Phosphorylation of protein inhibitor of activated STAT1 (PIAS1) by MAPK-activated protein kinase-2 inhibits endothelial inflammation via increasing both PIAS1 transrepression and SUMO E3 ligase activity

Abstract

Objective: Protein inhibitor of activated signal transducer and activator of transcription-1 (PIAS1) is known to function as small ubiquitin-like modifier (SUMO) E3 ligase as well as transrepressor. The aim of the study is to elucidate the regulatory mechanisms for these 2 different functions, especially with respect to endothelial inflammation.

Methods and results: The mitogen-activated protein kinase (MAPK)-activated protein kinase-2 is a proinflammatory kinase and phosphorylates PIAS1 at the Ser522 residue. Activation of MAPK-activated protein kinase-2 enhances p53-SUMOylation, but a PIAS1 phosphorylation mutant, PIAS1-S522A, abolished this p53-SUMOylation, suggesting a critical role for PIAS1-S522 phosphorylation in its SUMO ligase activity. Because nuclear p53 can inhibit Kruppel-like factor 2 promoter activity, we investigated the roles for PIAS1 phosphorylation and p53-SUMOylation in the Kruppel-like factor 2 and endothelial NO synthase expression. Both MAPK-activated protein kinase-2 and PIAS1 overexpression increased Kruppel-like factor 2 promoter activity and endothelial NO synthase expression, which were inhibited by expressing a p53-SUMOylation defective mutant, p53-K386R, and PIAS1-S522A. PIAS1-S522A also abolished the anti-inflammatory effect of wild-type PIAS1 in vitro and also in vivo, which was examined by leukocyte rolling in microvessels of skin grafts transduced by adenovirus encoding PIAS1-WT or - S522A mutant.

Conclusions: Our study has identified a novel negative feedback regulatory pathway through which MAPK-activated protein kinase-2 limits endothelial inflammation via the PIAS1 S522 phosphorylation-mediated increase in PIAS1 transrepression and SUMO ligase activity.

Figure 1. MK2-mediated PIAS1 phosphorylation enhances NF-κB Transactivation under TNF stimulation

HUVECs were transfected with an NF-κB-luc reporter construct and kinase dead wild-type PIAS1 (A) or PIAS1 siRNA (B), stimulated with TNF, and then assayed for firefly and Renilla luciferase activities. For experiments A–B, quantitative data are shown with n=3, values are mean ± S.D.; *P < 0.05, **P < 0.01. (C) PIAS1 was initially divided into three ~200 amino acid fragments (1–257, 231–456, 431–651) and analyzed by in vitro kinase assay. Note strong phosphorylation of the PIAS1 fragment 431–651 by MK2. A representative autoradiogram is shown along with a Ponceau stain to show GST-PIAS1 expression levels. (D) The PIAS1 431–651 fragment was further divided in half (431–550 and 551–651) and analysis was repeated. (E) PIAS1 phosphorylation mutants were constructed and used to evaluate their effects on NF-κB transactivation under TNF stimulation using dual-luciferase reporter assays. *P < 0.05, **P < 0.01 (n=3 mice; mean ± S.D.). (F) HUVECs were transfected for 48 hrs with either MK2 or control siRNA as indicated and then the cells were stimulated with TNF (10 ng/ml) as indicated. PIAS1 S522 phosphorylation was detected by immunoblotting with anti-phospho-PIAS1 S522 (top). The MK2 and PIAS1 expressions were detected by immnoblotting with anti-MK2 and -PIAS1, respectively. Values are mean ± S.D. (lower panel, n=3); **P < 0.01 compared to the sample without TNF stimulation, ^##P < 0.01 compared to each concentration of TNF stimulation in the control siRNA. (G) Endogenous PIAS1 in HUVECs was depleted using siRNA, and after 48 hrs the cells were transduced with adenovirus containing PIAS1-WT or PIAS1- S522A mutant. After 16 hrs of transduction cells were stimulated by TNF for 10 min, and PIAS1 was immuno-precipitated by anti-PIAS1 and immunoblotted with anti-phospho-PIAS1 S522 (top). PIAS1 and tubulin expression were detected by immnoblotting with anti-PIAS1 and -tubulin, respectively. Values are mean ± S.D. (lower panel, n=3); *P < 0.05, **P < 0.01. (H) NF-κB transactivation under TNF stimulation was determined using dual-luciferase reporter assays in PIAS1 S522A mutant knock-in experiments. HUVECs were transfected with PIAS1 siRNA or control siRNA and after 48 hrs transduced with Ad-PIAS1-WT, Ad-PIAS1-S522A mutant, or Ad-LacZ. After 16 hrs of transduction the NF-κB-luc reporter was transfected, and cells were stimulated with TNF and then assayed for firefly and Renilla luciferase activities. Values are mean ± S.D. (n=3); *P < 0.05, **P < 0.01.

Figure 1. MK2-mediated PIAS1 phosphorylation enhances NF-κB Transactivation under TNF stimulation

HUVECs were transfected with an NF-κB-luc reporter construct and kinase dead wild-type PIAS1 (A) or PIAS1 siRNA (B), stimulated with TNF, and then assayed for firefly and Renilla luciferase activities. For experiments A–B, quantitative data are shown with n=3, values are mean ± S.D.; *P < 0.05, **P < 0.01. (C) PIAS1 was initially divided into three ~200 amino acid fragments (1–257, 231–456, 431–651) and analyzed by in vitro kinase assay. Note strong phosphorylation of the PIAS1 fragment 431–651 by MK2. A representative autoradiogram is shown along with a Ponceau stain to show GST-PIAS1 expression levels. (D) The PIAS1 431–651 fragment was further divided in half (431–550 and 551–651) and analysis was repeated. (E) PIAS1 phosphorylation mutants were constructed and used to evaluate their effects on NF-κB transactivation under TNF stimulation using dual-luciferase reporter assays. *P < 0.05, **P < 0.01 (n=3 mice; mean ± S.D.). (F) HUVECs were transfected for 48 hrs with either MK2 or control siRNA as indicated and then the cells were stimulated with TNF (10 ng/ml) as indicated. PIAS1 S522 phosphorylation was detected by immunoblotting with anti-phospho-PIAS1 S522 (top). The MK2 and PIAS1 expressions were detected by immnoblotting with anti-MK2 and -PIAS1, respectively. Values are mean ± S.D. (lower panel, n=3); **P < 0.01 compared to the sample without TNF stimulation, ^##P < 0.01 compared to each concentration of TNF stimulation in the control siRNA. (G) Endogenous PIAS1 in HUVECs was depleted using siRNA, and after 48 hrs the cells were transduced with adenovirus containing PIAS1-WT or PIAS1- S522A mutant. After 16 hrs of transduction cells were stimulated by TNF for 10 min, and PIAS1 was immuno-precipitated by anti-PIAS1 and immunoblotted with anti-phospho-PIAS1 S522 (top). PIAS1 and tubulin expression were detected by immnoblotting with anti-PIAS1 and -tubulin, respectively. Values are mean ± S.D. (lower panel, n=3); *P < 0.05, **P < 0.01. (H) NF-κB transactivation under TNF stimulation was determined using dual-luciferase reporter assays in PIAS1 S522A mutant knock-in experiments. HUVECs were transfected with PIAS1 siRNA or control siRNA and after 48 hrs transduced with Ad-PIAS1-WT, Ad-PIAS1-S522A mutant, or Ad-LacZ. After 16 hrs of transduction the NF-κB-luc reporter was transfected, and cells were stimulated with TNF and then assayed for firefly and Renilla luciferase activities. Values are mean ± S.D. (n=3); *P < 0.05, **P < 0.01.

Figure 2. MK2 increases p53-SUMOylation via PIAS1 phosphorylation

(A) HUVECs were transduced with Ad-LacZ, Ad-MK2, or Ad-DN-MK2 for 18 hrs and then transfected for 24 hrs as indicated with Flag-tagged p53, HA-tagged SUMO3, and Ubc9. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-HA (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag and the MK2, Ubc9, and SUMO expressions were detected by indicated antibodies. Mono-SUMOylation band (≈74kDa) and poly-SUMOylation bands (> 78kDa) were detected. (B) HUVECs were transfected for 48 hrs with either MK2 or control siRNA as indicated and then the cells were stimulated with TNF (10 ng/ml) for 3 hrs. p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO (top). The MK2, p53, and SUMO expressions were confirmed by immnoblotting with anti-MK2, - p53, and -SUMO, respectively. The quantitative data are shown with n=3 (A and B, lower panel), values are mean ± S.D.; **P < 0.01. (C) HUVECs were transduced for 18 hrs with Ad-PIAS1 or Ad-PIAS1-S522A with Ad-MK2 or Ad-LacZ as indicated, then the cells were stimulated with TNF for 3 hrs. p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO (top). PIAS1, MK2, p53, and SUMO expression was confirmed by immnoblotting with anti-PIAS1, MK2, -p53, and -SUMO2/3, respectively. (D) Values are mean ± S.D. (Fig. 2C, n=3); *P < 0.05, **P < 0.01.

Figure 3. MK2-mediated PIAS1 phosphorylation regulates p53-SUMOylation and subsequently enhances KLF2 promoter activity and eNOS mRNA expression

(A, B) Effects of p53 SUMOylation on KLF2 promoter activity (A) and eNOS mRNA expression (B) in HUVECs transduced with Ad-LacZ, Ad-MK2, or Ad-PIAS1 were evaluated using dual-luciferase reporter assays (A) and real-time quantitative PCR (B). HUVECs were transduced with Ad-LacZ, -MK2, -PIAS1, or -p53-KR for 18 hrs and further transfected with the KLF2 promoter activity reporter construct for 18 hrs, then assayed for firefly and Renilla luciferase activities (A). After 18 hrs of the adenovirus transduction, HUVECs were assayed for eNOS mRNA levels (B). (C) HUVECs were transduced with Ad-LacZ, -PIAS1-WT, -PIAS1-S522A, or -MK2 for 18 hrs, and KLF2 promoter activity (C) or eNOS mRNA levels (D) were detected as described for A–B. *P < 0.05, **P < 0.01 (n=3 mice; mean ± S.D.). (E) A signaling scheme describing the relationship between the inhibitory effect of p53 on KLF2-eNOS expression and MK2-induced PIAS1-S522 phosphorylation which leads to p53 SUMOylation.

Figure 4. PIAS1 phosphorylation mutant decreases EC inflammation

HUVECs were transduced with Ad-LacZ, Ad-PIAS1-WT, or Ad-PIAS1-S522A for 18 hrs followed by treatment with TNF. Cells were evaluated for mRNA expression for E-selectin (A), ICAM-1 (B), VCAM-1(C), and MCP-1 (D) by real-time quantitative PCR. Values are mean ± S.D. (n=3); **P < 0.01. (E) Protein expression of PIAS1-WT and PIAS1-S522A was evaluated by Western blotting using anti-PIAS1. (F) A scheme describing the effects of TNF and subsequent PIAS1-S522 phosphorylation-mediated negative feedback on inflammatory gene expression in ECs.

Figure 5. PIAS1 Ser522 phosphorylation inhibits TNF-mediated leukocyte rolling in the skin transplants

After 7 days of transplantation of skin grafts that were transduced with Ad-LacZ, Ad-PIAS1-WT, or Ad-PIAS1-S522A, the rolling and velocity of leukocytes (A–B) or fluorescent beads coated with anti-VCAM-1 (D–E) in the grafts were determined by intravital microscopy with and without 3 hrs of TNF injection. (A) Representative images with indications of distance traveled by leukocytes between two adjacent frames. (B) Quantification of leukocyte rolling and velocity in the skin graft’s micro-vessels. A total of ≈100 leukocytes was counted from two different fields in each skin graft. *P < 0.05 and **P < 0.01 (n=4 mice; mean ± S.D.). (C) Protein expression of VCAM-1, ICAM-1, PIAS1, and tubulin was determined by immunoblotting using whole tissue lysates from skin grafts as indicated. The figure is representative of two independent experiments. (D) Quantification of the roling velocity of anti-VCAM-1 antibody-coated fluorescent beads in the skin graft’s micro-vessels. A total of ≈ 60–100 beads were counted from two different microscope fields in each skin graft. **P < 0.01 (n=3; mean ± S.D.). (E) Captured images at each 15 ms show movement of anti-VCAM-1 antibody-coated fluorescent beads in the skin graft’s micro-vessel. Grey and black arrows indicate an anti-VCAM-1 antibody-coated fluorescent bead in the vessel. Grey arrow and circle: the position of the bead at 0 ms. Bars; 20 μm.

Figure 6. A schematic drawing showing how stimulation by TNF activates MK2-mediated NF-κB transactivation independent of the canonical IKKα-IκBα-NF-κB pathway in endothelial cells

(A) TNF-mediated activation of IKKα not only induces NF-κB transactivation through the canonical TNF-IKKα-NF-κB signaling pathway but also negatively regulates this event by phosphorylating PIAS1 at the S90 residue. Similar to IKKα, TNF-mediated activation of MK2 leads to NF-κB transactivation and also activates a negative feedback mechanism through which MK2 phosphorylates PIAS1 at the S522 residue to enhance its transrepression of NF-κB and p53 SUMOylation, p53 nuclear export, and subsequent KLF2-mediated eNOS expression. (B) The domafin structure of PIAS1. SAP domain: scaffold attachment factor A and B. PINIT: Pro-Ile-Asn-Ile-Thr motif. RLD: RING-finger-like zinc binding domain, protein-protein interactions, interacts with the SUMO conjugase Ubc9, sumoylation. AD: highly acidic domain. S/T: serine/threonine rich region. PIAS1 S90 site lie in the NF-κB interacting region, whereas PIAS1 S522 site does not lie in any of PIAS1’s functional domains involved in NF-κB binding or its SUMO E3 ligase activity (RLD) domain.