Muon pairs and associated hadrons in pion-nucleon interactions

Imperial Users onl

The role of c-Jun in controlling the EPAC1-dependent induction of the SOCS3 gene in HUVECs

The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63

Genomic analysis of the role of transcription factor C/EBPδ in the regulation of cell behaviour on nanometric grooves

C/EBPδ is a tumour suppressor transcription factor that induces gene expression involved in suppressing cell migration. Here we investigate whether C/EBPδ-dependent gene expression also affects cell responses to nanometric topology. We found that ablation of the C/EBPδ gene in mouse embryonal fibroblasts (MEFs) decreased cell size, adhesion and cytoskeleton spreading on 240 nm and 540 nm nanometric grooves. ChIP-SEQ and cDNA microarray analyses demonstrated that many binding sites for C/EBPδ, and the closely related C/EBPβ, exist throughout the mouse genome and control the upregulation or downregulation of many adjacent genes. We also identified a group of C/EBPδ-dependent, trans-regulated genes, whose promoters contained no C/EBPδ binding sites and yet their activity was regulated in a C/EBPδ-dependent manner. These genes include signalling molecules (e.g. SOCS3), cytoskeletal components (Tubb2, Krt16 and Krt20) and cytoskeletal regulators (ArhGEF33 and Rnd3) and are possibly regulated by cis-regulated diffusible mediators, such as IL6. Of particular note, SOCS3 was shown to be absolutely required for efficient cell spreading and contact guidance on 240 nm and 540 nm nanometric grooves. C/EBPδ is therefore involved in the complex regulation of multiple genes, including cytoskeletal components and signalling mediators, which influence the nature of cell interactions with nanometric topology

Genome-Wide Mapping Defines a Role for C/EBPβ and c-Jun in Non-Canonical Cyclic AMP Signalling

The novel exchange protein activated by cyclic AMP (EPAC1) activator, I942, induces expression of the suppressor of cytokine signalling 3 (SOCS3) gene, thereby inhibiting interleukin 6 (IL6) inflammatory processes in human umbilical vein endothelial cells (HUVECs). Here we use RNA-SEQ and ChIP-SEQ to determine global gene responses to I942, in comparison with cyclic AMP production promoted by forskolin and rolipram (F/R). We found that I942 promoted significant changes in the RNA expression of 1413 genes, largely associated with microtubule stability and cell cycle progression, whereas F/R regulated 197 genes linked to endothelial cell function, including chemokine production and platelet aggregation. A further 108 genes were regulated by both treatments, including endothelial regulatory genes involved in purinergic signalling and cell junction organization. ChIP-SEQ demonstrated that F/R induced genome-wide recruitment of C/EBPβ and c-Jun transcription factors, whereas I942 promoted recruitment of c-Jun to genes associated with IL6 signalling, with little effect on C/EBPβ activation. Despite this, certain key inflammatory genes, including IL6, VEGF, CCL2/MCP1, VCAM1, SELE and ICAM1 were regulated by I942 without significant c-Jun recruitment, suggesting an additional, indirect mode of action for I942. In this regard, SOCS3 induction by I942 was found to require c-Jun and was associated with suppression of IL6-promoted ERK MAP kinase and AKT activity and induction of ICAM1. Pharmacological inhibition of ERK and AKT also potentiated ICAM1 induction by I942. We therefore propose that c-Jun activation by I942 regulates endothelial gene expression in HUVECs through direct mechanisms, involving recruitment of c-Jun or, as for ICAM1, through indirect regulation of tertiary regulators, including SOCS3

Vascular smooth muscle cells enhance immune/vascular interplay in a 3-cell model of vascular inflammation

Atherosclerosis is a serious cardiovascular disease that is characterised by the development of atheroma, which are lipid-laden plaques that build up within arterial walls due to chronic inflammatory processes. These lesions are fundamentally attributed to a complex cellular crosstalk between vascular smooth muscle cells (VSMCs), vascular endothelial cells (VECs) and central immune cells, such as macrophages (Mɸs), which promote vascular inflammation. The presence of VSMCs exerts both positive and negative effects during atheroma development, which can be attributed to their phenotypic plasticity. Understanding the interactions between these key cell types during the development of vascular inflammation and atheroma will enhance the scope for new therapeutic interventions. This study aims to determine the importance of VSMCs for shaping the extracellular cytokine/chemokine profile and transcriptional responses of VECs (human coronary artery endothelial cells; HCAECs) to activated lipopolysaccharide (LPS)-stimulated THP1 Mɸs, in a 3-cell model of human vascular inflammation. It is evident that within the presence of VSMCs, enhanced cytokine production was associated with up-regulation of genes associated with vascular inflammation t. Results demonstrate that the presence of VSMCs in co-culture experiments enhanced cytokine production (including CXCL1/GROα, IL-6, IL-8 and CCL2/MCP1) and inflammatory gene expression (including genes involved in JAK/STAT, Jun and NFκB signalling) in HCAECs co-cultured with LPS-stimulated THP1 Mɸs. Our results highlight the importance of VSMCs in immune/endothelial cell interplay and indicate that 3-cell, rather than 2-cell co-culture, may be more appropriate for the study of cellular crosstalk between immune and vascular compartments in response to inflammatory and atherogenic stimuli

Non-cyclic nucleotide EPAC1 activators suppress lipopolysaccharide-regulated gene expression, signalling and intracellular communication in differentiated macrophage-like THP-1 cells

This study explores the anti-inflammatory effects of non-cyclic nucleotide EPAC1 activators, PW0577 and SY007, on lipopolysaccharide (LPS)-induced responses in differentiated THP-1 macrophage-like cells. Both activators were found to selectively activate EPAC1 in THP-1 macrophages, leading to the activation of the key down-stream effector, Rap1. RNA sequencing analysis of LPS-stimulated THP-1 macrophages, revealed that treatment with PW0577 or SY007 significantly modulates gene expression related to fibrosis and inflammation, including the suppression of NLRP3, IL-1β, and caspase 1 protein expression in LPS-stimulated cells. Notably, these effects were independent of p65 NFκB phosphorylation at Serine 536, indicating a distinct mechanism of action. The study further identified a shared influence of both activators on LPS signalling pathways, particularly impacting extracellular matrix (ECM) components and NFκB-regulated genes. Additionally, in a co-culture model involving THP-1 macrophages, vascular smooth muscle cells, and human coronary artery endothelial cells, EPAC1 activators modulated immune-vascular interactions, suggesting a broader role in regulating cellular communication between macrophages and endothelial cells. These findings enhance our understanding of EPAC1's role in inflammation and propose EPAC1 activators as potential therapeutic agents for treating inflammatory and fibrotic conditions through targeted modulation of Rap1 and associated signalling pathways

The protein kinase C inhibitor, Ro-31-7459, is a potent activator of ERK and JNK MAP kinases in HUVECs and yet inhibits cyclic AMP-stimulated <i>SOCS-3</i> gene induction through inactivation of the transcription factor c-Jun

Induction of the suppressor of cytokine signalling 3 (SOCS-3) gene is vital to the normal control of inflammatory signalling. In order to understand these processes we investigated the role of the proto-oncogene component of the AP-1 transcription factor complex, c-Jun, in the regulation of SOCS-3 gene induction. We found that cyclic AMP stimulation of HUVECs promoted phosphorylation and activation of JNK MAP kinase and its substrate c-Jun. The JNK responsive element of the human SOCS-3 promoter mapped to a putative AP-1 site within 1000 bp of the transcription start site. The PKC inhibitors, GF-109203X, Gö-6983 and Ro-317549, were all found to inhibit AP-1 transcriptional activity, transcriptional activation of this minimal SOCS-3 promoter and SOCS-3 gene induction in HUVECs. Interestingly, Ro-317549 treatment was also found to promote PKC-dependent activation of ERK and JNK MAP kinases and promote JNK-dependent hyper-phosphorylation of c-Jun, whereas GF-109203X and Gö-6983 had little effect. Despite this, all three PKC inhibitors were found to be effective inhibitors of c-Jun DNA-binding activity. The JNK-dependent hyper-phosphorylation of c-Jun in response to Ro-317549 treatment of HUVECs does therefore not interfere with its ability to inhibit c-Jun activity and acts as an effective inhibitor of c-Jun-dependent SOCS-3 gene induction

Non-cyclic nucleotide EPAC1 activators suppress lipopolysaccharide-regulated gene expression, signalling and intracellular communication in differentiated macrophage-like THP-1 cells

This study explores the anti-inflammatory effects of non-cyclic nucleotide EPAC1 activators, PW0577 and SY007, on lipopolysaccharide (LPS)-induced responses in differentiated THP-1 macrophage-like cells. Both activators were found to selectively activate EPAC1 in THP-1 macrophages, leading to the activation of the key down-stream effector, Rap1. RNA sequencing analysis of LPS-stimulated THP-1 macrophages, revealed that treatment with PW0577 or SY007 significantly modulates gene expression related to fibrosis and inflammation, including the suppression of NLRP3, IL-1β, and caspase 1 protein expression in LPS-stimulated cells. Notably, these effects were independent of p65 NFκB phosphorylation at Serine 536, indicating a distinct mechanism of action. The study further identified a shared influence of both activators on LPS signalling pathways, particularly impacting extracellular matrix (ECM) components and NFκB-regulated genes. Additionally, in a co-culture model involving THP-1 macrophages, vascular smooth muscle cells, and human coronary artery endothelial cells, EPAC1 activators modulated immune-vascular interactions, suggesting a broader role in regulating cellular communication between macrophages and endothelial cells. These findings enhance our understanding of EPAC1's role in inflammation and propose EPAC1 activators as potential therapeutic agents for treating inflammatory and fibrotic conditions through targeted modulation of Rap1 and associated signalling pathways

Osteogenic lineage restriction by osteoprogenitors cultured on nanometric grooved surfaces – the role of focal adhesion maturation

The differentiation of progenitor cells is dependent on more than biochemical signalling. Topographical cues in natural bone extracellular matrix guide cellular differentiation through the formation of focal adhesions, contact guidance, cytoskeletal rearrangement and ultimately gene expression. Osteoarthritis and a number of bone disorders present as growing challenges for our society. Hence, there is a need for next generation implantable devices to substitute for, or guide, bone repair in vivo. Cellular responses to nanometric topographical cues need to be better understood in vitro in order to ensure the effective and efficient integration and performance of these orthopaedic devices. In this study, the FDA approved plastic polycaprolactone, was embossed with nanometric grooves and the response of primary and immortalised osteoprogenitor cells observed. Nanometric groove dimensions were 240 nm or 540 nm deep and 12.5 μm wide. Cells cultured on test surfaces followed contact guidance along the length of groove edges, elongated along their major axis and showed nuclear distortion, they formed more focal complexes and a lower proportions of mature adhesions relative to planar controls. Down-regulation of the osteoblast marker genes RUNX2 and BMPR2 in primary and immortalised cells was observed on grooved substrates. Down-regulation appeared to directly correlate with focal adhesion maturation, indicating the involvement of ERK 1/2 negative feedback pathways following integrin mediated FAK activation