Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa
Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa
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DOI：
10.1073/pnas.1803154115
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发表时间：
2018-07-10
影响因子：
11.1
通讯作者：
Chen, Mei
中科院分区：
文献类型：
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作者：
Lincoln, Vadim;Cogan, Jon;Chen, Mei
赫利茨交界性大疱性表皮松解症(H-Jeb)是一种无法治愈的、毁灭性的、大多是致命的遗传性皮肤病，目前只有支持性治疗。H-Jeb是由LAMA3、LAMB3或LAMC2功能缺失突变引起的，导致层粘连蛋白332完全丧失，层粘连蛋白332是锚定细丝的主要成分，介导表皮-真皮黏附。LAMB3(层粘连蛋白β3)突变占H-Jeb患者的80%，与H-Jeb相关的LAMB3突变中95%是导致提前终止密码子(PTCs)的无义突变。在这项研究中，我们评估了庆大霉素在H-Jeb层粘连蛋白β3缺失角质形成细胞中诱导PTC通读的能力，该细胞转染了编码八种不同LAMB3无义突变的表达载体。我们发现庆大霉素在所有测试的八个无义突变中都能诱导PTC通读。接下来，我们使用慢病毒载体产生了具有R635x和c290x无义突变的稳定转导的H-Jeb细胞。这些细胞系与不同浓度的庆大霉素孵育后，以剂量依赖和持续的方式合成和分泌全长层粘连蛋白β3。重要的是，庆大霉素诱导的层粘连蛋白β3恢复了层粘连蛋白332在真皮/表皮交界处的组装、分泌和沉积，并通过免疫印迹分析、免疫荧光显微镜和体外三维皮肤等效模型评估了基底角质形成细胞中α6β4整合素的适当极化。最后，新修复的层粘连蛋白332通过逆转H-Jeb细胞异常的细胞形态、生长能力差、细胞-基质粘附性差和运动过度，纠正了H-Jeb细胞的异常细胞表型。因此，庆大霉素可能为H-Jeb和其他由PTC突变引起的遗传性皮肤病提供一种治疗方法。
Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin beta 3) mutations account for 80% of patients with H-JEB, and similar to 95% of H-JEB-associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin beta 3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin beta 3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin beta 3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of alpha 6 beta 4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.