Regulation of melanocyte chemokine expression by folliculin protein in vitiligo patients / 中华医学美学美容杂志

Objective:To investigate the effect of human tumor suppressor folliculin (FLCN) on the expression of melanocyte chemokines (MC) mediated by immune factors in vitiligo.Methods:The MC of vitiligo patients that received autologous melanocyte transplantation in the Department of Dermatology, Hangzhou Third People′s Hospital from January to April 2019 were collected. The blister fluid of the white spot and the normal part was taken. Western blot was used to analyze the expression difference of MC and FLCN protein in normal, vitiligo patients and that induced by immune factors; FLCN shRNA lentivirus was constructed by shRNA and transfected into normal MC (FLCN shRNA MC) to interfere with the expression of silenced FLCN gene. The effect of immune factors on chemokines in FLCN shRNA MC was detected by ELISA.Results:The results of Western blot showed that FLCN protein was highly expressed in melanocytes of vitiligo patients, immune factors stimulated FLCN protein expression in normal melanocytes significantly increased ( t=1.27; P<0.001), chemokine CXCL10 and CCL20 also significantly increased ( t=104.53 and 60.21, respectively; P<0.001). The expression of FLCN in FLCN shRNA MC was significantly decreased ( F=1.95, P<0.001); and the high expression of CXCL10 and CCL20 induced by immune factors was significantly inhibited ( F=93.676 and 74.096, all P<0.001). Conclusions:Immune factors can stimulate the expression of CXCL10 and CCL20, which are closely related to vitiligo, while FLCN is a key protein involved in immune factors inducing melanocyte chemokine expression.

Role of folliculin in interferon-γ-mediated apoptosis of and chemokine secretion by melanocytes / 中华皮肤科杂志

Objective:To investigate the role of folliculin in apoptosis of and chemokine secretion by melanocytes mediated by interferon-γ (IFN-γ) .Methods:Normal primary melanocytes were isolated from circumcised foreskin tissues from a healthy male child, and primary vitiliginous melanocytes were isolated from normally pigmented suction-blistered epidermis from patients with vitiligo after suction blister epidermal grafting. Western blot analysis was performed to determine the folliculin protein expression in normal primary melanocytes, primary vitiliginous melanocytes and a human primary melanocyte line PIG1. PIG1 cells stimulated with 10 ng/ml IFN-γ for 48 hours served as induction group, and untreated PIG1 cells served as control group. Real-time quantitative RCR (qRT-PCR) was performed to determine the mRNA expression of folliculin, autophagy-related microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ and Beclin genes, and Western blot analysis to determine the protein expression of folliculin, Beclin1 and LC3Ⅱ/Ⅰ, as well as phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in the above cells. Furthermore, the melanocytes stimulated with 10 ng/ml IFN-γ for 48 hours were divided into several groups: negative control group infected with an empty lentiviral vector, folliculin inhibition group infected with a folliculin-inhibiting lentivirus, autophagy enhancement group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with a mTOR inhibitor, autophagy inhibition group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with an AMPK inhibitor. Then, flow cytometry was conducted to detect apoptosis of PIG1 cells, and enzyme-linked immunosorbent assay to measure the concentration of chemokines CXCL10 and CCL20 in the culture supernatant of PIG1 cells in the above groups. Measurement data were compared among multiple groups by using one-way analysis of variance, and multiple comparisons were carried out by using least significant difference- t test. Results:The relative protein expression level of folliculin significantly differed among the normal primary melanocytes (0.850 ± 0.120) , primary vitiliginous melanocytes (1.507 ± 0.170) and PIG1 cells (0.697 ± 0.130; F = 50.09, P < 0.001) , and was significantly higher in the primary vitiliginous melanocytes than in the normal primary melanocytes and PIG1 cells ( t = 4.06, 5.89, respectively, both P < 0.01) . Compared with the control group, the induction group showed significantly increased relative mRNA and protein expression levels of folliculin (both P < 0.01) , but significantly decreased relative mRNA and protein expression levels of LC3Ⅱ and Beclin (all P < 0.01) ; moreover, the induction group showed significantly decreased LC3Ⅱ/Ⅰ levels (0.72 ± 0.02) and AMPK phosphorylation levels (0.714 ± 0.023) in the PIG1 cells compared with the control group (1.13 ± 0.02, 1.176 ± 0.002, t = 7.34, 6.67, respectively, both P < 0.01) , but significantly increased mTOR phosphorylation levels (1.051 ± 0.023) compared with the control Group (0.451 ± 0.016, t = 3.81, P = 0.009) . There were significant differences in the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 among the control group, induction group and other treatment groups (all P < 0.001) ; specifically, the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 were significantly higher in the induction group than in the control group, lower in the folliculin inhibition group than in the negative control group, lower in the autophagy enhancement group than in the folliculin inhibition group, and higher in the autophagy inhibition group than in the folliculin inhibition group (all P < 0.05) . Conclusions:Folliculin is highly expressed in vitiliginous melanocytes. Folliculin expression and downstream signaling pathways are regulated by IFN-γ, and folliculin may participate in IFN-γ-mediated melanocyte apoptosis and chemokine secretion via regulating autophagy.

Two Cases of Birt-Hogg-Dube Syndrome with Pulmonary Cysts / 대한내과학회지

Birt-Hogg-Dube (BHD) syndrome is a rare autosomal-dominant disease caused by germline folliculin (FLCN) mutations, characterized by fibrofolliculoma or trichodiscoma, renal tumors, and multiple lung cysts with or without spontaneous pneumothorax. Here, we report two cases of BHD syndrome that presented with bilateral pulmonary cysts. One patient was a 39-year-old woman who had a history of pneumothorax, multiple papules on her cheeks, and a family history of the same skin lesions and renal cell carcinoma in her father. BHD syndrome was confirmed by molecular tests that revealed a missense mutation in FLCN gene (exon 4, c.31T > C). The other patient was a 56-year-old man who showed FCLN gene polymorphism and typical radiopathological features of multiple cysts in the lung, but apparently no other manifestation.

Birt-Hogg-Dube Syndrome, a Rare Case in Korea Confirmed by Genetic Analysis

Simple benign tumors can present as part of a syndrome with substantial mortality. Fibrofolliculomas are benign skin tumors most often associated with the Birt-Hogg-Dube syndrome (BHDS). The most life-threatening complication of this syndrome is renal cancer and other major features include multiple lung cysts and spontaneous pneumothorax. We present the case of a 54 year-old man with multiple flesh-colored papules on his face confirmed histologically as fibrofolliculomas. He had a history of recurrent pneumothorax and chest computed tomography showed multiple lung cysts. To confirm the diagnosis of BHDS, we conducted gene analysis that revealed a single nucleotide duplication in the folliculin (FLCN) gene (Exon 11, C.1285dupC). BHDS confirmed by the FLCN gene mutation is rarely reported in Korea. Appropriate investigation is recommended whenever a patient with benign skin tumors is encountered.