To identify and characterize new possible molecular targets
To identify and characterize new possible molecular targets, the mechanisms of various kinds of TCDD toxicities that occur by abnormal regulation in the downstream of AhR signals have been studied (Yoshioka et al., 2011). Experimental evidence has revealed that prostanoids (i.e., prostaglandins and thromboxane) could be the molecular targets of TCDD-induced developmental toxicity in rodents and zebrafish. The thromboxane receptor (TP) is responsible for pericardial edema during organogenesis of zebrafish embryos (Nijoukubo et al., 2016; Teraoka et al., 2009). On the other hand, cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase-1 (mPGES-1), which are key I-BET-762 synthesis responsible for prostanoid synthesis, play critical roles in TCDD-induced neonatal hydronephrosis in rodent models (Aida-Yasuoka et al., 2014; Nishimura et al., 2008; Yoshioka et al., 2012, 2014; Yoshioka et al., 2016). In rodent models, neonates exposed to TCDD via lactation developed hydronephrosis during the first two weeks after birth (Couture-Haws et al., 1991). Two unique characteristics of neonatal hydronephrosis are that (i) there is no anatomical or functional obstruction in the ureter and (ii) cotreatment of a COX-2-selective inhibitor prevents neonatal onset of TCDD-induced neonatal hydronephrosis (Nishimura et al., 2008). Furthermore, studies also showed that the lack of mPGES-1, which converts prostaglandin H2 (PGH2) to prostaglandin E2 (PGE2), also protects mice from TCDD-induced neonatal hydronephrosis, showing that COX-2 and mPGES-1 critically affect the development of TCDD-induced neonatal hydronephrosis (Aida-Yasuoka et al., 2014; Nishimura et al., 2008; Yoshioka et al., 2012, 2014). PGE2 receptors are classified into four subtypes, EP1 – EP4 (Boie et al., 1997; Breyer and Breyer, 2000a, b), and each receptor subtype has unique and complex histological localization and physiological roles in the nephrons (Antonucci et al., 2007; Guan et al., 2002; Jensen et al., 2001; Taniguchi et al., 1994). Since PGE2 plays a predominant role in TCDD-induced neonatal hydronephrosis, it would be logical to postulate that PGE2 signals abnormally transmitted via one or more of the four subtypes cause this disease. Thus, in this study, we aimed to clarify which PGE2 receptor subtype is the causal factor for TCDD-induced neonatal hydronephrosis onset.
Materials and methods
Hydronephrosis in mouse pups induced by exposure to TCDD via lactation is characterized by a lack of anatomical obstruction of the ureter and is significantly associated with polyuria due to a urine concentration defect (Yoshioka et al., 2016; Nishimura et al., 2008). The most significant finding of this study was that EP1, not EP2, EP3, or EP4, of the four PGE2 receptor subtypes, is predominantly responsible for neonatal hydronephrosis. As long as EP1 exists, the contribution of EP2, EP3, or EP4, if any, in TCDD-induced neonatal hydronephrosis onset is minimal. This conclusion was supported by our results showing that TCDD exposure almost completely induces hydronephrosis in EP2–/– or EP3–/– pups and EP4 antagonist–treated pups (Table 2, Table 3, Table 4). However, EP1 cannot be deemed the sole cause of TCDD-induced hydronephrosis because of at least three reasons. First, the incidence of TCDD-induced hydronephrosis in EP1+/+ mice used as controls in this study with each EP-subtype-null mice was not 100%, but 80% for EP1+/+ (Table 1), 80% for EP2+/+ (Table 2), 88.9% for EP3+/+ (Table 3), and 88.9% EP4+/+ (Table 4). These data are consistent with our previous results using 14-day-old C57Bl/6 J male pups (Nishimura et al., 2008). Second, even the EP1–/– group harboring the other three EP subtypes developed TCDD-induced hydronephrosis: the incidence was 28.6%, and the severity degree in individual pups was 2 or less (Table 1, Fig. 5C). This result suggested that the possible involvement of other EP subtypes in TCDD-induced hydronephrosis pathogenesis cannot be excluded. Third, another possible factor that might interfere with the role of EP1 in TCDD-induced hydronephrosis onset is protein kinase N (PKN), which is encoded at the EP1 locus on the antiparallel DNA strand (Batshake and Sundelin, 1996) and is highly likely to be disrupted when EP1–/– mice are produced by a conventional method (Guan et al., 2007). TCDD exposure can activate cytosolic phospholipase A2α (cPLA2α) to release arachidonate and develop neonatal hydronephrosis (Yoshioka et al., 2014); arachidonate as well as other fatty acids activate PKN (Guan et al., 2007). Thus, the limitation due to such gene manipulation may be reflected by incomplete onset of TCDD-induced toxicity in both EP1–/– and PKN–/– mice.