Immunolocalization of Low-Affinity Prostaglandin E 2 Receptors, EP 1 and EP 2 , in Adult Human Epidermis Raymond L. Konger, à w Steven D. Billings, à Angela B. Thompson, à Akira Morimiya, à Jack H. Ladenson, z Yvonne Landt,z Alice P. Pentland,y and Sunil Badve à Departments of à Pathology & Laboratory Medicine and wDermatology, Indiana University School of Medicine, Indianapolis, Indiana, USA; zDivision of Laboratory Medicine, Washington University School of Medicine, St Louis, Missouri, USA; yDepartment of Dermatology, University of Rochester School of Medicine & Dentistry, Rochester, New York, USA Four prostaglandin (PG)E 2 receptors have been described, termed E-series prostaglandin receptors (EP 1 –EP 4 ), that can be further subclassified as low-affinity (EP 1 and EP 2 ) or high-affinity (EP 3 and EP 4 ) receptors. Activation of the low-affinity PGE 2 receptors is likely to be important in mediating the actions of the high levels of PGE 2 found in various pathologic processes. The pattern of expression of these receptors in epidermis, however, is unknown. We therefore examined the immunolocalization of the EP 1 and EP 2 receptors in human epidermis. The EP 1 and EP 2 receptors demonstrated both plasma membrane and perinuclear or nuclear staining within the basal and spinous layers. Within the granular layer, both receptors were expressed in the cytoplasm with a grainy or granular ap- pearance. The major differences were that the EP 2 receptor demonstrated a zone of decreased to absent plasma membrane staining in the superficial spinous layer and only scattered cellular staining within the granular layer. In contrast, the EP 1 receptor was prominently expressed throughout the stratum granulosum and the plasma mem- brane staining pattern was seen throughout the spinous layer. In cultured primary human keratinocytes, we also verified the presence of functional EP 1 receptor coupled to intracellular calcium mobilization and EP 2 receptor coupled to cAMP production. Key words: keratinocytes/immunolocalization/prostaglandin E 2 /EP 1 receptor/EP 2 receptor/second messengers J Invest Dermatol 124:965–970, 2005 Prostaglandin E 2 (PGE 2 ) has an important role in regulating keratinocyte growth, differentiation, and apoptosis (Lo et al, 1998; Goldyne, 2000). PGE 2 is formed by cleavage of arachidonic acid from cellular phospholipids, followed by its conversion to PGH 2 by one of two cyclooxygenases (COX-1 and -2). Increased COX-2 activity and expression has been shown to mediate pathologic skin changes associated with wound repair, inflammatory diseases, ultraviolet irradiation, and neoplasia (Goldyne, 2000; Iversen and Kragballe, 2000; Lee et al, 2003). The presence of both COX-1 and COX-2 has been demonstrated in both normal and neoplastic hu- man skin (Goldyne, 2000). PGE 2 acts by binding to one of four different het- erotrimeric G-protein coupled receptors (GPCR), termed E- series prostaglandin receptors (EP 1 –EP 4 ) (Breyer et al, 2001). These receptors differ in the second messenger pathways activated upon PGE 2 binding. The receptors can be roughly broken into two receptor classes based on their PGE 2 -binding affinities. High-affinity receptors, EP 3 and EP 4 , bind PGE 2 at subnanomolar levels, whereas low-affin- ity EP 1 and EP 2 receptors have dissociation constants in the low nanomolar range. EP 2 and EP 4 receptors are coupled to adenylate cyclase activation, whereas EP 1 receptors are coupled to cytosolic and nuclear calcium mobilization and activation of phospholipase C. EP 3 receptors have multiple splice variants that have been shown to signal through Ga i , Ga s ,Ga 13 , and Gbg subunits (Hatae et al, 2002). The presence of multiple receptor subtypes with different PGE 2 -binding affinities and signaling pathways demon- strate the diversity of PGE 2 -mediated signaling. The ability to mechanistically define the role of PGE 2 in epidermal physiology and pathology is therefore dependent on first establishing the expression and function of the individual PGE 2 receptors in skin. We have previously demonstrated that primary human keratinocytes (PHK) in culture express transcripts for EP 2À4 receptors and that the low-affinity EP 2 receptor stimulates growth in non-confluent PHK in vitro (Konger et al, 1998). Compared with high-affinity receptors that would be ex- pected to be fully activated at low levels of PGE 2 produc- tion, the low-affinity PGE 2 receptors are likely to be of relatively greater importance under pathologic conditions associated with increased PGE 2 production. Thus, in order to better understand how PGE 2 functions in human skin, we examined the expression and localization of both low-af- finity EP receptor subtypes in normal adult human skin. To verify that these receptors are expressed and functionally active in keratinoctyes, we also examined their expression Abbreviations: COX, cyclooxygenase; EP, E-series prostaglandin receptor; GPCR, G-protein coupled receptor; HEK, human em- bryonic kidney; HIER, heat-induced epitope retrieval; IHC, immunohistochemistry; PBS, phosphate-buffered saline; PGE 2 , prostaglandin E 2 ; PHK, primary human keratinocyte Copyright r 2005 by The Society for Investigative Dermatology, Inc. 965