Role of PGE2 Receptors in Mouse Skin Cancer

采用小鼠皮肤启动/促进模型，研究PGE2受体EP1、EP2、EP3和EP4在乳头状瘤发生发展中的作用。吲哚美辛用于抑制表皮PGE2的产生，并使启动/促进小鼠的乳头状瘤形成减少约60%。在TPA/吲哚美辛治疗的小鼠中，EP2激动剂(Butaprost)将乳头状瘤的形成恢复到TPA单独治疗的水平，但EP1、EP3和EP4激动剂无效。在乳头状瘤中，EP2激动剂增加了RAS和PKA的激活，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还可抑制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-arrestin1/2可以促进EP2的内化，也可以促进GPCR非依赖的EP2介导的信号转导。因此，数据表明，在启动/促进模型中，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。 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起重要作用。由于角质形成细胞的复制对肿瘤的发展至关重要，我们研究了EP2激活的信号通路对角质形成细胞复制的影响。外用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；抑制EGFR或PKA(AG1478或H89)可使Butaprost诱导的角质形成细胞复制减少约70%。由于先前的几项研究表明，EGFR的GPCR激活涉及一个-arrestin/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。在本研究中，观察到布他前列素剂量和时间递增地增加了EGFR和Src的激活，并诱导了-arrestin1/Src复合体的形成，而-arrestin1缺乏则降低了Src和EGFR的激活。与导致Src和随后的EGFR激活的B-arrestin1一致，对Src和EGFR抑制剂(PP2和AG1478)的研究表明，Src位于p-EGFR的上游。Butaprost还诱导Akt、ERK1/2和STAT3的激活，抑制EGFR使它们的激活减少约60%，表明这些效应器可能部分地被EGFR激活。除了EGFR的激活外，通过p-GSK3和p-CREB的形成来衡量，Butaprost还增加了小鼠皮肤中cAMP水平和PKA的激活。抑制PKA(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，结果表明EP2通过G蛋白依赖的PKA的激活和G蛋白不依赖的、抑制素1依赖的EGFR的激活来促进小鼠皮肤角质形成细胞的复制。

Using the mouse skin initiation/promotion model the contributions of the PGE2 receptors, EP1, EP2, EP3 and EP4 in papilloma development were investigated. Indomethacin was used to inhibit epidermal PGE2 production and reduced papilloma formation about 60% in initiated/promoted mice. In TPA/indomethacin treated mice the EP2 agonist (butaprost) restored papilloma formation to TPA alone treatment levels, but EP1, EP3 and EP4 agonists were ineffective. In papillomas, the EP2 agonist increased Ras and PKA activation, 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-arrestin1/2 can contribute to EP2 internalization as well as facilitate GPCR independent EP2-mediated signaling. Thus, the data indicate that PGE2 acting via EP2 activates signal transduction pathways that can contribute to mouse skin papilloma development in the initiation/promotion model. The prostaglandin E2 (PGE2) G protein coupled receptor (GPCR), EP2, plays an important role in mouse skin tumor development. Because keratinocyte replication is essential for tumor development, we investigated EP2-activated signaling pathways that contributed to keratinocyte replication. Topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication and activated EGFR and PKA; and inhibition of EGFR or PKA (AG1478 or H89) decreased butaprost-induced keratinocyte replication about 70%. Because several previous studies indicated that GPCR activation of EGFR involved a β-arrestin/Src complex, the possibility of EP2 acting via this mechanism in keratinocytes was investigated. In the present study, it was observed that butaprost dose and time-debendently increased EGFR and Src activation and induced β-arrestin1/Src complex formation, and that β-arrestin1-deficiency reduced Src and EGFR activation. In agreement with B-arrestin1 leading to Src and subsequently EGFR activation, studies with Src and EGFR inhibitors (PP2 and AG1478) indicated Src to be upstream of p-EGFR. Butaprost also induced the activation of Akt, ERK1/2 and STAT3; and inhibition of EGFR reduced their activation about 60%, indicating these effectors to be activated, in part, by EGFR. In addition to EGFR activation, butaprost increased cAMP levels and PKA activation in mouse skin as measured by p-GSK3β and p-CREB formation. PKA inhibition (H89) decreased butaprost-induced GSK3B, CREB and ERK activation, but did not affect EGFR activation. Thus, the results indicated that EP2 contributed to mouse skin keratinocyte replication by G protein dependent activation of PKA, and G protein independent, β-arrestin1 dependent, activation of EGFR.