Epidermal melanocytes (EM) and keratinocytes (K) produce prostaglandin E₂ (PGE₂) in response to H₂O₂. Hydrogen peroxide (50 μM for 24 h) increased PGE₂ production in three of five primary EM cultures tested and in one keratinocyte culture tested. B (basal conditions), H₂O₂ (50 μM H₂O₂ for 24 h). Cell cultures: M20, 23, 37, 42, 48 and K. Error bars expressed as mean ± SEM (n = 3). P < 0.05 = *, P < 0.01 = **.

Basal melanin levels and melanogenic enzyme expression/activity in primary epidermal melanocyte (EM) cultures. (A) Basal melanin levels in primary EM cultures, (B) basal dopa-oxidase activity of tyrosinase and expression of tyrosinase, tyrosinase-related protein-1 (TRP-1) and dopachrome tautomerase (DCT) in primary EM cultures, (C) densitometric analysis of tyrosinase, TRP-1 and DCT expression normalized to actin expression. SPT (skin phototype). All lanes are from the same blot (i.e. experiment) and so are directly comparable. Cell cultures: 23, 24, 29, 37, 40, 42, 45 and 50. M = modified by glycosylation; N = native.

Epidermal melanocytes (EM) and keratinocytes (K) produce prostaglandin-E₂ (PGE₂) under basal and arachidonic acid (AA) stimulated conditions. (A) AA (3 μM) incubation for 24 h increased PGE₂ production from basal levels in all 10 primary EM cultures and 1 primary K culture. (B) EM data shown in figure (A) pooled by skin phototype. B (basal conditions), AA (3 μM AA for 24 h), SPT (skin phototype). Cell cultures: M20, 23, 24, 29, 37, 40, 42, 45, 48, 50 and K. Error bars expressed as mean ± SEM: (A) n = 3, (B) n = 15. P < 0.01 = **, P < 0.001 = ***.

Indomethacin and NS-398 reduce prostaglandin E₂ (PGE₂) production in epidermal melanocytes (EM) and keratinocytes (K). (A) Incubation of EM with indomethacin or NS-398 reduced PGE₂ production from basal or arachidonic acid (AA) stimulated levels in five primary EM cultures and (B) a primary keratinocyte culture. B (basal conditions), B + I (100 μM indomethacin for 1 h prior to 24 h stimulation period), B + NS (50 μM NS-398 for 1 h prior to 24 h stimulation period), AA (3 μM AA for 24 h), AA + I (100 μM indomethacin for 1 h prior to stimulation with AA and for 24 h during stimulation), AA + NS (50 μM NS-398 for 1 h prior to stimulation with AA and for 24 h during stimulation). Cell cultures: M20, 23, 37, 42, 48 and K. Error bars expressed as mean ± SEM: (A) n = 3, (B) n = 15. P < 0.05 = *, P < 0.01 = **.

Prostaglandin E₂ (PGE₂) production in epidermal melanocytes (EM) and keratinocytes (K) is modulated by 73 mJ/cm² ultraviolet B (UVB). (A) UVB increased PGE₂ production in 6 of 10 primary EM cultures (4 at statistical significance) and 1 keratinocyte culture but had no effect in 4 of 10 EM cultures. (B) PGE₂ production in response to UVB is increased significantly only in EM derived from individuals with skin phototype-1 (SPT-1). (C) Incubation with both indomethacin and NS-398 reduced PGE₂ production in response to UVB in 1 of 4 EM cultures tested and 1 keratinocyte culture tested. B (basal conditions), UVR (73 mJ/cm² UVB), UVR + I (1 h preincubation with 100 μM indomethacin then irradiation with 73 mJ/cm² UVB and then post-incubation with 100 μM indomethacin for 24 h), UVR + NS (1 h preincubation with 50 μM NS-398 then irradiation with 73 mJ/cm² UVB and then post-incubation with 50 μM NS-398 for 24 h). Cell cultures: M20, 23, 42, 48 and K. In (A) and (C) error bars are expressed as mean ± SEM (n = 3), in (B) error bars are expressed as mean ± SEM (n = 15). P < 0.05 = *, P < 0.01 = **.

Epidermal melanocytes (EM) under basal conditions express the cellular machinery required to produce prostaglandin-E₂ (PGE₂). (A) Immunocytochemical detection of PGE₂ enzyme machinery in EM. (A1) gp-100 protein (positive control for EM), (A2) cPLA₂ protein, (A3) cyclooxygenase-1 (COX-1) protein, (A4) very weak or undetectable COX-2 protein in EM, inset positive control for COX-2 in keratinocytes, (A5) cPGES protein, (A6) mPGES-1 protein, (A7) mPGES-2 protein, (A8) negative control (no primary antibody). (B) PCR of COX-2 enzyme in EM. (B1) molecular weight marker, (B2) negative control (no cDNA), (B3) actin mRNA, (B4) COX-2 mRNA. (C) Western Blotting analysis of COX-2 enzyme in EM. (C1) molecular weight marker, (C2) negative control (no primary antibodies), (C3) actin and COX-2 protein in FM3 melanoma cells (COX-2 positive control), (C4) actin protein but lack of COX-2 protein expression under basal conditions, (C5) actin protein but lack of COX-2 protein expression 24 h post 73 mJ/cm² ultraviolet B (UVB), (C6) actin protein but lack of COX-2 protein expression 48 h post 50 μM H₂O₂. EM used in figure (A) was derived from individual 37 skin phototype-1 (SPT-1) but is representative of results obtained from 10 different primary EM cultures. EM used in figures (B) and (C) were derived from individual 37 but is representative of results obtained from three different primary EM cultures (i.e. 23, 37 and 48). All lanes are from the same blot (i.e. experiment) and so are directly comparable.