Exploring the association of POSTN+ cancer-associated fibroblasts with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) play a critical role in regulating TNBC tumor development. This study aimed to identify and characterize a specific subtype of CAFs associated with TNBC. Initially, using high-throughput bulk transcriptomic data in two cohorts, we identified three CAF-related subtypes (CS1, CS2, CS3) in TNBC samples. These three CAFs subtypes were closely linked to the tumor microenvironment. The CS1 subtype exhibited a relatively immune-rich microenvironment and a favourable prognosis, whereas the CS3 subtype displayed an immune-deprived tumor microenvironment and an unfavourable prognosis. Through WGCNA analysis, POSTN was identified as a key biomarker for CAFs associated with TNBC. Then, POSTN+CAFs was identified and characterized. Both POSTN and POSTN+CAFs showed significant positive correlations with stromal molecules HGF and MET at both the transcriptional and protein levels. Specifically co-localized with CAFs in the tumor stromal area, POSTN, produced by POSTN+CAFs, could modulate the HGF-MET axis, serving as a bypass activation pathway to regulate tumor cell proliferation in response to EGFR inhibitor and MET inhibitor. This study underscores the significance of POSTN and POSTN+CAFs as crucial targets for the diagnosis and treatment of TNBC.

Caffeic acid derivative WSY6 protects melanocytes from oxidative stress by reducing ROS production and MAPK activation.

Purpose: Vitiligo is a chronic depigmentation disease caused by a loss of functioning melanocytes and melanin from the epidermis. Oxidative stress-induced damage to melanocytes is key in the pathogenesis of vitiligo. WSY6 is a caffeic acid derivative synthesized from epigallocatechin-3-gallate (EGCG). This study is to investigate whether the new chemical WSY6 protected melanocytes from H2O2-induced cell damage and to elucidate the underlying molecular mechanism. Patients and methods: The present study compared the antioxidative potential of WSY6 with EGCG in hydrogen peroxide (H2O2)-treated PIG1 cells. Western blotting was used to study the protein expression of cyto-c, cleaved-caspase3, cleaved-caspase9, and the activation of MAPK family members, including p38, ERK1/2, JNK and their phosphorylation in melanocytes. ROS assay kit to detect intracellular reactive oxygen species production; CCK8 and lactate dehydrogenase leak assay to detect cytotoxicity. Results: EGCG and WSY6 ameliorated H2O2-induced oxidative stress damage in PIG1 cells in a does-dependent manner, while WSY6 was much more effective. WSY6 reduced cellular ROS production, cytochrome c release, downregulated caspase-3 and caspase-9 activation. MAPK pathway signaling including phosphorylated p38, ERK and JNK were observed under oxidative stress and can be much protected by pre-treatment of WSY6. Conclusion: These results indicated that WSY6 could be a more powerful antioxidant than EGCG and protect melanocytes against oxidative cytotoxicity.

EGR1 Mediated Reduction of Fibroblast Secreted-TGF-ß1 Exacerbated CD8+ T Cell Inflammation and Migration in Vitiligo.

Vitiligo is a T cell-mediated depigment skin disease caused by the complex interplay between melanocyte dysfunction, environmental stimulation, and dysregulated immune signals. Transforming growth factor-ß1 (TGF-ß1), which typically derives from regulatory T cells, has long been identified at low levels in the peripheral system of vitiligo patients. Here, through RNA-sequencing and transcription factor enrichment, we revealed that in response to CD8+ T cell-secreted interferon-gamma (IFN-γ), stromal fibroblast downregulates early growth response 1 (EGR1) activity, leading to TGF-ß1 deficiency. The defective immune regulation loop further exacerbated local CD8+ T cell inflammation and promoted inflammatory cell migration in vitiligo. Thus, fibroblast-derived TGF-ß1 plays an important stromal signal in vitiligo pathogenesis.

Pathogenic Th2 Cytokine Profile Skewing by IFN-γ-Responding Vitiligo Fibroblasts via CCL2/CCL8.

PURPOSE: Vitiligo is a T cell-mediated skin depigmentation disease. Though treatments arresting disease progression and inducing repigmentation are available, the efficacy of these options is often limited and poorly sustained. How stromal signals contribute to the interferon-γ-dominant skin niches is unclear. This study aims to determine how fibroblasts participate in the IFN-γ-dominant vitiligo niche. PATIENTS AND METHODS: Mouse vitiligo models were established. Fibroblasts from control and vitiligo mice were extracted for RNA sequencing. In vitro IFN-γ stimulation was performed to verify the JAK-STAT pathway by qPCR and Western blot. T cell polarization with chemokines was measured by flow cytometry. Protein levels in tissues were also examined by IHC. RESULTS: The vitiligo mouse model recapitulates the human CD8-IFN-γ pathway. RNA sequencing revealed elevated chemokine CCL2 and CCL8 in vitiligo fibroblast, which may be regulated by the JAK-STAT signaling. Such phenomenon is verified by JAK inhibitor peficitinib in vitro. Moreover, CCL2 addition into the naïve T polarization system promoted type 2 cytokines secretion, which represents a hallmark of vitiligo lesions. CONCLUSION: Dermal fibroblasts, a principal constituent of skin structure, respond to IFN-γ by skewing T cells towards a type 2 cytokine profile via CCL2 and CCL8, which can be abrogated by JAK inhibitor peficitinib.

Narrowband Ultraviolet B Combined with Topical Simvastatin Solution in the Treatment of Vitiligo: A Case Report.

Background: Phototherapy is one of the treatments for vitiligo. To be specific, the combination of narrowband ultraviolet B (NB-UVB) with topical preparations has currently become the most common therapeutic modality. Moreover, the research on new topical drug has been a hot issue in the field of vitiligo. Objective: At present, simvastatin has been considered as a potential therapeutic agent for the treatment of vitiligo. To the best of our knowledge, this is the first case report concerning the successful application of NB-UVB combined with topical simvastatin in the treatment of vitiligo. Methods: In this article, a clinical case report was presented, where the patient was not responsive to NB-UVB but was markedly responsive to the treatment of UVB combined with topical simvastatin. Results: A 34-year-old Chinese female patient with vitiligo was cured by NB-UVB combined with topical simvastatin solution. Conclusions: NB-UVB combined with topical simvastatin may be a potential treatment against vitiligo. This research was approved by the Medical Ethics Committee of Third People's Hospital of Hangzhou.

[Corrigendum] The role of VIT1/FBXO11 in the regulation of apoptosis and tyrosinase export from endoplasmic reticulum in cultured melanocytes.

Following the publication of the above article, an interested reader drew to the authors' attention that the Transwell cell migration assay data shown in Fig. 4A appeared to be partly overlapping with data presented for experiments performed under different experimental conditions in Figs. 4D and E. The authors independently examined the figure and realized that inadvertent errors had been made during the assembly of Fig. 4; furthermore, owing to the time that has elapsed since this paper was published, the authors no longer had access to the original data. Accordingly, to further verify the conclusions reported in the study, the authors repeated these experiments, and the results obtained were found to be consistent with the original findings. The new version of Fig. 4 is shown below. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 26: 57-65, 2010; DOI: 10.3892/ijmm_00000435].

Inhibition of Fam114A1 protects melanocytes from apoptosis through higher RACK1 expression.

Fam114A1 is a gene closely related to the development of nerve cells, melanocytes, and nerve cells that originate from the neural crest of the embryonic ectoderm. Recent studies showed that Fam114A1 has a role in the occurrence of ankylosing myelitis spondylitis and autoimmune enteritis; still, its cellular function remains poorly understood. In this study, we investigated the effect of Fam114A1 on the biological activity of melanocytes. We found that the expression of Fam114A1 in vitiligo melanocytes (MCV-L, MCV-N, PI3V) was higher than that in normal melanocytes, and the biological function of melanocytes was significantly affected when the Fam114A1 gene was silenced. Inhibition of Fam114A1 increased proliferation, migration, and melanin synthesis proteins, decreased apoptosis, while its overexpression reversed this process. Mechanistically, we discovered that RACK1 is a target protein of Fam114A1 and that RACK1 can be negatively regulated by Fam114A1. Further study showed that Fam114A1 inhibition could not protect melanocytes from apoptosis once the expression of RACK1 protein was silenced. In summary, Fam114A1 is an effective regulatory protein for regulating the function of melanocytes. Inhibition Fam114A1 protects melanocytes from apoptosis through increasing RACK1.

CXCL9 as a key biomarker of vitiligo activity and prediction of the success of cultured melanocyte transplantation.

This study aimed to investigate the potential biomarkers of vitiligo by evaluating the disease activity and curative effect of autologous cultured pure melanocyte transplantation (CMT) on patients. Altogether, 36patients with stable vitiligo were treated with CMT. Blister fluid samples were collected from patients with stable vitiligo. Patients with active vitiligo were matched with healthy controls. The chemokine levels in the serum and blister fluid samples were measured using Luminex. The curative effect on patients with stable vitiligo was evaluated 6 months after treatment. Treatment responses were defined according to the extent of repigmentation as effective (if 50% or more repigmentation was achieved) or ineffective (if less than 50% or worse repigmentation was achieved). Patients received re-transplantation if the initial treatment was ineffective. The levels of C-X-C motif chemokine ligand (CXCL)9 and CXCL10 in blister fluid samples were significantly lower in stable patients than in active participants. Receiver operating characteristic analysis revealed that the levels of CXCL9 and CXCL10 were sensitive and specific in diagnosing active vitiligo. Further, 65.6% (21/32) of patients who received CMT had effective treatment responses. The high CXCL9 level in the blister fluid was a significant predictor of ineffective treatment responses. The treatment response was significantly enhanced after treatment. Four patients with ineffective treatment responses received anti-inflammatory treatment and re-transplantation. The CXCL9 and CXCL10 levels in the blister fluid were related to the presence of active vitiligo. Also, the CXCL9 level was a predictor of the effectiveness of CMT in treating vitiligo.

Topical epigallocatechin-3-gallate in the treatment of vitiligo.

OBJECTIVE: To preliminarily assess the efficacy and safety of the topical application of Epigallocatechin-3-gallate (EGCG) in treating vitiligo, a 6-month clinical trial was carried out. METHOD: Patients were randomly given topical application of EGCG on the assigned lesions, with pimecrolimus being used as the control for twice a day over a 6-month treatment period. Responses to treatment were assessed based on the changes in VASI score for percentage reduction in body surface area and the PGA scores. RESULTS: According to our results, both drugs were discovered to be markedly effective on repigmentation. The VASI of lesion had diminished from 1.19 ± 0.42 to 0.63 ± 0.38, in the EGCG-treated lesions, while from 1.18 ± 0.43 to 0.61 ± 0.36 in the pimecrolimus-treated lesions, and there was no statistically significant difference in VASI score between the EGCG-treated lesions and pimecrolimus-treated lesions (P = 0.755). Meanwhile, the mean PGA score on the EGCG applied side was 4.39 ± 2.23, while that was 4.43 ± 2.02 on the pimecrolimus applied side (P = 0.886). Furthermore, difference in the improvement degree between pimecrolimus side and EGCG side was not statistically significant (P = 0.845). Notably, no serious side effects were observed throughout the study. CONCLUSION: Findings of the study indicate that topical EGCG can be effective on treating vitiligo.

Decreased SUMOylation of the retinoblastoma protein in keratinocytes during the pathogenesis of vitiligo.

The role of SUMOylation in the pathogenesis of vitiligo has not been reported previously. The present study aimed to reveal abnormalities in small ubiquitin­like modifier (SUMO) conjugation in keratinocytes from depigmented lesions of patients with vitiligo and confirm the role of SUMOylation in keratinocytes from patients with vitiligo. Skin samples used for immunohistochemistry were obtained by punch biopsy from the depigmented lesions of 6 patients. Blisters were produced by vacuum and the roofs were collected for keratinocyte culture. HaCaT cells were transduced with SUMO1 knockdown vectors. The protein expression of SUMO1, SUMO­specific peptidase 1 (SENP1), ubiquitin­conjugating enzyme E2 I (Ubc9), SUMO­activating enzyme subunit 1 (SAE1), cyclin­dependent kinase (CDK)2, CDK6, proliferating cell nuclear antigen (PCNA), retinoblastoma protein (Rb), phosphorylated Rb (pRb) and ß­actin was assessed by western blotting. The SUMOylation status of proteins was assessed by immunoprecipitation. Cell cycle analysis was performed by flow cytometry and cell proliferation rate was investigated using a Cell Counting Kit­8. The results demonstrated that the levels of SUMO1­conjugated proteins were decreased in vitiligo lesions and vitiligo keratinocytes compared with normal controls. The protein expression of Ubc9 was decreased and SENP1 was increased in vitiligo keratinocytes compared with normal keratinocytes, with no alterations in SAE1 expression. Following knockdown of SUMO1 in HaCaT cells, the proliferation of HaCaT cells was reduced and the cell cycle was arrested in G1 phase. Furthermore, the protein expression levels of PCNA, CDK2, CDK6 and pRb were reduced in SUMO1­knockdown HaCaT cells, and SUMOylated Rb was also decreased markedly in keratinocytes from lesions of patients with vitiligo compared with normal keratinocytes. In conclusion, vitiligo lesions in the present study exhibited dysregulated SUMOylation and deSUMOylation balance and dysregulation of cell cycle progression may be present in SUMO1 knockdown HaCaT cells. These results indicate that deSUMOylation of Rb of keratinocytes may serve an important role in vitiligo, providing a novel direction for the study into the mechanism of vitiligo.

Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes.

Swollen endoplasmic reticulum (ER) is commonly observed in the melanocytes of vitiligo patients; however, the cause and proteins involved in this remain to be elucidated. Oxidative stress has been reported to be involved in the pathogenesis of vitiligo and previous studies have demonstrated that hydrogen peroxide (H2O2) induced melanocyte apoptosis, whereas quercetin exhibited cytoprotective activities against the effects of H2O2. The aim of the present study was to further investigate the role of H2O2 in the ER of melanocytes as well as its role in the export of tyrosinase from ER; in addition, the present study aimed to determine the mechanism by which quercetin protects against the effects of H2O2. The results demonstrated that melanocyte cells treated with H2O2 presented with swollen ER; however, a normal ER configuration was observed in untreated cells as well as quercetin/H2O2­treated cells. Furthermore, H2O2 inhibited tyrosinase export from the ER and decreased expression levels of tyrosinase; however, quercetin was found to attenuate the effects induced by H2O2. In conclusion, the results of the present study confirmed the hypothesis that H2O2 induced ER dilation and hindered functional tyrosinase export from the ER of melanocytes. It was also found that quercetin significantly weakened these effects mediated by H2O2, therefore it may have the potential for use in the treatment of vitiligo.

Interferon-γ induces senescence in normal human melanocytes.

BACKGROUND: Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear. OBJECTIVE: To investigate the effects of IFN-γ on viability and cellular functions of melanocytes. METHODS: Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. ß-galactosidase staining was utilized to evaluate melanocyte senescence. RESULTS: Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of ß-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes. CONCLUSION: IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.

[Impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome].

OBJECTIVE: To determine the impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome. METHODS: Western blot was used to detect 26S proteasome from 8 vitiligo patients and 4 healthy controls. Melanocytes were incubated with proteasome inhibitor (lactacystin) and further detected as follows: cell survival by MTT assay, proteasome activity with fluorescence, ultrastructure observation with electron microscope, co-localization of tyrosinase and calreticulin (endoplasmic reticulum marker) by confocal laser scanning microscopy and 26S proteasome and tyrosinase with Western blot. RESULTS: The 26S proteasome expression level from lesions of vitiligo (1.05 ± 0.40) was significantly lower than the donor sites (1.82 ± 0.88) and the healthy controls (1.88 ± 0.16) (P < 0.05). But no significant difference existed between the latter two groups (P > 0.05). Compared to the untreated group, a 12-h incubation of 10 µmol/L lactacystin showed inhibitory effects on melanocytes (0.999 ± 0.110 vs 1.372 ± 0.127, P < 0.05) and significantly decreased proteasome activity (0.234 ± 0.019 vs 1, P < 0.01). Expansion rate of endoplasmic reticulum in the lactacystin group (1.91 ± 0.17) was significantly higher than that of the untreated cells (1.17 ± 0.11) (P < 0.05). More tyrosinase co-localized with calreticulin in endoplasmic reticulum in lactacystin-treated cells was observed than that of the untreated group. Compared with the untreated group, significantly decreased levels of tyrosinase (146 ± 10 vs 269 ± 8, P < 0.01) and tyrosinase activity (0.159 ± 0.017 vs 0.221 ± 0.019, P < 0.01) were shown in the lactacystin group (P < 0.05). CONCLUSIONS: Significantly decrease of 26S proteasome is found in lesions of vitiligo patients. Inhibition of 26S proteasome may lead to expansion of endoplasmic reticulum of melanocytes, impact export of tyrosinase from melanocyte endoplasmic reticulum and expression of tyrosinase.

Dermal mesenchymal stem cells (DMSCs) inhibit skin-homing CD8+ T cell activity, a determining factor of vitiligo patients' autologous melanocytes transplantation efficiency.

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients' autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.

CD8+ T cells from vitiligo perilesional margins induce autologous melanocyte apoptosis.

Cell-mediated autoimmunity has been suggested to be involved in the melanocyte apoptosis that occurs in vitiligo. We investigated the cytotoxicity to autologous melanocytes of CD8+ T cells from the perilesional margins and peripheral blood samples of vitiligo patients. CD8+ T cells isolated from skin biopsied from the edges of depigmented skin patches of vitiligo patients or from peripheral blood samples of the same donors were proliferated in culture medium. The primary cultures of CD8+ T cells and autologous melanocytes were mixed at ratios of 1:1, 1:2 or 1:5 and incubated for 3 days. The apoptosis of the melanocytes was analyzed by flow cytometry. Secreted cytokines in selected samples were measured by cytokine arrays. The results show that the CD8+ T cells were successfully isolated from the vitiligo perilesional margins. This cell population showed a significantly higher percentage of CD69 expression (56.13±3.55 versus 29.93±2.35%, p<0.01) and CD137 expression (41.74±1.06 versus 25.97±1.63%, p<0.01) compared with CD8+ T cells in peripheral blood from the same donors. The co-culturing of CD8+ T cells from lesional skin with autologous melanocytes induced apoptosis in the melanocytes (16.63±1.21, 16.71±0.63 and 18.32±1.60% for CD8+ T cells and autologous melanocytes at ratios of 1:1, 1:2 and 1:5, respectively). IL-6 levels were much higher in the co-culture (3.01-fold higher than in a melanocyte monoculture and 17.32-fold higher than in a CD8+ T-cell monoculture). The CD8+ T cells were also demonstrated to secrete more IL-13. Taken together, our data demonstrate that the infiltration of active CD8+ T cells takes place in the vitiligo perilesional margins. Those CD8+ T cells present significantly higher activation levels and higher cytotoxicity to autologous melanocytes than their counterparts from peripheral blood samples. These data suggest that CD8+ T cells are likely to be involved in the pathogenesis of vitiligo.

Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes.

Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data have shown that AKT activation is involved in the cell survival process. Yet, the potential mechanism of niacin in protection against UV-induced skin damage has thus far not fully been eluvidated. We observed that niacin pretreatment enhances UV induced activation of AKT (Ser473 phosphorylation) as well as that of the downstream signal mTOR (S6 and 4E-BP1 phosphorylation). The PI3K/AKT inhibitor, LY294002, and the mTOR inhibitor, rapamycin, largely neutralized the protective effects of niacin, suggesting that AKT and downstream signaling mTOR/S6 activation are necessary for the niacin-induced protective effects against UV-induced cell death and cell apoptosis. Collectively, our data suggest that niacin may be utilized to prevent UV-induced skin damage and provide a novel mechanism of its photoprotective effects against the UV radiation of sunlight by modulating both AKT and downstream mTOR signaling pathways.

Immunodetection of the MCHR1 antibody in vitiligo patient sera.

Human vitiligo is an acquired depigmenting skin disorder characterized by milk-white skin macules resulting from a chronic and progressive loss of melanocytes. It has been suggested that autoimmune mechanisms are involved in this disorder. We undertook this project to obtain an MCHR1-C linear peptide containing four main MCHR1 B-cell epitopes in an attempt to detect the IgG antibody against MCHR1 in vitiligo. The target gene encoding the MCHR1-C peptide was cloned into a pGEX-4T-2 expression vector, expressed in Escherichia coli BL21, and purified using a GST column. The molecular weight was analyzed by SDS-PAGE and western blotting. The IgG antibody response to MCHR1 was detected by ELISA with MCHR1-C as a coated antigen, and the absorption experiment was used to assess the binding ability of the Ab detected by MCHR1-C with a membrane protein in human epidermal melanocytes. The purified peptide MCHR1-C with a molecular weight of 33 kDa was obtained, and could bind to the MCHR1 antibody in human sera. Of the vitiligo patient sera examined, 24 of 145 (16.55%) tested positive for the MCHR1 antibody, and the frequency in the vitiligo patient group was significantly greater compared to the normal control. However, no significant difference between gender, disease duration, clinical subtype or family history between the two groups was observed. In addition, the Ab detected by MCHR1-C in sera was specifically absorbed by the membrane protein obtained from human epidermal melanocytes. Collectively, our data suggest that the MCHR1-C peptide can be used to detect the MCHR1 antibody in vitiligo patients as a coated antigen instead of MCHR1 protein. We conclude that the level of MCHR1 antibody in active vitiligo patients is significantly higher than that in healthy individuals, while gender, disease duration, clinical subtype and family history have no association with the level of MCHR1 antibody in vitiligo patients.

[Expression of InnVit/FBXO11 in vitiligo and its role in tyrosinase export from endoplasmic reticulum].

OBJECTIVE: To investigate the roles of InnVit (FBX011) gene in melanocytes by detecting the expression of InnVit gene in vitiligo and analyzing the impact of InnVit gene on morphology of endoplasmic reticulum (ER) and the tyrosinase export from ER. METHODS: The lesion tissues and the donor tissues were collected from 10 vitiligo patients to examine the InnVit gene expression by immunohistochemistry. Synthesized specific siRNA and constructed plasmid P3XF-P120 were separately transfected into cells for the silence and over-expression of InnVit gene with lipofectamine(TM) 2000. The untreated cells were used as control. Morphology of ER of cells from the above three groups was observed under electron microscope. Co-localization of tyrosinase and calreticulin was identified by confocal laser scanning microscopy. InnVit, tyrosinase and calreticulin were examined by Western blot. RESULTS: In vitiligo patients, the expression of InnVit gene in the lesions was markedly lower than that in the donor tissues. The normal morphology of ER was found in the untreated and the plasmid groups whereas inflated ER was shown in siRNA group. And the relative inflation rate in siRNA group (1.97 +/- 0.48) was higher than that in the untreated group (1.28 +/- 0.09) and plasmid group (1.24 +/- 0.13) (both P = 0.001). In the untreated and the plasmid groups, tyrosinase was expressed beyond the scope marked by ER marker protein calreticulin partly, but co-localized with calreticulin in ER in the siRNA group. Western blot showed that, contrast to the untreated group (0.320 +/- 0.020), a lower expression level of InnVit in the siRNA group (0.030 +/- 0.004, P = 0.001) and a higher expression of InnVit in the plasmid group were shown (0.710 +/- 0.040, P = 0.001). No significant difference about the expression level of calreticulin was observed among the three groups (P > 0.05). As compared with the untreated group (0.350 +/- 0.030), a higher tyrosinase level in the siRNA group (1.040 +/- 0.060, P = 0.001) and in the plasmid group (0.720 +/- 0.030, P = 0.001) was found. And the former was higher than the latter (P = 0.001). CONCLUSION: A lower expression of InnVit is observed in the lesion tissues than in the donor tissues from vitiligo patients. The InnVit gene can have an impact on the morphology of ER and tyrosinase export from ER. And it may further affect the function of melanocytes.

The role of VIT1/FBXO11 in the regulation of apoptosis and tyrosinase export from endoplasmic reticulum in cultured melanocytes.

Our previous study has shown that VIT1 gene in Chinese vitiligo patients is de facto the FBXO11 gene, and the silencing of that gene has an impact on the ultrastructure of melanocytes. In this study, we further identified the role of the FBXO11 gene in melanocytes and the relationship between dilated endoplasmic reticulum (ER) and tyrosinase by inhibition and overexpression of FBXO11 gene. Cell proliferation, apoptosis, cycle and migration of melanocytes were examined when the FBXO11 gene was silenced or overexpressed. The results showed that FBXO11 gene promoted cell proliferation and suppressed cell apoptosis, and yet had little effect on cell migration. Obvious swelling of ER was found in the cells transfected with siRNA of FBXO11 gene. Interestingly, protein level of tyrosinase was extraordinarily high following inhibition of FBXO11 gene. Further examination revealed that tyrosinase and calreticulin were co-localized in ER of transfected cells following siRNA of FBXO11 gene, suggesting that tyrosinase could not be exported from ER effectively. Collectively, our results support the notion that FBXO11 plays an important role in regulating proliferation and apoptosis of melanocytes, and functional export of tyrosinase from ER in vitiligo melanocytes.

[Abnormal nuclear translocation of nuclear factor-E2 related factor 2 in the lesion of vitiligo].

OBJECTIVE: To investigate whether abnormal translocation of nuclear factor-E2 related factor 2 (Nrf2) exists in the lesion of vitiligo. METHODS: Skin specimens from 8 vitiligo patients and 3 healthy controls were collected, half of them underwent laser co-focal microscopy to detect the Nrf2 location and half of them underwent cell culture. Blister fluid was collected form the 8 vitiligo patients and skin donor sites to detect the levels of serum superoxide dismutase (SOD), catalase (CAT), and malonyldialdehyde (MDA) by using detection kit. Expression of Nrf2 in epidermal cell of the 8 vitiligo patients and primary epidermal cell of the 3 healthy controls was identified with cell immunofluorescence histochemistry method. The nuclear and cytoplasmic proteins of all above samples were isolated to be identified by Western blotting. RESULTS: The levels of SOD and CAT in the lesion tissue were significantly lower than those in the skin donor site. The levels of MDA in the lesion tissue were significantly higher than those in the skin donor sites (both P < 0.05). Immunofluorescence histochemistry, showed that Nrf2 was predominantly cytoplasmic in the epidermal cells in the lesion, while Nrf2 expression could be seen in both the cytoplasm and nucleus in the epidermal cells in the normal skin donor sites and skins of the healthy controls. Western blotting showed that the nuclear Nrf2 level in the vitiligo skin lesion was (0.11 +/- 0.03), significantly lower than that in the normal skin donor site (0.27 +/- 0.06) and in the skins of the healthy controls (0.32 +/- 0.02) (both P < 0.01). However, there was no significant difference in the Nrf2 level of in cytoplasm among the three types of tissues (0.63 +/- 0.04, 0.61 +/- 0.03, and 0.65 +/- 0.04, all P > 0.05). CONCLUSION: Nrf2 does not translocate from cytoplasm into the nucleus in the lesion of vitiligo patients.