[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.

[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.

[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.

[Association of single nucleotide polymorphisms of Nrf2 promoter region with susceptibility to vitiligo].

OBJECTIVE: To investigate the association of the single nucleotide polymorphisms (SNPs) in Nrf2 promoter region with the susceptibility to risk of vitiligo. METHODS: Samples of peripheral blood were collected from 300 vitiligo patients and 300 healthy persons. The genotypes of -686A/G, -684G/A, and -650C/A were detected by direct-sequencing. Genotyping of variable number of tandem repeat (VNTR) was performed by gene scan analysis with an ABI 310 Sequencer. Genetic and allelic frequencies were analyzed by Chi-square test and the risk was evaluated by calculating OR and 95% CI. RESULTS: There was statistical significant difference in genotypic and allelic frequencies of -650C/A between the vitiligo group and healthy control group (P < 0.05), and A -650 allele was associated with risk for vitiligo statistically significantly (OR = 1.724, 95% CI: 1.345-2.211, chi2 = 18.096, P < 0.01). Homozygote of A allele increased the risk for vitiligo obviously (OR = 2.902, 95% CI: 1.624-5.188, P < 0.01). No significant difference was found in other three polymorphisms between the two groups. CONCLUSION: polymorphism of Nrf2 promoter region -650C/A was associated with the development of vitiligo and A -650 allele may be one of risk factors for vitiligo.

The susceptibility to vitiligo is associated with NF-E2-related factor2 (Nrf2) gene polymorphisms: a study on Chinese Han population.

Vitiligo is an acquired pigmentary disorder and its pathogenesis remains unclear. Oxidative stress is considered to be the initial pathogenic event in the melanocyte destruction. NF-E2-related factor2 (Nrf2) is a transcription factor regulating the expression of detoxifying and antioxidant genes. To investigate the association of the Nrf2 gene promoter polymorphisms with vitiligo in Chinese Han population, the genotypes of -686A/G, -684G/A and -650C/A and the genotyping of variable number of tandem repeat were detected. The data were analysed by the chi-square test and the risk was evaluated by calculating OR and 95% CI. There was statistically significant difference in genotypic and allelic frequencies of -650C/A between the two groups (P < 0.05). A(-650) allele was significantly associated with the risk for vitiligo (OR = 1.724, chi(2) = 18.096). Polymorphism of the Nrf2 gene promoter at -650C/A was associated with the development of vitiligo and A(-650) allele may be one of the risk factors.

[Expression and purification of epitope peptide of human tyrosinase and its antigenicity in vitiligo patients].

OBJECTIVE: To express the epitope peptide of human tyrosinase (TYR), and discuss the application of the peptide in detecting autoantibody of the vitiligo patients. METHODS: The epitope areas 240 - 255, 289 - 294, 295 - 300, 435 - 447, and 461 - 479 of human TYR were synthesized and connected to the vector pGEM-T. The target gene was cloned to the prokaryotic expression vector pGEX-4T-2, which was then transferred to Escherichia coli BL21 host cells. Isopropy-beta-D-thiogalactoside (IPTG) was used to induce the protein expression that was examined with SDS-PAGE and Western blotting. Indirect ELISA was conducted to detect the antigenicity of the peptide in 100 blood specimens of active vitiligo patients and 30 healthy controls. RESULTS: The recombinant expression vector was constructed successfully. The SDS-PAGE and Western blotting results showed expression of the recombinant protein in E. coli. The amount of the recombinant protein reached about 70% of the total mass of bacterial protein with PAGE analysis system. With the glutathione S-transferase (GST) purification kit, the purity of recombinant protein reached over 90%. Indirect ELISA showed that reaction with the target protein was negative in all the 30 healthy controls and was positive in 64 of the 100 active vitiligo patients. CONCLUSION: The epitope peptide of human TRY is expressed successfully, and it has antigenicity in the serum of vitiligo patients.

[Coding-sequence mutation and polymorphism analysis of EDNRB gene in patients with Hirschsprung's disease from Zhejiang region].

To determine the characters of EDNRB gene in patients with sporadic Hirschsprung's disease (HD) and discuss the relationship between mutation of EDNRB gene and HD, seventy five sporadic HD cases and forty normal cases as control were collected and DNA was extracted from peripheral white blood cells by standard method. All seven exons of EDNRB gene were analyzed by single strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP) and direct sequencing. In result, A G --> A transition in codon 277 was observed in exon 4 in six patients. This variation leads to a nonsense mutation (L277L) and is a previously described polymorphism, the rate of variation is 8% (6/75). A G --> A transition in codon185 was detected in exon 2 in two patients, this is a novel heterozygous mutation, leading to a missense mutation (V185M), and the rate of mutation is 2.7% (2/75). So we can conclude that mutation of EDNRB gene can be detected in Chinese patients with sporadic HD and EDNRB gene might play an important role in the pathogenesis of HD.