Page 172 - Haematologica June

K. Kurakula et al.
cules such as tumor necrosis factor-α (TNF-α) and lipopolysaccharides (LPS), which induce TF expression via activating protein 1 (AP-1) and nuclear factor-kappa B (NF- κB) signaling.1,9,10 Consistently, inflammation-induced coag- ulation is completely abrogated by inhibition of TF activity in vivo.11
The TF protein contains two fibronectin type-III ectodomains, a single type-I transmembrane domain, and a twenty-amino acid cytoplasmic domain (TFCD), which plays an important role in several of the above-mentioned pathologies that involve TF.6-8 The TFCD can undergo mul- tiple post-translational modifications, including palmitoyla- tion at residue Cys245, phosphorylation at residues Ser253 and Ser258 by PKC-α and p38α-kinase, respectively, and ubiquitination at residue Lys255.12-15 These modifications act in concert to attenuate the interaction of the TFCD with the membrane lipid bilayer, as well as make it accessible to other interacting proteins that regulate TF activity.13,14,16
Peptidyl-prolyl cis-trans isomerase, NIMA-Interacting 1 (Pin1) is an enzyme that catalyzes cis-trans isomerization of proline residues that are preceded by a phosphorylated ser- ine or threonine (a pSer/pThr-Pro motif) within its target proteins. The C-terminal isomerase domain of Pin1 binds the motif and catalyzes proline cis-trans isomerization, while the N-terminal WW-domain is responsible for medi- ating protein-protein interactions and target specificity.17-19 Conformational changes induced by Pin1-catalyzed proline isomerization have been shown to alter the phosphoryla- tion, localization, stability, protein-protein interactions, and transcriptional activity of its target proteins, which include c-Jun, NF-κB, AP-1, p53, β-catenin, and the nuclear recep- tors PPARg and Nur77.20-22
Here, we report that Pin1 enhances TF gene expression in activated vascular cells, and directly interacts with TF pro- tein through a pSer258-Pro259 motif in the TFCD. We pro- vide the solution structure of the TFCD in complex with the WW-domain of Pin1, which shows that this interaction requires both phosphorylation of Ser258 as well as trans- configuration of the pSer258-Pro259 peptide bond in the TFCD. Functionally, we demonstrate that Pin1 increases the protein half-life and pro-coagulant activity of TF in human vascular cells.
Methods
Mice
Mice in which the cytoplasmic domain of endogenous TF is deleted (TFDCD mice)23 and wild-type litter mates were maintained under pathogen-free conditions at the Scripps Research Institute Animal Facility with approved protocols of the institutional Animal Care and Use Committee (protocols #08-0008 and #08- 0009).
Cell culture
Human umbilical vein endothelial cells (HUVECs) and smooth muscle cells (SMCs) were isolated from umbilical cords anony- mously collected at the Academic Medical Center, as previously described,22 in accordance with the institute’s ethical guidelines and with approval from the institute's Medical Ethical Committee. Please see the Online Supplementary Methods for a detailed descrip- tion of cell culture conditions.
Lentiviral transductions
Recombinant lentiviral particles encoding Pin1, shPin1, or back-
bone control constructs were produced as previously described.22 Cells were transduced at a multiplicity of infection of 100 for 24 hours (h), after which the medium was refreshed and cells were cultured for an additional 24 h before starting experiments.
Transfections and luciferase assays
HEK293T cells and SMCs were transfected using the CalPhos Mammalian Transfection Kit (Clontech) or Lipofectamine 3000 (Invitrogen), respectively. Cells were transfected according to the manufacturer’s instructions with wild-type, AP-1 binding site mutated, or NF-kB binding site mutated TF promoter luciferase reporter constructs (a gift from Nigel Mackman;24 Addgene #15442-15444) together with Pin1 or Pin1 mutants (described by van Tiel et al.22). The pRL-TK Renilla-reporter was co-transfected as internal control. After 24 h, cells were stimulated with 100 ng/mL 12-O-Tetradecanoylphorbol-13-acetate (PMA; Sigma) for 6 h. For NF-κB or AP-1 signaling inhibition, cells were treated with vehicle (DMSO), 10 mM BAY-117085 (Calbiochem), or 10 mM SP600125 (SelleckChem). Assays were performed using the dual-luciferase reporter assay and a Glomax Multi detection system (Promega).
Nuclear magnetic resonance spectroscopy
Nuclear magentic resonance (NMR) experiments were per- formed on Bruker DRX 600 MHz and 800 MHz spectrometers equipped with triple resonance 1H/13C/15N optimized for proton detection at 300K. Acquired data were converted from Bruker XWINNMR format to NMRVIEW v.5.3 format using NMRpipe software.25,26 All spectra were analyzed using NMRVIEW. All experiments were performed at the UH NMR Facility at the University of Houston. Please see the Online Supplementary Methods for a detailed description of NMR spectroscopy proce- dures.
TF protein half-life assays
Smooth muscle cells (SMCs) or HEK293T cells were transfected with Pin1 or Pin1 mutants and either TF or TFDCD constructs. After 24 h, transfected cells were treated with 50 mg/mL cycloheximide (Sigma) for times indicated. TF protein levels were quantified by western blotting.
TF activity assays
Tissue factor activity was determined in SMCs, HUVECs, and EC-RF24 cells as previously described.27 Briefly, transduced cells were serum-starved overnight followed by stimulation with 50 ng/mL TNF-α (Peprotech) for 3 h. Cells were washed with PBS and incubated with 1 nM human Factor VIIa and 100 nM human Factor X (Kordia) at 37°C. Supernatant samples were collected in 100 mM EDTA, 50 mM Tris after 10, 20, and 30 minutes (min), incubated with 0.4 mM of FXa chromogenic substrate S-2222, and absorbance was measured at 405 nm.
Statistical analysis
Data are presented as mean±Standard Error of Mean. Significance was determined by unpaired two-tailed Student’s t- test or one- or two-way ANOVA with Bonferroni post-hoc correc- tion as indicated in the figure legends. P<0.05 was considered sta- tistically significant.
Results
Pin1 enhances TF gene expression via activation of NF-κB and AP-1
Pin1 modulates the activity of various transcription fac- tors involved in TF gene expression.14,15 Therefore, we ini-
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haematologica | 2018; 103(6)