- Email: [email protected]
Cyclooxygenase isoforms in human skin
Prostaglandins & other Lipid Mediators 63 (2000) 15–23
Cyclooxygenase isoforms in human skin Marc E. Goldyne* Department of Dermatology, University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA
1. Introduction The skin performs critical homeostatic functions including thermoregulation, sensory perception, protection against microbial invasion, and protection against absorption of potentially toxic environmental agents. To perform these functions, the skin must maintain a structural and functional integrity that derives from an array of complex cellular and biochemical interactions. The structural homeostasis of the skin is partly the result of regulated cell proliferation and differentiation within the topmost segment of the skin called the epidermis (see Fig. 1). The epidermis is a stratified epithelium. It has a basal layer of cells (basal keratinocytes) that have the capacity to proliferate. The suprabasal daughter cells (prickle cell layer), while losing the ability to proliferate under normal conditions, undergo differentiation and apoptosis as they migrate toward the surface. These cells begin to synthesize complex new proteins and to secrete a complex glycolipid “barrier” between the granular and cornified layers that regulates both water loss as well as permeability of exogenous substances (e.g. topically applied medications). Ultimately, the cornified layer of keratin proteins is formed as a major structural and functional interface between the host’s internal and external environments. Alterations in the proliferating and differentiating compartments of the epidermis, whether from endogenous and/or exogenous triggers, are associated with a number of skin diseases including two types of skin cancer. It is therefore important to identify and understand the function of enzymes that may influence cell proliferation and differentiation within the epidermis. By doing so, we should establish a more rational basis for clinical therapeutics. The prostaglandins, comprising one of the major classes of eicosanoids, are synthesized in humans primarily from arachidonic acid, an essential fatty acid, via activity that initially * Tel.: ⫹1-530-752-9765; fax: ⫹1-530-752-9766. E-mail address: [email protected] (M.E. Goldyne). 0090-6980/00/$ – see front matter © 2000 Published by Elsevier Science Inc. PII: S 0 0 9 0 - 6 9 8 0 ( 0 0 ) 0 0 0 9 4 - 0
16
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
Fig. 1. Human skin consists of the epidermis and underlying dermis. The epidermis has 4 regions (layers). The basal layer rests on a basement membrane that separates it from the underlying dermis. The basal keratinocytes constitute the proliferative pool of the epidermis. The overlying daughter cells in the prickle cell layer begin a process of differentiation with generation of keratin, as well as other structural proteins that will allow them to form the topmost cornified layer. In the so-called granular layer, cells begin to undergo apoptosis while at the same time making more keratin proteins and releasing so-called lamellar bodies that provide horizontally dispersed sheets of complex extracellular lipids critical to the overall barrier function of the overlying cornified layer.
was thought to reflect a single enzyme called cyclooxygenase. From the time that prostaglandin E2 (PGE2) was identified in rat [1] and in human skin [2] in the 1970s, numerous studies have attempted to identify the roles of PGE2 as well as other prostaglandins in both normal and pathologic skin responses. One of the obstacles to achieving this understanding has been the fact that almost all cell types synthesize some profile of prostaglandins and human skin is no exception; consequently identifying the cellular source(s) and the cellular target(s) of prostaglandin generation in the context of specific skin responses is paramount. While this process of identification is far from complete, recent developments in the enzymology of prostaglandin generation as well as in the elucidation of prostaglandin receptors provide new avenues to explore in the attempt to define how prostaglandins modulate human skin function. This review will focus on studies of the cyclooxygenase enzymes in human and other
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
17
mammalian epidermis and their participation in epidermal homeostasis and disease. Although not addressed in this review, there is a sizeable body of data that examines mutual regulatory influences between the lower skin compartment, called the dermis, and the overlying epidermis. Exclusion of this data from the present review, however, should not prevent the reader from grasping the potential regulatory and pathophysiologic importance of cyclooxygenases in the skin.
2. Cyclooxygenases– general information Two isoforms of cyclooxygenase, commonly referred to as COX-1 and COX-2 are currently recognized [3,4]. The genes for these two isozymes exist on different chromosomes in humans as well as in mice [5]. Structurally, the two isozymes share about 60% homology in overall amino acid sequences and about 90% in the active site region where arachidonic acid as well as COX inhibitors bind [6]. In the binding site region, the COX-2 gene encodes for a valine rather than an isoleucine as found in COX-1 [7]. This single amino acid substitution alters the inhibitor binding domain in such a way that inhibitor selectivity becomes possible. Early studies on the cyclooxygenase isozymes focused on inflammatory cells. Investigators found that whereas COX-1 was constitutively expressed, COX-2 required stimulusbased induction [3,4,8]. Consequently, it was postulated that the prostaglandins (as well as thromboxane) that participate in homeostatic regulation derive from the constitutive COX-1 whereas the prostaglandins that participate in inflammation derive from the inducible COX-2. Indeed, the rush by the pharmaceutical industry to develop COX-2 inhibitors derived from the theory that major therapeutic benefits would accompany inhibiting only the production of inflammatory prostaglandins without affecting the synthesis of homeostatic prostaglandins. In keeping with this theory, studies on specific COX-2 inhibitors show that they provide anti-inflammatory effects without inducing the gastric ulcerations associated with the use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) [9]. Consequently, by not inhibiting COX-1 activity that provides cytoprotective prostaglandins for the gastric mucosa, the use of specific COX-2 inhibitors should prevent the estimated 107,000 hospitalizations each year and about 16,000 deaths that result from traditional NSAIDinduced gastroduodenal injury [10]. On the other hand, the assumptions made by investigators in the field of inflammation regarding the nature and roles of the two recognized cyclooxygenase isozymes cannot be automatically extrapolated to other cell, tissue, and organ functions. More specifically, research on other tissues and organs challenges the concept of COX-2 functioning only as an inducible enzyme under pathophysiological circumstances. In fact, COX-2 appears to be constitutively expressed in the kidney, bladder, intestine, lung, heart, and brain of rodents and rabbits (reviewed in [11]). A study done using mice showed that disruption of the COX-2 gene resulted in a severe nephropathy without affecting inflammatory responses to traditionally used stimuli (e.g. tetradecanoyl phorbol acetate) [12]. Furthermore, as will be reviewed subsequently, studies on the expression of COX-2 in human and mammalian skin
18
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
suggest that this isozyme may participate in homeostatic function of the epidermis and moreover, overexpression may be a feature of certain types of skin dysplasia and cancer.
3. Cyclooxygenases in mammalian and human skin Early studies of arachidonic acid deficiency in animals and later in humans receiving hyperalimentation documented profound effects on skin structure and function. The epidermis became more thick (hyperkeratosis), hair loss ensued, and barrier function was lost (reviewed in [13]). The observation that topical application of PGE2 could reverse epidermal hyperkeratosis in the rat [14], although not restore barrier integrity [15], was probably the first indication that prostaglandins and, in turn, cyclooxygenase activity, contributed to skin homeostasis. Other studies in mouse and rat skin showed that the NSAID indomethacin could suppress epidermal cell proliferation and that PGE2 could override this suppression and, in fact, induce proliferation of epidermal keratinocytes [16]. In vitro, human skin keratinocytes have been shown to modulate their proliferation through prostaglandin synthesis [17]. 3.1. COX isozymes in mouse skin COX-1 and COX-2 isozymes can both be expressed in mouse epidermis [18]. When evaluating homogenized frozen epidermal tissue from both neonatal and adult mice, only COX-1 mRNA as well as COX-1 protein were found. COX-1 protein was already present in epidermis of 1-day-old animals and remained constant for the next 10 days of evaluation when the adult skin phenotype was developing. When keratinocytes were isolated from mouse epidermis and subjected to density gradient centrifugation, investigators found a different set of results. In both neonatal and adult mice, COX-1 mRNA increased with the relative level of cell differentiation. In neonatal skin, however, the COX-1 protein was increasingly expressed with differentiation whereas it was equally expressed in all layers in adult skin. In contrast to the frozen tissue studies, COX-2 mRNA was now found in all neonatal keratinocyte fractions; however there was no expression of corresponding protein. In the adult keratinocytes, COX-2 mRNA was barely detectable and no COX-2 protein could be demonstrated. In contrast to the above findings, mouse epidermis stimulated to undergo hyperplastic transformation (e.g. stripping the stratum corneum or applying the phorbol ester PMA) expressed both COX-2 mRNA and protein. A stimulus (4 – 0-methyl-PMA) that caused balanced hyperproliferation (i.e. proliferation balanced by cell desquamation resulting in no hyperplasia) failed to induce expression of COX-2 mRNA or protein. Based on these results, it appears that in mouse skin, COX-2 expression seems to follow the inducible pattern found in inflammatory cells. These findings of inducible expression of COX-2 were substantiated in a subsequent study from our laboratory. Expression of COX-2 protein appeared in the basal layer of mouse epidermis following treatment with acetone that disrupts the barrier function of the epidermis [19]. Cultured mouse keratinocytes, unlike freshly isolated cells or frozen tissue, express both COX isozymes constitutively as do a number of murine keratinocyte cell lines. These results show that, in culture, murine keratinocytes assume a phenotype different from that of resting
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
19
cells. In fact expression of COX-2 protein, in mice at least, may serve as a marker for stimulated keratinocytes. These findings also constitute a caveat to investigators seeking to extrapolate observations from cell culture experiments to in vivo cell function. 3.2. COX isozymes in human skin Human epidermis, unlike that of the mouse, appears to express both COX isozymes. This was initially demonstrated using immunohistochemistry as well as Western blotting of protein extracts from cultured human keratinocytes [19]. In a study from our laboratory, COX-1 immunostaining was observed throughout the epidermis whereas COX-2 immunostaining was more prominent in the more differentiated layers of the epidermis. Another group of investigators also observed this latter finding [20]. In contrast, a more recent study found COX-2 immunostaining only in a few keratinocytes in the basal compartment of interfollicular skin and in more differentiated parts of the hair follicle [21]. The reasons for this discrepancy in results are unclear but may derive from differences in sample preparation, sites sampled, and/or antibody specificities. Thus, unlike mouse skin, COX-2 protein in human skin may be constitutively expressed in the more differentiated layers of the epidermis. In keeping with this concept, increased expression of COX-2 was present in biopsies of squamous cell carcinomas that derive from more differentiated keratinocytes; in contrast, little if any expression of COX-2 was observed in basal cell carcinomas that derive from the least differentiated basal cells [19 –21]. One study that also evaluated keratoacanthomas, another type of squamous proliferation that has many features of squamous cell carcinoma, showed increased expression of COX-2 [21].
4. Physiologic and pathophysiologic implications 4.1. Physiologic implications The extracellular concentration of calcium seems to be critical for induction of keratinocyte differentiation [22,23]. In fact, an extracellular calcium gradient exists in normal human skin with the lowest concentration present in the basal cell region and the highest concentration evident around the more differentiated keratinocytes higher in the epidermis [24]. A relationship between COX-2, the epidermal calcium gradient, and keratinocyte differentiation is suggested by two studies from our laboratory. Working with human keratinocyte cultures, we observed that increasing extracellular calcium resulted in: 1) increased expression of COX-2 mRNA and protein, but not COX-1 protein [19], 2) increased PGE2 synthesis by the keratinocytes [25], and 3) induction of cornified envelope formation which is a recognized marker of keratinocyte differentiation [26]. In the presence of indomethacin, however, cornified envelope formation was significantly suppressed, but could be reestablished by adding exogenous PGE2 [25]. Our finding of a possible link between PGE2, COX-2 expression, and keratinocyte differentiation is in keeping with a study by other investigators looking at the effect of cholcalciferol on human keratinocytes [27]. A similar link between COX-2 expression and differentiation is also suggested in several other human cell systems and in murine kidney [28 –30]. It is important to point out that there are types of cells in which differentiation appears to
20
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
involve COX-1, rather than COX-2, induction [31]. Consequently, the physiologic roles of the COX isozymes are most likely cell and tissue specific, and to extrapolate the findings from one type of cell or tissue study to untested cells and tissues needs to be undertaken with caution. While this approach appears self-evident, COX-2 is still often described in the general literature as the inducible form of cyclooxygenase when, as cited previously, it is now recognized as constitutively expressed in a number of tissues [11,32]. 4.2. Pathophysiologic implications One of the more intensively studied areas of cyclooxygenase function is that relating to carcinogenesis. In studies on colon cancer, the overexpression of COX-2 in human and animal colon cancer as well as the beneficial effects of cyclooxygenase inhibitors on colon cancer development in a murine model have been documented [33–36]. These studies have stimulated interest in the possible roles of cyclooxygenases in promotion of other types of cancer. In a murine skin model of multistage carcinogenesis, COX-2 expression is specifically stimulated with phorbol ester tumor promoters [18], and it has previously been demonstrated that inhibitors of COX-2 have an anti-tumor effect in this model [37]. Another tumor promoting agent, UV light, also induces cyclooxygenase activity in mouse skin and two recent studies document the ability of a specific COX-2 inhibitor to suppress UV-induced skin cancer formation in hairless mice [38,39]. In human skin, UV light induces expression of COX-2 in the keratinocytes [20,40]. UV-light also increases expression of the arachidonoyl-specific high molecular weight, calcium-dependent phospholipase A2 (cPLA2) which appears to mediate the increased PGE2 synthesis in keratinocytes after UV exposure [41]. Consequently, at least two points of pharmacological attack could be developed if further studies substantiate the participation of increased PGE2 synthesis in non-melanoma skin carcinogenesis. Whereas a role for altered COX-2 expression and activity in human squamous cell carcinogenesis is supported by the data heretofore reviewed, other control points in eicosanoid generation and function need further elucidation before the relative contribution of COX-2 to skin carcinogenesis can be more accurately defined. For example, recent studies have documented heterogeneity among prostaglandin E (EP) receptors [42,43], and one study on human keratinocytes suggests that the effects of PGE2 on keratinocyte growth appear to be mediated in part through the EP2 and EP4 receptors [44]. The possibility therefore exists that alterations in EP receptors may be a feature in some forms of skin carcinogenesis. In fact, a study of human breast cancer cell lines suggests both loss and/or dysfunction of EP receptors as factors in tumor propagation [45]. In a murine model of colon cancer, EP1 knockout mice display a significant decrease in the appearance of precancerous lesions and an EP1 antagonist produces a decrease in precancerous lesions among wild type mice [46].
5. Summary The reviewed data document expression of both COX-1 and COX-2 in human and mammalian skin as well as up-regulation of COX-2 in specific types of epidermally- derived
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
21
precancerous and cancerous lesions. In a physiologic context, some evidence suggests that COX-2 expression may be involved in homeostatic regulation of normal keratinocyte differentiation. In a pathophysiologic context, increased COX-2 expression in keratinocytes occurs following exposure to UV light and chronic exposure to UV light is associated with the generation of both actinic keratoses, and squamous cell carcinomas (reviewed in [47]). It is worth noting that both actinic keratoses and squamous cell carcinomas also show mutations in the p53 tumor suppressor gene [48]. Consequently, at least several gene alterations, one of which involves the COX-2 gene, appear to be required for the phenotypic epidermal squamous carcinoma cell. The fact that UV –induced basal cell carcinomas do not appear to express COX-2 to the same extent as the squamous cell carcinomas suggests that COX-2 expression or up-regulation is not necessarily a required property of the carcinoma phenotype. At the same time, the more aggressive behavior of squamous cell carcinomas compared to basal cell carcinomas may have something to do with COX-2 expression since a relationship between COX-2 expression and metastatic potential has been demonstrated in human colon cancer cells [49]. In a therapeutic context, the data suggest that COX-2 inhibitors may have a role in suppressing the appearance of epidermal neoplasms (e.g. actinic keratoses, squamous cell carcinomas, and keratoacanthomas) that demonstrate increased COX-2 expression. This has already been shown in a murine model. However, the observed suppression of carcinogenesis has never been complete, and this finding suggests, as already mentioned, that other factors may be more seminal to the ultimate appearance of the different types of skin cancers. Nevertheless, other control points in the generation and action of PGE2 or other cyclooxygenase-derived eicosanoids may merit attempts to develop other types of chemotherapy or chemoprevention; these would include inhibitors of cytosolic phospholipase A2 as well as EP receptor antagonists. Future studies will no doubt document the validity, or lack thereof, of these investigative approaches. References [1] Ziboh VA, Shia SL. Prostaglandin E2: biosynthesis and effects on glucose and lipid metabolism in rat skin. Arch Biochem 1971;146:100 –9. [2] Jonsson C-E, Anggard E. Biosynthesis and metabolism of prostaglandin E2 in human skin. Scand J Clin Lab Invest 1972;29:289 –96. [3] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and –2. Adv Immunol 1996;62:167–215. [4] Herschman HR, Reddy ST, Xie W. Function and regulation of prostaglandin synthase-2. Adv Exp Med Biol 1997;62:61– 6. [5] Crofford L. COX-1 and COX-2 tissue expression: implications and predictions. J Rheumatol 1997;24(suppl 49):15–9. [6] Kurumbail RG, Stevens AM, Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature 1996;384:644 – 8. [7] Gierse JK, McDonald JJ, Hauser SD, et al. A single amino acid difference between cyclooxygenase-1 (COX-1) and –2 (COX-2) reverses the selectivity of COX-2 specific inhibitors. J Biochem 1996;271: 15810 – 4. [8] Needleman P, Isakson PC. The discovery and function of COX-2. J Rheumatol 1997;24(suppl 49):6 – 8. [9] Lefkowith JB. Cyclooxygenase-2 specificity and its clinical implications. Am J Med 1999;106(5B):43S– 50S.
22
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
[10] Singh G, Rosen-Ramey D. NSAID induced gastrointestinal complications: the ARAMIS perspective 1997. J Rheumatol Suppl 1998;51:8 –16. [11] Lipsky PE. The clinical potential of cyclooxygenase-2-specific inhibitors. Am J Med 1999;106(5B):51S– 57S. [12] Morham SG, Langenbach R, Loftin CD, et al. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473– 82. [13] Prendiville JS, Manfredi LN. Skin signs of nutritional disorders. Semin Dermatol 1992;11(1):88 –97. [14] Ziboh VA, Hsia SL. Effects of prostaglandin E2 on rat skin: inhibition of sterol ester biosynthesis and clearing of scaly lesions in essential fatty acid deficiency. J Lipid Res 1972;13:458 – 67. [15] Elias PM, Brown B, Ziboh VA. The permeability barrier in essential fatty acid deficiency: evidence for a direct role for linoleic acid in barrier function. J Invest Dermatol 1980;74:230 –3. [16] Furstenberger G, Marks F. Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin E2 in mouse epidermis in vivo. Biochem Biophys Res Commun 1978;84(4): 1103–11. [17] Pentland AP, Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 1986;77:246 –51. [18] Scholz K, Furstenberger G, Muller-Decker K, et al. Differential expression of prostaglandin-H synthase isoenzymes in normal and activated keratinocytes in vivo and in vitro. Biochem J 1995;309:263– 69. [19] Leong J, Hughes-Fulford M, Rakhlin N, et al. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation. Exp Cell Res 1996;224:79 – 87. [20] Buckman SY, Gresham A, et al. COX-2 expression is induced by UVB exposure in human skin: Implications for the development of skin cancer. Carcinogenesis 1998;19:723–9. [21] Muller-Decker K, Reinerth G, Krieg P, et al. Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin. Int J Cancer 1999;82:648 –56. [22] Yuspa SH, Kilkenny AE, Steinert PM, et al. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 1989;109:1207–17. [23] Pillai S, Bickle D, Mancianti ML, et al. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium. J Cell Physiol 1990;143:294 –302. [24] Menon GK, Elias PM. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol 1991;127:57– 63. [25] Evans CB, Pillai S, Goldyne ME. Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes. Prostaglandins Leukot Essen Fatty Acids 1993;49:777– 81. [26] Sun TT, Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell 1976;9:511–21. [27] Kanekura T, Laulederkind SJ, Kirtikara K, et al. Cholecalciferol induces prostaglandin E2 biosynthesis and transglutaminase activity in human keratinocytes. J Invest Dermatol 1998;111(4):634 –9. [28] Hume R, Kelly R, Cossar D, et al. Exp Cell Res 1991;194:111–7. [29] Hoff T, DeWitt D, Volkhard K, et al. FEBS Lett 1993;320:38 – 42. [30] Zhang MZ, Wang JL, Cheng HF, et al. Cyclooxygenase-2 in rat nephron development. Am J Physiol 1997;273(6 Pt 2):F994 –F1002. [31] Smith CJ, Morrow JD, Roberts LJ, et al. Biochem Biophys Res Commun 1993;182:787–93. [32] O’Neill GP, Ford-Hutchinson AW. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett 1993;330:156 – 60. [33] Eberhart CE, Coffey RJ, Radhika A, et al. Up-regulation of cyclooxygenase-2 expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107:1183–7. [34] DuBois RN, Radhika A, Reddy BS, et al. Increased cyclooxygenase–2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 1996;110:1259 – 62. [35] Jacoby RF, Marshall DJ, Newton MA, et al. Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal antiinflammatory drug piroxicam. Cancer Res 1996;56:710 – 4.
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
23
[36] Kawamori T, Rao CV, Seibert K, et al. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res 1998;58:409 –12. [37] Muller-Decker K, Kopp-Schneider A, Marks F, et al. Localization of prostaglandin H synthase isoenzymes in murine epidermal tumors: suppression of skin tumor promotion by PGHS-2 inhibition. Mol Carcinog 1998;22:36 – 44. [38] Fischer SM, Lo HH, Gordon G, et al. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol Carcinog 1999;25(4): 231– 40. [39] Pentland AP, Schoggins JW, Scott GA, et al. Reduction of UV-induced skin tumors in hairless mice by selective COX-2 inhibition. Carcinogenesis 1999;20(10):1939 – 44. [40] Soriani M, Luscher P, Tyrrell RM. Direct and indirect modulation of ornithine decarboxylase and cyclooxygenase by UVB radiation in human skin cells. Carcinogenesis 1999;20(4):727–32. [41] Chen X, Gresham A, Morrison A, Pentland A. Oxidative stress mediates synthesis of cytosolic phospholipase A2 after UVB injury. Biochim Biophys Acta Lipids Lipid Metab 1996;1299:22–33. [42] Ichikawa A, Sugimoto Y, Negishi M. Molecular aspects of the structures and functions of the prostaglandin E receptors. J Lipid Mediat Cell Signal 1996;14(1–3):83–7. [43] Jin J, Mao GF, Ashby B. Constitutive activity of human prostaglandin E receptor EP3 isoforms. Br J Pharmacol 1997;121(2):317–23. [44] Konger RL, Malaviya R, Pentland AP. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim Biophys Acta 1998;1401(2):221–34. [45] Planchon P, Veber N, Magnien V, Israel L, Starzec AB. Alteration of prostaglandin E receptors in advanced breast tumor cell lines. Mol Cell Endocrinol 1995;111(2):219 –23. [46] Watanabe K, Kawamori T, Nakatsugi S, Ohta T, Ohuchida S, Yamamoto H, Maruyama T, Kondo K, Ushikubi F, Narumiya S, Sugimura T, Wakabayashi K. Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res 1999;59(20):5093– 6. [47] Salasche SJ. Epidemiology of actinic keratoses and squamous cell carcinoma. J Am Acad Dermatol (Suppl) 2000;42(1, part 2):S4 –S7. [48] Ziegler A, Jonason AS, Leffell DJ, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994;372: 773– 6. [49] Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997;94:3336 – 40.
Cyclooxygenase isoforms in human skin Marc E. Goldyne* Department of Dermatology, University of California San Francisco, 4150 Clement Street, San Francisco, CA 94121, USA
1. Introduction The skin performs critical homeostatic functions including thermoregulation, sensory perception, protection against microbial invasion, and protection against absorption of potentially toxic environmental agents. To perform these functions, the skin must maintain a structural and functional integrity that derives from an array of complex cellular and biochemical interactions. The structural homeostasis of the skin is partly the result of regulated cell proliferation and differentiation within the topmost segment of the skin called the epidermis (see Fig. 1). The epidermis is a stratified epithelium. It has a basal layer of cells (basal keratinocytes) that have the capacity to proliferate. The suprabasal daughter cells (prickle cell layer), while losing the ability to proliferate under normal conditions, undergo differentiation and apoptosis as they migrate toward the surface. These cells begin to synthesize complex new proteins and to secrete a complex glycolipid “barrier” between the granular and cornified layers that regulates both water loss as well as permeability of exogenous substances (e.g. topically applied medications). Ultimately, the cornified layer of keratin proteins is formed as a major structural and functional interface between the host’s internal and external environments. Alterations in the proliferating and differentiating compartments of the epidermis, whether from endogenous and/or exogenous triggers, are associated with a number of skin diseases including two types of skin cancer. It is therefore important to identify and understand the function of enzymes that may influence cell proliferation and differentiation within the epidermis. By doing so, we should establish a more rational basis for clinical therapeutics. The prostaglandins, comprising one of the major classes of eicosanoids, are synthesized in humans primarily from arachidonic acid, an essential fatty acid, via activity that initially * Tel.: ⫹1-530-752-9765; fax: ⫹1-530-752-9766. E-mail address: [email protected] (M.E. Goldyne). 0090-6980/00/$ – see front matter © 2000 Published by Elsevier Science Inc. PII: S 0 0 9 0 - 6 9 8 0 ( 0 0 ) 0 0 0 9 4 - 0
16
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
Fig. 1. Human skin consists of the epidermis and underlying dermis. The epidermis has 4 regions (layers). The basal layer rests on a basement membrane that separates it from the underlying dermis. The basal keratinocytes constitute the proliferative pool of the epidermis. The overlying daughter cells in the prickle cell layer begin a process of differentiation with generation of keratin, as well as other structural proteins that will allow them to form the topmost cornified layer. In the so-called granular layer, cells begin to undergo apoptosis while at the same time making more keratin proteins and releasing so-called lamellar bodies that provide horizontally dispersed sheets of complex extracellular lipids critical to the overall barrier function of the overlying cornified layer.
was thought to reflect a single enzyme called cyclooxygenase. From the time that prostaglandin E2 (PGE2) was identified in rat [1] and in human skin [2] in the 1970s, numerous studies have attempted to identify the roles of PGE2 as well as other prostaglandins in both normal and pathologic skin responses. One of the obstacles to achieving this understanding has been the fact that almost all cell types synthesize some profile of prostaglandins and human skin is no exception; consequently identifying the cellular source(s) and the cellular target(s) of prostaglandin generation in the context of specific skin responses is paramount. While this process of identification is far from complete, recent developments in the enzymology of prostaglandin generation as well as in the elucidation of prostaglandin receptors provide new avenues to explore in the attempt to define how prostaglandins modulate human skin function. This review will focus on studies of the cyclooxygenase enzymes in human and other
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
17
mammalian epidermis and their participation in epidermal homeostasis and disease. Although not addressed in this review, there is a sizeable body of data that examines mutual regulatory influences between the lower skin compartment, called the dermis, and the overlying epidermis. Exclusion of this data from the present review, however, should not prevent the reader from grasping the potential regulatory and pathophysiologic importance of cyclooxygenases in the skin.
2. Cyclooxygenases– general information Two isoforms of cyclooxygenase, commonly referred to as COX-1 and COX-2 are currently recognized [3,4]. The genes for these two isozymes exist on different chromosomes in humans as well as in mice [5]. Structurally, the two isozymes share about 60% homology in overall amino acid sequences and about 90% in the active site region where arachidonic acid as well as COX inhibitors bind [6]. In the binding site region, the COX-2 gene encodes for a valine rather than an isoleucine as found in COX-1 [7]. This single amino acid substitution alters the inhibitor binding domain in such a way that inhibitor selectivity becomes possible. Early studies on the cyclooxygenase isozymes focused on inflammatory cells. Investigators found that whereas COX-1 was constitutively expressed, COX-2 required stimulusbased induction [3,4,8]. Consequently, it was postulated that the prostaglandins (as well as thromboxane) that participate in homeostatic regulation derive from the constitutive COX-1 whereas the prostaglandins that participate in inflammation derive from the inducible COX-2. Indeed, the rush by the pharmaceutical industry to develop COX-2 inhibitors derived from the theory that major therapeutic benefits would accompany inhibiting only the production of inflammatory prostaglandins without affecting the synthesis of homeostatic prostaglandins. In keeping with this theory, studies on specific COX-2 inhibitors show that they provide anti-inflammatory effects without inducing the gastric ulcerations associated with the use of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) [9]. Consequently, by not inhibiting COX-1 activity that provides cytoprotective prostaglandins for the gastric mucosa, the use of specific COX-2 inhibitors should prevent the estimated 107,000 hospitalizations each year and about 16,000 deaths that result from traditional NSAIDinduced gastroduodenal injury [10]. On the other hand, the assumptions made by investigators in the field of inflammation regarding the nature and roles of the two recognized cyclooxygenase isozymes cannot be automatically extrapolated to other cell, tissue, and organ functions. More specifically, research on other tissues and organs challenges the concept of COX-2 functioning only as an inducible enzyme under pathophysiological circumstances. In fact, COX-2 appears to be constitutively expressed in the kidney, bladder, intestine, lung, heart, and brain of rodents and rabbits (reviewed in [11]). A study done using mice showed that disruption of the COX-2 gene resulted in a severe nephropathy without affecting inflammatory responses to traditionally used stimuli (e.g. tetradecanoyl phorbol acetate) [12]. Furthermore, as will be reviewed subsequently, studies on the expression of COX-2 in human and mammalian skin
18
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
suggest that this isozyme may participate in homeostatic function of the epidermis and moreover, overexpression may be a feature of certain types of skin dysplasia and cancer.
3. Cyclooxygenases in mammalian and human skin Early studies of arachidonic acid deficiency in animals and later in humans receiving hyperalimentation documented profound effects on skin structure and function. The epidermis became more thick (hyperkeratosis), hair loss ensued, and barrier function was lost (reviewed in [13]). The observation that topical application of PGE2 could reverse epidermal hyperkeratosis in the rat [14], although not restore barrier integrity [15], was probably the first indication that prostaglandins and, in turn, cyclooxygenase activity, contributed to skin homeostasis. Other studies in mouse and rat skin showed that the NSAID indomethacin could suppress epidermal cell proliferation and that PGE2 could override this suppression and, in fact, induce proliferation of epidermal keratinocytes [16]. In vitro, human skin keratinocytes have been shown to modulate their proliferation through prostaglandin synthesis [17]. 3.1. COX isozymes in mouse skin COX-1 and COX-2 isozymes can both be expressed in mouse epidermis [18]. When evaluating homogenized frozen epidermal tissue from both neonatal and adult mice, only COX-1 mRNA as well as COX-1 protein were found. COX-1 protein was already present in epidermis of 1-day-old animals and remained constant for the next 10 days of evaluation when the adult skin phenotype was developing. When keratinocytes were isolated from mouse epidermis and subjected to density gradient centrifugation, investigators found a different set of results. In both neonatal and adult mice, COX-1 mRNA increased with the relative level of cell differentiation. In neonatal skin, however, the COX-1 protein was increasingly expressed with differentiation whereas it was equally expressed in all layers in adult skin. In contrast to the frozen tissue studies, COX-2 mRNA was now found in all neonatal keratinocyte fractions; however there was no expression of corresponding protein. In the adult keratinocytes, COX-2 mRNA was barely detectable and no COX-2 protein could be demonstrated. In contrast to the above findings, mouse epidermis stimulated to undergo hyperplastic transformation (e.g. stripping the stratum corneum or applying the phorbol ester PMA) expressed both COX-2 mRNA and protein. A stimulus (4 – 0-methyl-PMA) that caused balanced hyperproliferation (i.e. proliferation balanced by cell desquamation resulting in no hyperplasia) failed to induce expression of COX-2 mRNA or protein. Based on these results, it appears that in mouse skin, COX-2 expression seems to follow the inducible pattern found in inflammatory cells. These findings of inducible expression of COX-2 were substantiated in a subsequent study from our laboratory. Expression of COX-2 protein appeared in the basal layer of mouse epidermis following treatment with acetone that disrupts the barrier function of the epidermis [19]. Cultured mouse keratinocytes, unlike freshly isolated cells or frozen tissue, express both COX isozymes constitutively as do a number of murine keratinocyte cell lines. These results show that, in culture, murine keratinocytes assume a phenotype different from that of resting
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
19
cells. In fact expression of COX-2 protein, in mice at least, may serve as a marker for stimulated keratinocytes. These findings also constitute a caveat to investigators seeking to extrapolate observations from cell culture experiments to in vivo cell function. 3.2. COX isozymes in human skin Human epidermis, unlike that of the mouse, appears to express both COX isozymes. This was initially demonstrated using immunohistochemistry as well as Western blotting of protein extracts from cultured human keratinocytes [19]. In a study from our laboratory, COX-1 immunostaining was observed throughout the epidermis whereas COX-2 immunostaining was more prominent in the more differentiated layers of the epidermis. Another group of investigators also observed this latter finding [20]. In contrast, a more recent study found COX-2 immunostaining only in a few keratinocytes in the basal compartment of interfollicular skin and in more differentiated parts of the hair follicle [21]. The reasons for this discrepancy in results are unclear but may derive from differences in sample preparation, sites sampled, and/or antibody specificities. Thus, unlike mouse skin, COX-2 protein in human skin may be constitutively expressed in the more differentiated layers of the epidermis. In keeping with this concept, increased expression of COX-2 was present in biopsies of squamous cell carcinomas that derive from more differentiated keratinocytes; in contrast, little if any expression of COX-2 was observed in basal cell carcinomas that derive from the least differentiated basal cells [19 –21]. One study that also evaluated keratoacanthomas, another type of squamous proliferation that has many features of squamous cell carcinoma, showed increased expression of COX-2 [21].
4. Physiologic and pathophysiologic implications 4.1. Physiologic implications The extracellular concentration of calcium seems to be critical for induction of keratinocyte differentiation [22,23]. In fact, an extracellular calcium gradient exists in normal human skin with the lowest concentration present in the basal cell region and the highest concentration evident around the more differentiated keratinocytes higher in the epidermis [24]. A relationship between COX-2, the epidermal calcium gradient, and keratinocyte differentiation is suggested by two studies from our laboratory. Working with human keratinocyte cultures, we observed that increasing extracellular calcium resulted in: 1) increased expression of COX-2 mRNA and protein, but not COX-1 protein [19], 2) increased PGE2 synthesis by the keratinocytes [25], and 3) induction of cornified envelope formation which is a recognized marker of keratinocyte differentiation [26]. In the presence of indomethacin, however, cornified envelope formation was significantly suppressed, but could be reestablished by adding exogenous PGE2 [25]. Our finding of a possible link between PGE2, COX-2 expression, and keratinocyte differentiation is in keeping with a study by other investigators looking at the effect of cholcalciferol on human keratinocytes [27]. A similar link between COX-2 expression and differentiation is also suggested in several other human cell systems and in murine kidney [28 –30]. It is important to point out that there are types of cells in which differentiation appears to
20
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
involve COX-1, rather than COX-2, induction [31]. Consequently, the physiologic roles of the COX isozymes are most likely cell and tissue specific, and to extrapolate the findings from one type of cell or tissue study to untested cells and tissues needs to be undertaken with caution. While this approach appears self-evident, COX-2 is still often described in the general literature as the inducible form of cyclooxygenase when, as cited previously, it is now recognized as constitutively expressed in a number of tissues [11,32]. 4.2. Pathophysiologic implications One of the more intensively studied areas of cyclooxygenase function is that relating to carcinogenesis. In studies on colon cancer, the overexpression of COX-2 in human and animal colon cancer as well as the beneficial effects of cyclooxygenase inhibitors on colon cancer development in a murine model have been documented [33–36]. These studies have stimulated interest in the possible roles of cyclooxygenases in promotion of other types of cancer. In a murine skin model of multistage carcinogenesis, COX-2 expression is specifically stimulated with phorbol ester tumor promoters [18], and it has previously been demonstrated that inhibitors of COX-2 have an anti-tumor effect in this model [37]. Another tumor promoting agent, UV light, also induces cyclooxygenase activity in mouse skin and two recent studies document the ability of a specific COX-2 inhibitor to suppress UV-induced skin cancer formation in hairless mice [38,39]. In human skin, UV light induces expression of COX-2 in the keratinocytes [20,40]. UV-light also increases expression of the arachidonoyl-specific high molecular weight, calcium-dependent phospholipase A2 (cPLA2) which appears to mediate the increased PGE2 synthesis in keratinocytes after UV exposure [41]. Consequently, at least two points of pharmacological attack could be developed if further studies substantiate the participation of increased PGE2 synthesis in non-melanoma skin carcinogenesis. Whereas a role for altered COX-2 expression and activity in human squamous cell carcinogenesis is supported by the data heretofore reviewed, other control points in eicosanoid generation and function need further elucidation before the relative contribution of COX-2 to skin carcinogenesis can be more accurately defined. For example, recent studies have documented heterogeneity among prostaglandin E (EP) receptors [42,43], and one study on human keratinocytes suggests that the effects of PGE2 on keratinocyte growth appear to be mediated in part through the EP2 and EP4 receptors [44]. The possibility therefore exists that alterations in EP receptors may be a feature in some forms of skin carcinogenesis. In fact, a study of human breast cancer cell lines suggests both loss and/or dysfunction of EP receptors as factors in tumor propagation [45]. In a murine model of colon cancer, EP1 knockout mice display a significant decrease in the appearance of precancerous lesions and an EP1 antagonist produces a decrease in precancerous lesions among wild type mice [46].
5. Summary The reviewed data document expression of both COX-1 and COX-2 in human and mammalian skin as well as up-regulation of COX-2 in specific types of epidermally- derived
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
21
precancerous and cancerous lesions. In a physiologic context, some evidence suggests that COX-2 expression may be involved in homeostatic regulation of normal keratinocyte differentiation. In a pathophysiologic context, increased COX-2 expression in keratinocytes occurs following exposure to UV light and chronic exposure to UV light is associated with the generation of both actinic keratoses, and squamous cell carcinomas (reviewed in [47]). It is worth noting that both actinic keratoses and squamous cell carcinomas also show mutations in the p53 tumor suppressor gene [48]. Consequently, at least several gene alterations, one of which involves the COX-2 gene, appear to be required for the phenotypic epidermal squamous carcinoma cell. The fact that UV –induced basal cell carcinomas do not appear to express COX-2 to the same extent as the squamous cell carcinomas suggests that COX-2 expression or up-regulation is not necessarily a required property of the carcinoma phenotype. At the same time, the more aggressive behavior of squamous cell carcinomas compared to basal cell carcinomas may have something to do with COX-2 expression since a relationship between COX-2 expression and metastatic potential has been demonstrated in human colon cancer cells [49]. In a therapeutic context, the data suggest that COX-2 inhibitors may have a role in suppressing the appearance of epidermal neoplasms (e.g. actinic keratoses, squamous cell carcinomas, and keratoacanthomas) that demonstrate increased COX-2 expression. This has already been shown in a murine model. However, the observed suppression of carcinogenesis has never been complete, and this finding suggests, as already mentioned, that other factors may be more seminal to the ultimate appearance of the different types of skin cancers. Nevertheless, other control points in the generation and action of PGE2 or other cyclooxygenase-derived eicosanoids may merit attempts to develop other types of chemotherapy or chemoprevention; these would include inhibitors of cytosolic phospholipase A2 as well as EP receptor antagonists. Future studies will no doubt document the validity, or lack thereof, of these investigative approaches. References [1] Ziboh VA, Shia SL. Prostaglandin E2: biosynthesis and effects on glucose and lipid metabolism in rat skin. Arch Biochem 1971;146:100 –9. [2] Jonsson C-E, Anggard E. Biosynthesis and metabolism of prostaglandin E2 in human skin. Scand J Clin Lab Invest 1972;29:289 –96. [3] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and –2. Adv Immunol 1996;62:167–215. [4] Herschman HR, Reddy ST, Xie W. Function and regulation of prostaglandin synthase-2. Adv Exp Med Biol 1997;62:61– 6. [5] Crofford L. COX-1 and COX-2 tissue expression: implications and predictions. J Rheumatol 1997;24(suppl 49):15–9. [6] Kurumbail RG, Stevens AM, Gierse JK, et al. Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents. Nature 1996;384:644 – 8. [7] Gierse JK, McDonald JJ, Hauser SD, et al. A single amino acid difference between cyclooxygenase-1 (COX-1) and –2 (COX-2) reverses the selectivity of COX-2 specific inhibitors. J Biochem 1996;271: 15810 – 4. [8] Needleman P, Isakson PC. The discovery and function of COX-2. J Rheumatol 1997;24(suppl 49):6 – 8. [9] Lefkowith JB. Cyclooxygenase-2 specificity and its clinical implications. Am J Med 1999;106(5B):43S– 50S.
22
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
[10] Singh G, Rosen-Ramey D. NSAID induced gastrointestinal complications: the ARAMIS perspective 1997. J Rheumatol Suppl 1998;51:8 –16. [11] Lipsky PE. The clinical potential of cyclooxygenase-2-specific inhibitors. Am J Med 1999;106(5B):51S– 57S. [12] Morham SG, Langenbach R, Loftin CD, et al. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473– 82. [13] Prendiville JS, Manfredi LN. Skin signs of nutritional disorders. Semin Dermatol 1992;11(1):88 –97. [14] Ziboh VA, Hsia SL. Effects of prostaglandin E2 on rat skin: inhibition of sterol ester biosynthesis and clearing of scaly lesions in essential fatty acid deficiency. J Lipid Res 1972;13:458 – 67. [15] Elias PM, Brown B, Ziboh VA. The permeability barrier in essential fatty acid deficiency: evidence for a direct role for linoleic acid in barrier function. J Invest Dermatol 1980;74:230 –3. [16] Furstenberger G, Marks F. Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin E2 in mouse epidermis in vivo. Biochem Biophys Res Commun 1978;84(4): 1103–11. [17] Pentland AP, Needleman P. Modulation of keratinocyte proliferation in vitro by endogenous prostaglandin synthesis. J Clin Invest 1986;77:246 –51. [18] Scholz K, Furstenberger G, Muller-Decker K, et al. Differential expression of prostaglandin-H synthase isoenzymes in normal and activated keratinocytes in vivo and in vitro. Biochem J 1995;309:263– 69. [19] Leong J, Hughes-Fulford M, Rakhlin N, et al. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation. Exp Cell Res 1996;224:79 – 87. [20] Buckman SY, Gresham A, et al. COX-2 expression is induced by UVB exposure in human skin: Implications for the development of skin cancer. Carcinogenesis 1998;19:723–9. [21] Muller-Decker K, Reinerth G, Krieg P, et al. Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin. Int J Cancer 1999;82:648 –56. [22] Yuspa SH, Kilkenny AE, Steinert PM, et al. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 1989;109:1207–17. [23] Pillai S, Bickle D, Mancianti ML, et al. Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium. J Cell Physiol 1990;143:294 –302. [24] Menon GK, Elias PM. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol 1991;127:57– 63. [25] Evans CB, Pillai S, Goldyne ME. Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes. Prostaglandins Leukot Essen Fatty Acids 1993;49:777– 81. [26] Sun TT, Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope. Cell 1976;9:511–21. [27] Kanekura T, Laulederkind SJ, Kirtikara K, et al. Cholecalciferol induces prostaglandin E2 biosynthesis and transglutaminase activity in human keratinocytes. J Invest Dermatol 1998;111(4):634 –9. [28] Hume R, Kelly R, Cossar D, et al. Exp Cell Res 1991;194:111–7. [29] Hoff T, DeWitt D, Volkhard K, et al. FEBS Lett 1993;320:38 – 42. [30] Zhang MZ, Wang JL, Cheng HF, et al. Cyclooxygenase-2 in rat nephron development. Am J Physiol 1997;273(6 Pt 2):F994 –F1002. [31] Smith CJ, Morrow JD, Roberts LJ, et al. Biochem Biophys Res Commun 1993;182:787–93. [32] O’Neill GP, Ford-Hutchinson AW. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett 1993;330:156 – 60. [33] Eberhart CE, Coffey RJ, Radhika A, et al. Up-regulation of cyclooxygenase-2 expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994;107:1183–7. [34] DuBois RN, Radhika A, Reddy BS, et al. Increased cyclooxygenase–2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 1996;110:1259 – 62. [35] Jacoby RF, Marshall DJ, Newton MA, et al. Chemoprevention of spontaneous intestinal adenomas in the Apc Min mouse model by the nonsteroidal antiinflammatory drug piroxicam. Cancer Res 1996;56:710 – 4.
M.E. Goldyne / Prostaglandins & other Lipid Mediators 63 (2000) 15–23
23
[36] Kawamori T, Rao CV, Seibert K, et al. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, against colon carcinogenesis. Cancer Res 1998;58:409 –12. [37] Muller-Decker K, Kopp-Schneider A, Marks F, et al. Localization of prostaglandin H synthase isoenzymes in murine epidermal tumors: suppression of skin tumor promotion by PGHS-2 inhibition. Mol Carcinog 1998;22:36 – 44. [38] Fischer SM, Lo HH, Gordon G, et al. Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis. Mol Carcinog 1999;25(4): 231– 40. [39] Pentland AP, Schoggins JW, Scott GA, et al. Reduction of UV-induced skin tumors in hairless mice by selective COX-2 inhibition. Carcinogenesis 1999;20(10):1939 – 44. [40] Soriani M, Luscher P, Tyrrell RM. Direct and indirect modulation of ornithine decarboxylase and cyclooxygenase by UVB radiation in human skin cells. Carcinogenesis 1999;20(4):727–32. [41] Chen X, Gresham A, Morrison A, Pentland A. Oxidative stress mediates synthesis of cytosolic phospholipase A2 after UVB injury. Biochim Biophys Acta Lipids Lipid Metab 1996;1299:22–33. [42] Ichikawa A, Sugimoto Y, Negishi M. Molecular aspects of the structures and functions of the prostaglandin E receptors. J Lipid Mediat Cell Signal 1996;14(1–3):83–7. [43] Jin J, Mao GF, Ashby B. Constitutive activity of human prostaglandin E receptor EP3 isoforms. Br J Pharmacol 1997;121(2):317–23. [44] Konger RL, Malaviya R, Pentland AP. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes. Biochim Biophys Acta 1998;1401(2):221–34. [45] Planchon P, Veber N, Magnien V, Israel L, Starzec AB. Alteration of prostaglandin E receptors in advanced breast tumor cell lines. Mol Cell Endocrinol 1995;111(2):219 –23. [46] Watanabe K, Kawamori T, Nakatsugi S, Ohta T, Ohuchida S, Yamamoto H, Maruyama T, Kondo K, Ushikubi F, Narumiya S, Sugimura T, Wakabayashi K. Role of the prostaglandin E receptor subtype EP1 in colon carcinogenesis. Cancer Res 1999;59(20):5093– 6. [47] Salasche SJ. Epidemiology of actinic keratoses and squamous cell carcinoma. J Am Acad Dermatol (Suppl) 2000;42(1, part 2):S4 –S7. [48] Ziegler A, Jonason AS, Leffell DJ, et al. Sunburn and p53 in the onset of skin cancer. Nature 1994;372: 773– 6. [49] Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential. Proc Natl Acad Sci USA 1997;94:3336 – 40.