Raymond L. Konger

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

Microsomal prostaglandin E2 synthase-1 in breast cancer: a potential target for therapy

The anti‐tumour actions of cyclooxygenases (COX) are thought to be mediated by inhibition of prostaglandin E2 (PGE2) synthesis. However, COX‐2 inhibition also alters cellular production of other prostaglandins such as prostacyclin (PGI2). The latter action is believed to be important for the development of adverse cardio‐vascular events. Microsomal PGES (mPGES‐1) is an enzyme downstream to COX‐2 and affects PGE2 production only. It is possible that targeting mPGES‐1 could decrease PGE2 production without affecting PGI2 production. In order to assess the potential of mPGES‐1 as a target for therapy, we analysed its expression in breast cell lines and normal and malignant breast tissues. The expression of mPGES‐1 and COX‐2 was correlated in tumour cells and vascular endothelium, and with prognostic parameters in breast cancer. Although not detectable in normal epithelial cells, expression was noted in areas of fibrocystic change and in situ carcinoma. mPGES‐1 expression was noted in 79% of breast cancer tissues. Its expression did not correlate with COX‐2 overexpression or with prognostic markers of breast cancer. Endothelial cells did not show mPGES‐1 expression. Upregulation of mPGES‐1 is therefore frequent in pre‐malignant and malignant breast disease. In this study, coordinate over‐expression of COX‐2 and mPGES‐1 was not observed, particularly in the endothelial cells of blood vessels. Targeting mPGES‐1 might prove to be an alternative therapeutic strategy to inhibit PGE2 production. Copyright

UVB Radiation-Mediated Inhibition of Contact Hypersensitivity Reactions Is Dependent on the Platelet-Activating Factor System

Through its ability to both induce immunosuppression and act as a carcinogen, UVB radiation plays a major role in cutaneous malignancies. Recent studies have indicated that UVB-mediated inhibition of delayed-type hypersensitivity reactions is mediated, in part, by the lipid mediator platelet-activating factor (PAF). The objective of this study was to further define the mechanism by which UVB inhibits contact hypersensitivity (CHS) reactions. UVB irradiation resulted in an inhibition of subsequent CHS to the chemical DNFB in wild-type, but not in PAF-R-deficient mice. UVB-mediated inhibition of CHS was also blocked by a cyclooxygenase-2 (COX-2) inhibitor or a neutralizing antibody directed against IL-10. UVB irradiation upregulated IL-10 mRNA levels in lymph nodes and spleen only to significant levels in PAF-R-expressing mice. Bone marrow transplantation studies demonstrated that UVB-mediated immunomodulatory effects were dependent on PAF-R-positive bone marrow. These studies suggest that UVB irradiation results in epidermal production of PAF agonists, which then act on PAF-R-positive bone marrow-derived cells to upregulate IL-10 through COX-2-generated prostaglandins.

Ultraviolet B Radiation Generated Platelet-Activating Factor Receptor Agonist Formation Involves EGF-R-Mediated Reactive Oxygen Species

Recent studies have implicated the lipid mediator platelet-activating factor (PAF) in UVB-mediated systemic immunosuppression known to be a major cause for skin cancers. Previously, our group has demonstrated that UVB irradiation triggers the production of PAF and oxidized glycerophosphocholines that act as PAF-receptor (PAF-R) agonists. The present studies explored the mechanisms by which UVB generates PAF-R agonists. UVB irradiation of human epidermal KB cells resulted in both increased levels of reactive oxygen species (ROS) and PAF-R agonistic activity. Pretreatment of KB cells with antioxidants vitamin C and N-acetylcysteine or the pharmacological inhibitor PD168393 specific for the epidermal growth factor receptor all inhibited UVB-induced ROS as well as PAF-R agonists, yet had no effect on fMLP-mediated PAF-R agonist production. In addition, in vivo production of PAF-R agonists from UVB-irradiated mouse skin was blocked by both systemic vitamin C administration and topical PD168393 application. Moreover, both vitamin C and PD168393 abolished UVB-mediated but not the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine-mediated immunosuppression as measured by the inhibition of delayed type contact hypersensitivity to the chemical dinitrofluorobenzene. These studies suggest that UVB-induced systemic immunosuppression is due to epidermal growth factor receptor-mediated ROS which results in PAF-R agonist formation.

Loss of the EP2 Prostaglandin E2 Receptor in Immortalized Human Keratinocytes Results in Increased Invasiveness and Decreased Paxillin Expression

Prostaglandin E(2) (PGE(2)) receptor subtype EP(2), which is coupled to cAMP metabolism, is known to mediate proliferation of primary human keratinocytes in vitro. The effect of gain or loss of EP(2) receptors in immortalized human keratinocytes (HaCat cells) was examined. HaCat keratinocytes were transfected with sense or anti-sense constructs of the EP(2) receptor. Loss or gain of EP(2) expression was documented by immunoblot and associated changes in agonist-stimulated cAMP production. Loss or gain of EP(2) receptor expression correlated with alterations in plating efficiencies but with modest affects on growth. When cell lines were studied in an organ culture model, anti-sense clones were highly invasive compared with vector controls and sense transfectants. A marked increase in prostaglandin production is commonly seen in malignant lesions. Because prostaglandin receptors are known to undergo ligand-induced receptor down-regulation, we sought to determine whether EP(2) receptor down-regulation results in increased invasiveness. In vector controls, invasiveness was reproduced by ligand-dependent EP(2) receptor down-regulation as assessed by immunohistochemistry. In addition, loss of EP(2) receptor expression was associated with decreased paxillin expression, a critical component of focal adhesion assembly. Thus, down-regulation of EP(2) receptors represents a potential mechanism for neoplastic progression to an invasive phenotype.

The environmental stressor ultraviolet B radiation inhibits murine antitumor immunity through its ability to generate platelet-activating factor agonists

Ubiquitous pro-oxidative stressor ultraviolet B radiation (UVB) to human or mouse skin generates platelet-activating factor (PAF) and novel oxidatively modified glycerophosphocholines (Ox-GPCs) with PAF-receptor (PAF-R) agonistic activity. These lipids mediate systemic immunosuppression in a process involving IL-10. The current studies sought to determine the functional significance of UVB-mediated systemic immunosuppression in an established model of murine melanoma. We show that UVB irradiation augments B16F10 tumor growth and is dependent on host, but not melanoma cell; PAF-R-expression as UVB or the PAF-R agonist, carbamoyl PAF (CPAF), both promote B16F10 tumor growth in wild-type (WT) mice, independent of whether B16F10 cells express PAF-Rs, but do not augment tumor growth in Pafr -/- mice. UVB-mediated augmentation of experimental murine tumor growth was inhibited with antioxidants, demonstrating the importance of Ox-GPC PAF-R agonists produced non-enzymatically. Host immune cells are required as CPAF-induced augmentation of tumor growth which is not seen in immunodeficient NOD SCID mice. Finally, depleting antibodies against IL-10 in WT mice or depletion of CD25-positive cells in FoxP3(EGFP) transgenic mice block UVB and/or CPAF-induced tumor growth supporting a requirement for IL-10 and Tregs in this process. These findings indicate that UVB-generated Ox-GPCs with PAF-R agonistic activity enhance experimental murine melanoma tumor growth through targeting host immune cells, most notably Tregs, to mediate systemic immunosuppression.

Ultraviolet B Radiation of Human Skin Generates Platelet-activating Factor Receptor Agonists

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor‐alpha (TNF‐α), UVB generates bioactive lipids including platelet‐activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB‐mediated production of PAF agonists is due primarily to the pro‐oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF‐receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB‐generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF‐α) production, these studies indicate that this novel activity could be involved in human disease.

The EP3 receptor stimulates ceramide and diacylglycerol release and inhibits growth of primary keratinocytes

Abstract:  Primary human keratinocytes (PHKs) are known to express the EP3 subtype of prostaglandin E2 receptor. To better understand the role of EP3 receptors in regulating epidermal function, we characterized their expression, localization, and signaling effects in human skin. Three different splice variants of the EP3 receptor (EP3A1, EP3C, and EP3D) were found to be expressed. Immunohistochemical analysis of human skin demonstrated that EP3 receptors were most prominently expressed in the basal and lower spinous layers of the epidermis. The EP3 receptor agonist sulprostone was then used to examine EP3 receptor‐dependent keratinocyte signaling pathways and functional effects. We observed that sulprostone inhibits keratinocyte growth at doses between 0.02 and 2 nM and induces sn‐1,2‐diacylglycerol (DAG) and ceramide production. Concurrent expression of the cell‐cycle inhibitory protein p21WAF1 also occurred. These data suggest that EP3 receptors produce epidermal growth inhibition through the action of DAG and ceramide second messengers.

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

Ultraviolet light radiation (UVR) has profound effects upon human skin. Yet, the exact targets for UVR are unclear. Inasmuch as UVR is a known pro-oxidative stressor, one potential target for UVR could be oxidatively modified glycerophosphocholines (GPC). Importantly, recent studies demonstrate that these oxidized GPCs (ox-GPC) are potent agonists for the platelet-activating factor receptor and peroxisome proliferator-activated receptor gamma. This review discusses these new biologically active lipids and their down-stream receptor targets that provide a unique system of biosensors for detecting and responding to UVR photo-oxidation.

Prostanoid receptor EP1 expression in breast cancer

Cyclooxygenase enzymes play an important role in carcinogenesis, and increased expression of cyclooxygenase enzymes has been reported in cancers arising at a number of different sites. Most, if not all of these actions are thought to be mediated by prostaglandin E2 (PGE2). The actions of PGE2 are mediated via four main prostanoid receptors, designated EP1, EP2, EP3 and EP4, based on their different pharmacological properties and secondary messenger pathways. Recently, expression of EP1 has been reported in rat mammary gland and the inhibition of this receptor has been documented to have chemopreventive effect in this animal model. EP1 has also been shown to decrease the incidence of colon cancer in mouse models. In this study, we analysed the expression of EP1 in normal and malignant breast tissues. Expression of EP1 was analysed in breast (benign and cancer) cell lines by reverse-transcriptase polymerase chain reaction and by western blot analyses. Expression was also analysed by immunohistochemistry in normal breast tissues and in 89 cases of breast cancer. Semiquantitative analysis of the staining was performed. The data were compared with and correlated with other prognostic factors like tumour size, tumour grade, lymph node status, oestrogen receptor, progesterone receptor (PR), HER2/neu and cyclooxygenase-2. EP1 expression was demonstrated in human breast cancer by immunohistochemistry. Expression of EP1 was seen both in the cytoplasm and/or in the nuclear membrane in majority of cases. Nuclear EP1 expression correlated with PR (P=0.032) and inversely with node positivity (P=0.025). However, EP1 expression did not correlate with expression of cyclooxygenase-2 (P=0.059). Expression of EP1 is frequently seen in human breast cancers. Nuclear expression of EP1 correlates with good prognosis markers like node negative status and PR expression.