Regulation of MAPK ERK1/2 Signaling by Phosphorylation: Implications in Physiological and Pathological Contexts

Protein phosphorylation represents a rapid and reversible post-translational regulation that enables a fast control of protein activation that play key roles in cell signaling. For instance, Mitogen Activated Protein Kinase (MAPK) pathways are activated upon sequential phosphorylations, resulting in phosphorylation of cytosol and nuclear targets. We focus here on MAPK ERK1/2 signaling that accounts for diverse cellular responses such as cell cycle progression, proliferation, differentiation, senescence, migration, formation of GAP junctions, cell adhesion, cell motility, survival and apoptosis. We review the role of protein phosphorylation in MAPK ERK1/2 activation, in its regulation in time and space and how its dysregulation can lead to tumorigenesis

Transcriptomic and Functional Evidence for Differential Effects of MCF-7 Breast Cancer Cell-Secretome on Vascular and Lymphatic Endothelial Cell Growth

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-β signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC

MicroRNAs in Tumor Endothelial Cells: Regulation, Function and Therapeutic Applications

Tumor endothelial cells (TECs) are key stromal components of the tumor microenvironment, and are essential for tumor angiogenesis, growth and metastasis. Accumulating evidence has shown that small single-stranded non-coding microRNAs (miRNAs) act as powerful endogenous regulators of TEC function and blood vessel formation. This systematic review provides an upto-date overview of these endothelial miRNAs. Their expression is mainly regulated by hypoxia, pro-angiogenic factors, gap junctions and extracellular vesicles, as well as long non-coding RNAs and circular RNAs. In preclinical studies, they have been shown to modulate diverse fundamental angiogenesis-related signaling pathways and proteins, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) pathway; the rat sarcoma virus (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; the phosphoinositide 3-kinase (PI3K)/AKT pathway; and the transforming growth factor (TGF)-β/TGF-β receptor (TGFBR) pathway, as well as krüppel-like factors (KLFs), suppressor of cytokine signaling (SOCS) and metalloproteinases (MMPs). Accordingly, endothelial miRNAs represent promising targets for future anti-angiogenic cancer therapy. To achieve this, it will be necessary to further unravel the regulatory and functional networks of endothelial miRNAs and to develop safe and efficient TEC-specific miRNA delivery technologies

Investigating the downstream signalling of the Rho GTPase RhoJ

RhoJ is an endothelial expressed Rho GTPase which regulates cell migration, protein trafficking and angiogenesis. Previous studies demonstrated that GTP-bound RhoJ activated MAP kinases, interacted with the GIT-PIX complex, caused a retarded electromobility shift of GIT2 and promoted focal adhesion disassembly. The aims of this study were to further characterise how RhoJ regulates MAP kinases and the GIT-PIX complex. The activity of RhoJ was manipulated in human umbilical vein endothelial cells (HUVECs) by either lentiviral transduction of constitutively active mutant form of RhoJ (daRhoJ) or transfection with RhoJ-specific si-RNA duplexes, to activate and inhibit it, respectively. Expression of daRhoJ resulted in a small increase in ERK1/2, but not JNK, phosphorylation in HUVECs cultured in complete media. In a time-course experiment, si-RNA-mediated RhoJ knockdown significantly reduced levels of phospho-ERK1/2, this was particularly evident after 15 min stimulation with VEGFA, FGF2 or complete growth media. Other groups had shown that the GIT-PIX complex interacted with and regulated the activity of MEK1 and ERK1/2, and that ERK1/2 localised to focal adhesions; we therefore aimed to test how RhoJ affected this. Experiments demonstrated that very low levels of GIT1 co-precipitated with either MEK1 or ERK1/2, and there was no evident co-localisation of ERK1/2 with vinculin, a focal adhesion protein. This low level of co-precipitation combined with some technical difficulties hindered the assessment of how RhoJ affected this interaction. This, combined with the lack of focal adhesion localisation of ERK1/2 led to our hypothesising that the GIT-PIX complex was not involved in RhoJ’s regulation of ERK1/2 in endothelial cells. One potential alternative mechanism is via p21-activated kinases (PAK), kinases known to be both downstream of RhoJ and able to regulate RAF1. The role of PAKs in the daRhoJ-mediated change in the electrophoretic mobility of GIT2 was assessed. Two PAK inhibitors were tested, and PF-3758309, but not IPA-3, abrogated GIT2’s mobility shift in daRhoJ expressing endothelial cells. This suggests that the electromobility shift of GIT2 is due to serine and threonine phosphorylation and the differential effect of the inhibitors suggests that PAK4 acts downstream of RhoJ to affect phosphorylation of GIT2. Data in this thesis further delineates the molecular pathways downstream of RhoJ and offers insight into the mechanisms by which RhoJ regulates its endothelial functions

Neuroprotection: Rescue from Neuronal Death in the Brain

Dear Colleagues, The brain is vulnerable to injury. Following injury in the brain, apoptosis or necrosis may occur easily, leading to various functional disabilities. Neuronal death is associated with a number of neurological disorders including hypoxic ischemia, epileptic seizures, and neurodegenerative diseases. The brain subjected to injury is regarded to be responsible for the alterations in neurotransmission processes, resulting in functional changes. Oxidative stress produced by reactive oxygen species has been shown to be related to the death of neurons in traumatic injury, stroke, and neurodegenerative diseases. Therefore, scavenging or decreasing free radicals may be crucial for preventing neural tissues from harmful adversities in the brain. Neurotrophic factors, bioactive compounds, dietary nutrients, or cell engineering may ameliorate the pathological processes related to neuronal death or neurodegeneration and appear beneficial for improving neuroprotection. As a result of neuronal death or neuroprotection, the brain undergoes activity-dependent long-lasting changes in synaptic transmission, which is also known as functional plasticity. Neuroprotection implying the rescue from neuronal death is now becoming one of global health concerns. This Special Issue attempts to explore the recent advances in neuroprotection related to the brain. This Special Issue welcomes original research or review papers demonstrating the mechanisms of neuroprotection against brain injury using in vivo or in vitro models of animals as well as in clinical settings. The issues in a paper should be supported by sufficient data or evidence. Prof. Bae Hwan Lee Guest Edito