The myeloperoxidase-derived oxidant HOSCN inhibits protein tyrosine phosphatases and modulates cell signalling via the mitogen-activated protein kinase (MAPK) pathway in macrophages

Abstract

MPO (myeloperoxidase) catalyses the oxidation of chloride, bromide and thiocyanate by hydrogen peroxide to HOCl (hypochlorous acid), HOBr (hypobromous acid) and HOSCN (hypothiocyanous acid) respectively. Specificity constants indicate that SCN- is a major substrate for MPO. HOSCN is also a major oxidant generated by other peroxidases including salivary, gastric and eosinophil peroxidases. While HOCl and HOBr are powerful oxidizing agents, HOSCN is a less reactive, but more specific, oxidant which targets thiols and especially low pKa species. In the present study we show that HOSCN targets cysteine residues present in PTPs (protein tyrosine phosphatases) with this resulting in a loss of PTP activity for the isolated enzyme, in cell lysates and intact J774A.1 macrophage-like cells. Inhibition also occurs with MPO-generated HOCl and HOBr, but is more marked with MPO-generated HOSCN, particularly at longer incubation times. This inhibition is reversed by dithiothreitol, particularly at early time points, consistent with the reversible oxidation of the active site cysteine residue to give either a cysteine-SCN adduct or a sulfenic acid. Inhibition of PTP activity is associated with increased phosphorylation of p38a and ERK2 (extracellular-signal-regulated kinase 2) as detected by Western blot analysis and phosphoprotein arrays, and results in altered MAPK (mitogen-activated protein kinase) signalling. These data indicate that the highly selective targeting of some protein thiols by HOSCN can result in perturbation of cellular phosphorylation and altered cell signalling. These changes occur with (patho)physiological concentrations of SCN- ions, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage, particularly in smokers who have elevated plasma levels of SCN-.

Figure 1. Inhibition of isolated PTP1B activity and loss of cysteine residues on treatment with HOSCN

Isolated PTP1B was washed with buffer using a 3000 Da molecular mass cut-off filter to remove DTT present in the commercial preparation before use. The protein (0.5 μM, 18.7 μg·ml⁻¹) was subsequently exposed to HOSCN at the concentrations indicated for 5 min at 22 °C before determination of residual enzymatic activity and remaining thiols. (A) PTP activity (0.090 μM, 3.3 μg·ml⁻¹ PTP1B final assay concentration). (B) Thiol groups present in isolated PTP1B (0.25 μM, 9.35 μg·ml⁻¹ PTP1B final assay concentration) were measured using ThioGlo-1™ with GSH used to construct a standard curve. Values are means±S.E.M. of duplicate determinations from three separate experiments. Statistical analyses compared all concentrations with the 0 μM HOSCN condition using one-way ANOVA with Bonferroni's post-hoc test; *P<0.05.

Figure 2. Inhibition of PTP activity in J774A.1 cell lysates by HOSCN

PTP activity in cell lysates (0.33±0.03 mg of cell protein/ml) was determined after incubation with: (A) HOSCN at the concentrations indicated for 5 min; (B) phosphate buffer (■) or 25 μM HOSCN (▲) for the times indicated. Values are means±S.D. of triplicate determinations from each of four separate experiments. Statistical analyses in (A) compared all concentrations with the 0 μM oxidant condition using one-way ANOVA with Bonferroni's post-hoc test; in (B) analyses compared the control with HOSCN-treatment at each time point using two-way ANOVA with Bonferroni's post-hoc test. In both cases * indicates P<0.05.

Figure 3. Inhibition of PTP activity in J774A.1 cell lysates by HOSCN is reversible with short exposure times, but irreversible on prolonged oxidant treatment

PTP activity in cell lysates (0.33±0.03 mg of cell protein/ml) was determined after incubation with phosphate buffer or 25 μM HOSCN for 15 min (solid bars) and 120 min (open bars) at 22 °C followed by treatment with 10 mM DTT for 10 min at 37 °C. Values are means±S.E.M. of triplicate determinations from each of four separate experiments. Statistical analyses were carried out using two-way ANOVA with Bonferroni's post-hoc test, *P<0.05. NS, not significantly different.

Figure 4. Inhibition of PTP activity in intact J774A.1 cells by HOSCN

PTP activity (expressed per mg of cell protein) in cells (plated at 0.5×10⁶ cells/ml with assay performed at 0.32±0.06 mg of cell protein/ml) was determined after incubation with HOSCN, at the concentrations indicated, for either 15 min (solid bars) or 60 min (open bars) at 22 °C. Values are means±S.E.M. of duplicate determinations from each of two wells from three separate experiments. Statistical analyses compared all concentrations with the 0 μM oxidant conditions using one-way ANOVA with Bonferroni's post-hoc test; *P<0.05. ND, not determined.

Figure 5. Concentration-dependent inhibition of PTP activity in J774A.1 cell lysates by MPO-derived oxidants

PTP activity in cell lysates (0.33±0.03 mg of cell protein/ml) was determined after incubation with 100 nM MPO and 100 mM Cl⁻ (open bars), 100 μM Br⁻ (solid bars) or 100 μM SCN⁻ (grey bars) with 25 μM H₂O₂ for the times indicated at 22 °C. After incubation the samples were filtered using 10000 Da molecular mass cut-off filters to remove MPO before quantification of the p-nitrophenolate anion released from the p-nitrophenylphosphate. Controls were incubated in the absence of H₂O₂. Values are means±S.D. of triplicate determinations from each of three separate experiments. Statistical analyses were carried using two-way ANOVA with Bonferroni's post-hoc test. *P<0.05 for each of the data sets at each time point compared with the time zero control. #P<0.05 for the 100 μM SCN⁻ condition compared with the Cl⁻ and Br⁻ conditions at the same time point. †P<0.05 for the 60 min compared with 15 min time points for the 100 μM SCN⁻ condition.

Figure 6. Concentration-dependent inhibition of PTP activity in J774A.1 cell lysates by MPO-derived HOCl and HOSCN

PTP activity in cell lysates (0.33±0.03 mg of cell protein/ml) was determined after incubation with 100 nM MPO, 100 mM Cl⁻ and increasing concentrations of SCN⁻ with 25 μM H₂O₂ for 15 min at 22 °C. Control samples contained cell lysates, MPO and H₂O₂ incubated under the same conditions, but in the absence of any added Cl⁻ or SCN⁻. After incubation MPO was removed before quantification of the released p-nitrophenolate anion as described in the legend to Figure 5. Values are means±S.D. of four determinations from two separate experiments. Statistical analyses were carried out using a repeated measures one-way ANOVA with Bonferroni's post-hoc test. *P<0.05 for the absence of both Cl⁻ and SCN⁻ compared with the presence of 100 mM Cl⁻. #P<0.05 for increasing concentrations of SCN⁻ in the presence of 100 mM Cl⁻ compared with the absence of SCN⁻.

Figure 7. Effect of HOSCN on the extent of protein phosphorylation in J774A.1 cells

(A) MAPK array for phosphorylated proteins detected in J774A.1 cells (1×10⁶) treated with either (a) PBS or (b) 50 μM HOSCN in PBS, pH 7.4, for 15 min at 37 °C. After oxidant (or control) exposure, cells were lysed using the buffer provided with the kit, and 300 μg of protein (determined using the Bio-Rad protein assay) used for each array. Following binding, the arrays were washed and reacted with the supplied antibody cocktail as specified by the manufacturer. Chemiluminescent detection was performed with ECL with the signals detected using a Bio-Rad ChemiDoc XRS system and Bio-Rad Quantity One software (version 4.6.1). Pairs of protein spots marked ‘1’ are positive controls. Duplicate spots from phosphorylated ERK2 (MAPK1; spots marked ‘2’) and p38α (spots marked ‘3’) are indicated. (B) Western blots of total and phosphorylated p38α, and corresponding gel densitometry (ratio of phosphorylated to total, n=3 independent experiments), from cells exposed to HOSCN at the stated concentrations as described for (A). (C) Western blots of total and phosphorylated MKK3 (solid bars) and MKK6 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for (A). (D) Western blots of total and phosphorylated ERK1 (solid bars) and ERK2 (open bars), and corresponding gel densitometry (ratio of phosphorylated to total, from three independent experiments), from cells exposed to HOSCN at the stated concentrations as described for (A). Statistical analyses compared the HOSCN-treated samples with the untreated control (PBS-treated) cells using one-way ANOVA with Bonferroni's post-hoc test, *P<0.05.

Figure 8. Effect of HOSCN exposure on gene expression in J774A.1 cells

MAPK gene array following treatment of J774A.1 cells with HOSCN. Cells (plated at 0.5×10⁶ ml⁻¹) were exposed to 50 μM HOSCN (open bars) or 100 μM HOSCN (solid bars) in PBS at 37 °C for 15 min. Total RNA was subsequently extracted, used to synthesize cDNA and used in the MAPK signalling pathway PCR array as described in the Experimental section. Histograms show data for: (A) transcription factors, (B) cyclin-related proteins, (C) MAPKs and (D) other genes. All genes were normalized against five reference genes and data are expressed relative to control (PBS-treated) cells, and are means±S.E.M. for triplicate determinations from three independent experiments. Statistical analyses compared the HOSCN-treated samples with the no-oxidant (PBS-treated) control cells using one-way ANOVA with Bonferroni's post-hoc test, *P<0.05.