SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo

Abstract

Mitochondrial dysregulation has been implicated in oxidative stress-induced melanocyte destruction in vitiligo. However, the molecular mechanism underlying this process is merely investigated. Given the prominent role of nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylase Sirtuin3 (SIRT3) in sustaining mitochondrial dynamics and homeostasis and that SIRT3 expression and activity can be influenced by oxidative stress-related signaling, we wondered whether SIRT3 could play an important role in vitiligo melanocyte degeneration by regulating mitochondrial dynamics. Methods: We initially testified SIRT3 expression and activity in normal and vitiligo melanocytes via PCR, immunoblotting and immunofluorescence assays. Then, cell apoptosis, mitochondrial function and mitochondrial dynamics after SIRT3 intervention were analyzed by flow cytometry, immunoblotting, confocal laser microscopy, transmission electron microscopy and oxphos activity assays. Chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), immunoblotting and immunofluorescence assays were performed to clarify the upstream regulatory mechanism of SIRT3. Finally, the effect of honokiol on protecting melanocytes and the underlying mechanism were investigated via flow cytometry and immunoblotting analysis. Results: We first found that the expression and the activity of SIRT3 were significantly impaired in vitiligo melanocytes both in vitro and in vivo. Then, SIRT3 deficiency led to more melanocyte apoptosis by inducing severe mitochondrial dysfunction and cytochrome c release to cytoplasm, with Optic atrophy 1 (OPA1)-mediated mitochondrial dynamics remodeling involved in. Moreover, potentiated carbonylation and dampened peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) activation accounted for SIRT3 dysregulation in vitiligo melanocytes. Finally, we proved that honokiol could prevent melanocyte apoptosis under oxidative stress by activating SIRT3-OPA1 axis. Conclusions: Overall, we demonstrate that SIRT3-dependent mitochondrial dynamics remodeling contributes to oxidative stress-induced melanocyte degeneration in vitiligo, and honokiol is promising in preventing oxidative stress-induced vitiligo melanocyte apoptosis.

Keywords: SIRT3; apoptosis; melanocyte; mitochondrial dynamics; vitiligo.

Figure 1

Impaired SIRT3 expression and activity in vitiligo melanocytes under oxidative stress. (A) The relative mRNA level of SIRT3 in PIG1 and PIG3V cells after the treatment of 1.0 mM H₂O₂ for 24 h. Data represent mean ± SD (n = 3). (B) The protein level of SIRT3 in PIG1 and PIG3V cells after 1.0 mM H₂O₂ treatment. β-Actin was detected as loading control. Data represent mean ± SD (n = 3). (C) Immunofluorescence staining analysis of SIRT3 expression in PIG1 and PIG3V cells after 1.0 mM H₂O₂ treatment, Nuclei were counterstained with DAPI (blue). Data are representative of three independently performed experiments. Scale bar = 50 μm (magnification: 600 ×). Intensity of SIRT3 signal in melanocytes was quantified using Image J software. (D) SIRT3 activity in PIG1 and PIG3V cells after 1.0 mM H₂O₂ treatment. Data represent mean ± SD (n = 3). (E) Acetylation of mitochondiral protein in PIG1 and PIG3V cells after H₂O₂ treatment. TOMM20 was detected as loading control. Data represent mean ± SD (n = 3). (F) Acetylation of SOD2 in PIG1 and PIG3V cells after 1.0 mM H₂O₂ treatment for 24 h. β-Actin was detected as loading control. Data represent mean ± SD (n = 3). p value was calculated by two-tailed Student's t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant.

Figure 2

Impaired SIRT3 expression and activity in melanocytes of vitiligo perilesional skin. (A) Representative images of the expression of SIRT3 (green) in healthy skin (n = 8) and vitiligo perilesional skin (n = 8) detected by immunofluorescence. Melanocytes in the epidermis were stained with antibody to Melan-A (red). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm (Magnification: the left = 600 ×; the right = 1800 ×). EP, epidermis; DE, dermis. Bar graphs represent the mean values of 8 samples and 10 fields of view per sample. Mean ± SD is shown. (B) Representative images of the expression of Ac-SOD2 (green) in healthy skin (n = 8) and vitiligo perilesional skin (n = 8) detected by using immunofluorescence. Melanocytes in the epidermis were stained with antibody to Melan-A (red). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm (Magnification: the left = 600 ×; the right = 1800 ×). Intensity of SIRT3 signal in melanocytes was quantified using Image J software. Bar graphs represent the mean values of 8 samples and 10 fields of view per sample. Mean ± SD is shown. p value was calculated by two-tailed Student's t-test. ^**p < 0.01.

Figure 3

SIRT3 deficiency contributes to cell apoptosis and mitochondrial dysfunction in melanocytes. (A) PIG1 cells transfected with si-NC or si-SIRT3 were treated by different concentrations of H₂O₂ (0, 0.5, 1.0 mM), and the cell viability was determined by CCK-8. si-NC refers to negative control small interfering RNA, and si-SIRT3 refers to small interfering RNA against SIRT3. Data represent mean ± SD (n = 3). (B) The apoptotic level of PIG1 cells transfected with si-NC or si-SIRT3 after H₂O₂ treatment for 24 h was examined by flow cytometry. Bar graphs represent mean ± SD (n = 3). (C) The level of apoptosis-related proteins in PIG1 cells was detected by immunoblotting. Data are representative of three independently experiments. Quantitative analysis on the apoptosis-related proteins of interest as indicated (mean ± SD, n = 3). (D) The mitochondrial ROS level of PIG1 cells was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. Bar graphs represent mean ± SD (n = 3). (E) Assessment of ATP level in PIG1 cells with treatment as indicated. Data represent mean ± SD of triplicates. (F) The mitochondrial membrane potential level of PIG1 cells was examined by JC-1 staining. The scatter plot of the flow cytometry analysis shows the distribution of JC-1 aggregates (Red) and JC-1 monomer (Green) cell population. Histogram calculated the relative ratio of Red against Green fluorescence (mean ± SD, n=3). p value was calculated by two-tailed Student's t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant.

Figure 4

SIRT3-dependent mitochondrial dynamics remodeling coordinates cytochrome c release and respiratory complex activities under oxidative stress. (A, B) PIG1 cells were transfected with si-NC or si-SIRT3 following the treatment of 1.0 mM H₂O₂ for 24 h. si-NC refers to negative control small interfering RNA, and si-SIRT3 refers to small interfering RNA against SIRT3. PIG3V cells were transfected with plasmid OE-NC or OE-SIRT3 following treatment with 1.0 mM H₂O₂ for 24 h. OE-NC refers to transfection with control plasmids, and OE-SIRT3 refers to transfection with SIRT3 plasmids. The expression of cytochrome c in mitochondria or cytoplasm was detected. TOMM20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. β-Actin was detected as loading control for the whole cell lysate. Data are representative of three independent experiments. (C) Relative enzyme activity of respiratory complexes I, II, III, IV and V were measured in PIG1 and PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates. (D) Representative confocal microscope images of the mitochondrial network in PIG1 and PIG3V cells with treatment as indicated. Scale bar = 50 μm (Magnification: the upper = 600 ×; the under = 1800 ×). The proportion of PIG1 and PIG3V cells (n = 100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. Data are representative of three independent experiments. (E) Representative transmission electron microscopy images of the mitochondrial network in PIG1 and PIG3V cells. Scale bar = 500 nm. Mitochondrial length was quantified using Image J software in PIG1 and PIG3V cells. One hundred mitochondria were randomly selected in each treatment group. Data are representative of three independent experiments. Mean ± SD is shown. p value was calculated by two-tailed Student's t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

SIRT3 deficiency contributes to cell apoptosis and mitochondrial dysfunction via OPA1. (A) The acetylation level of OPA1 was determined in PIG1 cells transfected with si-NC or si-SIRT3 following the treatment with H₂O₂ by co-immunoprecipitation analysis. Data are representative of three independent experiments. (B) PIG1 cells were co-transfected with si-SIRT3 and OPA1 plasmids (OE-OPA1) or control plasmids (OE-NC), and then were treated with 1.0 mM H₂O₂ for 24 h. si-NC refers to negative control small interfering RNA, and si-SIRT3 refers to small interfering RNA against SIRT3. Representative confocal microscope images of the mitochondrial network in PIG1 cells were shown. Scale bar = 50 μm (magnification: the upper = 600 ×; the lower = 1800 ×).The proportion of PIG1 and PIG3V cells (n = 100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. Data are representative of three independent experiments. (C) The expression of cytochrome c in mitochondrial or cytoplasmic compartments in PIG1 cells with indicated treatment. TOMM 20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. β-Actin was detected as loading control for the whole cell lysate. (D) Relative enzyme activity of respiratory complexes I, II, III, IV and V were measured in PIG1 cells with indicated treatment. Data represent mean ± SD of triplicates. (E) The apoptotic level of PIG1 cells was examined by annexin V-FITC/PI staining (mean ± SD, n = 3). (F) The level of apoptosis-related proteins in PIG1 cells with indicated treatment. (G) The mitochondrial ROS level of PIG1 cells with indicated treatment was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. Bar graphs represent mean ± SD (n = 3). (H) The mitochondrial membrane potential level of PIG1 cells was analyzed by JC-1 staining. The scatter plot of the flow cytometry analysis shows the distribution of JC-1 aggregates (Red) and JC-1 monomer (Green) cell population. Histogram calculated the relative ratio of Red against Green fluorescence (mean ± SD, n = 3). (I) Assessment of ATP level in PIG1 cells with treatment as indicated. Data represent mean ± SD of triplicates. p value was calculated by two-tailed Student's t-test. ^**p < 0.01, ^***p < 0.001.

Figure 6

Oxidative stress simultaneously impairs SIRT3 activity and transcription. (A) Representative images of the expression of 8-OHdG (green) in healthy skin (n = 8) and vitiligo perilesional skin (n = 8) detected by using immunofluorescence. Melanocytes were stained with antibody to Melan-A (red). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm (Magnification: the left = 600 ×; the right = 1800 ×). Intensity of 8-OHdG signal in melanocytes was quantified using Image J software. Bar graphs represent mean values of 8 samples and 10 fields of view per sample. Mean ± SD is shown. (B) PIG1 and PIG3V cells were treated with 1.0 mM H₂O₂ for 24 h. Cell extracts were subjected to testify the carbnylation of SIRT3 via examing the interation between SIRT3 and DNPH. Data represent mean ± SD (n = 3). (C, D) PIG1 and PIG3V cells were treated with 1.0 mM H₂O₂ for 24 h. The mRNA and the protein levels of PGC1α in PIG1 and PIG3V cells were detected. Data represent mean ± SD (n = 3). (E) Enrichment of PGC1α to the promoter of SIRT3 after indicated treatment. Data are presented as mean ± SD. (F) Representative images of the expression of PGC1α (green) in healthy skin (n = 8) and vitiligo perilesional skin (n = 8) detected by using immunofluorescence. Melanocytes were stainied with antibody to Melan-A (red). Nuclei were counterstained with DAPI (blue). Scale bar = 50 μm (Magnification: the left = 600 ×; the right = 1800 ×). Intensity of PGC1α signal in melanocytes was quantified using Image J software. Bar graphs represent mean values of 8 samples and 10 fields of view per sample. Mean ± SD is shown. p value was calculated by two-tailed Student's t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, NS, non-significant.

Figure 7

HKL protects vitiligo melanocytes against oxidative stress by activating SIRT3-OPA1 axis. (A-B) PIG3V cells were pre-treated with 5 μM HKL for 24 h and then treated with 1.0 mM H₂O₂ for 24 h. The mRNA and the protein levels of SIRT3 were detected by qRT-PCR and immunoblotting. (C) SIRT3 activity of PIG3V cells with treatment as indicated was measured based on an enzymatic reaction using a SIRT3 activity assay kit. Data represent mean ± SD (n = 3). (D) The protein level of SOD2 and Ac-SOD2 in PIG3V cells with treatment as indicated were detected. Mean ± SD is shown (n = 3). (E) Representative confocal microscope images of the mitochondrial network in PIG3V cells with treatment as indicated. Scale bar = 50 μm (Magnification: the upper = 600 ×; the under = 1800 ×). The proportion of PIG3V cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. (F) The apoptosis level of PIG3V cells with treatment as indicated was examined by annexin V-FITC/PI staining. The mitochondrial ROS level of PIG3V cells with treatment as indicated was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. The mitochondrial membrane potential level of PIG3V cells with treatment as indicated was analyzed by JC-1 staining. Assessment of ATP level in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates. (G) Representative confocal microscope images of the mitochondrial network in PIG3V cells with treatment as indicated. si-NC refers to negative control small interfering RNA, si-OPA1 refers to small interfering RNA against OPA1. Scale bar = 50 μm (Magnification: the upper = 600 ×; the under = 1800 ×). The proportion of PIG3V cells (n=100 cells for each sample) with tubulated, intermediate and fragmented mitochondria was quantified. (H) The apoptosis level of PIG3V cells with treatment as indicated was examined by annexin V-FITC/PI staining. The mitochondrial ROS level of PIG3V cells with treatment as indicated was examined by MitoSOX™ Red mitochondrial superoxide indicator staining. The mitochondrial membrane potential level of PIG3V cells with treatment as indicated was analyzed by JC-1 staining. Assessment of ATP level in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates.(I) The level of apoptosis-related proteins in PIG3V cells with treatment as indicated was detected by immunoblotting. (J) The level of cytochrome c in mitochondria or cytoplasm in PIG3V cells with treatment as indicated was detected by western blotting. TOMM20 was detected as loading control for mitochondrial fraction. Tubulin was detected as loading control for cytosolic fraction. β-Actin was detected as loading control for the whole cell lysate. (K) Relative enzyme activity of respiratory complexes I, II, III, IV and V were measured in PIG3V cells with treatment as indicated. Data represent mean ± SD of triplicates. p value was calculated by two-tailed Student's t-test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 8

SIRT3-OPA1 pathway regulates mitochondrial dynamics and cell apoptosis in melanocytes under oxidative stress. Oxidative stress potentiates SIRT3 expression and activity in normal human melanocytes, but suppresses both the expression and the activity of SIRT3 in vitiligo melanocytes. SIRT3 deficiency in vililigo melanoncytes contributes to mitochondiral fission via inducing hyperacetylation of OPA1, leading to cytochrome c release from mitochondria to cytoplasm and deficient respiratory complex activities, which ultimately activates apoptotic pathway and results in melanocyte destruction in vitiligo.