Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

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Mammary cancer is one of the leading causes of death in both the human and companion animal population. There are many histological and pathophysiological similarities between human and feline mammary cancer, therefore investigating the molecular pathways of disease progression in one species may provide useful information for both. The EGFR/MAPK signal transduction pathway is upregulated in many human breast tumors, and both EGFR and MAPK have been implicated as independent prognostic indicators for decreased survival times in human breast cancer patients. We report here that active MAPK levels can be detected in both peripheral white blood cells (WBC) and mammary adipose tissue in cats. Adipose tissue levels of active MAPK were similar to those observed in peripheral WBC, suggesting that WBC MAPK might serve as a useful biomarker in the diagnosis or follow-up treatment of disease. PUFA have been reported to influence breast cancer risk in humans, and may modulate the EGFR/MAPK pathway through a variety of mechanisms. Dietary PUFA n-6-to-n-3 ratio in cats was reflected in mammary adipose tissue and resulted in altered active MAPK levels in both adipose tissue and peripheral white blood cells, suggesting that PUFA may have similar effects on the feline and human MAPK pathway. In human breast tumor cell line studies, rather than having opposing effects, as was hypothesized, it was demonstrated that n-6 and n-3 PUFA exerted similar effects on EGFR+ breast tumor cell proliferation and activity of the EGFR/MAPK pathway. Slightly more than twice the concentration of n-3 PUFA was needed to elicit the same response as n-6 in cells. These results indicate n-6 and n-3 PUFA can modulate proliferation of EGFR+ tumor cells similarly and may be exerting their effects, in part, through the EGFR/MAPK pathway. Decreasing total PUFA intake, while increasing the n-3-to-n-6 PUFA ratio, may be practical as preventative or adjuvant therapy for breast cancer. / Ph. D.

No / Cryptolepis sanguinolenta and its bioactive alkaloid, cryptolepine have shown anti-inflammatory activity. However, the underlying mechanism of anti-inflammatory action in neuronal cells has not been investigated. In the present study we evaluated an extract of C. sanguinolenta (CSE) and cryptolepine (CAS) on neuroinflammation induced with IL-1β in SK-N-SH neuroblastoma cells. We then attempted to elucidate the mechanisms underlying the anti-neuroinflammatory effects of CAS in SK-N-SH cells. Cells were stimulated with 10 U/ml of IL-1β in the presence or absence of different concentrations of CSE (25–200 μg/ml) and CAS (2.5–20 μM). After 24 h incubation, culture media were collected to measure the production of PGE2 and the pro-inflammatory cytokines (TNFα and IL-6). Protein and gene expressions of cyclooxygenase (COX-2) and microsomal prostaglandin synthase-1 (mPGES-1) were studied by immunoblotting and qPCR, respectively. CSE produced significant (p < 0.05) inhibition of TNFα, IL-6 and PGE2 production in SK-N-SH cells. Studies on CAS showed significant and dose-dependent inhibition of TNFα, IL-6 and PGE2 production in IL-1β-stimulated cells without affecting viability. Pre-treatment with CAS (10 and 20 μM) was also found to inhibit IL-1β-induced protein and gene expressions of COX-2 and mPGES-1. Further studies to determine the mechanism of action of CAS showed inhibition of NF-κBp65 nuclear translocation, but not IκB phosphorylation. At 10 and 20 μM, CAS inhibited IL-1β-induced phosphorylation of p38 MAPK. Studies on the downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK2) showed that CAS produced significant (p < 0.05) and dose dependent inhibition of MAPKAPK2 phosphorylation in IL-1β-stimulated SK-N-SH cells. This study clearly shows that cryptolepine (CAS) inhibits neuroinflammation through mechanisms involving inhibition of COX-2 and mPGES-1. It is suggested that these actions are probably mediated through NF-κB and p38 signalling.

Yes / Extracellular-signal-regulated kinase 5 (ERK5), also termed big MAPK1 (BMK1), is the most recently discovered member of the mitogen-activated protein kinase (MAPK) family. It is expressed in a variety of tissues and is activated by a range of growth factors, cytokines and cellular stresses. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade is critical for normal cardiovascular development and vascular integrity. In vitro studies have revealed that, in endothelial cells, ERK5 is required for preventing apoptosis, mediating shear-stress signalling and regulating tumour angiogenesis. The present review focuses on our current understanding of the role of ERK5 in regulating endothelial cell function. / Biotechnology and Biological Sciences Research Council, the Medical Research Council and the Wellcome Trust

Yes / The antidiuretic hormone arginine vasopressin (AVP) regulates water homeostasis, blood pressure and a range of stress responses. It is synthesized in the hypothalamus and released from the posterior pituitary into the general circulation upon a range of stimuli. While the mechanisms leading to AVP secretion have been widely investigated, the molecular mechanisms regulating AVP gene expression are mostly unclear. Here we investigated the neurotransmitters and signal transduction pathways that activate AVP gene expression in the paraventricular nucleus (PVN) of the rat using acute brain slices and quantitative real-time PCR. We show that stimulation with l-glutamate robustly induced AVP gene expression in acute hypothalamic brain slices containing the PVN. More specifically, we show that AVP transcription was stimulated by NMDA. Using pharmacological treatments, our data further reveal that the activation of ERK1/2 (PD184352), CaMKII (KN-62) and PI3K (LY294002; 740 Y-P) is involved in the NMDA-induced AVP gene expression in the PVN. Together, this study identifies NMDA-mediated cell signalling pathways that regulate AVP gene expression in the rat PVN. / Supported by a generous donation from Jonathan Feuer.