top right). Synthetic peptides carrying Ala substitutions were used in the kinase assay to determine the residues in these peptides that serve as target(s) of PKC- phosphorylation. Neither single nor double mutations of Thr668, Ser670, or Thr681 in peptide 663/686 altered PKC-–mediated phosphorylation in comparison with the wild-type peptide (Figure 2C), indicating that multiple residues in the peptide are targets of the kinase.
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Nanoelectrospray Mass Spectrometric Phosphopeptide Analysis of 665/674 and 677/686
Mass spectrometry was performed with in vitro PKC-–phosphorylated and nonphosphorylated peptides 665/674 or 677/686, decapeptides spanning Thr668 and Ser670 or Thr681, respectively. Static nanoelectrospray using the ion-trap full mass scan of the in vitro–phosphorylated peptide 665/674 showed the unphosphorylated double-charged peptide mass of 640.4 m/z, as well as 689.3 m/z corresponding to the double-charged monophosphorylated peptide (Figure IA in the online data supplement). Further evidence of peptide phosphorylation was obtained with neutral loss scans of +49 m/z and +98 m/z producing clear ions at 689.8 m/z and 1378.5 m/z for the +2 and +1 charge states, respectively (supplemental Figure IB and IC). Although ions corresponding to the addition of more than 1 phosphate moiety to this peptide were not observed, multiple phosphorylated species are often under-represented in mass spectrometric analyses.36,37 MS/MS fragmentation was optimized for 689.8 (44 V) and 1378.5 (50 V) by ramping the collision energy from 10 to 90 V in increments of 2 V. However, the MS/MS spectra for both 689.8 and 1378.5 were relatively featureless and yielded little sequence information useful in assigning the site of phosphorylation. Poor fragmentation was attributed to both the absence of a C-terminal basic residue necessary for strong y ion production and the presence of several basic residues that may compromise peptide cleavage.38 Static nanoelectrospray analysis of the in vitro PKC- reaction for peptide 677/686 indicated the presence of a monophosphorylated peptide. Despite being undetected in the full mass scan (supplemental Figure ID), the phosphopeptide (+2, 665.5 m/z) was clearly observed using a neutral loss scan (+49 m/z) specific for phosphopeptides (supplemental Figure IE). MS/MS fragmentation of the phosphopeptide at 40 V showed a distinct loss of phosphate from the double-charged parent ion (supplemental Figure 1F). These data thus demonstrate that peptides 665/674 and 677/686 are PKC- substrates. The exact site of phosphorylation could not be accurately deciphered by this method because of the absence of key diagnostic fragment ions.
Mutation of Thr668/Ser670/Thr681 Abolishes PKC-–Dependent Ser and Thr Phosphorylation in the ZNF of Sp1
To verify the importance of Thr668/Ser670/Thr681 as targets of PKC-, we cloned wild-type and triple mutant Sp1 ZNF cDNA into plasmid pQE-82L and produced recombinant His-tagged protein. These proteins were then used as substrates in an in vitro kinase reaction with 32P-ATP. Figure 3 demonstrates that wild-type Sp1 ZNF is phosphorylated by PKC-, whereas the triple mutant is not a substrate for the kinase (Figure 3, left). We next performed an in vitro kinase reaction using unlabeled ATP, to produce "cold" wild-type and triple mutant Sp1 ZNF. We immunoblotted for pSer, pThr, pTyr, Sp1, and His tag by Western blot analysis using the respective antibodies. Mutation of Thr668/Ser670/Thr681 abolished PKC-–dependent Ser and Thr phosphorylation in the ZNF of Sp1 (Figure 3, right), consistent with findings using native Sp1 (Figure 1B).
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Generation of Phospho-Specific Sp1 Antibodies and Demonstration That Phospho-Specific Sp1 Antibodies Bind Phosphorylated Sp1 Peptide and Protein
Based on the preceding data, we raised polyclonal phospho-specific antibodies to PKC-–modified Sp1 in NZW rabbits using previously described strategies.39,40 Synthetic peptides and phosphopeptides corresponding to 664/675 (containing Thr668 and Ser670) and 676/686 (containing Thr681) with N-terminal Cys residues were conjugated to keyhole limpet hemocyanin. To demonstrate the phospho-specificity of our p664/675 (containing Thr668 and Ser670) and p676/686 (containing Thr681) antibodies following anti-serum subtraction for nonphosphorylated-peptide specificity, we performed dot blot analysis. Nitrocellulose paper was impregnated with increasing amounts of unphosphorylated (664/675 or 676/686) and phosphorylated (p664/675 or p676/686) peptide or BSA, and the membranes were incubated separately with each antibody before chemiluminescence detection. Anti-p664/675 recognized p664/675 in a dose-dependent manner but failed to interact with 676/686 or BSA (Figure 4A, left). Conversely, anti-p676/686 bound p676/686 but not 664/675 nor BSA (Figure 4A, right). Western blotting was performed with anti-p664/675, anti-p676/686, and anti-Sp1 to confirm antibody recognition of phosphorylated endogenous Sp1. Sp1 was phosphorylated by NOG and CAM, with no change in levels of non–phospho-specific Sp1 (Figure 4B, left and right).
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Ang II Stimulates PKC- and Sp1 Phosphorylation
Ang II, the effector peptide hormone of the renin–angiotensin system involved in blood pressure control, vascular tone, and growth factor induction41,42 has previously been shown to activate PKC- in SMCs.43 However, whether Ang II stimulates Sp1 phosphorylation and subsequently gene expression through this pathway is unknown. Western blot analysis using SMCs treated with Ang II (10–7 mol/L) for various times confirmed that Ang II stimulates PKC-(Thr410) phosphorylation, as band intensity increased within 30 minutes and continued to do so up to 3 hours (Figure 5A). In contrast, non–phospho-specific PKC- levels remained unchanged (Figure 5A). Western blot analysis with anti-p676/686, anti-p664/675, or Sp1 antibodies performed with SMCs treated with or without Ang II (10–7 mol/L) for 1 hour revealed that Ang II induced Sp1 phosphorylation within 1 hour, without influencing levels of non–phospho-specific Sp1 (Figure 5B). Both Western blot analysis (Figure 5C, top) and immunoprecipitation analysis (Figure 5C, bottom) revealed that losartan (1 µmol/L) inhibits Ang II–inducible phosphorylation of PKC- (at Thr410), whereas the same concentration of PD123319 had no effect. Further experiments revealed that Ang II stimulates both Thr668/Ser670 and Thr681 phosphorylation in an Ang II type 1 (AT1) (but not Ang II type 2 [AT2])-receptor dependent manner (Figure 5B). These data demonstrate that Ang II stimulation of PKC- and Sp1 phosphorylation is mediated via the AT1 receptor.
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Ang II Increases Sp1 Phosphorylation at the PDGF-D Promoter
Recent studies by our group have demonstrated that Sp1 interacts with the PDGF-D promoter and that Ang II (10–7 mol/L) induces PDGF-D mRNA expression in vascular SMCs.29 Our generation of phospho-specific Sp1 antibodies led us to investigate the status of phosphorylated Sp1 physically bound to the PDGF-D promoter in cells exposed to Ang II. ChIP analysis using these antibodies revealed that phosphorylated Sp1 was associated with at least 3 sites in the PDGF-D promoter under basal conditions (Figure 6A) and that Ang II increased phospho-Sp1 occupancy of the PDGF-D promoter spanning site –472/–469 (Figure 6A). We performed electrophoretic mobility shift analysis (EMSA) to provide supportive evidence. Figure 6B demonstrates that the ability of Sp1 to bind to a fragment of the PDGF-D promoter is enhanced by exposure of the cells to Ang II, which stimulates Sp1 phosphorylation (Figures 6B and 7
C, right) without affecting levels of non–phospho-specific Sp1 (Figures 5B and 6B, left). The identities of Sp1 and phospho-Sp1 bound to DNA are indicated by positive supershift/elimination analyses (Figure 6B, right), and nonspecificity is ruled out by negative supershifts with Sp3 and YY1 antibodies. These data demonstrate increased DNA binding when Sp1 is phosphorylated in cells treated with Ang II. Alterations in the ability of a transcription factor to interact with DNA as a function of phosphorylation has previously been reported for Ikaros,44 which controls G1-to-S transition, and the Ets family member PU.1.45
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Ang II–Inducible PDGF-D Expression Involves PKC-–Dependent Sp1 Phosphorylation at Thr668, Ser670, and Thr681
To demonstrate the dependence of Ang II–inducible PDGF-D expression on PKC-, we performed real-time PCR on extracts of SMCs treated with or without Ang II (10–7 mol/L) and a pseudosubstrate (PS) inhibitor of PKC-. Ang II–inducible PDGF-D mRNA expression at 2 hours was blocked by prior incubation with 100 µmol/L of the PKC- inhibitor (Figure 7A) but not 50 or 75 µmol/L (Figure 7A). Because Ang II activates PKC- and Sp1 phosphorylation (Figure 5A through 5C), we examined the effect of PKC- knockdown on Ang II–inducible PDGF-D expression. Western blotting and quantitative real-time PCR analysis revealed that PKC- small interfering RNA inhibits levels of PKC-(pThr410) (Figure 7B, right) and PDGF-D mRNA expression (Figure 7B, left) stimulated by Ang II. In contrast, a size-matched double-stranded RNA with irrelevant sequence failed to influence levels of Ang II stimulation of PKC-(pThr410) and PDGF-D (Figure 7B). To demonstrate the contribution of Thr668, Ser670, and Thr681 in Ang II–inducible PDGF-D transcription, we introduced Ala substitutions into CMV-Sp1, a CMV-based expression vector generating exogenous full-length Sp1. Western blot analysis with anti-p676/686 revealed that Ang II stimulated Sp1 phosphorylation (pThr681) and that this increased recognition was blocked by mutation of Thr681 (Figure 7C, right). This suggests further that the antibody detects basal and inducible endogenous phosphorylated Sp1. Transient transfection analysis demonstrated that Ang II induction of PDGF-D promoter–reporter activity in the presence of Sp1 was abolished by the triple mutant (CMV-Sp1m668/670/681) (Figure 7C, left) but was not compromised by CMV-Sp1m641/668/670, CMV-Sp1m641/670/681, or single or double Ala substitutions of these and other residues (Figure 7C, left, and data not shown). Thus, PDGF-D promoter activity was stimulated by Ang II and Sp1, and when Ang II was combined with a series of mutant Sp1 constructs, except the triple mutant (CMV-Sp1m668/670/681). EMSA with 32P-Oligo D using nuclear extracts of cells transfected with wild-type Sp1 or the triple mutant revealed that Ala mutations at Thr668, Ser670, and Thr681 did not compromise its ability to bind DNA (Figure 7D). To further demonstrate that the triple mutation does not perturb Sp1 ZNF interaction with a fragment of the PDGF-D promoter, we cloned wild-type and mutant cDNA into plasmid pQE-82L and produced recombinant His-tagged protein. No difference in DNA-binding intensity was observed between wild-type and triple mutant ZNF (Figure 7D, bottom right). These data thus demonstrate that the triple mutant does not alter the ability of Sp1 to bind to DNA, whether in the absence or presence of endogenous Sp1. Finally, Western blot analysis was performed to determine whether the potent vasoconstrictor ET-1, like Ang II, could influence Sp1 phosphorylation. PKC- is activated by ET-1.31,32 Figure 7E shows that ET-1 increased Sp1 phosphorylation without affecting levels of total Sp1, demonstrating that vascular growth factors other than Ang II can also influence Sp1 phosphorylation.
Detection of Phosphorylated Sp1 in Human Atherosclerotic, Balloon-Injured, and Permanently Ligated Rat Carotid Arteries
We used the newly generated anti-p676/686 antibody to demonstrate, for the first time, the existence of this modified form of Sp1 in 2 vascular settings in which SMCs play a prominent role. Immunohistochemical analysis with formalin-fixed tissue revealed that phospho-Sp1, like non–phospho-specific Sp1, was expressed in human atherosclerotic lesions (Figure 8A). Previous studies by our group demonstrated the presence of active PKC-(pThr410) in these lesions.30 We next used the antibody to determine whether Sp1 can be inducibly phosphorylated in response to acute vascular injury, building on recent studies that have demonstrated increased angiotensin-converting enzyme, PKC-, and PDGF-D expression in injured rat carotid arteries.46–48 We injured the carotid arteries of Sprague–Dawley rats in 2 separate models and performed immunohistochemical analysis on cross-sections at various times after injury. Sp1 was basally phosphorylated in the uninjured vessel wall and inducibly stimulated by injury within 1.5 hour (Figure 8B). Incubating the sections with calf intestinal alkaline phosphatase49 before the addition of primary antibody abrogated anti-p676/686 immunoreactivity without affecting total Sp1 staining (data not shown). Sp1 was also phosphorylated in rat carotid arteries within hours of permanent ligation injury (Figure 8C). To temporally link Sp1 phosphorylation with PDGF-D expression, we stained sister sections for the growth factor and found that elevated PDGF-D levels followed the induction of Sp1 phosphorylation (Figure 8D). Finally, we probed for anti-p676/686 immunoreactivity in extracts of SMCs isolated from human atheromatous carotid plaque or rat SMCs 1 hour after injury. Phospho-specific Sp1 immunoreactivity was higher in atheromatous plaque SMCs and injured SMCs compared with their control SMC counterparts (Figure 8E). These data demonstrate dynamic Sp1 phosphorylation in the injured artery wall as well as Sp1 phosphorylation in the comparatively chronic pathological setting of atherosclerosis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sp1 has long been known to be a phosphoprotein.3 Here, we demonstrate that the PKC-
–Sp1 phosphorylation axis is a key regulatory pathway in Ang II–inducible PDGF-D expression and that amino acid residues Thr668, Ser670, and Thr681 in the second ZNF region of Sp1 are specifically modified by PKC-. We have generated novel phospho-specific Sp1 antibodies and have demonstrated their use as immunoreagents. This is the first identification of residues in Sp1 modified by a kinase also regulating PDGF transcription in response to Ang II. This is, to the best of our knowledge, the first time Sp1 phosphorylation has been documented in the injured artery wall (Figure 8).
The structure of the DNA-binding domain of Sp1 has been resolved by solution NMR techniques by Oka et al.50 Ser670 and Thr681 (designated Ser581 and Thr592, respectively, in the study by Oka et al50) reside within the second of 3 ZNFs at each edge of the -helical DNA recognition region, whereas Thr668 (designated Thr579) lies between the second β-turn and the -helix.50 The short antiparallel β-sheet is formed via 2 hydrogen bonds, whereas the -helix is stabilized by 2 hydrogen bonds in its amino terminus. Our findings demonstrate that PKC- phosphorylation of all 3 residues in Sp1 (Thr668, Ser670, and Thr681) is necessary for Ang II induction of the PDGF-D promoter. Neither single nor double mutations to Ala in any of these residues were sufficient to ablate Ang II regulation of PDGF-D transcription (data not shown). Examination of their positions by 3D modeling reveals the 3 amino acids lie in close proximity to each other, spaced apart maximally by only 13 residues in a protein comprising more than 750 amino acids. The 3 residues are predicted to reside on the same side in the folded protein (Figure 7F); Ser670 and Thr681 face the same direction at either end of an -helical DNA-recognition domain. Thr668, being only 2 residues away from Ser670, is aligned in the same general direction as Ser670 and Thr681, despite a hairpin bend after the β-sheet. Ser641, on the other hand, is located distally upstream on the first ZNF. The close proximity and orientation of Thr668, Ser670, and Thr681 in Sp1 are consistent with the possibility of simultaneous phosphorylation by PKC-.
It is unclear whether phosphorylation causes a conformational change in Sp1 or the promoter it binds51 or if its modification engages and facilitates assembly of a transcriptional cofactor complex52 favoring increased Sp1-dependent gene expression. Lessons in subsequent studies may be drawn from other transcription factors. CREB, for example, undergoes a conformational change when phosphorylated at Ser133 by PKA, inducing bends and twists in the bound DNA.51 Moreover, steroidogenic factor-1–dependent transcription involves extracellular signal-regulated kinase (ERK)-dependent Ser203 phosphorylation in steroidogenic factor-1, which recruits nuclear receptor cofactors such as GRIP1 and SMRT.53 Additionally, Sp1 phosphorylation may regulate gene expression via mechanisms involving histone deacetylase. A recent study, for example, demonstrates derecruitment of the p107 repressor from the luteinizing hormone receptor promoter in a PKC-–induced Sp1 phosphorylation-dependent manner and identified Ser641 as a phosphorylation site,17 although we could not confirm this in the present study. Larouche et al identified 2 residues in Sp1, namely Thr453 and Thr739, that are phosphorylated by ERK.8 We recently demonstrated that both these residues in Sp1 are required for ERK-dependent fibroblast growth factor-2 repression of PDGF receptor- transcription in SMCs.54 Moreover, these residues are required for ERK-dependent galectin-1 regulation of p27 transcription in carcinoma cells.55 The question of whether Sp1 phosphorylation increases or decreases its affinity for DNA is controversial and is likely to be gene-specific and dependent on the local microenvironment.56,57 This study demonstrates that Ang II–inducible Sp1 phosphorylation results in increased DNA binding without increased Sp1 expression. Other examples of genes whose expression is a function of Sp1 phosphorylation include, from our own work PDGF-B,14 FasL,13 and PDGF receptor-,54,58 as well as gastrin, dihydrofolate reductase, matrix metalloproteinase-2, nitric oxide synthase, and cyclin D3.56 These genes play important roles in a wide variety of pathophysiological processes, including cell cycle regulation, tumor growth and metastasis, SMC growth and blood vessel thickening, angiogenesis, and apoptosis. The present study shows for the first time that phosphorylated Sp1 is expressed in SMCs of human atherosclerotic plaques and is dynamically expressed together with PDGF-D in SMCs of the injured rat carotid artery wall. Sp1 phosphorylation may thus represent a biologically important posttranslational regulatory mechanism. Delineation of particular amino acid residues in Sp1 modified by phosphorylation, as in the present study, will underpin the future development of specific pharmacological agents that would interfere with kinase-Sp1–dependent gene expression axes and serve as novel tools to prevent or treat vascular and other disease.
This study defines specific amino acids in Sp1 that are phosphorylated by PKC- and demonstrates transcriptional changes as a consequence of the posttranslational modification. The functional importance of the PKC-–Sp1 axis is borne out by mutations in 3 critical residues in the ZNF domain of Sp1 (Thr668, Ser670, and Thr681) and the switch in Sp1 affinity for DNA stimulated by Ang II. The phospho-specific Sp1 antibodies generated in this study will serve as important molecular tools to provide further new insights on the roles that the PKC-–Sp1 axis plays in normal physiological and pathophysiologic processes.
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Acknowledgments |
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We thank Matthew McKay (Australian Proteome Analysis Facility) for mass spectrometry assistance and Yuri Bobryshev (School of Medical Sciences, University of New South Wales) for archival tissue and immunohistochemical analysis.
Sources of Funding
This work was supported by grants from the Australian National Health and Medical Research Council and the Australian Research Council.
Disclosures
None.
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Footnotes |
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Original received November 5, 2007; revision received December 11, 2007; accepted January 15, 2008.
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References |
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