CHK1-driven SENP2 S344 phosphorylation under laminar flow prevents endothelial activation-regulated fibrosis and atherogenesis while preserving endothelial lineage
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ABSTRACT: BACKGROUND: The deSUMOylase SENP2 exerts athero-protective effects by inhibiting endothelial cell (EC) activation through attenuating ERK5 and p53 SUMOylation. Publicly available datasets show that SENP2 S344 is phosphorylated by Checkpoint Kinase 1 (CHK1), but the functional role remains unknown. METHODS: Mouse SENP2 S343A (human S344A) phosphodeficient knock in (KI) mutant was generated by CRISPR/Cas9, and vascular-specific function was assessed via bone marrow transplantation (BMT). ECs from KI and wild type (WT) mice were exposed to smooth (laminar flow; l-flow) or grooved (disturbed flow; d-flow) cone-and-plate devices and characterized by RNA sequencing (RNA-seq). RESULTS: L-flow increased CHK1 S280 and SENP2 S344 phosphorylation, which inhibited ERK5 and p53 SUMOylation and atherogenesis in vivo. BMT-generated vascular specific SENP2 S344A KI showed more atherogenesis but thinner fibrous cap formation specifically in the aortic arch area (d-flow) compared to that of WT mice. Ionizing radiation (IR) decreased CHK1 expression and SENP2 S344 phosphorylation, which might account for differences between systemic and BMT-generated vascular specific SENP2 S344A KI models. RNA-seq data analysis showed that SENP2 S344 phosphorylation in ECs in response to l-flow inhibited EC activation and fibrotic changes without interfering EC lineage phenotype. Lastly, l-flow-induced expression of genes was regulated by SENP2 S344 phosphorylation through ERK5 activation and inhibited EC apoptosis. CONCLUSIONS: We uncovered a novel mechanism by which l-flow inhibits EC activation, including proliferation, migration, inflammation, and fibrotic changes, via upregulating CHK1-mediated SENP2 S344 phosphorylation to attenuate atherogenesis. We also uncovered a unique expression pattern of fibrotic changes without affecting EC lineage, which is distinct from endothelial-to-mesenchymal transition and therefore should be considered a unique type of EC activation for its potential role in vulnerable plaque formation.
Project description:ERK5 is a dual kinase-transcription factor, which contains an N-terminal kinase domain and transactivation domains in the C-terminal half. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions regarding the functional role of ERK5 kinase inhibitors. We aimed to investigate how ERK5 reprograms myeloid cells (MC) to the senescence-associated secretory phenotype (SASP), consequently leading to atherosclerosis. We showed that atherosclerosis was inhibited in ERK5 S496A (dephosphorylation mimic) knock-in (KI) mice. Furthermore, ERK5 S496 phosphorylation was required for not only SASP but also senescence-associated cell growth (SACG) observed in the plaque via upregulation of aryl hydrocarbon receptor (AHR). We also discovered a key effect of ERK5 S496 phosphorylation on SUMOylation at a novel site of NRF2 (i.e., K518), which inhibited NRF2 transcriptional activity without affecting ERK5 kinase activity and antagonized oxidized LDL (oxLDL)-induced SASP. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) both inhibited oxLDL-induced ERK5 S496 phosphorylation, suggesting that ERK5 S496 phosphorylation was involved at least in part of the effects of these inhibitors. We have discovered a novel mechanism, in which ERK5 S496 phosphorylation directly inhibits NRF2 activity by upregulating NRF2 K518 SUMOylation, and induces SACG and atherosclerosis.
Project description:Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation upregulates the expression of inflammatory mediators and of TLR2 itself, and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in ECs, and tested the hypothesis that TLR2 signaling differs in ECs and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in HUVEC, primary human lung microvascular ECs and human monocytes. Additionally, we observed that whereas MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the upregulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5 and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in ECs and monocytes, and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.
Project description:Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation upregulates the expression of inflammatory mediators and of TLR2 itself, and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in ECs, and tested the hypothesis that TLR2 signaling differs in ECs and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in HUVEC, primary human lung microvascular ECs and human monocytes. Additionally, we observed that whereas MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the upregulation of intracellularly expressed TLR2 and inflammatory mediators via NF-M-NM-:B, JNK and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5 and NF-M-NM-:B promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in ECs and monocytes, and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes. qPCR gene expression profiling. HUVEC monolayers were grown to confluency in EGM-2 media. THP1 suspension cells were grown RPMI 1640 supplemented with 10% FBS, L-glutamine, and antibiotics. Neither cell line was treated with any agonists. RQ values were calculated using the 2-M-NM-^TM-NM-^TCt method. Two biological replicates and one technical replicate were analyzed for both cell lines.
Project description:We expressed a constitutively active mutant of MEK5 (MEK5D) in human primary endothelial cells (EC) to study the transcriptional and functional responses to Erk5 activation under static conditions.
Project description:We expressed a constitutively active mutant of MEK5 (MEK5D) in human primary endothelial cells (EC) to study the transcriptional and functional responses to Erk5 activation under static conditions. HUVEC were infected with either empty vector or constitutively active MEK5D and RNA was processed for microarray analysis 40 h post infection.
Project description:Targeted deletion of TRAF7 revealed that it is a crucial part of shear stress-responsive MEKK3-MEK5-ERK5 signaling pathway induced in endothelial cells by blood flow. Similarly, to Mekk3-, Mek5- or Erk5-deficient mice, Traf7-deficient embryos died in utero around midgestation due to impaired endothelial cell integrity. They displayed significantly lower expression of transcription factor Klf2, an essential regulator of vascular hemodynamic forces downstream of the MEKK3-MEK-ERK5 signaling pathway. Deletion of Traf7 in endothelial cells of postnatal mice was also associated with severe cerebral hemorrhage. Here, we show that besides MEKK3 and MEK5, TRAF7 associates with a planar cell polarity protein SCRIB. SCRIB binds with an N-terminal region of TRAF7, while MEKK3 associates with the C-terminal WD40 domain. Downregulation of TRAF7 as well as SCRIB inhibited fluid shear stress-induced phosphorylation of ERK5 in cultured endothelial cells. These findings suggest that TRAF7 and SCRIB may comprise an upstream part of the MEKK3-MEK5-ERK5 signaling pathway. Objective: to present first in vivo experimental evidence of TRAF7 function by using global and endothelium-specific TRAF7 knockout mice and comparing transcriptomes of developing embryos.
Project description:Endothelial function and integrity are compromised after allogeneic bone marrow transplantation (BMT) but how this affects immune responses broadly remains unknown. Using a preclinical model of cytomegalovirus (CMV) reactivation after BMT, we found compromised antiviral humoral responses induced by IL-6 signaling. IL-6 signaling in T cells maintained Th1 cells resulting in sustained IFN secretion which promoted endothelial cell (EC) injury, loss of the neonatal Fc receptor (FcRn) responsible for immunoglobulin G (IgG) recycling, and rapid IgG loss. In contrast, recipient IgG producing cells are rapidly eliminated after BMT. T cell-specific deletion of IL-6R led to persistence of recipient-derived CMV-specific IgG and inhibited CMV reactivation. Deletion of IFN in donor T cells also eliminated EC injury and FcRn loss. In a phase III clinical trial, blockade of IL-6R with tocilizumab promoted CMV-specific IgG persistence and significantly attenuated early HCMV reactivation. In sum, IL-6 invokes IFN-dependent EC injury and consequent IgG loss leading to CMV reactivation. Hence, cytokine inhibition represents a logical strategy to prevent endothelial injury thereby preserving humoral immunity after immunotherapy.
Project description:Fluid shear stress (FSS) from blood flow sensed by vascular endothelial cells (ECs) determines vessel behavior but regulatory mechanisms are only partially understood. We used cell State Transition Assessment and Regulation (cSTAR), a powerful new computational method, to elucidate EC transcriptomic states under low shear stress (LSS), physiological shear stress (PSS), high shear stress (HSS), and oscillatory shear stress (OSS) that induce vessel inward remodeling, stabilization, outward remodeling or disease susceptibility, respectively. Combined with publicly available EC transcriptomic responses to drug treatments from the LINCS database, this approach inferred a regulatory network controlling EC states and made several novel predictions. Particularly, inhibiting cell cycle dependent kinase (CDK) 2 was predicted to initiate inward vessel remodeling and promote atherogenesis. In vitro, PSS activated CDK2 and induced late G1 cell cycle arrest. In mice, EC deletion of CDK2 triggered inward artery remodeling, pulmonary and systemic hypertension and accelerated atherosclerosis. These results validate use of cSTAR and identify key determinants of normal and pathological artery remodeling.
Project description:This study reports the evolution of the donor CD8 effector T cell transcriptomes at multiple sites and time points within the same host following allo-SCT. Donor derived CD8+ T cells were flow sorted from multiple organs in mice developing GVHD in two clinically relevant murine models of H-2b MHC-matched minor antigen-mismatched BMT: (1) B6>129 model – transfer of C57BL/6 polyclonal CD4+ and CD8+ T cells into 129/Sv BMT recipients; (2) F>M – transfer of monoclonal HY-specific TCR-transgenic MataHari CD8+ T cells into C57BL/6 male BMT recipients.