Role of PGE2 Receptors in Mouse Skin Cancer

利用小鼠皮肤起始/促进模型，研究了PGE 2受体EP 1、EP 2、EP 3和EP 4在乳头状瘤发生中的作用。吲哚美辛用于抑制表皮PGE 2的产生，并在启动/促进小鼠中减少乳头状瘤形成约60%。在TPA/吲哚美辛处理的小鼠中，EP 2激动剂（布他前列素）将乳头状瘤形成恢复至单独TPA处理水平，但EP 1、EP 3和EP 4激动剂无效。在乳头状瘤中，EP 2激动剂增加Ras和PKA活化，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位于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-arrestin 1/2可以促进EP 2内化以及促进GPCR非依赖性EP 2介导的信号传导。因此，数据表明，PGE 2通过EP 2激活信号转导途径，可以有助于小鼠皮肤乳头状瘤的发展中的启动/促进模型。 前列腺素E2（PGE 2）G蛋白偶联受体（GPCR），EP 2，在小鼠皮肤肿瘤的发生中起重要作用。 由于角质形成细胞的复制是肿瘤发展所必需的，我们研究了EP 2激活的信号通路，有助于角质形成细胞的复制。局部应用EP 2激动剂布他前列素，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（AG 1478或H89）使布他前列素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活化涉及α-抑制蛋白/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。在本研究中，观察到布他前列素剂量和时间依赖性地增加EGFR和Src激活，并诱导-arrestin 1/Src复合物的形成，和-arrestin 1-缺陷减少Src和EGFR激活。与B-抑制蛋白1导致Src和随后的EGFR激活一致，Src和EGFR抑制剂（PP 2和AG 1478）的研究表明Src位于p-EGFR的上游。布他前列素还诱导Akt、ERK 1/2和STAT 3的活化; EGFR的抑制使其活化降低约60%，表明这些效应物部分由EGFR活化。除EGFR活化外，通过p-GSK 3和p-CREB形成测定，布他前列素还可增加小鼠皮肤中cAMP水平和PKA活化。PKA抑制（H89）降低了布他前列素诱导的GSK 3B、CREB和ERK活化，但不影响EGFR活化。因此，结果表明，EP 2通过G蛋白依赖性PKA活化和G蛋白非依赖性、-arrestin 1依赖性EGFR活化促进小鼠皮肤角质形成细胞复制。

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.