Aquaporin-3-mediated hydrogen peroxide transport is required for NF-κB signalling in keratinocytes and development of psoriasis

Abstract

Aquaporin 3 (AQP3), a water/glycerol channel protein, has been found to transport hydrogen peroxide (H2O2). Here, we show that H2O2, imported via AQP3, is involved in nuclear factor-κB (NF-κB) signalling in keratinocytes and in the pathogenesis of psoriasis. IL-23-mediated induction of psoriasis is reduced in AQP3 knockout mice (AQP3(-/-)), and is accompanied by impaired NF-κB activation and intracellular H2O2 accumulation. In primary keratinocyte cultures, cellular import of H2O2 produced by membrane NADPH oxidase 2 (Nox2) in response to TNF-α is facilitated by AQP3 and required for NF-κB activation by regulation of protein phosphatase 2A. As AQP3 associates with Nox2, we propose that this interplay constitutes H2O2-mediated signalling in response to TNF-α stimulation. Collectively, these data indicate that AQP3-facilitated H2O2 transport is required for NF-κB activation in keratinocytes in the development of psoriasis.

Figure 1. Impaired IL-23-induced psoriasis-like skin in AQP3 ^−/− mice

(a) Left: representative immunohistochemical staining for AQP3 in the skin from five healthy volunteers and 18 psoriatic patients. Right: immunostaining with anti-AQP3 (cy3, red) and anti-CD3 (FITC, green) in psoriatic skin. Scale bar, 100 μm. Epi, epidermis; der, dermis. (b–f) IL-23 (500 ng) or vehicle control (PBS) was intradermally injected daily into the ear skin of WT and AQP3 ^−/− mice for 4 days. Skin samples were excised at 24 h after the final IL-23 injection. (b) Left: haematoxylin and eosin staining of ears from WT and AQP3 ^−/− mice. Scale bar, 100 μm. Arrow head, infiltrating lymphocytes. Right: whole skin and epidermal thickness determined from haematoxylin and eosin staining (s.e., n =5, *P<0.01 by t-test).(c) Immunostaining with anti-AQP3 (FITC, green) and anti-CD3 (cy3, red) in WT mouse skin. Scale bar, 100 μm (left), 20 μm (right). (d) Left: representative immunohistochemical staining for Ki67. Right: the ratio of Ki67 positive cells in the epidermis (s.e., n =3, *P<0.01 by t-test). (e) CD3 ⁺ γδTCR ⁺ cell numbers in epidermis and dermis from WT and AQP3 ^−/− mice analysed by flow cytometry (s.e., n =4–5, *P<0.01 by t-test). (f) mRNA expression of IL-17A, IL-17F, IL-22 and IL-19 in skin tissues determined by real-time RT–PCR (s.e., n =4–5,*P<0.01 by t-test). Data are expressed as the ratio to GAPDH.

Figure 2. Impaired IL-23-induced psoriasis is dependent on AQP3 expression in both keratinocytes and T cells

(a,b) IL-23-induced psoriatic model using bone marrow (BM) cell-transferred mice. Recipient WT and AQP3 ^−/− mice (Rec) received transplants of BM cells from donor WT and AQP3 ^−/− mice (Don). (a) Ear thickness at 24 h after final injections (s.e., n =5, *P<0.01 by t-test). (b) Representative haematoxylin and eosin staining. Scale bar, 100 μm. (c) Chemotaxis assay. The migration efficiency of CD3 ⁺ T cells toward the ligands CCL20 (100 ng ml⁻¹) or CXCL9 (100 ng ml ⁻¹) was determined using a transwell chamber with 5 μm pores. Data are expressed as the percentage of applied cells (s.e., n =5, *P<0.01 by t-test). (d) mRNA expression levels of CCR6 in epidermis (left) and CXCR3 in dermis (right) by real-time RT–PCR (s.e., n =5, *P<0.01 by t-test). (e) mRNA expression levels of IL-17A and IL-17F by real-time RT–PCR in sorted T cells incubated with IL-23 in the presence of CD3/28 for 3 days (s.e., n =5, *P<0.01 by t-test). Data are expressed as the IL-17/GAPDH ratio. (f) IL-22 level by ELISA in the culture medium with IL-23 in the presence of anti-IL-4 and anti-IFNγ for 3 days (s.e., n =5, *P<0.01 by t-test).

Figure 3. AQP3 deficiency impairs H₂O₂ elevation and NF-κB activation in IL-23-treated skin

(a,b) Mice were injected with IL-23 into the ear as described in Fig. 1. (a) Immunostaining of phospho-p65 in PBS (control) or IL-23 injected WT and AQP3 ^−/− skin. Scale bar, 100 μm. (b) Representative immunoblot analysis using antibodies against phospho-Iκβα, Iκβα and β-actin. Experiments were performed in two other independent experiments with similar results. (c–e) Haematoxylin and eosin staining (c), ear thickness (d) and phospho-p65 immunostaining (e) of ears from WT mice treated daily with IL-23 or PBS (control) in the presence of anti-TNF-α blocking monoclonal antibody or isotype control. (f) H₂DCFDA fluorescence in the skin treated with IL-23 (three continuous applications) with or without anti-TNF-α antibody. Anti-TNF-α antibody was injected 1 h before sampling. (g) H₂DCFDA fluorescence in WT and AQP3 ^−/− skin treated daily with PBS (control) or IL-23 by fluorescence microscopy. (h) The mean fluorescence intensity (MFI) of H₂DCFDA by FACS analysis in epidermal cells (s.e., n = 5, *P<0.05, **P<0.01 by t-test).

Figure 4. Impaired TNF-α-induced NF-κB activation in AQP3-deficient keratinocytes

(a,b) Primary keratinocytes from WT and AQP3 ^−/− mice were incubated with TNF-α (100 ng ml ⁻¹) for 5 min to 1 h. (a) Representative immunoblot using antibodies against phospho-IKKβ, IKKβ, phospho-Iκβα, Iκβα, phospho-p65, p65 and β-actin at indicated times. (b) Cell lysate (TNF-α, 100 ng ml ⁻¹, 5 min) was immunoprecipitated with anti-TNFR1. Immunoblot was performed with anti-Traf2, anti-RIP1 and anti-TNFR1. Ct, control; T, TNF-α. Experiments in a and b were performed in two other independent experiments with similar results. (c) mRNA expression in primary keratinocytes by real-time RT–PCR. Cells from WT and AQP3^−/− mice were incubated with TNF-α (100 ng ml ⁻¹) for 24 h. Data are expressed as the ratio to GAPDH (s.e., n =5, *P<0.05, **P<0.01 by t-test). (d) Primary keratinocytes from WT and AQP3 ^−/− mice were incubated with IL-22 (100 ng ml ⁻¹, 10 min) or IFNγ (100 ng ml ⁻¹, 10 min). Representative immunoblot analysis with anti-phospho-Stat3, -Jak2, Stat3 or Jak2. A second set of experiments gave similar results.

Figure 5. AQP3-dependent H₂O₂ permeability in keratinocytes

(a, b) H₂O₂ uptake into primary cultured keratinocytes. Keratinocytes were incubated with H₂O₂ (10 to 300 μM), and cellular H₂O₂ was detected using CM-H₂DCFDA fluorescence using a plate reader. (a) Representative fluorescence intensity of CM-H₂DCFDA. (b) Increased fluorescence intensity at 15 s after addition of H₂O₂ (s.e., n = 5, *P<0.01, H₂O₂ added versus control cells by t-test). (c–d) Intracellular H₂O₂ was monitored by CM-H₂DCFDA fluorescence with TNF-α stimulation (100 ng ml^{− 1}). (c) Representative fluorescence intensity in WT and AQP3 ^−/− cells. (d) Cells were incubated with DPI (5 μM), catalase (2,000 U ml ⁻¹) or vehicle (PBS) for 30 min, and followed by TNF-α (100 ng ml ⁻¹) for 30 s (s.e., n =5, *P<0.01 by t-test). (e) WT and AQP3 ^−/− cells transfected with HyPer were incubated with TNF-α (100 ng ml ⁻¹) or H₂O₂ (100 μM) for 3 min. Representative immunofluorescence. Scale bar, 20 μm. (f) Cellular H₂O₂ after stimulation by TNF-α (100 ng ml ⁻¹) or H₂O₂ (100 μM) in primary keratinocytes from WT, AQP3^−/−, or Nox2^−/− mice using CM-H₂DCFDA fluorescence (1 min, s.e., n = 7, *P<0.01 by t-test). (g) Left: immunoblot of AQP3 and Nox2 in membrane-rich fraction from WT, AQP3 ^−/− and Nox2 ^−/− keratinocytes. Right: cell lysates by RIPA were immunoprecipitated with anti-AQP3 or anti-Nox2 showing the interaction between endogenous AQP3 and Nox2.

Figure 6. AQP3-dependent H₂O₂ accumulation in keratinocytes regulates NF-κB activation

(a) Left: representative immunofluorescence of p65 in keratinocytes from WT and AQP3 ^−/− mice. Cells were stimulated with TNF-α (100 ng ml ⁻¹) for 1 h. Some cells were incubated with catalase (2,000 U ml⁻¹) or DPI (5 μM) for 30 min before TNF-α stimulation. Scale bar, 20 μm. Right: numbers of p65 positive cells in the nucleus (s.e., over 100 cells from four different fields, *P<0.01 by t-test). (b,c) AQP3 ^−/− primary keratinocytes were incubated with TNF-α (100 ng ml ⁻¹) and/or H₂O₂ (300 μM). (b) Cellular H₂O₂ level (1 min, s.e., n = 7, *P<0.01 by t-test). (c) Representative immunoblot with phospho-IKKα/β, IKKβ, phospho-p65, p65 and β-actin stimulated for indicated times. (d–f) AQP3 ^−/− primary keratinocytes were transfected with mouse AQP3 cDNA or empty vector (pCMV6). (d) Left: mRNA analysed by quantitative RT–PCR. Right: immunoblotting with AQP3 antibody. (e) Cellular H₂O₂ after stimulation with TNF-α (100 ng ml⁻¹) or H₂O₂ (100 μM; 1 min, s.e., n = 6, *P<0.05, **P<0.01 by t-test). (f) Immunoblot with phospho-IKKα/β or IKKβ stimulated with TNF-α (100 ng ml⁻¹) for indicated times. Experiments in c and f were performed in two independent sets of experiments with similar results.

Figure 7. TNF-α-mediated H₂O₂ inactivates PP2A and regulates NF-κB activation

(a) PP2A activity in WT and AQP3^−/− keratinocytes stimulated with TNF-α (100 ng ml ⁻¹) or H₂O₂ (300 μM) for 10 min. Some cells were incubated with catalase (2,000 U ml⁻¹) for 30 min before stimulation. PP2A activity was calculated from the rate of dephosphorylation of a radioactive substrate per minute per microgram protein in the presence and absence of okadaic acid (OA, 10 μM), an inhibitor of PP2A (s.e., n =5, *P<0.01 by t-test). (b) WT keratinocytes were incubated with OA (10 μM, 30 min) and stimulated with TNF-α (100 ng ml⁻¹, 15 min). Immunoblot with anti-phospho-IKKα/β, IKKβ, phospho-p65, p65 and β-actin. (c–e) Ppp2ca knockdown in WT and AQP3 ^−/− keratinocytes by siRNA transfection. (c) PP2A expression by quantitative RT–PCR and immunoblotting (s.e., n =4, *P<0.01 by t-test). (d) Cells were stimulated with TNF-α (100 ng ml ⁻¹). Representative immunoblot with phospho-IKKα/β, IKKβ, phospho-p65 and p65. (e) WT cells with control- or Ppp2ca-siRNA transfection were incubated with catalase (2,000 U ml ⁻¹, 30 min) before TNF-α stimulation. (f,g) WT keratinocytes were transfected with empty vector (pCMV6) or plasmid-expressing mouse Ppp2ca. (f) PP2A overexpression was analysed by quantitative RT–PCR and immunoblotting (s.e., n =4, *P<0.01 by t-test). (g) Representative immunoblot of phospho-IKKα/β, IKKβ, phospho-p65 and p65 with TNF-α (100 ng ml ⁻¹) stimulation. (h) Mice were injected IL-23 into the ear as described in Fig. 1 and PP2A activity was measured in epidermal homogenates. Some WT mice were injected DPI (2 μg g ⁻¹ weight) intravenously 1 h before IL-23 injection (s.e., n =3–5, *P<0.01 by t-test). Experiments in d, e and g were performed in two additional sets of independent experiments with similar results.

Figure 8. TNF-α induced NF-κB activation depends on AQP3 in human primary keratinocytes

NHEK were transfected with AQP3 or non-targeting (Ct) siRNA. (a) Left: relative mRNA expression of AQP3/GAPDH (s.e., n =4, *P<0.01 by t-test). Right: immunoblot of membrane fraction with anti-AQP3 and anti-Na⁺/K ⁺-ATPase. (b) Cellular H₂O₂ levels determined by CM-H₂DCFDA after TNF-α (100 ng ml⁻¹) or H₂O₂ (10 μM) stimulation (30 s, s.e., n = 5, *P<0.01 by t-test). (c) Immunoblot with phospho-IKKβ, IKKβ, phospho-p65 and p65 after TNF-α stimulation. (d) mRNA expression of IL-17C and IL-6 by real-time RT–PCR. Cells were incubated with TNF-α (50 ng ml ⁻¹) for 24 h. Data are expressed as the ratio to GAPDH (s.e., n =5, *P<0.05, **P<0.01 by t-test). (e) Ppp2ca knockdown in NHEK by siRNA transfection (s.e., n =4, *P<0.01 by t-test). (f) Immunoblot with phospho-p65 and p65 after TNF-α stimulation in AQP3 and/or Ppp2ca knockdown cells. Experiments in c and f were performed in two additional sets of independent experiments with similar results. (g) NHEK cell lysates were immunoprecipitated with anti-AQP3 or anti-Nox2 antibodies, showing interaction between AQP3 and Nox2.

Figure 9. Model of AQP3-mediated NF-κB activation

TNF-α binds to TNFR1 in keratinocytes and induces the production of H₂O₂ by Nox2. Extracellular H₂O₂ is rapidly transported intracellularly through AQP3. H₂O₂ modifies PP2A, regulating IKKβ and/or NF-κB/p65 activation.