Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

Abstract

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D3. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP+ over NAD+ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE-->5-HETE) is NADPH-dependent and is maximal at pH 6. Although the K(m) for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the V(max) is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP(+) (K(m) 139 nM), inhibited by NADPH (K(i) 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP+ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP+ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP+ to NADPH.

Figure 1. Effects of various differentiation agents on 5-oxo-ETE synthesis by U937 cells

(A) U937 cells were cultured for 3 days (black bars) in the presence of vehicle (Veh), 18 nM PMA, 50 nM dh-VitD₃ (VitD), 1.3% DMSO or 0.3 μM RA. Following pre-incubation with 100 μM PMS for 6 min, the cells (2.5×10⁶/ml) were incubated with 4 μM 5-HETE for 20 min at pH 7.4 and the amounts of 5-oxo-ETE determined by RP-HPLC. The amounts of 5-oxo-ETE produced by peripheral blood monocytes (Mono) and neutrophils (Neu) (hatched bars) are shown for comparison. (B) Time course for induction of 5-HEDH activity in U937 cells by PMA. U937 cells were treated with either vehicle (○) or 18 nM PMA (●). After different times the cells were harvested and 5-oxo-ETE formation was determined as described above. *P<0.05; **P<0.01; ***P<0.001; n≥4. (C) Lineweaver–Burk plots for the formation of 5-oxo-ETE by U937 cell microsomes. Microsomal fractions obtained from U937 cells treated with either vehicle (○) or PMA (18 nM) (●) for 4 days were incubated for 5 min at pH 7.4 with 100 μM NADP⁺ and various concentrations of 5-HETE, after which time 5-oxo-ETE was measured by RP-HPLC. The results are one representative out of a total of five experiments with similar results (see Table 1).

Figure 2. Effects of various agents on 5-oxo-ETE synthesis by HL-60 cells

(A) HL-60 cells were cultured for 3 days (black bars) in the presence of vehicle (Veh), 18 nM PMA, 50 nM dh-VitD₃ (VitD), 1.3% DMSO or 0.3 μM RA, and the rates of 5-oxo-ETE synthesis were determined as described in the legend for Figure 1. The amounts of 5-oxo-ETE produced by peripheral blood monocytes (Mono) and neutrophils (Neu) (hatched bars) are shown for comparison. (B) Time course for the induction of 5-HEDH activity in HL-60 cells by dh-VitD₃. HL-60 cells were treated with either vehicle (○) or 50 nM dh-VitD₃ (●). After different times the cells were harvested and the rates of 5-oxo-ETE formation were determined as described in the legend for Figure 1. The values are means±S.E.M. (n≥4). *P≤0.05; **P≤0.01; ***P≤0.001.

Figure 3. Effects of NADP⁺ and NAD⁺ on 5-HEDH activity in U937 cell microsomes

U937 cell microsomes (7.5 μg of protein/ml) were incubated for 10 min at 37 °C at pH 7.4 with 5-HETE (4 μM) in the presence of different concentrations of NADP⁺ (●) or NAD⁺ (○). 5-Oxo-ETE was quantified by RP-HPLC. All values are means±S.E.M. (n=4).

Figure 4. Interconversion of 5-oxo-ETE and 5-HETE by U937 cells

(A) Lineweaver–Burk plots for the interconversion of 5-HETE (5h) and 5-oxo-ETE (5o). Different concentrations of 5-HETE were incubated with U937 cell microsomes (50 μg of protein/ml) in the presence of NADP⁺ (100 μM) for 5 min at pH 7.4 and 37 °C and 5-oxo-ETE was quantified by RP-HPLC (5h→5o; ●). Alternatively, different concentrations of 5-oxo-ETE were incubated with U937 cell microsomes (100 μg of protein/ml) in the presence of NADPH (100 μM) for 5 min at 37 °C and 5-HETE was quantified (5o→5h; ○). (B) Effects of pH on the interconversion of 5-HETE (5h) and 5-oxo-ETE (5o). U937 cell microsomes (50 μg of protein/ml) in 50 mM Mes (circles), HEPSSO (triangles), or Caps (squares), adjusted to different pH values, were incubated for 5 min with either 1 μM 5-HETE and 100 μM NADP⁺ (closed symbols) or 1 μM 5-oxo-ETE and 100 μM NADPH (open symbols). The values are means±S.E.M. (n=5).

Figure 5. Mechanism for oxidation of 5-oxo-ETE by 5-HEDH

Different concentrations of 5-HETE were incubated with a microsomal fraction from U937 cells (50 μg of protein/ml) for 5 min at 37 °C at pH 7.4 in the presence of different concentrations of NADP⁺. (A) Lineweaver–Burk plots for the oxidation of 5-HETE by U937 cell microsomes in the presence of 0.1 μM (●), 0.17 μM (○), 0.3 μM (■), 0.6 μM (□), 1.5 μM (▼) and 10 μM (▽) NADP⁺. (B) The slope of each of the curves shown in (A) against the reciprocal of the concentration of NADP⁺. (C) The intercept (i.e. 1/V_max for 5-HETE→5-oxo-ETE) for each of the curves shown in (A) against the reciprocal of the concentration of NADP⁺. Data are for a single representative of seven independent experiments with similar results.

Figure 6. Lineweaver–Burk plot for NADPH

U937 cell microsomes (100 μg of protein/ml) were incubated with 5-oxo-ETE (3 μM) in the presence of different concentrations of NADPH for 10 min at 37 °C at pH 7.4 and the amount of 5-HETE formed was measured by HPLC. The data are for a single representative of three separate experiments with similar results.

Figure 7. Inhibition of 5-oxo-ETE formation by NADPH

Microsomal fractions (7.5 μg of protein/ml) from U937 cells were incubated with 5-HETE (4 μM) in the presence of different concentrations of NADP⁺ and NADPH for 10 min at 37 °C at pH 7.4. The amounts of 5-oxo-ETE formed were measured by RP-HPLC. (A) Inhibitory effects of different concentrations of NADPH in the presence of 3 (●), 10 (▽) or 30 μM (▲) NADP⁺. The amounts of 5-oxo-ETE are shown as percentages of the maximal amounts formed in the absence of NADPH and have been plotted against the ratios of NADP⁺ to NADPH. The values are means±S.E.M. (n=3). (B) Lineweaver–Burk plots for NADP⁺ in the presence of different concentrations of vehicle (Veh; ●), 3 μM NADPH (△), or 10 μM NADPH (▲). The data are for a single representation of four separate experiments with similar results.

Figure 8. Effects of tBuOOH on 5-oxo-ETE synthesis and pyridine nucleotide levels in intact U937 cells

(A) U937 cells (10⁶ cells/ml) were incubated with 5-HETE (1 μM) for different times at pH 7.4 in the presence of either vehicle (○) or 100 μM tBuOOH (●), and 5-oxo-ETE was measured by RP-HPLC. (B) U937 cells were incubated for different times in the presence of 100 μM tBuOOH, and NADP⁺ (●) and NADPH (△) were quantified by RP-HPLC as described in the Materials and methods section. (C) The ratios of NADP⁺ to NADPH (from B; ●) are plotted along with the average rates of formation of 5-oxo-ETE (○) over different time periods (from A) following the addition of tBuOOH to U937 cells. The values are means±S.E.M. (n=5), except for the NADP⁺/NADPH ratios shown in (C), in which case S.E.M. could not be calculated because the NADP⁺ and NADPH measurements were not from the same cell preparations.

Figure 9. 5-Oxo-ETE synthesis by U937 cell microsomes at physiological levels of pyridine nucleotides

5-HETE (4 μM) was incubated for 10 min at 37 °C and pH 7.4 with U937 cell microsomes (15 μg of protein/ml; n=3) in the presence of concentrations of NADP⁺ and NADPH comparable with those found in resting (25 and 300 μM respectively) and tBuOOH-stimulated (200 and 65 μM respectively) U937 cells. 5-Oxo-ETE was quantified by RP-HPLC as described in the Materials and methods section.