IDH2 deficiency accelerates skin pigmentation in mice via enhancing melanogenesis

Abstract

Melanogenesis is a complex biosynthetic pathway regulated by multiple agents, which are involved in the production, transport, and release of melanin. Melanin has diverse roles, including determination of visible skin color and photoprotection. Studies indicate that melanin synthesis is tightly linked to the interaction between melanocytes and keratinocytes. α-melanocyte-stimulating hormone (α-MSH) is known as a trigger that enhances melanin biosynthesis in melanocytes through paracrine effects. Accumulated reactive oxygen species (ROS) in skin affects both keratinocytes and melanocytes by causing DNA damage, which eventually leads to the stimulation of α-MSH production. Mitochondria are one of the main sources of ROS in the skin and play a central role in modulating redox-dependent cellular processes such as metabolism and apoptosis. Therefore, mitochondrial dysfunction may serve as a key for the pathogenesis of skin melanogenesis. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. Downregulation of IDH2 expression resulted in an increase in oxidative DNA damage in mice skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also promoted pigmentation on the dorsal skin of mice, as evident from the elevated levels of melanin synthesis markers. Furthermore, pretreatment with mitochondria-targeted antioxidant mito-TEMPO alleviated oxidative DNA damage and melanogenesis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in skin melanogenesis in association with mitochondrial ROS and suggest unique therapeutic strategies for the prevention of skin pigmentation.

Keywords: IDH2; Melanogenesis; Mito-TEMPO; Mitochondria; α-MSH.

Graphical abstract

Fig. 1

Effects of IDH2 downregulation on melanogenesis in JB6 cells. (A) Intracellular α-MSH concentration in JB6 keratinocytes measured with α-MSH assay kit. (B) Immunoblot analysis of the secreted α-MSH in JB6-conditioned media. After 48 h of JB6 cultivation, the supernatant was collected and used as the conditioned medium. Media were concentrated and processed for immunoblot analysis. (C) Immunoblot analysis of α-MSH in JB6 keratinocytes. (D) Immunofluorescence image of α-MSH in JB6 keratinocytes. (E) Immunoblot analysis of POMC in JB6 keratinocytes. (F) Immunofluorescence image of POMC in JB6 keratinocytes. (G) Immunoblot analysis of proteins related to ATM-mediated DNA damage signal activation in JB6 keratinocytes. Actin was used as a loading control. The proteins levels were normalized to actin level. (H) Representative immunofluorescence images of 8-OH-dG (green) analysis to measure the oxidative DNA damage. DCFH-DA fluorescence levels for the evaluation of the intracellular hydrogen peroxide level in JB6 keratinocytes after the inhibition of IDH2 expression. Mitochondrial membrane potential (MP) in JB6 keratinocytes was evaluated by measuring JC-1 fluorescence level. The histogram represents the quantification of MP as a ratio of JC-1 (green/red) in different treatment groups. MitoSOX fluorescence level for used for the evaluation of mitochondrial ROS generation in JB6 keratinocytes. All fluorescence images were counterstained with Hoechst 33342 for nuclear morphology. All histograms represent quantification of fluorescence intensity. (I) Immunoblot analysis of Prx-SO₃ levels in JB6 keratinocytes. (J) Masson-Fontana staining for melanin content evaluation in B16F10 cells. Cells were cultured with JB6-conditioned media for 48 h. The medium was changed every day. (K) Intracellular melanin content of B16F10 melanoma cells. Cells were cultured with conditioned media for 48 h. (L) Immunoblot analysis of melanin synthesis markers MITF, tyrosinase, TRP1, and TRP2 in B16F10 melanoma cells. Cells were cultured with conditioned media for 48 h. Actin was used as a loading control. The proteins levels were normalized to actin level. (M) Analysis of tyrosinase activity and cAMP content in B16F10 melanoma cells. Cells were cultured with conditioned media for 48 h. All histograms represent the quantification of fluorescence intensity. In A-N, results are shown as the mean ± SD (n = 3). * p < 0.05 versus control cells.

Fig. 2

Mito-TEMPO alleviates the increased level of melanogenesis via IDH2 downregulation in vitro. JB6 cells were incubated for 24 h after seeding and treated with mito-TEMPO (200 nM) for 24 h. (A) Intracellular α-MSH concentration in JB6 keratinocytes was measured with α-MSH assay kit. (B) Immunoblot analysis of the secreted α-MSH in JB6 conditioned media. Media were concentrated and processed for immunoblot analysis. (C) Immunoblot analysis of $\alpha$-MSH in JB6 keratinocytes. (D) Immunoblot analysis of POMC in JB6 keratinocytes. (E) Immunoblot analysis of proteins related to ATM-mediated DNA damage signal activation in JB6 keratinocytes. Actin was used as a loading control. The proteins levels were normalized to the actin level. (F) Representative immunofluorescence images of 8-OH-dG (Green) analysis to evaluate the oxidative DNA damage. DCFH-DA fluorescence levels for the evaluation of the intracellular hydrogen peroxide level. Mito-SOX fluorescence level for the evaluation of mitochondrial ROS generation in JB6 keratinocytes. All fluorescence images were counterstained with Hoechst 33342 for nuclear morphology. All histograms represent quantification of fluorescence intensity. (G) Masson-Fontana staining for melanin content evaluation in B16F10 cells. Cells were cultured with JB6 conditioned media for 48 h. For mito-TEMPO-treated conditioned media, JB6 cells seeded for 24 h were treated with 200 nM mito-TEMPO for 24 h. The medium was changed every day. (H) Intracellular melanin contents of B16F10 cultured with conditioned media. (I) Immunoblot analysis of melanin synthesis markers such as MITF, tyrosinase, TRP1, and TRP2 in B16F10 melanoma cells. Cells were cultured with conditioned media for 48 h. Actin was used as a loading control. The proteins levels were normalized to actin level. All histograms represent the quantification of fluorescence intensity. In A-I, results are shown as the mean ± SD (n = 3). * p < 0.05 versus IDH2 shRNA-transfected cells.

Fig. 3

Effects of IDH2 deficiency on the skin melanogenesis of mice. The dorsal skin of mice (10–12 weeks old) was shaved with a trimmer, followed by their euthanization. (A) Dorsal skin images of WT and IDH2^−/− KO mice. (B) Hematoxylin & eosin staining for the evaluation of the histological image of WT and KO mice skin. (C) Masson-Fontana staining of the dorsal skin for the analysis of skin melanin content. (D) Melanin content of skin from WT and KO mice. (E) Tyrosinase activity assay was used to detect tyrosinase activity of both WT and KO mice skin. (F) Representative immunohistochemical images of melanin synthesis markers such as MITF, tyrosinase, TRP1, and TRP2 in mice skin. All sections were counterstained with anti-melanoma antibody (red) for pan-melanocytes. Nuclei were counterstained with Hoechst 33342. (G) Immunofluorescence image of α-MSH and POMC in mice skin. All sections were counterstained with anti-pan-keratinocyte antibody (red) for pan-keratinocytes and Hoechst 33342 (blue) for nuclear morphology. (H) Immunofluorescence image of DNA damage markers such as p53, p-Chk2, p-ATM, and γ-H2AX in mice skin. All sections were counterstained with anti-pan-keratinocyte antibody (red) for pan-keratinocytes and Hoechst 33342 (blue) for nuclear morphology. (I) Immunofluorescence image of 8-OH-dG (green) and MitoSOX (red) in mice skin. Sections were stained with pan-keratinocyte and Hoechst 33342 (blue). All histograms represent the quantification of fluorescence intensity. In D-I, results are shown as the mean ± SD (n = 3–6 mice in each group). *p < 0.05 between the two genotypes indicated.

Fig. 4

Mito-TEMPO alleviates the increased level of melanogenesis in IDH2-deficient mice. Male juvenile C57BL/6 mice (6-week old) received a daily injection of mito-TEMPO (0.7 mg/kg, i.p.) for 30 days. (A) Dorsal skin images of untreated KO mice and KO mice treated with mito-TEMPO. (B) Hematoxylin & eosin staining for the evaluation of the histological image. (C) Masson-Fontana staining of skin for the analysis of the melanin content. (D) The melanin content was measured to evaluate melanin production in untreated KO mice and KO mice treated with mito-TEMPO. (E) Tyrosinase activity assay was used to detect tyrosinase activity in mice skin. (F) Immunofluorescence image of mice skin for MITF. Sections were stained with anti-melanoma antibody (red) for pan-melanocytes and Hoechst 33342 for nuclear morphology. (F) Immunofluorescence image of mice skin for α-MSH. (H) Immunofluorescence image of mice skin for p53. (I) Immunofluorescence image of mice skin for γ-H2AX. All sections were counterstained with anti-pan-keratinocyte antibody (red) for pan-keratinocytes and Hoechst 33342 (blue) for nuclear morphology. (J) Immunofluorescence image of mice skin for 8-OH-dG (green). (K) Immunofluorescence image of mice for MitoSOX (red). Sections were stained with pan-keratinocyte and Hoechst 33342 (blue). (L) Schematic diagram summarizing that IDH2 deficiency promotes melanogenesis. In D-I, results are shown as the mean ± SD (n = 3–6 mice in each group). *p < 0.05 versus KO mice untreated with mito-TEMPO.