Mitogen activated protein kinases (MAPK), such as ERK1/2 and p38 MAPK and phosphatidylinositol-3 phosphate kinase (PI-3K) play a major role in the development of cardiac hypertrophy. Recently, we have shown their crucial role in the regulation of the myocardial function through their effects on crucial ion channels. It is the focus of this study to resolve the interaction between these pathways and its implication on the function of the normal and hypertrophied cardiomyocytes. To that end, we created arteriovenous fistula in the adult rat that developed volume-overload eccentric cardiac hypertrophy over a 3-week period. We measured the relative activity of ERK1/2, p38 MAPKs and Akt through western blot analysis and assessed the functional density of the outward rectifier potassium current (IK) using the patch-clamp technique. The results showed a mutual negative autoregulation between ERK1/2 and p38 in normal cardiomyocytes, which disappears during cardiac hypertrophy. In addition, PI-3K seems to assume a greater role in mediating IGF-1 effects on the MAPKs during cardiac hypertrophy. This was also relevant to IK functional density which was reduced by activation of both MAPKs and Akt by angiotensin II (ANG II) and insulin-like growth factor-1 (IGF-1), respectively; however, this reduction was reversed by inhibition of PI-3K alone in hypertrophied myocytes but not in normal ones. This raises an important implication relative to the role of IGF-1-dependent activation of PI-3K, which may translate into a differential prognostic for cardiac hypertrophy among ethnic groups. This is true in African Americans, having higher circulating IGF-1 levels, and especially true for the athletes among them.

Cardiac fibroblasts play an important role in myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Estrogen has been reported to have a number of cardioprotective properties. However, it is unclear whether estrogen affects cardiac fibroblast differentiation. The purpose of the present study was to investigate the effect of estrogen on angiotensin II-induced cardiac fibroblast proliferation and differentiation. Cardiac fibroblasts were stimulated with angiotensin II (1 μM) in the presence or absence of 17β-estradiol (100 nM). Pretreatment of cardiac fibroblasts with 17β-estradiol significantly inhibited angiotensin II-induced cardiac fibroblast proliferation and differentiation (indicated by a reduction in alpha-smooth muscle actin (α-SMA) expression) by 25% and 20%. Pretreatment of 17β-estradiol significantly reduced angiotensin II-increased levels of phospho-p38 mitogen-activated protein kinase (MAPK) by 40% and nuclear factor-κB (NF-κB) binding activity in cardiac fibroblasts by 55%. Our data suggests estrogen could have an anti-fibrotic effect through limiting cardiac fibroblast proliferation and differentiation, which are the critical steps in the pathogenesis of cardiac fibrosis.

We have recently reported that antioxidant liposomes can be used as antidotes for mustard gas induced lung injury in guinea pigs. The maximum protection was achieved with a liposome composed of tocopherols (α, γ, δ) and N-acetylcysteine (NAC) when administered after 5 min of exposure of 2-chloroethyl ethyl sulfide (CEES), a half sulfur mustard gas. We also reported an association of mustard gas-induced lung injury with an activation of MAPK/AP-1 signaling pathway and cell proliferation. The objective of the present study was to investigate whether CEES-induced MAPKs/AP-1 signaling pathway is influenced by antioxidant liposome therapy. A single dose (200 μl) of the antioxidant liposome was administered intratracheally after 5 min of exposure of CEES (0.5 mg/kg). The animals were sacrificed after 1 h and 30 days of CEES exposure. Although the liposome treatment did not have any significant effect on the activation of the MAPKs family (ERK1/2, p38 and JNK1/2), it significantly counteracted the CEES-induced activation of AP-1 transcription factors and corresponding increase in the protein levels of Fos, ATF and Jun family members. The liposome treatment significantly blocked the CEES-induced increase in the protein levels of cyclin D1, a cell cycle protein and PCNA, a cell differentiation marker. Furthermore, it protected lung against CEES-induced inflammation and infiltration of neutrophils, eosinophils and erythrocytes in the alveolar space. This suggests that the protective effect of antioxidant liposome against CEES-induced lung damage is mediated via control of AP-1 signaling.

Genetic changes acquired in the pigment producing cells of the skin, called melanocytes, can lead to formation of the deadly cancer melanoma. Mutations or amplifications leading to the activation of the RAS/MAPK pathway occur in more than 90% of melanomas. Melanocyte development and survival requires the stimulation of this pathway by the receptor tyrosine kinase (RTK) KIT. In ~2% of melanomas, oncogenic KIT mutations drive tumor formation; however, the majority of melanomas lose wild-type KIT expression, suggesting that KIT could suppress melanoma formation. In human melanoma patients of The Cancer Genome Atlas (TCGA), we found an association between BRAFV600E mutations and low KIT mRNA expression, so we tested whether KIT loss would affect BRAFV600E-driven tumor onset by crossing a kit(lf) mutant allele into melanoma-prone Tg(mitfa:BRAFV600E); p53(lf) zebrafish. We observed that kit(lf)-mutant zebrafish experienced accelerated tumor onset and their tumors had increased RAS/MAPK pathway activation. In BRAFV600E-mutant melanoma cells, KIT activity reduced RAS/MAPK signaling by promoting activation of wild-type BRAF (BRAFWT). Furthermore, we found that overexpression of BRAFWT delayed tumor onset in Tg(mitfa:BRAFV600E); p53(lf); mitfa(lf) zebrafish, but had no effect in kit(lf); Tg(mitfa:BRAFV600E); p53(lf); mtifa(lf) zebrafish and a cohort of TCGA BRAFV600E-mutant melanoma patients with high KIT expression and high BRAFWT allele ratios experienced a reduced likelihood of metastasis and extended overall survival. These studies indicate that wild-type KIT acts to suppress melanoma formation through activation of BRAFWT, causing reduced signaling output of BRAFV600E-mutant cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20243号 / 医博第4202号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 原田 浩, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13206号 / 論医博第2160号 / 新制||医||1031(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 岩田 想, 教授 秋山 芳展 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.