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Activation of EGFR and stimulation of arginase in pulmonary hypertension.

机译:肺动脉高压中EGFR的激活和精氨酸酶的刺激。

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摘要

Pulmonary hypertension is a life-threatening disease characterized by progressive vascular dysfunction and remodeling. Pulmonary hypertension can be a primary disorder, or it can be secondary to an underlying condition. There is currently no cure for pulmonary hypertension and available therapies remain essentially supportive (52). The hallmark of pulmonary hypertension is vascular remodeling, which has recently been postulated to represent a type of malignant transformation (54). Epidermal growth factor receptor tyrosine kinase (EGFR) is critical for promoting growth, survival, and differentiation of many types of cells. EGFR is activated by phosphorylation, which initiates signal transduction cascades leading to a variety of cellular responses including proliferation. EGFR tyrosine kinase has been recognized as a potential therapeutic target to prevent tumor cell proliferation in various types of cancer associated with local hypoxia. Pulmonary hypertension is seen in lung diseases characterized by hypoxia such as chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). We hypothesized that hypoxia causes EGFR activation leading to greater arginase expression and thereby to greater cellular proliferation in human pulmonary microvascular endothelial cells (hPMVEC).;Arginase metabolizes L-arginine to urea and L-ornithine (L-orn). The L-orn produced by arginase can be further metabolized by ornithine decarboxylase and/or ornithine aminotransferase to ultimately produce polyamines and/or proline, respectively. Polyamines and proline are vital to the cellular proliferation necessary for pulmonary vascular remodeling. Our laboratory has previously reported that cytokine-induced arginase up-regulation in bovine pulmonary arterial endothelial cells was dependent on EGFR activation. Mitogen-activated protein kinases (MAPK) are a group of evolutionarily conserved proteins linking cell surface receptors to critical intracellular targets. Activity of the MAPK is regulated through successive activation of a three-tiered cascade of kinases. The extracellular signal-regulated kinases (ERKs), a family member of the MAPK, are activated by the sequential activation of Raf and MEK kinases. A part of the intracellular pool of activated ERK translocates to the nucleus where it phosphorylates and activates transcription factors involved in mitogenic signaling. We hypothesized that EGFR tyrosine kinase would be increased in hypoxia and that arginase would be downstream of this. We further hypothesized that ERK would be involved in hypoxia-induced EGFR activation and subsequent induction of arginase in human pulmonary microvascular endothelial cells.;To test our hypotheses we studied human pulmonary microvascular endothelial cells (hPMVEC). For specific aim 1, the hPMVEC were incubated in either normoxia or hypoxia. The levels of EGFR, proliferating cell nuclear antigen (PCNA), arginase I and arginase II were determined by immunoblotting. EGFR activity assays were performed. Increases in cell proliferation due to EGFR activity were assessed using pharmacological inhibitors of EGFR and arginase. For specific aim 2, pharmacological inhibitors of EGFR or the MAPK (ERK and JNK) were employed in the various experiments. For specific aim 3, we utilized both male and female adult mice and exposed them to hypoxia for a period of 2 weeks while being treated with vehicle or the EGFR inhibitor AG1478. We harvested tissue from the animals to assay mRNA and protein levels of specific genes, as well as performed physiological measurements on the animals.
机译:肺动脉高压是一种威胁生命的疾病,其特征是进行性血管功能障碍和重塑。肺动脉高压可以是原发性疾病,也可以继发于潜在疾病。目前尚无治愈肺动脉高压的方法,可用的治疗方法仍然基本上是支持性的(52)。肺动脉高压的标志是血管重塑,最近被认为代表了一种恶性转化(54)。表皮生长因子受体酪氨酸激酶(EGFR)对于促进多种类型细胞的生长,存活和分化至关重要。 EGFR通过磷酸化激活,从而引发信号转导级联反应,从而导致多种细胞反应,包括增殖。 EGFR酪氨酸激酶已被认为是预防与局部缺氧有关的各种类型癌症中肿瘤细胞增殖的潜在治疗靶标。在以低氧为特征的肺部疾病(如慢性阻塞性肺疾病(COPD)和间质性肺部疾病(ILD))中可见肺动脉高压。我们假设缺氧会导致EGFR活化,从而导致更大的精氨酸酶表达,从而导致人肺微血管内皮细胞(hPMVEC)的细胞增殖更大。精氨酸酶将L-精氨酸代谢为尿素和L-鸟氨酸(L-orn)。精氨酸酶产生的L-orn可以进一步被鸟氨酸脱羧酶和/或鸟氨酸氨基转移酶代谢以最终分别产生多胺和/或脯氨酸。多胺和脯氨酸对于肺血管重塑所需的细胞增殖至关重要。我们的实验室以前曾报道过,牛肺动脉内皮细胞中细胞因子诱导的精氨酸酶上调依赖于EGFR的激活。丝裂原激活的蛋白激酶(MAPK)是一组将细胞表面受体连接至关键细胞内靶标的进化保守蛋白。 MAPK的活性是通过连续激活三层级联的激酶来调节的。 MAPK的家族成员,细胞外信号调节激酶(ERKs)通过Raf和MEK激酶的顺序激活而被激活。激活的ERK的细胞内池的一部分易位至细胞核,在细胞中磷酸化并激活有丝分裂信号传导中涉及的转录因子。我们假设低氧时EGFR酪氨酸激酶会增加,而精氨酸酶将在此下游。我们进一步假设,ERK将参与缺氧诱导的人肺微血管内皮细胞中的EGFR激活和精氨酸酶的诱导。对于特定目标1,将hPMVEC在常氧或低氧条件下孵育。通过免疫印迹测定EGFR,增殖细胞核抗原(PCNA),精氨酸酶I和精氨酸酶II的水平。进行EGFR活性测定。使用EGFR和精氨酸酶的药理学抑制剂评估了由于EGFR活性引起的细胞增殖增加。对于特定目标2,在各种实验中使用了EGFR或MAPK的药理抑制剂(ERK和JNK)。对于特定目的3,我们同时使用了成年雄性和雌性小鼠,并在接受媒介物或EGFR抑制剂AG1478治疗的情况下将它们暴露于缺氧状态2周。我们从动物中收集组织以测定特定基因的mRNA和蛋白质水平,并对动物进行生理测量。

著录项

  • 作者

    Toby, Inimary.;

  • 作者单位

    The Ohio State University.;

  • 授予单位 The Ohio State University.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 152 p.
  • 总页数 152
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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