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（PGE 2）受体（EP）在化学和紫外线诱导的小鼠皮肤肿瘤发生中的作用。人类的流行病学研究 证实了PGs，包括PGE 2，在包括皮肤癌在内的多种癌症中的重要作用。通过环氧合酶（考克斯-1/考克斯-2）产生的PGE 2作用于四种不同的细胞表面受体（EP 1、EP-2、EP-3、EP-4），它们属于GPCR家族。 在一系列研究中，小鼠皮肤起始/促进模型用于研究PGE 2受体EP 2和EP 4在乳头状瘤发展中的作用。吲哚美辛被用来抑制表皮PG的产生，减少乳头状瘤形成约60%。在吲哚美辛处理的小鼠中，EP 2激动剂使乳头状瘤形成恢复到TPA处理水平，但EP 4激动剂无效。在乳头状瘤中，EP 2激动剂增加Ras活化和水平以及c-AMP、p-Src、p-EGFr、p-Erk和p-Akt水平。用EGFr（Ag 1478）或Src（PP 2）抑制剂治疗乳头状瘤证明p-EGFr在p-Src的下游。Ag 1478还抑制Ras、Erk和AKT的激活。EP 2-/-小鼠的乳头状瘤数量减少了60%，Ras、Src、AKT和Erk的活化减少，这一观察结果证实了EP 2在乳头状瘤发展中的作用。p-Src或EP 2的免疫沉淀表明野生型乳头状瘤中存在EP 2-b-arrestin 1/2-p-Src复合物。B-抑制蛋白可以促进EP 2的内化，也可以作为对接蛋白，促进EP 2信号传导。这些数据表明，PGE 2通过EP 2激活信号转导途径，可以促进小鼠皮肤乳头状瘤的发展。 在第二系列研究中，研究了EP 2和EP 4在UV诱导的皮肤损伤中的作用。用UVB（5 kJ/m2）急性处理野生型（WT）和考克斯-2-/-小鼠，并比较凋亡信号传导途径。暴露后，与WT小鼠相比，考克斯-2-/-中的细胞凋亡高2.5倍。由于前列腺素（PG）E2是主要的UV诱导的PG，并通过四种受体（EP 1至4）显示其活性，因此在WT和考克斯-2-/-小鼠中研究了EP信号传导的可能差异。UVB暴露后，WT小鼠中EP 1、EP 2和EP 4的蛋白水平升高，但考克斯-2-/-小鼠中EP 2和EP 4水平降低50%。活化的cAMP依赖性蛋白激酶（PKA）和Akt在EP 2和EP 4信号传导中处于下游，并且它们的水平在UVB暴露的考克斯-2-/-小鼠中降低。此外，p-Bad（Ser 136和Ser 155），激活Akt和PKA的抗凋亡产物，分别在UVB暴露的考克斯-2-/-小鼠中显著减少。为了进一步研究EP 2和EP 4的作用，用吲哚美辛局部处理UVB暴露的CD-1小鼠以阻断内源性PGE 2产生，并应用PGE 2、EP 2激动剂或EP 4激动剂。吲哚美辛使PKA和Akt活化降低约60%，但PGE 2和激动剂恢复了它们的活性。此外，两种激动剂均使考克斯-2-/-小鼠的细胞凋亡减少50%。这些数据表明，考克斯-2产生的PGE 2在UVB暴露的小鼠皮肤中具有抗凋亡作用，其涉及EP 2和EP 4介导的信号传导，其减少角质形成细胞凋亡。