Role of PGE2 Receptors in Mouse Skin Cancer

我们先前报道了前列腺素E2(PGE2)G蛋白偶联受体EP2在小鼠皮肤肿瘤的发生发展中发挥重要作用(Chun等人，Carcinenesis，30：1620,2009)。由于角质形成细胞的增殖对皮肤肿瘤的发展至关重要，因此我们研究了EP2介导的促进角质形成细胞增殖的信号通路。单独局部应用EP2激动剂Butaprost可剂量依赖性地增加角质形成细胞的复制，并激活EGFR和PKA；而EGFR或PKA(分别为AG1478或H89)的抑制可使Butaprost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-arrestin1/Src复合体，因此我们研究了EP2在角质形成细胞中通过这一机制发挥作用的可能性。结果表明，布他前列素可增加细胞内Src和EGFR的活性，并诱导b-arrestin1/Src复合体的形成。B-arrestin1缺乏降低了Src和EGFR的激活，证实了b-arrestin1s在它们的激活中的作用。通过对Src和EGFR抑制剂(分别为PP2和AG1478)的研究表明，Src位于EGFR的上游，这与b-arrestin1对Src和EGFR激活的作用一致。Butaprost还可诱导Akt、ERK1/2和STAT3的激活，b-arrestin1缺乏或EGFR抑制均可降低它们的激活，证实了b-arrestin1和EGFR在它们的激活中的作用。根据p-GSK3b和p-CREB的形成，除了EGFR的激活外，Butaprost还增加了cAMP水平和PKA的激活。PKA抑制剂H89可降低Butaprost诱导的GSK3b、CREB和ERK的激活，但不影响EGFR的激活。因此，我们最近的结果表明，EP2通过G蛋白不依赖于G蛋白、Barrest 1依赖于EGFR的激活以及G蛋白依赖的PKA激活来促进小鼠角质形成细胞的增殖。 除了EP2在启动/促进模型中促进皮肤肿瘤的形成外(Chun等人，癌症发生，30：1620,2009)，我们以前报道过EP2在UVB暴露的小鼠皮肤中起到抗凋亡反应的作用(Chun等人)。癌症研究，67：2015,2007)。由于Survivin是细胞存活的调节因子，因此我们研究了COX-2和EP2对UVB照射小鼠皮肤中Survivin的可能调节作用。在野生型小鼠皮肤中，UVB可时间依赖地增加COX-2、Survivin和调节Survivin表达的转录因子磷酸化信号转导和转录激活子3(p-STAT3)的水平，而COX-2或EP2缺乏显著降低了它们的诱导。局部应用COX-2选择性抑制剂塞来昔布也能降低UVB诱导的Survivin水平。为了进一步研究PGE2和EP2在Survivin调控中的作用，用吲哚美辛抑制UVB诱导的内源性PG的产生。吲哚美辛降低了UVB诱导的Survivin水平，而PGE2和EP2激动剂Butaprost部分恢复了Survivin水平。表皮生长因子受体(EGFR)是EP2的下游效应因子，抑制EGFR(AG1478)可显著降低UVB对Survivin的诱导和STAT3的激活。Butaprost可减少UVB诱导的COX-2-/-小鼠表皮细胞凋亡，EGFR抑制可阻断Butaprost的保护作用。此外，在没有UVB照射的情况下，Butaprost呈时间依赖性地增加NAVE小鼠皮肤中p-EGFR、p-STAT3和Survivin的水平，而EP4激动剂PGE1酒精并不显著增加p-STAT3或Survivin的水平。这些结果表明，COX-2产生的PGE2通过EP2介导的EGFR/STAT3通路调节小鼠皮肤中Survivin的表达，抑制EP2/Survivin通路可能为UVB诱导的皮肤癌的化学预防/化疗提供治疗策略。

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.