Role of PGE2 Receptors in Mouse Skin Cancer

我们先前报道了前列腺素E2（PGE 2）G蛋白偶联受体（GPCR），EP 2，使用起始促进模型在小鼠皮肤肿瘤发展中起重要作用（Chun et al.，Carcinogenesis，30：1620，2009）。由于角质形成细胞增殖是皮肤肿瘤发展所必需的，因此研究了可能有助于角质形成细胞增殖的EP 2介导的信号传导途径。EP 2激动剂布他前列素的单次局部应用剂量依赖性地增加角质形成细胞复制和激活EGFR和PKA;抑制EGFR或PKA（分别为AG 1478或H89）可使布他前列素诱导的角质形成细胞增殖减少70%。由于先前的研究表明EGFR的GPCR激活涉及b-抑制蛋白1/Src复合物，因此研究了EP 2通过该机制在角质形成细胞中起作用的可能性。据观察，布他前列素增加Src和EGFR激活，并诱导b-抑制蛋白1/Src复合物的形成。b-arrestin 1缺陷减少Src和EGFR的激活，证实了b-arrestin 1在其激活中的作用。与b-抑制蛋白1促进Src和EGFR活化一致，Src和EGFR抑制剂（分别为PP 2和AG 1478）的研究表明Src位于EGFR的上游。布他前列素还诱导Akt、ERK 1/2和STAT 3的活化; b-抑制蛋白1缺陷或EGFR抑制均降低其活化，证实了b-抑制蛋白1和EGFR在其活化中的作用。除EGFR活化外，布他前列素还增加cAMP水平和PKA活化，通过p-GSK 3b和p-CREB形成进行测量。PKA抑制剂H89可降低布他前列素诱导的GSK 3b、CREB和ERK活化，但不影响EGFR活化。因此，我们最近的研究结果表明，EP 2通过G蛋白非依赖性，barrestin 1依赖性EGFR激活和G蛋白依赖性PKA激活促进小鼠角质形成细胞增殖。 除了EP 2在起始/促进模型中有助于皮肤肿瘤形成之外（Chun等人，Carcinogenesis，30：1620，2009），我们先前报道了EP 2在UVB暴露的小鼠皮肤中的抗肿瘤反应中起作用（Chun等人，Cancer Res.，67：2015，2007）。由于生存素是细胞存活的调节因子，因此研究了UVB暴露小鼠皮肤中考克斯-2和EP 2对生存素的可能调节。在野生型小鼠皮肤中，UVB时间依赖性地增加了考克斯-2、生存素和磷酸化信号转导子和转录激活子3（p-STAT 3）（一种调节生存素表达的转录因子）的水平;而考克斯-2或EP 2缺乏显著降低了它们的诱导作用。局部应用考克斯-2选择性抑制剂塞来昔布，也降低了UVB诱导的生存素水平。为了进一步研究PGE 2和EP 2在Survivin调节中的作用，使用吲哚美辛抑制UVB诱导的内源性PG产生。吲哚美辛降低UVB诱导的生存素水平，而PGE 2和EP 2激动剂，布他前列素，部分恢复生存素水平。表皮生长因子受体（EGFR）是EP 2的下游效应子，抑制EGFR（AG 1478）可显著降低UVB对生存素的诱导和STAT 3的激活。在考克斯-2-/-小鼠中，UVB诱导的表皮细胞凋亡被布他前列素减少，EGFR抑制阻断了布他前列素的保护作用。此外，在没有UVB暴露的情况下，布他前列素时间依赖性地增加了未处理小鼠皮肤中的p-EGFR、p-STAT 3和生存素水平，而EP 4激动剂PGE 1乙醇没有显著增加p-STAT 3或生存素水平。这些数据表明，考克斯-2产生的PGE 2通过EP 2介导的EGFR/STAT 3途径调节小鼠皮肤中的生存素表达，并且抑制EP 2/生存素途径可能为UVB诱导的皮肤癌的化学预防/化学治疗提供治疗策略。

We previously reported that the prostaglandin E2 (PGE2) G protein coupled receptor (GPCR), EP2, plays important roles in mouse skin tumor development using the initiation promotion model (Chun et al., Carcinogenesis, 30:1620, 2009). Because keratinocyte proliferation is essential for skin tumor development, EP2-mediated signaling pathways that could contribute to keratinocyte proliferation were investigated. A single topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication and activated EGFR and PKA; and inhibition of either EGFR or PKA (AG1478 or H89, respectively) decreased butaprost-induced keratinocyte proliferation by 70%. Because previous studies indicated that GPCR activation of EGFR involved a b-arrestin1/Src complex, the possibility of EP2 acting via this mechanism in keratinocytes was investigated. It was observed that butaprost increased Src and EGFR activation and induced b-arrestin1/Src complex formation. b-arrestin1-deficiency reduced Src and EGFR activation, confirming b-arrestin1s role in their activation. In agreement with b-arrestin1 contributing to Src and EGFR activation, studies with Src and EGFR inhibitors (PP2 and AG1478, respectively) indicated Src to be upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/2, and STAT3; and either b-arrestin1-deficiency or EGFR inhibition decreased their activation, confirming roles for b-arrestin1 and EGFR in their activation. In addition to EGFR activation, butaprost also increased cAMP levels and PKA activation, as measured by p-GSK3b and p-CREB formation. The PKA inhibitor, H89, decreased butaprost-induced GSK3b, CREB, and ERK activation, but did not affect EGFR activation. Thus, our recent results indicate that EP2 contributed to mouse keratinocyte proliferation by G protein independent, barrestin1 dependent activation of EGFR, and G protein dependent activation of PKA. In addition to EP2 contributing to skin tumor formation in the initiation/promotion model (Chun et al., Carcinogenesis, 30:1620, 2009), we previously reported that EP2 played a role in an anti-apoptotiic response in UVB-exposed mouse skin (Chun et al. Cancer Res., 67:2015, 2007). Because survivin is a regulator of cell survival, the possible regulation of survivin by COX-2 and EP2 in UVB-exposed mouse skin was investigated. In wild type mouse skin UVB time-dependently increased the levels of COX-2, survivin and phosphorylated-signal transducer and activator of transcription3 (p-STAT3), a transcription factor that regulates survivin expression; and COX-2- or EP2-deficiency significantly reduced their induction. Topical application of the COX-2 selective inhibitor, celecoxib, also reduced UVB-induced survivin levels. To further investigate the roles of PGE2 and EP2 in the regulation of survivin, indomethacin was used to inhibit UVB-induced endogenous PG production. Indomethacin reduced UVB-induced survivin levels, while PGE2 and the EP2 agonist, butaprost, partially restored survivin levels. The epidermal growth factor receptor (EGFR) is a downstream effector of EP2 and inhibition of EGFR (AG1478) significantly reduced UVB induction of survivin and activation of STAT3. UVB-induced epidermal apoptosis in COX-2-/- mice was reduced by butaprost and EGFR inhibition blocked butaprosts protective effects. Furthermore, butaprost in the absence of UVB exposure time-dependently increased p-EGFR, p-STAT3 and survivin levels in nave mouse skin, whereas the EP4 agonist, PGE1 alcohol, did not significantly increase p-STAT3 or survivin levels. These data suggest that COX-2-generated PGE2 regulates survivin expression in mouse skin via an EP2-mediated EGFR/STAT3 pathway, and inhibiting the EP2/survivin pathway may provide a therapeutic strategy for the chemoprevention/chemotherapy of UVB-induced skin cancer.