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

AbstractWe 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 mechanism, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways

Antigen-Presenting Cells in Essential Fatty Acid—Deficient Murine Epidermis: Keratinocytes Bearing Class II (Ia) Antigens May Potentiate the Accessory Cell Function of Langerhans Cells

Essential fatty acid deficiency (EFAD) is a useful model for studying the role of (n-6) fatty acid metabolism in normal physiology. Because cutaneous manifestations are among the earliest signs of EFAD and because abnormalities in the distribution and function of tissue macrophages have been documented in EFAD rodents, we studied the distribution and function of Class II MHC (Ia) antigen-bearing cells in EFAD CS7B1/6 mouse epidermis. Immunofluorescence studies revealed 1.9–9.6 (mean ± SEM = 5.2 ± 2.6) times more class II MHC (Ia) antigen-bearing epidermal cells in suspensions prepared from EFAD as compared to normal skin. Analysis of epidermal sheets demonstrated similar numbers of dendritic Ia+ and NLDC145+ cells in EFAD and normal epidermis, however. This discrepancy occurred because some keratinocytes in EFAD epidermal sheets expressed class II MHC (Ia) antigens, whereas keratinocytes in normal mouse epidermis did not. Two-color flow cytometry confirmed that all Ia+ cells in normal epidermis are Langerhans (Ia+ NLDC145+) cells, whereas Ia+ cells in EFAD epidermis are comprised of Langerhans cells and a subpopulation of keratinocytes (Ia+ NLDC145-. Similar levels of Ia antigens were expressed on EFAD and normal Langerhans cells. EFAD and normal epidermal cells were also compared in several in vitro assays of accessory cell function. Epidermal cells prepared from EFAD C57B1/6 mice present the protein antigen DNP-Ova to primed helper T cells more effectively than epidermal cells prepared from normal animals. EFAD epidermal cells are also more potent stimulators of T cells in primary and secondary allogeneic mixed lymphocyte-epidermal cell reactions than normal epidermal cells. The functional differences between EFAD and normal epidermal cells do not appear to result from increased cytokine release or decreased prostaglandin production by EFAD epidermal cells. In view of these findings and the observation that the antigen-presenting cell activity of EFAD epidermal cells correlates with the number of Ia+ keratinocytes in epidermal cell preparations, Ia+ keratinocytes (in the presenceof Langerhans cells) may potentiate cutaneous immune responses in vitro and perhaps in vivo as well. these results also suggest that (n-6) fatty acids or metabolites of (n-6) fatty acids are involved in regulating the expression of class II MHC (Ia) antigens by keratinocytes in vivo

The Role of AldoKeto Reductase 1C3 in Human Epidermis, Atopic Dermatitis and Cutaneous Squamous Cell Carcinoma

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2011.Aldoketo reductase 1C3 (AKR1C3) has been shown to mediate the metabolism of sex hormones and prostaglandins (PGs), both of which play a role in epidermal function and pathology. AKR1C3 expression has been initially characterized in normal human epidermis by immunofluorescence, revealing a strong expression in the differentiated epidermal layers compared with the proliferative compartment. In addition, AKR1C3 expression was up regulated during calciuminduced differentiation of primary human keratinocytes (PHKs). This suggested a possible function of AKR1C3 in keratinocyte differentiation, a tightly choreographed process that is crucial for proper function of the epidermis. The current work demonstrated that impairment of AKR1C3 during PHK differentiation altered the expression of the differentiation markers keratin 10 and loricrin, suggesting a role in keratinocyte differentiation. Prostaglandin D2 (PGD2), a preferred substrate of AKR1C3, is a lipid mediator that has been shown to be upregulated and promote inflammation in atopic dermatitis (AD). The current data showed that AKR1C3 expression is markedly upregulated by PHKs in response to PGD2, as well as in lesions of AD but of psoriasis, another inflammatory skin condition. These findings suggest a novel role for epidermal keratinocytes in regulating PGD2mediated inflammatory actions in AD. Data described in the current thesis show that AKR1C3 was overexpressed in squamous cell carcinoma (SCC), but its expression was completely undetectable in the tumor mass of basal cell carcinoma. Following this observation, several SCC cell lines have been derived from surgically excised human cutaneous SCC tumors. These cells demonstrated a defective terminal differentiation capacity compared with PHK and were able to generate SCClike tumors in immunocompromised mice, in vivo. In order to investigate the role of AKR1C3 overexpression in SCC growth and survival, one SCC cell line has been genetically manipulated to overexpress AKR1C3 and the proliferation of these cells has been assessed under various conditions. Taken together, this work characterizes the expression of AKR1C3 in normal epidermis, demonstrates a function for this enzyme in differentiation associated gene regulation, and suggests a role in supporting inflammation in AD. In addition, this work characterizes the expression patterns of AKR1C3 in SCC and suggests at least one possible role for this enzyme in SCC proliferation. Finally, human cutaneous SCC cell lines derived from tumors from sunexposed areas are currently unavailable; therefore, the establishment of these cell lines adds a significant contribution to this field of research

Mechanisms of UV-Induced Inflammation

The inflammation produced by exposure to ultraviolet (UV) light has been well documented clinically and histologically. However, the mechanisms by which mediators induce this clinical response remain poorly defined. It is clear that photochemistry occurring after UV absorption must be responsible for initiating these events. Some of these underlying mechanisms have been defined. After exposure to UV light, the formation of prostaglandins and the release of histamine are increased. In addition to an increase in the quantity of these mediators, an increase in sensitivity of irradiated tissue to agonist stimulation also occurs. This increased sensitivity may cause tissue to respond to agonist levels previously present. Phospholipase activity also increases, making more substrate available for prostaglandin formation. Oxygen radical - induced peroxidation of membrane lipids caused by irradiation may contribute to increased phospholipase activity. Oxygen-free radicals also participate in sunburn cell formation and in UV-induced decreases in Langerhans cell numbers. Several enzymatic and non-enzymatic mechanisms are present in skin for reducing these highly reactive oxygen species

Impact of Cosmetic Lotions on Nanoparticle Penetration through ex Vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study

Understanding the interactions of nanoparticles (NPs) with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD) NPs between freshly excised human and mouse skin. QDs were mixed in seven different vehicles, including five commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA) facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 h rest ex vivo. Resting human skin 24 h prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models

Effect of Gas Tension on Epidermal Keratinocyte DNA Synthesis and Prostaglandin Production

The gas phase partial pressure O2(PO2) overlying mouse keratinocyte cultures controls the rate of DNA synthesis of these cells by an undefined mechanism. In these studies, both PO2 and PCO2 tensions overlying primary cell cultures were varied within the physiologic range. The prostaglandin (PG) production of cells grown under several varying gas tensions was then determined using radioimmune assay. The cultures were grown under a PO2 of either 7.5% (physiologic for in vivo epidermis) or 21% (atmospheric; culture conditions and wound healing) for 5 days. The PCO2 was either 5 or 10%, 2 CO2 tensions routinely used in tissue culture studies. DNA synthesis was quantitated using [3H]dThd uptake into DNA and autoradiography. The PGE2 and PGF2α syntheses by the cultures over specific time periods were determined. Changing the PO2 from 21 to 7.5% decreased the rate of DNA synthesis, while PG production remained constant. When the PCO2 was varied from 5 to 10%, keratinocyte DNA synthesis remained unchanged but PG production was markedly stimulated. The PCO2 effect on PG production was greatest at the highest oxygen tension. The data indicated that under this set of variables, PG production by keratinocytes is not directly related to the rate of DNA synthesis of the cells. Apparently the amount of oxygen in the gas phase can have a permissive effect on epidermal keratinocyte PG production