Role of PGE2 Receptors in Mouse Skin Cancer

我们之前报道过前列腺素E2 (PGE2) G蛋白偶联受体（GPCR） EP2在小鼠皮肤肿瘤的发生发展中发挥重要作用（Chun et al., carcingenesis, 30:1620, 2009）。由于角质形成细胞增殖对皮肤肿瘤的发展至关重要，ep2介导的信号通路可能有助于角质形成细胞增殖。单次局部应用EP2激动剂，但他前列素，剂量依赖性地增加角化细胞复制并激活EGFR和PKA；抑制EGFR或PKA（分别为AG1478或H89）可使丁蛋白酶诱导的角质细胞增殖减少70%。由于先前的研究表明，GPCR激活EGFR涉及b-arrestin1/Src复合物，因此研究了EP2在角化细胞中通过该机制起作用的可能性。我们观察到，butaprost增加Src和EGFR的激活，并诱导b-arrestin1/Src复合物的形成。b-arrestin1缺乏降低了Src和EGFR的激活，证实了b-arrestin1在其激活中的作用。与b-arrestin1促进Src和EGFR激活的观点一致，Src和EGFR抑制剂（分别为PP2和AG1478）的研究表明Src位于EGFR的上游。但他前列素也诱导Akt、ERK1/2和STAT3的激活；b-arrestin1缺乏或EGFR抑制都会降低它们的激活，证实了b-arrestin1和EGFR在其激活中的作用。通过p-GSK3b和p-CREB的形成测量，除了EGFR激活外，butaprost还增加cAMP水平和PKA激活。PKA抑制剂H89降低了丁他前列腺素诱导的GSK3b、CREB和ERK的激活，但不影响EGFR的激活。因此，我们最近的研究结果表明，EP2通过G蛋白不依赖、barrestin1依赖的EGFR激活和G蛋白依赖的PKA激活来促进小鼠角化细胞增殖。

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. Recent studies have indicated that B-arrestin1 deficiency reduced skin tumor multiplicity and tumor size in the initiation/ promotion model, whereas B-arrestin2 deficiency increased tumor multiplicity and tumor sizes. The differing effects of B-arrestin1/2 deficiency are currently being investigated.