L-Ascorbyl-2-phosphate attenuates NF-κB signaling in SZ95 sebocytes without affecting IL-6 and IL-8 secretion

Abstract

Acne is the most common inflammatory skin disease. Interleukin-1 (IL-1) is at the beginning of the cytokine signaling cascade and may be involved in the pathogenesis of this disorder. It activates redox-sensitive transcription factors, which induce IL-6 and IL-8 expression. Interestingly, L-ascorbyl-2-phosphate (APS) was shown to have beneficial effects in patients with acne vulgaris. The mechanism of action of this agent remains unknown. Here, we investigated if APS attenuates IL-1β- or TNF-α-mediated IL-6 and IL-8 expression in SZ95 sebocytes, whereas TNF-α was used as control. We also explored NF-κB activation which is known to orchestrate IL-1β- and TNF-α-mediated cytokine expression in many cell types. Both IL-1β and TNF-α increased IL-6 and IL-8 mRNA expression in SZ95 sebocytes. However, only IL-1β induced IL-6 and IL-8 secretion. IL-1β but not TNF-α activated NF-κB canonical signaling as demonstrated by Iκ-Bα phosphorylation and degradation as well as by nuclear accumulation of NF-κB/p65. Concomitant treatment of SZ95 sebocytes with APS attenuated the effect of IL-1β and TNF-α on IL-6 and IL-8 gene expression as well as on IL-1β-mediated NF-κB signaling. In contrast, APS failed to reduce IL-1β-mediated IL-6 and IL-8 secretion, presumably by maintained IL-1β-mediated p38 activation, which is known to control IL-8 secretion. Our findings shed light into the impact of IL-1β on the inflammatory cytokine response and its molecular mechanisms in human sebocytes. Our data further suggest that the beneficial effect of APS in acne patients involves attenuation of NF-κB signaling but not reduction of IL-6 or IL-8 secretion.

Fig. 1

APS suppressed IL-1β- and TNF-α-mediated mRNA expression of IL-6 (a, c) and IL-8 (b, d) in SZ95 sebocytes. Cells were treated with IL-1β (0.1 ng/ml), TNF-α (10 ng/ml) alone or in combination with APS at indicated doses for 6–8 h followed by real-time RT-PCR analysis. ***p < 0.001, **p < 0.01, *p < 0.05 vs. IL-1β or TNF-α; n = 3

Fig. 2

Impact of IL-1β, TNF-α and APS on NF-κB activation in SZ95 sebocytes. Time kinetics of IL-1β- (a) and TNF-α- (b) mediated IκBα phosphorylation and protein degradation. Cells were treated with IL-1β (0.1 ng/ml) or TNF-α (10 ng/ml) as indicated followed by Western immunoblotting of total cell lysates with antibodies against phosphorylated and total IκBα. To ensure equal protein loading membranes were reprobed with an anti-tubulin-α antibody. Expression of phosphorylated and total IκBα was quantified by densitometry. Time kinetic analysis of nuclear accumulation of NF-κB/p65 after stimulation with IL-1β or TNF-α (c). Nuclear extracts were processed by Western immunoblotting using antibodies against NF-κB/p65. Nuclear protein loading was controlled by reprobing with an anti-TBP antibody. Expression of phosphorylated and total IκBα was quantified by densitometry. APS reduces nuclear translocation of NF-κB/p65 following stimulation with IL-1β (d). Nuclear translocation of NF-κB/p65 was assessed by immunofluorescence 30 min after stimulation. Panels depict representative images of at least three independent experiments with identical results. APS attenuates NF-κB activation as shown by reduced phosphorylation of IκBα (e). Phosphorylation of IκBα was determined by Western immunoblotting as outlined above. Expression of NF-κB/p65 was quantified by densitometry

Fig. 2

Impact of IL-1β, TNF-α and APS on NF-κB activation in SZ95 sebocytes. Time kinetics of IL-1β- (a) and TNF-α- (b) mediated IκBα phosphorylation and protein degradation. Cells were treated with IL-1β (0.1 ng/ml) or TNF-α (10 ng/ml) as indicated followed by Western immunoblotting of total cell lysates with antibodies against phosphorylated and total IκBα. To ensure equal protein loading membranes were reprobed with an anti-tubulin-α antibody. Expression of phosphorylated and total IκBα was quantified by densitometry. Time kinetic analysis of nuclear accumulation of NF-κB/p65 after stimulation with IL-1β or TNF-α (c). Nuclear extracts were processed by Western immunoblotting using antibodies against NF-κB/p65. Nuclear protein loading was controlled by reprobing with an anti-TBP antibody. Expression of phosphorylated and total IκBα was quantified by densitometry. APS reduces nuclear translocation of NF-κB/p65 following stimulation with IL-1β (d). Nuclear translocation of NF-κB/p65 was assessed by immunofluorescence 30 min after stimulation. Panels depict representative images of at least three independent experiments with identical results. APS attenuates NF-κB activation as shown by reduced phosphorylation of IκBα (e). Phosphorylation of IκBα was determined by Western immunoblotting as outlined above. Expression of NF-κB/p65 was quantified by densitometry

Fig. 3

Effect of APS on IL-1β- and TNF-α-mediated IL-6 (a) and IL-8 (b) secretion by SZ95 sebocytes. Cells were stimulated with IL-1β (0.1 ng/ml), TNF-α (10 ng/ml) alone or in combination with APS at indicated doses for 24 h followed by ELISA, n = 3

Fig. 4

Effects of IL-1β, TNF-α and APS on p38 MAPK (a, c) and p42/p44 ERK1/2 (b, d) phosphorylation in SZ95 sebocytes. Cells were stimulated with IL-1β (0.1 ng/ml) and TNF-α (10 ng/ml) alone or in combination with APS (1 mg/ml) for 30 min followed by Western immunoblotting with phosphospecific antibodies against p38 MAPK and p42/p44 ERK1/2. To ensure equal protein loading membranes were reprobed with antibodies against total p38 MAPK and p42/p44 ERK1/2. Expression of phosphorylated kinases was quantified by densitometry. Panels depict representative images of 3 independent experiments with similar results

Fig. 5

Impact of IL-1β, TNF-α and APS on COX2 mRNA (a, b) and protein expression (c) in SZ95 sebocytes. Expression of COX2 transcripts was determined by real-time RT-PCR following stimulation of cells with either IL-1β or TNF-α (10 ng/ml) alone or in combination with APS for 8 h. *p < 0.05 vs. control for IL-1β, n = 3; *p < 0.05 vs. TNF-α, n = 4. Expression of COX2 protein was assessed by Western immunoblotting following stimulation of cells with IL-1β or TNF-α as indicated. Expression of COX2 protein was quantified by densitometry. Panels are representative images of at least three independent experiments with identical results