We previously reported that the genetic deficiency of COX-1 or COX-2 decreased papilloma formation when the initiation/promotion mouse skin protocol was used (18
). Because PGE2
was the major PG formed in skin, we hypothesized that diminished PGE2
levels and therefore decreased PGE2
-induced signaling in the COX-deficient mice were responsible for decreased tumor formation. PGE2
manifests its activity via four GPCRs, EP1, EP2, EP3 and EP4 (2
); however, in the present study, we focused on the contribution of EP2 to mouse skin papilloma formation. Previous studies have reported that the levels of EP2 expression influenced mouse skin tumor formation in the initiation/promotion model (5
) and on ultraviolet exposure (37
). Using the initiation/promotion model, EP2 deficiency (5
) was shown to decrease and EP2 overexpression (36
) was shown to increase papilloma formation. However, in these studies, TPA-induced endogenous PGE2
was the likely activator of EP2. Indeed, exogenous PGE2
has been reported to activate the PKA and EGFR pathways in mouse keratinocytes and naive mouse skin, although a specific receptor was not identified (23
). In addition, PKA and EGFR have been reported to be activated in vitro
via EP2 in squamous cell carcinoma lines (24
). In the present report, we extend these studies by utilizing an EP2-selective agonist and EP2-deficient mice to identify EP2-mediated signaling pathways activated during tumor development in the mouse skin initiation/promotion model. In addition, in contrast to previous studies (39
), we report that EP2 formed a complex with β-arrestin1 and p-Src (), which based on observations for other GPCRs (13
), including EP4 (15
), could lead to EP2 desensitization and/or provide additional EP2-mediated signaling pathways.
Mutation and activation of H-Ras
are considered key events in papilloma development in the initiation/promotion model (16
), and the data show that EP2 influenced H-Ras activation in papillomas (). These findings suggested that increased levels of PGE2
in papillomas (18
), acting via EP2, could contribute to the activation of H-Ras. Because papillomas contain both WT and mutant H-Ras
), and mutant H-Ras
is considered to exist in the activated form, it is probably that EP2-mediated signaling is primarily influencing the activation of WT H-Ras. However, our data do not allow us to differentiate between EP2’s effects on WT and mutant H-Ras
. In support of EP2’s influence on Ras activation, it has been reported that PGE2
increased Ras activation in rodent and human intestinal cell lines (41
), and roles for EP1 and/or EP2 in the activation of Ras were indicated (42
). Additionally, the data indicated that EP2 contributed to the activation of two H-Ras effectors, ERK1/2 and AKT (), both known to be involved in papilloma formation (30
). However, quantitative differences exist between CAY’s ability to restore the level of these effectors and tumor formation in TPA/Indo-treated mice and the decreased level of these effectors and tumor formed in EP2−/− mice (compare ). In the former, it appears that EP2 is responsible for total restoration of effector activation and tumor formation, whereas in the latter, EP2 appears to only be responsible for 35–60% of effector activation and ~65% of the tumors formed. The reasons for CAY stimulation of EP2 showing a more pronounced effect than the extent to which EP2 deficiency decreases these effects are unclear. Strain difference may play a role as the TPA/Indo/CAY effects were observed in promotion-responsive CD-1 mice, whereas the EP2−/− mice were C57BL/6, a strain known to have decreased sensitivity to TPA promotion (43
). Notwithstanding, the data are in agreement with EP2 influencing the activation of H-Ras, ERK1/2 and AKT and significantly contributing to tumor formation in the initiation/promotion model.
EGFR is a receptor tyrosine kinase that is known to have a role in skin tumor formation (28
) and has been demonstrated to activate Ras, ERK1/2, AKT and other signaling effectors (45
). The data in demonstrate that AG1478 also reduced papilloma formation in the initiation/promotion model. Of relevance for EP2 contributing to TPA-induced EGFR activation, recent in vitro
studies have demonstrated that PGE2
causes the transactivation of EGFR (22
). In agreement with these effects of PGE2
on EGFR and reports demonstrating that other GPCRs can cause the transactivation of EGFR (46
), the present data showed that CAY stimulation of EP2 contributed to the activation of EGFR (), as well as the activation of H-Ras, ERK1/2 and AKT (). Because EGFR, Ras, ERK1/2 and AKT have been reported to play significant roles in skin tumor development (29
), our findings that EP2 contributes to their activation in the initiation/promotion model provides a link between TPA-induced COX-2/PGE2
), EP2-mediated signaling and papilloma development.
Because of the importance of EGFR signaling in papilloma development, studies were initiated to identify possible mechanisms by which EP2 contributed to EGFR activation. Several studies have implicated Src in the transactivation of EGFR by various GPCRs (14
). Based on the observation that CAY stimulation of EP2 contributed to Src activation and that Src was upstream of EGFR (), studies were conducted to identify a possible mechanism for EP2’s activation of Src. Several recent reports have indicated that Src can be activated as a result of the formation of a GPCR–β-arrestin1–Src complex (14
). The data in show that a complex involving EP2, β-arrestin1 and p-Src could be detected in CAY-treated skin and papillomas by immunoprecipitation of p-Src, suggesting that such a complex could contribute to Src activation in mouse skin. Furthermore, p-Src immunoprecipitation and western blotting of β-arrestin1 and p-Src from WT and EP2−/− mouse skin and papillomas () indicated that the p-Src–β-arrestin1 complex was decreased in EP2−/− mice, which further indicated EP2’s involvement in the formation of the complex. The immunoprecipitation data in indicate that p-EGFR and p-Src also form a complex, and the β-arrestin1 immunoprecipitation data () indicate that p-Src disassociates from the EP2–β-arrestin1–p-Src complex prior to interacting with EGFR. However, while our in vivo
data indicate the presence of the EP2–β-arrestin1 complex, they do not directly indicate that the complex is involved in Src activation and subsequent transactivation of EGFR or whether the complex is just a mechanism for the downregulation of EP2 by internalization/desensitization. To address this issue, studies with WT and β-arrestin1−/− mice are now in progress. Additionally, it should be stated that while our studies demonstrated the presence of the EP2–β-arrestin1–p-Src complex in mouse skin and papillomas, previous studies utilizing cultured cells suggested that EP2 did not form a complex with β-arrestin following ligand treatment (39
). Thus, the present data represent the first report of EP2 forming a complex with β-arrestin1 and offer the possibility that the EP2–β-arrestin1 complex may contribute to EP2-mediated signaling.
In addition to EP2 activating the EGFR pathway, we also observed that EP2 activation resulted in increased cAMP levels and the activation of PKA (). Regan et al.
) have previously reported that the activation of PKA was a major EP2 signaling pathway and involved activated Gαs
stimulation of AC. Our results indicated that EP2-mediated PKA activation led to increased p-CREB levels. Furthermore, PKA has also been shown to phosphorylate glycogen synthase kinase-3β that blocks phosphorylation-dependent β-catenin degradation, thereby allowing β-catenin/TCF-dependent transcription to occur (10
). Thus, a major effect of EP2-mediated activation of PKA may be the increased transcription of genes that contribute to transformed keratinocyte growth.
In summary, the results show that the PGE2 receptor, EP2, activates multiple signaling pathways/effectors that contribute to papilloma formation in the mouse skin initiation/promotion model. illustrates proposed signaling pathways activated by EP2. TPA-induced COX-2 and PGE2 cause the stimulation of EP2 leading to the Gαs-dependent activation of AC and PKA, as well as G protein-independent activation of Src/EGFR that involves an EP2–β-arrestin1–Src intermediate. While the data illustrate the importance of EP2-mediated signaling in papillomas, it does not identify the cell type in which the signaling was increased nor does it exclude TPA inducing PKA and EGFR activation by pathways independent of EP2 or that EP2 may activate additional pathways. However, the data described herein provide insight into the signaling pathways by which PGE2 activation of EP2 contributes to skin tumor formation.
Fig. 6. Proposed model for EP2-mediated signaling. EP2 stimulation by ligand binding leads to Gαs activation and dissociation, which then caused AC activation, cAMP production and PKA activation. EP2 ligand binding also causes EP2 association with β-arrestin1 (more ...)