The Expression And Significance Of Prostaglandin Receptors In Human Melanocytes

BackgroundProstaglandins（PG）are a group of physiologically active lipid compounds that are derivatives of arachidonic acid.The same prostaglandin may play different or even opposite effects in different tissues,which are generally autocrine or paracrine factors.Prostaglandins activate a variety of signaling pathways that regulate cell growth,differentiation and apoptosis.In the skin,prostaglandins are rapidly released by keratinocytes after UV radiation and are chronically present in a variety of inflammatory skin lesions,acting on melanocytes and causing post-inflammatory hyperpigmentation,but currently there is no in-depth study on whether prostaglandins are produced in and how prostaglandins act on melanocytes.Melanocytes are dendritic cells（DC）located in the stratum basale of the epidermis.The melanosomes contain tyrosinase,which is the key enzyme in the melanogenesis.Melanosomes can be released into the extracellular space from various areas of melanocyte dendrites through shedding vesicles.Then they can be captured by the microvilli of keratinocytes through the filopodia conduits and be phagocytosed.Melanosomes can be distributed around the perinuclear area in keratinocytes,forming perinuclear melanin caps to help their nuclei resist ultraviolet damage to nuclear DNA.Prostaglandin receptors refer to one or more of the major receptors of classicalnaturally occurring prostaglandins,namely prostaglandin D₂（ie PGD₂）,PGE₂,PGF_2α,prostacyclin（PGI₂）,thromboxane A₂（TXA₂）and PGH₂.They are named by the prostaglandin species that are preferentially bind and respond to.All prostaglandin receptors are G-protein coupled receptors belonging to the rhodopsin-like receptor family subfamily A14.It has been proved that prostaglandin PGE₂ acts on melanocytes to enlarge the cell body,increase dendrites,and promote melanosomes to transfer to keratinocytes,while PGF_2αupregulates melanocyte tyrosine activity and promote expression of the enzyme.It is therefore believed that melanocytes may express prostaglandin PGE2 and PGF2_αreceptors,including the PGE2 receptors EP1,EP2,EP3,EP4 and the PGF_2αreceptor FP.There are few studies on the expression of prostaglandin receptors in human epidermal melanocytes at home and abroad.In this study,the expressions of human epidermal melanocyte prostaglandin receptors were studied by immunofluorescence technique.The results are as following.ObjectiveIn this experiment,we investigated the expression of prostaglandin PGE₂ and PGF_2αreceptors in human immortalized melanocyte strain PIG1,human primary melanocytes and normal human foreskin frozen sections by immunofluorescence labeling.The study provides experimental evidence of the mechanism that prostaglandins are directly involved in the regulation of the pathophysiological function of melanocytes.Methods1.The human immortalized melanocyte strain PIG1 was given by the Dermatology Department of Xijing Hospital.2.The PIG1 cell line was inoculated in 254 medium containing melanocyte growth factor（HMGS）with 100 U/mL penicillin,100μg/mL streptomycin,and was cultured in a 37°C,5%CO₂ incubator.After 24 h,the culture medium was replaced to discard unattached cells,and then the solution was changed once every 2 to 3 days.Then,the cells were collected,seeded at a density of 1 x 10⁶/ml in a 6-well plate,and cultured until the cell adherence was about 80%.3.The children’s foreskin tissue is digested and separated in a sterile environment to obtain primary human melanocytes,which were cultured in selective medium for 3to 5 generations and used for later use.4.Human healthy foreskin tissue was from the Department of Pediatric Surgery of the First Affiliated Hospital of Zhengzhou University.There was no organic disease in the skin tissue,no pigmentary disease,no history of medical application in a month,and no family history of pigmentary diseases.Frozen slices were prepared for later use.5.The expressions of PGE2 and PGF2_αreceptor proteins in PIG1 cell line were detected by immunofluorescence single-labeling technique.6.Immunofluorescence multi-labeling technique was used to detect the expressions of PGE2 and PGF2_αreceptors in human primary melanocytes.7.Immunofluorescence multi-labeling technique was used to detect the expressions of PGE2 and PGF2_αreceptors in epidermal melanocytes of human foreskin frozen sections.8.Observe and collect images by fluorescence microscope.Results1.The immunofluorescence single-labeling detection of prostaglandin PGE₂receptor EP1,EP2,EP3,EP4 and PGF_2αreceptor FP protein in human immortalized melanocyte strain PIG1 was positive,the positive fluorescence was localized in PIG1cell membrane and cytoplasm,the EP1 and EP4 were stained with Alexa Fluor?488labeled secondary antibody and showed bright green fluorescence,the EP2,EP3,and FP were stained with Alexa Fluor?594-labeled secondary antibody and showed red fluorescence.2.Prostaglandin PGE₂ receptors EP1,EP2,EP3,EP4 and PGF_2αreceptor FP protein were positively expressed in human primary melanocytes and were detected by immunofluorescence multi-labeling,and the marker protein TYRP-1 was stained with Alexa Fluor?488-labeled secondary antibody,which provided bright green fluorescence.EP1,EP2,EP3,EP4,FP were stained with Alexa Fluor?594-labeled secondary antibody,which provided red fluorescence;merged positive fluorescence was pale yellow,localized to the membrane and cytoplasm of primary melanocytes,the nuclei were dyed by DAPI,showing blue-violet fluorescence.3.Immunofluorescence multi-labeling assay was positive of prostaglandin PGE2 receptors EP1,EP2,EP3,EP4 and PGF2_αreceptor FP protein in normal human foreskin frozen sections.Marker protein TYRP-1 was stained with Alexa Fluor?488-labeled secondary antibody showing bright green fluorescence;EP1,EP2,EP3,EP4,and FP were stained with Alexa Fluor?594-labeled secondary antibody,showing red fluorescence,and the positive fluorescence of the fusion was pale yellow.The results showed that the expressions of EP1 and EP4 receptor proteins were obvious,and the expressions of EP2,EP3 and FP receptor proteins were weak.Conclusions1.Prostaglandin PGE₂ receptor EP1,EP2,EP3,EP4 and PGF_2αreceptor FP proteins were positively expressed in PIG1 melanocytes,in human primary melanocytes and in frozen sections of healthy human foreskin tissues;2.It is suggested that prostaglandins can directly act on melanocytes through receptors,regulate their pathophysiological mechanism,and have positive significance for the prevention,treatment and new drug development of pigmented skin diseases.