Marthaler Anna M, Podgorska Marta, Feld Pascal, Fingerle Alina, Knerr-Rupp Katrin, Grässer Friedrich, Smola Hans, Roemer Klaus, Ebert Elke, Kim Yoo-Jin, Bohle Rainer M, Müller Cornelia S L, Reichrath Jörg, Vogt Thomas, Malejczyk Magdalena, Majewski Sławomir, Smola Sigrun
Institute of Virology, Saarland University, Homburg/Saar, Germany.
Hartmann AG, Heidenheim, Germany.
PLoS Pathog. 2017 Jun 22;13(6):e1006406. doi: 10.1371/journal.ppat.1006406. eCollection 2017 Jun.
Patients suffering from Epidermodysplasia verruciformis (EV), a rare inherited skin disease, display a particular susceptibility to persistent infection with cutaneous genus beta-human papillomavirus (beta-HPV), such as HPV type 8. They have a high risk to develop non-melanoma skin cancer at sun-exposed sites. In various models evidence is emerging that cutaneous HPV E6 proteins disturb epidermal homeostasis and support carcinogenesis, however, the underlying mechanisms are not fully understood as yet. In this study we demonstrate that microRNA-203 (miR-203), a key regulator of epidermal proliferation and differentiation, is strongly down-regulated in HPV8-positive EV-lesions. We provide evidence that CCAAT/enhancer-binding protein α (C/EBPα), a differentiation-regulating transcription factor and suppressor of UV-induced skin carcinogenesis, directly binds the miR-203 gene within its hairpin region and thereby induces miR-203 transcription. Our data further demonstrate that the HPV8 E6 protein significantly suppresses this novel C/EBPα/mir-203-pathway. As a consequence, the miR-203 target ΔNp63α, a proliferation-inducing transcription factor, is up-regulated, while the differentiation factor involucrin is suppressed. HPV8 E6 specifically down-regulates C/EBPα but not C/EBPβ expression at the transcriptional level. As shown in knock-down experiments, C/EBPα is regulated by the acetyltransferase p300, a well-described target of cutaneous E6 proteins. Notably, p300 bound significantly less to the C/EBPα regulatory region in HPV8 E6 expressing keratinocytes than in control cells as demonstrated by chromatin immunoprecipitation. In situ analysis confirmed congruent suprabasal expression patterns of C/EBPα and miR-203 in non-lesional skin of EV-patients. In HPV8-positive EV-lesions both factors are potently down-regulated in vivo further supporting our in vitro data. In conclusion our study has unraveled a novel p300/C/EBPα/mir-203-dependent mechanism, by which the cutaneous HPV8 E6 protein may expand p63-positive cells in the epidermis of EV-patients and disturbs fundamental keratinocyte functions. This may drive HPV-mediated pathogenesis and may potentially also pave the way for skin carcinogenesis in EV-patients.
疣状表皮发育不良（EV）患者患有一种罕见的遗传性皮肤病，对皮肤β-人乳头瘤病毒（β-HPV）属的持续性感染表现出特殊易感性，如8型人乳头瘤病毒（HPV）。他们在阳光暴露部位发生非黑素瘤皮肤癌的风险很高。在各种模型中，越来越多的证据表明皮肤HPV E6蛋白会扰乱表皮稳态并促进致癌作用，然而，其潜在机制尚未完全了解。在本研究中，我们证明微小RNA-203（miR-203）是表皮增殖和分化的关键调节因子，在HPV8阳性的EV病变中强烈下调。我们提供证据表明CCAAT/增强子结合蛋白α（C/EBPα）是一种调节分化的转录因子，也是紫外线诱导皮肤致癌作用的抑制剂，它直接结合miR-203基因的发夹区域，从而诱导miR-203转录。我们的数据进一步表明，HPV8 E6蛋白显著抑制这种新的C/EBPα/mir-203途径。结果，miR-203的靶标ΔNp63α（一种诱导增殖的转录因子）上调，而分化因子内披蛋白受到抑制。HPV8 E6在转录水平上特异性下调C/EBPα的表达，但不影响C/EBPβ的表达。如敲低实验所示，C/EBPα受乙酰转移酶p300调节，p300是皮肤E6蛋白的一个众所周知的靶标。值得注意的是，通过染色质免疫沉淀证明，在表达HPV8 E6的角质形成细胞中，p300与C/EBPα调节区域的结合明显少于对照细胞。原位分析证实了EV患者非病变皮肤中C/EBPα和miR-203在基底上层的一致表达模式。在HPV8阳性的EV病变中，这两种因子在体内均被有效下调，进一步支持了我们的体外数据。总之，我们的研究揭示了一种新的p300/C/EBPα/mir-203依赖性机制，通过该机制皮肤HPV8 E6蛋白可能在EV患者的表皮中扩增p63阳性细胞并扰乱基本的角质形成细胞功能。这可能驱动HPV介导的发病机制，并可能为EV患者的皮肤致癌作用铺平道路。
