Pathway Analysis for Genome-Wide Association Study of Basal Cell Carcinoma of the Skin

Recently, a pathway-based approach has been developed to evaluate the cumulative contribution of the functionally related genes for genome-wide association studies (GWASs), which may help utilize GWAS data to a greater extent.In this study, we applied this approach for the GWAS of basal cell carcinoma (BCC) of the skin. We first conducted the BCC GWAS among 1,797 BCC cases and 5,197 controls in Caucasians with 740,760 genotyped SNPs. 115,688 SNPs were grouped into gene transcripts within 20 kb in distance and then into 174 Kyoto Encyclopedia of Genes and Genomes pathways, 205 BioCarta pathways, as well as two positive control gene sets (pigmentation gene set and BCC risk gene set). The association of each pathway with BCC risk was evaluated using the weighted Kolmogorov-Smirnov test. One thousand permutations were conducted to assess the significance.Both of the positive control gene sets reached pathway p-values<0.05. Four other pathways were also significantly associated with BCC risk: the heparan sulfate biosynthesis pathway (p  =  0.007, false discovery rate, FDR  =  0.35), the mCalpain pathway (p  =  0.002, FDR  =  0.12), the Rho cell motility signaling pathway (p  =  0.011, FDR  =  0.30), and the nitric oxide pathway (p  =  0.022, FDR  =  0.42).We identified four pathways associated with BCC risk, which may offer new insights into the etiology of BCC upon further validation, and this approach may help identify potential biological pathways that might be missed by the standard GWAS approach

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: Peroxynitrite formation as a causative factor of neurotoxicity

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can he activated by ultraviolet B radiation and Ca2+ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm2), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 μM) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L- arginine (100 μM) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold alter treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm2). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm2) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl- amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Desferrioxamine (100 μM) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca2+-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm2) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 μM) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity

Inhibition of ultraviolet B-induced skin erythema by N-nitro-L-arginine and N-monomethyl-L-arginine

Ultraviolet B (UVB)-irradiated human keratinocytes and human endothelial cells release nitrogen oxides, i.e. nitric oxide (NO), S-nitrosothiols, hydroxylamine (H2NOH) as well as ammonia (NH3) formed from L-arginine. Generation of these compounds was time and concentration-dependent and decreased by both N-monomethyl-L-arginine (L-NMMA) and N-nitro-L-arginine (L-NA). WE radiation of the cells resulted in a concomitant increase of soluble guanylate cyclase (sGC) activity which was inhibited by L-NMMA and L-NA. S-nitrosothiols formed during the irradiation of the cells directly increased purified sGC activity by a mechanism characteristic of release of NO from a carried molecule. WE-irradiated cells promptly increased thiobarbituric acid reacting substance (TEARS) (estimated as malondialdehyde, MDA) production which were inhibited by desferrioxamine. In in vivo experiments using guinea pigs subjected to WE radiation, a Protection Factor (PF) of 2.25 +/- 0.75 was calculated when an emulsified cream formulation containing L-NMMA (1% w/w) and L-NA (1% w/w) was applied to their skin. In human volunteers subjected to UVB radiation a dose-dependent increase of PF was observed. When an emulsified cream formulation containing L-NMMA (1% w/w) and L-NA (1% w/w) was applied to their skin the PF was 2.15 +/- 0.80; by increasing the concentration of L-NMMA (2% w/w) and L-NA (2% w/w) the PF was 4.25 +/- 1.25. The present results indicate that WE radiation acts as a potent stimulator of human keratinocytes and endothelial cells to release nitrogen oxides that may diffuse out of the keratinocytes and endothelial cells, activating sGC in neighboring smooth muscle cells. This may be a major part of the integrated response of the skin leading to vasodilation and erythema. (C) 1997 Elsevier Science Ireland Ltd

Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes

Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to WE radiation. UVB irradiation (up to 20 mJ/cm(2)) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[H-3]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm(2)) of particulate NO synthase. A 5-fold increase in superoxide (O-2(-)) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm(2))-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm(2)) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation

RELEASE BY ULTRAVIOLET-B (UVB) RADIATION OF NITRIC-OXIDE (NO) FROM HUMAN KERATINOCYTES - A POTENTIAL ROLE FOR NITRIC-OXIDE IN ERYTHEMA PRODUCTION

1 The mechanism of human sunburn is poorly understood but its characteristic features include the development of erythema. In this study we attempted to determine whether human keratinocytes possess a nitric oxide (NO) synthase (NOS), if this enzyme could be activated to release NO following exposure to ultraviolet B (u.v.B) and to define whether this photo-induced response could be involved in the pathogenesis of sunburn erythema. 2 Treatment of human keratinocytes with various doses of u.v.B (290-320 nm) radiation (up to 100 mJ cm(-2)) resulted in a dose-dependent release of NO and cyclic GMP production that was reduced by NG-monomethyl-L-arginine (L-NMMA). 3 u.v.B irradiation of keratinocyte cytosol at varying doses (up to 50 mJ cm(-2)), resulted in a gradual rise in NO production, with a concomitant increase in soluble guanylate cyclase activity (sGC). 4 NOS isolated from the keratinocyte cytosol was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins. 5 In reconstitution experiments, when purified NOS was added to purified sGC, both isolated from keratinocyte cytosol, a four fold increase in cyclic GMP was observed. The GMP was increased by NO synthesized following u.v.B radiation (up to 20 mJ cm(-2)) of NOS. 6 In in vivo experiments, guinea-pigs were subjected to u.v.B light. A Protection Factor (PF) of 8.71+/-2.85 was calculated when an emulsified cream formulation containing L-NMMA (2%) was applied to their skin. 7 The present results indicate that u.v.B radiation acts as a potent stimulator of NOS in keratinocytes. NO is lipophilic and may diffuse out of the keratinocytes, activating sGC in endothelial cells and neighbouring smooth muscle cells. This may be a major part of the integrated response of the skin leading to vasodilatation and erythema

MODULATION OF PARTICULATE NITRIC-OXIDE SYNTHASE ACTIVITY AND PEROXYNITRITE SYNTHESIS IN CHOLESTEROL-ENRICHED ENDOTHELIAL-CELL MEMBRANES

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N-Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in superoxide (O-2(-)) and a 2.5-fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO-) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [(r(o)/r)-1](-1). Arrhenius plots of [(r(o)/r)- 1](-1) indicated that the lipid phase separation of the membranes at 26.2 +/- 1.5 degrees was elevated to 34.4 +/- 1.9 degrees in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 +/- 1.6 degrees, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 +/- 1.8 degrees which rose to 35.6 +/- 2.1 degrees in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 +/- 1.4 degrees in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties of plasma membrane-bound NO synthase inhibited by Mn2+ (as reflected by changes in the Hill coefficient) were changed by cholesterol, consistent with modulations of the fluidity of the lipid microenvironment of the enzyme. Our findings suggest that incorporation of high concentrations of cholesterol into endothelial cell membranes causes down-regulation of NO synthase by producing an increased packing of bulk lipids. In contrast, cholesterol incorporation at low concentrations up-regulates NO synthase by increasing the fluidity of the lipid microenvironment of the enzyme. The present studies concerning the behaviour of particulate NO synthase and rate of NO release with respect to the structure and function of the biomembranes provide important new clues as to the role of this fascinating molecule in atherosclerosis

Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation - Potential role for peroxynitrite in skin inflammation

In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O-2(-)) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm(2)) resulted in a dose-dependent increase in NO and ONOO- release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm(2)) resulted in an increase in XO activity that was inhibited by oxypurinol. UVB radiation (up to 100 mJ/cm(2)) of keratinocytes resulted in a 15-fold increase in S-nitrosothiol formation, which directly increased purified soluble guanylate cyclase (sGC) activity by a mechanism characteristic of release of NO from a carrier molecule. In reconstitution experiments, when UVB-irradiated (20 mJ/cm(2)) purified cNOS isolated from keratinocyte cytosol was combined with UVB-irradiated (20 mJ/cm(2)) purified XO, a 4-fold increase in ONOO- production, as compared to nonirradiated enzymes, was observed. ONOO- synthesized by NO and O-2(-) following UVB radiation of cNOS and XO was inhibited by oxypurinol (100 mu M) UVB radiation of keratinocyte cytosol resulted in an increase in oxygen free radical production, consistent with the increased production of ONOO- by UVB-irradiated keratinocyte cytosol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 +/- 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighboring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process

Nitric oxide and peroxynitrite released by ultraviolet B-irradiated human endothelial cells are possibly involved in skin erythema and inflammation

In this study we attempted to demonstrate whether endothelial cell nitric oxide synthase (eNOS) and xanthine oxidase (XO) could be activated to release nitric oxide (NO) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation and to define whether this light-induced response could be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human endothelial cells with UVB (290-320 nm) radiation (up to 100 mJ/cm2) resulted in an increase of both NO and ONOO- release that was inhibited by N(G)-monomethyl-L-arginine (L-NMMA). Treatment of cell cytosol with various doses of UVB radiation (up to 20 mJ/cm2) resulted in a threefold increase of XO activity that was inhibited (approximately 90%) by oxypurinol. In reconstitution experiments, when purified eNOS was added to purified XO, an almost fourfold increase in ONOO- production at 20 mJ/cm2 UVB radiation was observed. UVB radiation (100 mJ/cm2) decreased cell membrane fluidity, indicating changes in the physicochemical characteristics of the membranes. In in vivo experiments, when human volunteers were subjected to UVB Light, a protection factor (PF) of 3.90 ± 0.85 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA; 2%) and L-NMMA (2%) was applied to their skin. The present studies indicate that UVB radiation acts as a potent stimulator of eNOS and XO in human endothelial cells. The cytotoxic effects of NO and ONOO- may be the main factors in the integrated response of the skin leading to vasodilatation, the first key event of erythema production and the inflammation process

ULTRAVIOLET-IRRADIATED HUMAN ENDOTHELIAL-CELLS ELABORATE NITRIC-OXIDE THAT MAY EVOKE VASODILATORY RESPONSE

Human skin absorbs solar ultraviolet radiation which evokes vasodilation by a mechanism that is unknown. In this work, we show that ultraviolet (290-320 nm) irradiation of cultured human endothelial cells with doses (60 mJ/cm2) evoked nitric oxide (NO) release that was sustained for more than 60 min. In addition to augmenting NO, cyclic GMP production by human endothelial cells was also increased in a time-dependent manner. Ultraviolet stimulation of NO production was also observed with the enzyme NO synthase purified from the cytosol of human endothelial cells. These results indicate that ultraviolet radiation stimulates NO release, which stimulates cyclic GMP production by vascular smooth muscle, which results in a relaxation response

MET-ENKEPHALIN RECEPTOR-MEDIATED INCREASE OF MEMBRANE FLUIDITY MODULATES NITRIC-OXIDE (NO) AND CGMP PRODUCTION IN RAT-BRAIN SYNAPTOSOMES

The association of [H-3]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25 degrees C follows a sigmoid path with a Hill coefficient h = 1.25 +/- 0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33 +/- 0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5 +/- 7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r(o)/r) - 1](-1). Arrhenius-type plots of [(r(o)/r) - 1](-1) indicated that the lipid separation of the synaptosomal membranes at 23.4 +/- 1.2 degrees C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8 +/- 0.8 degrees C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10(-10) - 10(-4) M). These effects were reversed by naloxone (10(-4) M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin