Novel approaches to vitiligo treatment via modulation of mTOR and NF-κB pathways in human skin melanocytes

Abstract

Vitiligo is a skin depigmentation disorder with an increasing prevalence. Among recognized mechanisms is the oxidative stress that affects melanocytes which are responsible for skin pigmentation. Studies have shown that high concentration of hydrogen peroxide, or H₂O₂, induces apoptotic activities. Few studies have been done with lower doses of H₂O₂. Using human skin melanocytes, we investigated the effect of moderate concentration of H₂O₂ on melanocyte dendrites. Confocal data show that H₂O₂ at 250 µM induces loss of dendrites, as indicated by cytoskeletal proteins. α-melanocyte stimulating hormone or α-MSH pretreatment protects against H₂O₂-induced loss of dendrites, while α-MSH alone enhances dendrites. PI3K/AKT inhibitor LY294002 and mTORC1 inhibitor Rapamycin inhibit α-MSH-induced dendrites. In this study, we also investigated the effect of TNFα on cultured human skin melanocytes, since TNFα plays important roles in vitiligo. Confocal data demonstrate that TNFα induces NFκB activation. Western blot analysis shows that TNFα induces IκB phosphorylation and degradation. Interestingly, α-MSH does not have any effect of TNFα-induced IκB degradation and NF-κB activation. α-MSH, however, activates mTORC1 pathway. TNFα induces p38 but not AMPKα activation. Collectively, our data suggest that modulation of mTOR and NF-κB pathways may be a novel approach for better clinical management of vitiligo.

Keywords: TNFα; mTORC1; vitiligo; α-MSH.

Figure 1

H₂O₂ induces loss of dendrites and α-MSH protects against it in cultured human skin melanocytes. Cultured human skin melanocytes in eight well chamber slides were treated with 250 µM of H₂O₂ and fixed and stained with Hoechst for nucleus, stained with anti β-actin and α-tubulin as shown in (A), anti β-actin and vimentin as shown in (B) for cytoskeletal proteins. And cells were pretreated with α-MSH (10^-8M) for 1 hour and then treated with H₂O₂ for two hours. The cells were then fixed and stained with Hoechst for nucleus, stained with anti β-actin for cytoskeletal actin and stained with anti-phosphor-ribosomal protein S6 antibody for S6 phosphorylation as shown in (C). Dendricity was quantified based on β-actin staining in C, mean±SD, *p<0.05 (D). Scale bar=50µm.

Figure 2

α-MSH induces mTORC1 as measured as S6 phosphorylation but moderately AKT activation in cultured human skin melanocytes. Cells were cultured in eight well slides and pretreated with or without α-MSH for 1 hour and then treated with H₂O₂ for two hours. Cells were then fixed and stained with Hoechst for nucleus, stained with anti β-actin for cytoskeletal proteins and anti-phosphor-AKT for AKT activation as shown in (A). Cells were cultured in six well plates and treated with α-MSH (10^-8M). Cell lysates were collected at different time points post treatment for Western blot analysis, probed by anti-phosphor AKT and anti-phosphor S6 for mTORC1 activation, by anti-β-actin as loading control, as shown in (B). And cells were pretreated with PI3K/AKT inhibitor LY294002 (10µM), or mTORC1 inhibitor Rapamycin (10µM) for 1h, and then treated with α-MSH (10^-8M) for 1h. Cells were fixed and stained with Hoechst for nucleus and anti-phosphor-S6 for S6 activation as shown in (C). Scale bar=50µm.

Figure 3

TNFα but not IL1β induces NFκB activation and TNFα induces IκB phosphorylation and degradation in cultured human skin melanocytes. Cells were cultured in eight well chamber slides and treated with TNFα (10ng/ml) or IL1β (5ng/ml) at different time points. Cells were fixed with 4% formaldehyde in PBS and stained with anti-p65, then observed by confocal microscopy as shown in A. Cells were cultured in six well plates and treated with TNFα (10ng/ml) or IL1β (5ng/ml) at different time points. Cells were collected for SDS-PAGE/Western blot analysis using anti-IκB for IκB degradation by TNFα as shown in (B) or IL1β as shown in (C), or using anti-phosphor-IκB for IκB phosphorylation by TNFα as shown in (D). β-actin was probed as loading control. Scale bar=50µm.

Figure 4

α-MSH does not inhibit TNFα-induced IκB degradation but induces mTORC1 activation and TNFα induces p38 activation in cultured human skin melanocytes. Cells were cultured in six well plates, pre-treated with or without α-MSH (10^-8M) for 1 hour and treated with TNFα (10ng/ml) at different time points. Cells were collected for SDS-PAGE/Western blot analysis using anti-IκB or phosphor-S6 as shown in (A). Cells were cultured in six well plates and treated with TNFα (10ng/ml) at different time points. Cells were collected for SDS-PAGE/Western blot analysis using anti-phosphor p38 as shown in (B). β-actin was probed as a loading control.

Figure 5

A proposed model that α-MSH activates mTORC1 and protects against H₂O₂-Induced loss of dendrites in human skin melanocytes. Extracellular H₂O₂ activates p38 and NFκB leading to apoptosis and loss of dendrites. α-MSH activates mTORC1 pathway via cell surface receptor MC1R which is blocked by rapamycin, leading to protein synthesis, cell survival and more dendrites. TNFα activates cell surface receptor which induces IκB phosphorylation and degradation. This results in translocation of NF-κB subunits p65 and p50 from cytoplasm to nucleus, and expression of more cytokines, apoptosis, and loss of dendrites. TNFα may also induce intracellular H₂O₂ production that activates NF-κB pathway.