An ERK5-NRF2 Axis Mediates Senescence-Associated Stemness and Atherosclerosis.

BACKGROUND: ERK5 (extracellular signal-regulated kinase 5) is a dual kinase transcription factor containing an N-terminal kinase domain and a C-terminal transcriptional activation domain. Many ERK5 kinase inhibitors have been developed and tested to treat cancer and inflammatory diseases. However, recent data have raised questions about the role of the catalytic activity of ERK5 in proliferation and inflammation. We aimed to investigate how ERK5 reprograms myeloid cells to the proinflammatory senescent phenotype, subsequently leading to atherosclerosis. METHODS: A ERK5 S496A (dephosphorylation mimic) knock in (KI) mouse model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9), and atherosclerosis was characterized by hypercholesterolemia induction. The plaque phenotyping in homozygous ERK5 S496A KI and wild type (WT) mice was studied using imaging mass cytometry. Bone marrow-derived macrophages were isolated from hypercholesterolemic mice and characterized using RNA sequencing and functional in vitro approaches, including senescence, mitochondria reactive oxygen species, and inflammation assays, as well as by metabolic extracellular flux analysis. RESULTS: We show that atherosclerosis was inhibited in ERK5 S496A KI mice. Furthermore, ERK5 S496 phosphorylation mediates both senescence-associated secretory phenotype and senescence-associated stemness by upregulating AHR (aryl hydrocarbon receptor) in plaque and bone marrow-derived macrophages isolated from hypercholesterolemic mice. We also discovered that ERK5 S496 phosphorylation could induce NRF2 (NFE2-related factor 2) SUMOylation at a novel K518 site to inhibit NRF2 transcriptional activity without altering ERK5 catalytic activity and mediates oxidized LDL (low-density lipoprotein)-induced senescence-associated secretory phenotype. Specific ERK5 kinase inhibitors (AX15836 and XMD8-92) also inhibited ERK5 S496 phosphorylation, suggesting the involvement of ERK5 S496 phosphorylation in the anti-inflammatory effects of these ERK5 kinase inhibitors. CONCLUSIONS: We discovered a novel mechanism by which the macrophage ERK5-NRF2 axis develops a unique senescence-associated secretory phenotype/stemness phenotype by upregulating AHR to engender atherogenesis. The finding of senescence-associated stemness phenotype provides a molecular explanation to resolve the paradox of senescence in proliferative plaque by permitting myeloid cells to escape the senescence-induced cell cycle arrest during atherosclerosis formation.

A Potential Role for MAGI-1 in the Bi-Directional Relationship Between Major Depressive Disorder and Cardiovascular Disease.

PURPOSE OF REVIEW: Major Depressive Disorder (MDD) is characterized by persistent symptoms such as fatigue, loss of interest in activities, feelings of sadness and worthlessness. MDD often coexist with cardiovascular disease (CVD), yet the precise link between these conditions remains unclear. This review explores factors underlying the development of MDD and CVD, including genetic, epigenetic, platelet activation, inflammation, hypothalamic-pituitary-adrenal (HPA) axis activation, endothelial cell (EC) dysfunction, and blood-brain barrier (BBB) disruption. RECENT FINDINGS: Single nucleotide polymorphisms (SNPs) in the membrane-associated guanylate kinase WW and PDZ domain-containing protein 1 (MAGI-1) are associated with neuroticism and psychiatric disorders including MDD. SNPs in MAGI-1 are also linked to chronic inflammatory disorders such as spontaneous glomerulosclerosis, celiac disease, ulcerative colitis, and Crohn's disease. Increased MAGI-1 expression has been observed in colonic epithelial samples from Crohn's disease and ulcerative colitis patients. MAGI-1 also plays a role in regulating EC activation and atherogenesis in mice and is essential for Influenza A virus (IAV) infection, endoplasmic reticulum stress-induced EC apoptosis, and thrombin-induced EC permeability. Despite being understudied in human disease; evidence suggests that MAGI-1 may play a role in linking CVD and MDD. Therefore, further investigation of MAG-1 could be warranted to elucidate its potential involvement in these conditions.

Paradoxical effects of osteoprotegerin on vascular function: inhibiting inflammation while promoting oxidative stress?

Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor or tumor necrosis factor receptor superfamily member 11B, is well known as a modulator of bone remodeling. The contribution of OPG to cardiovascular disease (CVD) has been suggested, but its molecular mechanism is complex and remains unclear. In the present study, Alves-Lopes et al. (Clin. Sci. (Lond.) (2021) 135(20): https://doi.org/10.1042/CS20210643) reported the critical role of syndecan-1 (SDC-1, also known as CD138), a surface protein part of the endothelial glycocalyx, in OPG-induced vascular dysfunction. The authors found that in endothelial cells (ECs), through SDC-1, OPG increased eNOS Thr495 phosphorylation, thereby inhibiting eNOS activity. Furthermore, the OPG-SDC-1 interaction increased reactive oxygen species (ROS) production through NOX1/4 activation. Both the reduced eNOS activity and induced ROS production inhibited NO production and impaired EC function. In vascular smooth muscle cells (VSMCs), the OPG-SDC-1 interaction increased ROS production through NOX1/4 activation, subsequently increased MLC phosphorylation-mediated Rho kinase-MYPT1 regulation, leading to increased vascular contraction. Ultilizing wire myography and mechanistic studies, the authors nicely provide the evidence that SDC-1 plays a crucial role in OPG-induced vascular dysfunction. As we mentioned above, the molecular mechanism and roles of OPG in cardiovascular system are complex and somewhat confusing. In this commentary, we briefly summarize the OPG-mediated signaling pathways in cardiovascular system.

Free Cholesterol Bioavailability and Atherosclerosis.

PURPOSE OF REVIEW: As both a cholesterol acceptor and carrier in the reverse cholesterol transport (RCT) pathway, high-density lipoprotein (HDL) is putatively atheroprotective. However, current pharmacological therapies to increase plasma HDL cholesterol (HDL-c) concentration have paradoxically failed to prevent or reduce atherosclerosis and cardiovascular disease (CVD). Given that free cholesterol (FC) transfer between surfaces of lipoproteins and cells is reversible, excess plasma FC can be transferred to the cells of peripheral tissue sites resulting in atherosclerosis. Here, we summarize potential mechanisms contributing to this paradox and highlight the role of excess free cholesterol (FC) bioavailability in atherosclerosis vs. atheroprotection. RECENT FINDINGS: Recent findings have established a complex relationship between HDL-c concentration and atherosclerosis. Systemic scavenger receptor class B type 1 (SR-B1) knock out (KO) mice exhibit with increased diet-induced atherosclerosis despite having an elevated plasma HDL-c concentration compared to wild type (WT) mice. The greater bioavailability of HDL-FC in SR-B1 vs. WT mice is associated with a higher FC content in multiple cell types and tissue sites. These results suggest that dysfunctional HDL with high FC bioavailability is atheroprone despite high HDL-c concentration. Past oversimplification of HDL-c involvement in cholesterol transport has led to the failures in HDL targeted therapy. Evidence suggests that FC-mediated functionality of HDL is of higher importance than its quantity; as a result, deciphering the regulatory mechanisms by which HDL-FC bioavailability can induce atherosclerosis can have far-reaching clinical implications.

The Ser/Thr kinase p90RSK promotes kidney fibrosis by modulating fibroblast-epithelial crosstalk.

Healthy kidney structure and environment rely on epithelial integrity and interactions between epithelial cells and other kidney cells. The Ser/Thr kinase 90 kDa ribosomal protein S6 kinase 1 (p90RSK) belongs to a protein family that regulates many cellular processes, including cell motility and survival. p90RSK is predominantly expressed in the kidney, but its possible role in chronic kidney disease (CKD) remains largely unknown. Here, we found that p90RSK expression is dramatically activated in a classic mouse obstructive chronic kidney disease model, largely in the interstitial FSP-1-positive fibroblasts. We generated FSP-1-specific p90RSK transgenic mouse (RSK-Tg) and discovered that these mice, after obstructive injury, display significantly increased fibrosis and enhanced tubular epithelial damage compared with their wt littermates (RSK-wt), indicating a role of p90RSK in fibroblast-epithelial communication. We established an in vitro fibroblast-epithelial coculture system with primary kidney fibroblasts from RSK-Tg and RSK-wt mice and found that RSK-Tg fibroblasts consistently produce excessive H2O2 causing epithelial oxidative stress and inducing nuclear translocation of the signaling protein ß-catenin. Epithelial accumulation of ß-catenin, in turn, promoted epithelial apoptosis by activating the transcription factor forkhead box class O1 (FOXO1). Of note, blockade of reactive oxygen species (ROS) or ß-catenin or FOXO1 activity abolished fibroblast p90RSK-mediated epithelial apoptosis. These results make it clear that p90RSK promotes kidney fibrosis by inducing fibroblast-mediated epithelial apoptosis through ROS-mediated activation of ß-catenin/FOXO1 signaling pathway.

Senescent Phenotype Induced by p90RSK-NRF2 Signaling Sensitizes Monocytes and Macrophages to Oxidative Stress in HIV-Positive Individuals.

BACKGROUND: The incidence of cardiovascular disease is higher in HIV-positive (HIV+) patients than it is in the average population, and combination antiretroviral therapy (cART) is a recognized risk factor for cardiovascular disease. However, the molecular mechanisms that link cART and cardiovascular disease are currently unknown. Our study explores the role of the activation of p90RSK, a reactive oxygen species-sensitive kinase, in engendering senescent phenotype in macrophages and accelerating atherogenesis in patients undergoing cART. METHODS: Peripheral whole blood from cART-treated HIV+ individuals and nontreated HIV-negative individuals was treated with H2O2 (200 µmol/L) for 4 minutes, and p90RSK activity in CD14+ monocytes was measured. Plaque formation in the carotids was also analyzed in these individuals. Macrophage senescence was determined by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. The involvement of p90RSK-NRF2 signaling in cART-induced senescence was assessed by p90RSK-specific inhibitor (FMK-MEA) or dominant-negative p90RSK (DN-p90RSK) and NRF2 activator (NRF2A). Further, the severity of atherosclerosis was determined in myeloid cell-specific wild-type and DN-p90RSK transgenic mice. RESULTS: Monocytes from HIV+ patients exhibited higher levels of p90RSK activity and were also more sensitive to reactive oxygen species than monocytes from HIV-negative individuals. A multiple linear regression analysis involving cART, Reynolds cardiovascular risk score, and basal p90RSK activity revealed that cART and basal p90RSK activity were the 2 significant determinants of plaque formation. Many of the antiretroviral drugs individually activated p90RSK, which simultaneously triggered all components of the macrophage senescent phenotype. cART inhibited antioxidant response element reporter activity via ERK5 S496 phosphorylation. NRF2A reversed the H2O2-induced overactivation of p90RSK in cART-treated macrophages by countering the induction of senescent phenotype. Last, the data obtained from our gain- or loss-of-function mice conclusively showed the crucial role of p90RSK in inducing senescent phenotype in macrophages and atherogenesis. CONCLUSIONS: cART increased monocyte/macrophage sensitivity to reactive oxygen species- in HIV+ individuals by suppressing NRF2-ARE activity via p90RSK-mediated ERK5 S496 phosphorylation, which coordinately elicited senescent phenotypes and proinflammatory responses. As such, our report underscores the importance of p90RSK regulation in monocytes/macrophages as a viable biomarker and therapeutic target for preventing cardiovascular disease, especially in HIV+ patients treated with cART.

Flow signaling and atherosclerosis.

Atherosclerosis rarely develops in the region of arteries exposed to undisturbed flow (u-flow, unidirectional flow). Instead, atherogenesis occurs in the area exposed to disturbed flow (d-flow, multidirectional flow). Based on these general pathohistological observations, u-flow is considered to be athero-protective, while d-flow is atherogenic. The fact that u-flow and d-flow induce such clearly different biological responses in the wall of large arteries indicates that these two types of flow activate each distinct intracellular signaling cascade in vascular endothelial cells (ECs), which are directly exposed to blood flow. The ability of ECs to differentially respond to the two types of flow provides an opportunity to identify molecular events that lead to endothelial dysfunction and atherosclerosis. In this review, we will focus on various molecular events, which are differentially regulated by these two flow types. We will discuss how various kinases, ER stress, inflammasome, SUMOylation, and DNA methylation play roles in the differential flow response, endothelial dysfunction, and atherosclerosis. We will also discuss the interplay among the molecular events and how they coordinately regulate flow-dependent signaling and cellular responses. It is hoped that clear understanding of the way how the two flow types beget each unique phenotype in ECs will lead us to possible points of intervention against endothelial dysfunction and cardiovascular diseases.

Sub-cellular localization specific SUMOylation in the heart.

Although the majority of SUMO substrates are localized in the nucleus, SUMOylation is not limited to nuclear proteins and can be also detected in extra-nuclear proteins. In this review, we will highlight and discuss how SUMOylation in different cellular compartments regulate biological processes. First, we will discuss the key role of SUMOylation of proteins in the extra-nuclear compartment in cardiomyocytes, which is overwhelmingly cardio-protective. On the other hand, SUMOylation of nuclear proteins is generally detrimental to the cardiac function mainly because of the trans-repressive nature of SUMOylation on many transcription factors. We will also discuss the potential role of SUMOylation in epigenetic regulation. In this review, we will propose a new concept that shuttling of SUMO proteases between the nuclear and extra-nuclear compartments without changing their enzymatic activity regulates the extent of SUMOylation in these compartments and determines the response and fate of cardiomyocytes after cardiac insults. Approaches focused specifically to inhibit this shuttling in cardiomyocytes will be necessary to understand the whole picture of SUMOylation and its pathophysiological consequences in the heart, especially after cardiac insults. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

Coordination of Cellular Localization-Dependent Effects of Sumoylation in Regulating Cardiovascular and Neurological Diseases.

Sumoylation, a reversible post-transcriptional modification process, of proteins are involved in cellular differentiation, growth, and even motility by regulating various protein functions. Sumoylation is not limited to cytosolic proteins as recent evidence shows that nuclear proteins, those associated with membranes, and mitochondrial proteins are also sumoylated. Moreover, it is now known that sumoylation plays an important role in the process of major human ailments such as malignant, cardiovascular and neurological diseases. In this chapter, we will highlight and discuss how the localization of SUMO protease and SUMO E3 ligase in different compartments within a cell regulates biological processes that depend on sumoylation. First, we will discuss the key role of sumoylation in the nucleus, which leads to the development of endothelial dysfunction and atherosclerosis . We will then discuss how sumoylation of plasma membrane potassium channel proteins are involved in epilepsy and arrhythmia. Mitochondrial proteins are known to be also sumoylated, and the importance of dynamic-related protein 1 (DRP1) sumoylation on mitochondrial function will be discussed. As we will emphasize throughout this review, sumoylation plays crucial roles in different cellular compartments, which is coordinately regulated by the translocation of various SUMO proteases and SUMO E3 ligase. Comprehensive approach will be necessary to understand the molecular mechanism for efficiently moving around various enzymes that regulate sumoylation within cells.

Platelet Extracellular Regulated Protein Kinase 5 Is a Redox Switch and Triggers Maladaptive Platelet Responses and Myocardial Infarct Expansion.

BACKGROUND: Platelets have a pathophysiologic role in the ischemic microvascular environment of acute coronary syndromes. In comparison with platelet activation in normal healthy conditions, less attention is given to mechanisms of platelet activation in diseased states. Platelet function and mechanisms of activation in ischemic and reactive oxygen species-rich environments may not be the same as in normal healthy conditions. Extracellular regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase family member activated in hypoxic, reactive oxygen species-rich environments and in response to receptor-signaling mechanisms. Prior studies suggest a protective effect of ERK5 in endothelial and myocardial cells after ischemia. We present evidence that platelets express ERK5 and that platelet ERK5 has an adverse effect on platelet activation via selective receptor-dependent and receptor-independent reactive oxygen species-mediated mechanisms in ischemic myocardium. METHODS AND RESULTS: Using isolated human platelets and a mouse model of myocardial infarction (MI), we found that platelet ERK5 is activated post-MI and that platelet-specific ERK5(-/-) mice have less platelet activation, reduced MI size, and improved post-MI heart function. Furthermore, the expression of downstream ERK5-regulated proteins is reduced in ERK5(-/-) platelets post-MI. CONCLUSIONS: ERK5 functions as a platelet activator in ischemic conditions, and platelet ERK5 maintains the expression of some platelet proteins after MI, leading to infarct expansion. This demonstrates that platelet function in normal healthy conditions is different from platelet function in chronic ischemic and inflammatory conditions. Platelet ERK5 may be a target for acute therapeutic intervention in the thrombotic and inflammatory post-MI environment.

Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents.

Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.

De-SUMOylation enzyme of sentrin/SUMO-specific protease 2 regulates disturbed flow-induced SUMOylation of ERK5 and p53 that leads to endothelial dysfunction and atherosclerosis.

RATIONALE: Disturbed flow induces proinflammatory and apoptotic responses in endothelial cells, causing them to become dysfunctional and subsequently proatherogenic. OBJECTIVE: Although a possible link between SUMOylation of p53 and ERK5 detected during endothelial apoptosis and inflammation has been suggested, the mechanistic insights, especially under the proatherogenic flow condition, remain largely unknown. METHODS AND RESULTS: SUMOylation of p53 and ERK5 was induced by disturbed flow but not by steady laminar flow. To examine the role of the disturbed flow-induced p53 and ERK5 SUMOylation, we used de-SUMOylation enzyme of sentrin/Small Ubiquitin-like MOdifier (SUMO)-specific protease 2 deficiency (Senp2(+/-)) mice and observed a significant increase in endothelial apoptosis and adhesion molecule expression both in vitro and in vivo. These increases, however, were significantly inhibited in endothelial cells overexpressing p53 and ERK5 SUMOylation site mutants. Senp2(+/-) mice exhibited increased leukocyte rolling along the endothelium, and accelerated formation of atherosclerotic lesions was observed in Senp2(+/-)/Ldlr(-/-), but not in Senp2(+/+)/Ldlr(-/-), mice fed a high-cholesterol diet. Notably, the extent of lesion size in the aortic arch of Senp2(+/-)/Ldlr(-/-) mice was much larger than that in the descending aorta, also suggesting a crucial role of the disturbed flow-induced SUMOylation of proteins, including p53 and ERK5 in atherosclerosis formation. CONCLUSIONS: These data show the unique role of sentrin/SUMO-specific protease 2 on endothelial function under disturbed flow and suggest that SUMOylation of p53 and ERK5 by disturbed flow contributes to the atherosclerotic plaque formation. Molecules involved in this newly discovered signaling will be useful targets for controlling endothelial cells dysfunction and consequently atherosclerosis formation.

Streptococcus pneumoniae ClpL modulates adherence to A549 human lung cells through Rap1/Rac1 activation.

Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens.

A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis.

BACKGROUND: Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. METHODS AND RESULTS: Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR(-/-) mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE(-/-) mice. CONCLUSIONS: Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

p90RSK targets the ERK5-CHIP ubiquitin E3 ligase activity in diabetic hearts and promotes cardiac apoptosis and dysfunction.

RATIONALE: Cardiomyocyte apoptosis is one of the key events in the development and progression of heart failure, and a crucial role for ICER (inducible cAMP early repressor) in this process has been previously reported. ERK5 is known to inhibit cardiac apoptosis after myocardial infarction (MI), especially in hyperglycemic states, via association with CHIP ubiquitin (Ub) ligase and subsequent upregulation of CHIP ligase activity, which induces ICER ubiquitination and subsequent protein degradation. The regulatory mechanism governing ERK5/CHIP interaction is unknown. OBJECTIVE: We previously demonstrated increased p90RSK activation in the diabetic heart. As a logical extension of this work, we now investigate whether p90RSK activation inhibits ERK5-mediated CHIP activation, and subsequently increases ICER levels and apoptosis. METHODS AND RESULTS: p90RSK activation inhibits ERK5/CHIP association and CHIP Ub ligase activity. p90RSK and CHIP share a common binding site in the ERK5 C-terminal domain (aa571-807). Overexpression of either p90RSK or an ERK5 fragment (aa571-807) inhibits ERK5/CHIP association, suggesting that p90RSK and CHIP competes for ERK5 binding and that p90RSK activation is critical for inhibiting ERK5/CHIP interaction. We also identified ERK5-S496 as being directly phosphorylated by p90RSK and demonstrated that an ERK5-S496A mutant significantly impairs Angiotensin II-mediated inhibition of CHIP activity and subsequent increase in ICER levels. In vivo, either cardiac-specific depletion of ERK5 or overexpression of p90RSK inhibits CHIP activity and accelerates cardiac apoptosis after MI-a phenomenon fully reversible by activating ERK5. CONCLUSIONS: These data suggest a role for p90RSK in inhibiting CHIP activity and promoting cardiac apoptosis through binding to and phosphorylation of ERK5-S496.

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.

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.

A relay velocity model infers cell-dependent RNA velocity.

RNA velocity provides an approach for inferring cellular state transitions from single-cell RNA sequencing (scRNA-seq) data. Conventional RNA velocity models infer universal kinetics from all cells in an scRNA-seq experiment, resulting in unpredictable performance in experiments with multi-stage and/or multi-lineage transition of cell states where the assumption of the same kinetic rates for all cells no longer holds. Here we present cellDancer, a scalable deep neural network that locally infers velocity for each cell from its neighbors and then relays a series of local velocities to provide single-cell resolution inference of velocity kinetics. In the simulation benchmark, cellDancer shows robust performance in multiple kinetic regimes, high dropout ratio datasets and sparse datasets. We show that cellDancer overcomes the limitations of existing RNA velocity models in modeling erythroid maturation and hippocampus development. Moreover, cellDancer provides cell-specific predictions of transcription, splicing and degradation rates, which we identify as potential indicators of cell fate in the mouse pancreas.

MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation.

Actin filament remodeling regulates several endothelial cell (EC) processes such as contraction, migration, adhesion, and shape determination. Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)-mediated phosphorylation of heat-shock protein 27 kDa (HSP27) promotes actin filament remodeling, but little is known about the regulation of this event in ECs. We found that tumor necrosis factor-α (TNF-α) SUMOylated MK2 at lysine (K)-339 affected EC actin filament organization and migration. Loss of the MK2 SUMOylation site (MK2-K339R) increased MK2 kinase activity and prolonged HSP27 phosphorylation, enhancing its effects on actin filament-dependent events. Both TNF-α-mediated EC elongation and steady laminar shear stress-mediated EC alignment were increased by MK2-K339R. Moreover, kinase-dead dominant-negative MK2 (DN-MK2) inhibited these effects. Cell migration is a dynamic process regulated by actin filament remodeling. Both wild-type MK2 (WT-MK2) and DN-MK2 significantly enhanced TNF-mediated inhibition of EC migration, and MK2-K339R further augmented this effect. Interestingly, the p160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 reversed this effect by MK2-K339R, which strongly suggests that both excessive and insufficient levels of actin filament remodeling can block EC migration. Our study shows that MK2 SUMOylation is a new mechanism for regulating actin filament dynamics in ECs.

The significance of ERK5 catalytic-independent functions in disease pathways.

Extracellular signal-regulated kinase 5 (ERK5), also known as BMK1 or MAPK7, represents a recent addition to the classical mitogen-activated protein kinase (MAPK) family. This family includes well-known members such as ERK1/2, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), as well as atypical MAPKs such as ERK3, ERK4, ERK7 (ERK8), and Nemo-like kinase (NLK). Comprehensive reviews available elsewhere provide detailed insights into ERK5, which interested readers can refer to for in-depth knowledge (Nithianandarajah-Jones et al., 2012; Monti et al., Cancers (Basel), 2022, 14). The primary aim of this review is to emphasize the essential characteristics of ERK5 and shed light on the intricate nature of its activation, with particular attention to the catalytic-independent functions in disease pathways.