Decreased SUMOylation of the retinoblastoma protein in keratinocytes during the pathogenesis of vitiligo
Decreased SUMOylation of the retinoblastoma protein in keratinocytes during the pathogenesis of vitiligo
复制标题
白癜风发病过程中角质形成细胞中视网膜母细胞瘤蛋白的 SUMO 化减少
DOI：
10.3892/mmr.2018.9299
复制
发表时间：
2018-09-01
影响因子：
3.4
通讯作者：
Xu, Aie
中科院分区：
文献类型：
--
作者：
Zhou, Miaoni;Lin, Fuquan;Xu, Aie
SUMO化在白癜风发病机制中的作用尚未见报道。本研究旨在揭示白癜风患者色素脱失病变角质形成细胞中小泛素样修饰物（SUMO）结合的异常，并确认SUMO化在白癜风患者角质形成细胞中的作用。通过对6例患者的色素脱失病变进行穿刺活检，获得用于皮肤活检的皮肤样本。通过真空产生水泡，并收集顶部用于角质形成细胞培养。用SUMO 1敲低载体转导HaCaT细胞。Western blotting检测SUMO 1、SUMO特异性肽酶1（SENP 1）、泛素结合酶E2 I（Ubc 9）、SUMO激活酶亚基1（SAE 1）、细胞周期蛋白依赖性激酶（CDK）2、CDK 6、增殖细胞核抗原（PCNA）、视网膜母细胞瘤蛋白（Rb）、磷酸化Rb（pRb）和li-actin的蛋白表达。通过免疫沉淀法评估蛋白质的SUMO化状态。通过流式细胞术进行细胞周期分析，并使用Cell Counting Kit-8研究细胞增殖率。结果表明，与正常对照组相比，白癜风皮损和白癜风角质形成细胞中SUMO 1结合蛋白的水平降低。与正常角质形成细胞相比，白癜风角质形成细胞中Ubc 9蛋白表达减少，SENP 1蛋白表达增加，而SAE 1蛋白表达无变化。在HaCaT细胞中敲除SUMO 1后，HaCaT细胞的增殖降低，细胞周期被阻滞在G1期。此外，PCNA，CDK 2，CDK 6和pRb的蛋白表达水平在SUMO 1敲低的HaCaT细胞中降低，SUMO化的Rb在白癜风患者皮损的角质形成细胞中也明显低于正常角质形成细胞。总之，本研究中的白癜风病变表现出SUMO化和去SUMO化平衡失调，SUMO 1敲除HaCaT细胞中可能存在细胞周期进程失调。提示角质形成细胞Rb去SUMO化可能在白癜风发病中起重要作用，为白癜风发病机制的研究提供了新的方向。
The role of SUMOylation in the pathogenesis of vitiligo has not been reported previously. The present study aimed to reveal abnormalities in small ubiquitin-like 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 immunohistochcmistry 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, SUMO-specific peptidase 1 (SENP1), ubiquitin-conjugating enzyme E2 I (Ubc9), SUMO-activating enzyme subunit 1 (SAE1), cyclin-dependent kinase (CDK)2, CDK6, proliferating cell nuclear antigen (PCNA), retinoblastoma protein (Rb), phosphorylated Rb (pRb) and li-actin 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 Kit-8. The results demonstrated that the levels of SUMO1-conjugated 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 SUMO1-knockdown 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.