Role of PGE2 Receptors in Mouse Skin Cancer

PGE2 受体在小鼠皮肤癌中的作用

• 批准号: 7594028
• 负责人: Robert Langenbach
• 金额: $ 79.32万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594028
• 关键词: Agonist; Apoptosis; Apoptotic; CCL4 gene; Cell Surface Receptors; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Development; Dinoprostone; Docking; Epidemiologic Studies; Family; Goals; Human; Immunoprecipitation; Indomethacin; Malignant Neoplasms; Mediating; Modeling; Mus; Numbers; PTGS2 gene; Papilloma; Production; Prostaglandin Production; Prostaglandin-Endoperoxide Synthase; Prostaglandins; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; Role; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Skin; Skin Cancer; Skin Papilloma; UV induced; Ultraviolet B Radiation; Wild Type Mouse; arrestin B; cyclooxygenase 1; cyclooxygenase 2; human WFDC2 protein; inhibitor/antagonist; keratinocyte; progesterone 11-hemisuccinate-(2-iodohistamine); prostaglandin EP2 receptor; prostanoid receptor EP1; receptor; tumorigenesis

The major goal of the study was the elucidation of the role of prostaglandin E2 (PGE2) receptors (EP) in chemically and UV induced mouse skin tumorigenesis. Epidemiological studies in humans have confirmed an important role of PGs, including PGE2, in a variety of cancers including skin cancer. PGE2 produced via the cyclooxygenases (COX-1/COX-2) act on four different cell surface receptors (EP 1, -2, -3, -4), which belong to the family of GPCRs. In one series of studies, the mouse skin initiation/promotion model was used to investigate the contribution of the PGE2 receptors, EP2 and EP4, in papilloma development. Indomethacin was used to inhibit epidermal PG production and reduced papilloma formation about 60%. In indomethacin treated mice the EP2 agonist restored papilloma formation to TPA treatment levels, but the EP4 agonist was ineffective. In papillomas, the EP2 agonist increased Ras activation and levels as well as c-AMP, p-Src, p-EGFr, p-Erk and p-Akt levels. Treatment of papillomas with EGFr (Ag1478) or Src (PP2) inhibitors demonstrated that p-EGFr was downstream of p-Src. Ag1478 also inhibited the activation of Ras, Erk and AKT. EP2s role in papilloma development was confirmed by the observations that EP2 -/- mice showed a 60% reduction in papilloma numbers and reduced activation of Ras, Src, AKT and Erk. Immunoprecipitation of p-Src or EP2 indicated the presence of an EP2-b-arrestin1/2-p-Src complex in papillomas of wild type. B-arrestin can contribute to EP2 internalization as welll as serve as a docking protein and facilitate EP2 signaling. The data indicate that PGE2 acting via EP2 activates signal transduction pathways that can contribute to mouse skin papilloma development. In a second serries of studies, the roles of EP2 and EP4 on UV induced skin damage was investigated. Wild type (WT) and COX-2-/- mice were acutely treated with UVB (5 kJ/m2) and apoptotic signaling pathways compared. Following exposure, apoptosis was 2.5-fold higher in COX-2-/- compared to WT mice. Because prostaglandin (PG) E2 is the major UV-induced PG and manifests its activity via four receptors, EP1 to 4, possible differences in EP signaling were investigated in WT and COX-2-/- mice. Following UVB exposure, protein levels of EP1, EP2, and EP4 were elevated in WT mice, but EP2 and EP4 levels were 50% lower in COX-2-/- mice. Activated cAMP-dependent protein kinase (PKA) and Akt are downstream in EP2 and EP4 signaling, and their levels were reduced in UVB-exposed COX-2-/- mice. Furthermore, p-Bad (Ser136 and Ser155), anti-apoptotic products of activated Akt and PKA, respectively, were significantly reduced in UVB-exposed COX-2-/- mice. To further study EP2 and EP4s roles, UVB-exposed CD-1 mice were topically treated with indomethacin to block endogenous PGE2 production, and PGE2, an EP2 agonist or an EP4 agonist applied. Indomethacin reduced PKA and Akt activation about 60%, but PGE2 and the agonists restored their activities. Furthermore, both agonists decreased apoptosis in COX-2-/- mice by 50%. The data suggest that COX-2 generated PGE2 has anti-apoptotic roles in UVB-exposed mouse skin that involves EP2 and EP4 mediated signaling that reduced keratinocyte apoptosis.

本研究的主要目的是阐明前列腺素E2(PGE2)受体(EP)在化学和紫外线诱导的小鼠皮肤肿瘤形成中的作用。对人类的流行病学研究表明 证实了包括PGE2在内的PGs在包括皮肤癌在内的各种癌症中的重要作用。由环氧合酶(COX-1/COX-2)产生的PGE2作用于四种不同的细胞表面受体(EP1、-2、-3、-4)，属于GPCRs家族。 在一系列研究中，使用小鼠皮肤启动/促进模型来研究PGE2受体EP2和EP4在乳头状瘤发生中的作用。吲哚美辛可抑制表皮PG的生成，使乳头状瘤的形成减少约60%。在吲哚美辛治疗的小鼠中，EP2激动剂使乳头状瘤的形成恢复到TPA治疗水平，但EP4激动剂无效。在乳头状瘤中，EP2激动剂增加了RAS的激活和水平，以及c-AMP、p-Src、p-EGFR、p-Erk和p-Akt的水平。用EGFR(Ag1478)或Src(PP2)抑制剂治疗乳头状瘤表明，p-EGFR位于p-Src的下游。Ag1478还抑制RAS、ERK和AKT的激活。观察到EP2-/-小鼠的乳头状瘤数量减少了60%，Ras、Src、AKT和Erk的活性降低，证实了EP2s在乳头状瘤发生发展中的作用。P-Src或EP2的免疫沉淀表明野生型乳头状瘤中存在EP2-b-arrestin1/2-p-Src复合体。B-arrestin可以促进EP2的内化，也可以作为对接蛋白促进EP2信号转导。这些数据表明，PGE2通过EP2激活信号转导通路，从而促进小鼠皮肤乳头状瘤的发展。 在第二系列研究中，研究了EP2和EP4在紫外线所致皮肤损伤中的作用。用UVB(5kJ/m2)急性照射野生型(WT)小鼠和COX-2-/-小鼠，比较细胞凋亡信号转导途径。暴露后，COX-2-/-小鼠的细胞凋亡率是WT小鼠的2.5倍。由于前列腺素(PG)E2是紫外线诱导的前列腺素(PG)的主要受体，并通过EP1至EP4四种受体显示其活性，因此我们研究了WT和COX-2-/-小鼠EP信号的可能差异。经UVB照射后，WT小鼠EP1、EP2和EP4蛋白水平升高，而COX-2-/-小鼠EP2和EP4蛋白水平降低50%。活化的cAMP依赖的蛋白激酶(PKA)和Akt位于EP2和EP4信号的下游，在UVB暴露的COX-2-/-小鼠中，它们的水平降低。此外，在UVB暴露的COX-2-/-小鼠中，活化的Akt和PKA的抗凋亡产物p-Bad(Ser136和Ser155)显著减少。为了进一步研究EP2和EP4s的作用，用消炎痛局部治疗暴露于UVB的CD-1小鼠以阻断内源性PGE2的产生，并应用EP2激动剂或EP4激动剂PGE2。吲哚美辛使PKA和Akt活性降低约60%，而PGE2和激动剂则恢复其活性。此外，两种激动剂均可将COX-2-/-小鼠的细胞凋亡率减少50%。这些数据表明，COX-2产生的PGE2在UVB暴露的小鼠皮肤中具有抗凋亡作用，涉及EP2和EP4介导的信号转导，减少角质形成细胞的凋亡。