br Materials and methods br
Materials and methods
Discussion The EP4 NMS-E973 for PGE2 is similar to the EP1, EP2 and EP3 receptors in that they all have seven transmembrane segments and are coupled to Gα subunits of heterotrimeric G proteins, with a different Gα subunit for each receptor (Sugimoto and Narumiya, 2007). The EP4 receptor, however, has several unique structural features that suggest that it may have distinct biological functions. EP4 is also unique among the EP receptors in that it is rapidly internalized in response to PGE2 (Regan, 2003). The EP1 and EP2 receptors have been the most extensively studied with regard to cancer development and progression in several organ systems, including colon and breast cancer development (Kawamori et al., 2005, Kawamori et al., 2001, Watanabe et al., 1999). With regard to skin cancer, the EP1 receptor confers tumor promoting and even stronger tumor progression activity in carcinogen-initiated mice (Surh et al., 2011, Surh et al., 2012), and in UV-induced carcinogenesis (Tober et al., 2006). EP1 overexpression in transgenic mice resulted in protein kinase C activation and upregulation of COX-2, suggesting that its tumor promoting activity is via the same mechanism used by TPA (Surh et al., 2011, Surh et al., 2012). The EP2 receptor has also been linked to the growth and progression of a number of cancers (Baba et al., 2010, Castellone et al., 2005, Kuo et al., 2009, Sonoshita et al., 2001), including skin (Sung et al., 2005). EP2 null mice develop 50% fewer papillomas than WT mice (and no SCCs) in a DMBA/TPA protocol (Sung et al., 2005). Conversely, overexpression of EP2 resulted in significantly more skin tumors, including more SCCs, than WT mice (Sung et al., 2006). In a UV protocol EP2 null mice also developed 50% fewer tumors, but these tumors were larger and more aggressive than those in WT mice (Brouxhon et al., 2007), suggesting that the role of EP2 may be carcinogen-insult dependent. The limited number of studies on the EP3 receptor indicated that it can have both pro- and anti-inflammatory activity (Goulet et al., 2004, Honda et al., 2009). However, the EP3 receptor was not found to play a role in skin tumorigenesis, where inflammation is usually a driving factor (Shoji et al., 2005, Sung et al., 2005). Like the other EP receptors, EP4 was reported to be upregulated in skin tumors from both UV and DMBA/TPA protocols (Lee et al., 2005, Neumann et al., 2007, Tober et al., 2007). Because the EP4 receptor was also shown to contribute to breast, kidney, and head and neck cancers (Abrahao et al., 2010, Ma et al., 2013, Wu et al., 2011, Xin et al., 2012), we hypothesized that EP4 would also contribute to skin tumor development. As shown in this report, the skins of the BK5.EP4 mice are essentially identical to WT mice at the microscopic level, albeit their hair cycles are slightly different. When subjected to the two-stage DMBA/TPA protocol, the transgenic mice developed considerably more tumors than WT mice. More significantly, most of the tumors in the transgenic mice were SCCs, unlike in the wild type mice. Despite the fact that TPA treatment increased keratinocyte proliferation to a greater extent in the transgenic mice, the mechanism responsible for increased tumorigenesis is not clear. An unusual finding in the DMBA/TPA experiment was the development of a tumor in a transgenic mouse after DMBA but before TPA treatment was begun. This suggested that, like the EP1 receptor (Surh et al., 2012), EP4 could have endogenous tumor promoting activity. To test this, the response to DMBA alone was investigated. Using a single application of DMBA, it was visually apparent that the transgenic mice had a much more cytotoxic response than WT mice. Several avenues were investigated to attempt to elucidate the basis for this response. However, studies on the expression of enzymes that metabolize DMBA (CYP1A1, CYP1B1 and aromatase) as well as on keratinocyte stem cell (CD34 and ?6 integrin positive cells) numbers indicated no difference between transgenic and WT mice.