Abstract
To study the signal transduction mechanisms by which ultraviolet B (UVB) leads to increased prostaglandin E2 (PGE2) synthesis, human epidermal cultures were irradiated with 30 mJ/cm2 UVB and assayed for 6-h cumulative PGE2. Supernatants from irradiated cultures showed a 4-fold increase in PGE2 synthesis (113.6 +/- 26.8 pg/mg protein) when compared to supernatants from sham-irradiated cultures (25.6 +/- 3.9 pg/mg protein). Pretreatment of irradiated cultures with genistein (10 micrograms/ml) or tyrphostin-23 (50 microM), inhibitors of tyrosine kinases, blocked UVB-stimulated PGE2 synthesis. Treatment of nonirradiated cultures with epidermal growth factor (EGF), which acts through the receptor tyrosine kinase EGF-R, produced a 4-fold increase in PGE2 synthesis. However, addition of EGF to irradiated cultures did not further enhance their PGE2 synthesis, indicating irradiation rendered them refractory to EGF stimulation. In contrast, irradiated cultures could still significantly increase their PGE2 synthesis in response to the calcium ionophore A23187 or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate, suggesting that the lack of response to EGF was selective. Furthermore, anti-phosphotyrosine immunoblot analysis revealed UVB-induced phosphorylation of tyrosine residues of EGF-R, an indicator of receptor activation. Phosphorylation was maximal 30-60 min after irradiation and was blocked by the tyrosine kinase inhibitors, genistein and tyrphostin. The antioxidant N-acetylcysteine decreased UVB-induced EGF-R tyrosine phosphorylation and PGE2 synthesis to near-basal levels. Conversely, treatment of unirradiated cultures with the potent oxidant tert-butyl-hydroperoxide (100 microM) increased both PGE2 synthesis and EGF-R phosphorylation. Collectively, these data suggest that antioxidant depletion induced by UV results in tyrosine phosphorylation and activation of the EGF-R. This activation may subsequently activate epidermal phospholipase at early time points after UVB exposure.
Highlights
From the Division of Dermatology, Department of Medicine, Washington University School of Medicine, St
This suppression ofPGE2 synthesis correlates with the degree of epidermal growth factor (EGF)-R tyrosine phosphorylation occurring 30-60 min post-irradiation
Since deletion mutants involving tyrosine residues ofEGF-R demonstrate that autophosphorylation is a requirement for receptor activity (32,331 and isnecessary for EGF-Rinduced activation of phospholipase A2 [34], the enhanced tyrosine phosphorylation status of the epidermal growth factor receptor (EGF-R) observed post-irradiation is suggestive of receptor activation induced by unltraviolet B (UVB) exposure, which may lead to activation of phospholipaseA2
Summary
Phosphate, 200 p sodium orthovanadate, and 100 1.1~sodium molybdate. The cellular lysates were subsequently prepared by sonication. The tyrosine was incubated for 2 h with anti-phosphotyrosine (1:2000)followed by kinase inhibitor tyrphostin-23 was from Biomol (Philadelphia, PA); ge- incubation with protein A-horseradish peroxidase (1:2000; Amersham nistein was from Sigma, as was recombinant human EGF.All other Corp.) for 30min, both incubations were followedby two10-minwashes reagents were of highest research grade. Isolated epidermal cells were placed in DMEM containing 5% previously These cellular preparations were sonicated and centrifetal calf serum, penicillin (100unitdml), streptomycin(100pg/ml)and fuged at 16,000 x g a t 4 "C and normalized for protein. A similar increase in PGEz free medium.cultures treated with N-acetylcysteine were synthesis was observed in irradiated cultures during the same preincubated for 30 minprior to irradiation and thenreintroduced with interval (113.6 2 26.8 pdmg protein). By cultures were already synthesizing PGEz at a maximal rate, WB-inducedPGE, Is l)rosine Kinase-dependent t2 0.6
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