title: Immortalization of human melanocytes does not alter the de novo properties of nitric oxide to induce cell detachment from extracellular matrix components via cGMP.
abstract: Nitric oxide (NO) is an important mediator in many (patho)physiological processes including inflammation and skin cancer. A key transducer in NO signaling is the soluble guanylyl cyclase (sGC) that catalyzes the formation of guanosine 3',5'-cyclic monophosphate (cGMP). The basic mechanism of NO-cGMP signaling in melanocytic cells is, however, not well elucidated. A setback for such studies is the limited availability of patient-derived melanocytes. Here, we report that immortalized human normal and vitiliginous cell lines generated via cell transfection with human papilloma virus 16 genes E6 and E7 express NO synthase and guanylyl cyclase isoforms and the multidrug resistance-associated proteins 4 and 5 as selective cGMP exporters. Donors of NO (e.g., the NONOate (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA-NO) and reactive nitrogen oxygen species (RNOS) like 3-morpholino-sydnonimine (SIN-1) as a donor of peroxynitrite as well as YC-1 as a NO-independent sGC stimulator increased intracellular cGMP levels in immortalized melanocytes (up to eightfold over controls), indicating the expression of functional sGC in these cells. PAPA-NO and SIN-1 also reduced the attachment of immortalized melanocytes to extracellular matrix (ECM) components like fibronectin which was dependent on cellular melanin content and cGMP. Such effects on melanoma cells were positively related to metastatic potential and were cGMP independent. Intriguingly, nonpigmented metastatic melanoma cells were more sensitive to exogenous sources of RNOS than of NO. Thus, immortalized melanocytes can be used as a tool for further research on differences in cell signaling between the different melanocytic lineages in particular towards impairment of cell-ECM adhesion by NO or RNOS, which may be important in metastasis and vitiligo pathogenesis.
authors.affiliation: Institute of Aerospace Medicine, German Aerospace Center (DLR), Linder Hohe, 51170 Cologne, Germany. krassimira.ivanova@dlr.de
authors.forename: Krassimira
authors.initials: K
authors.lastname: Ivanova
authors.forename: Britta
authors.initials: B
authors.lastname: Lambers
authors.forename: Rene
authors.initials: R
authors.lastname: van den Wijngaard
authors.forename: I Caroline
authors.initials: IC
authors.lastname: Le Poole
authors.forename: Olga
authors.initials: O
authors.lastname: Grigorieva
authors.forename: Rupert
authors.initials: R
authors.lastname: Gerzer
authors.forename: Pranab K
authors.initials: PK
authors.lastname: Das
mesh_terms: D002448:Cell Adhesion; D006152:Cyclic GMP / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000378:metabolism; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D014162:Transfection
chemical_list: D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C059731:oncogene protein E7, Human papillomavirus type 16; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP