MVK在汗孔角化症发生发展过程中的作用及分子机制研究

Porokeratosis is recognized as an inherited epidermal keratinization disorder, which ruins the sufferers’ appearances and may also be prone to malignant tumor formation. Up to date, there is no effective treatment for porokeratosis. Mutations in MVK gene have been found to cause porokeratosis. We also detected a novel mutation P11S, which made the mutated protein less stable than the wild type (WT). Nevertheless, the stability alteration could not elucidate the pathogenesis of porokeratosis clearly. In our previous work, we found caspase-14, which degradated filaggrin to protect keratinocytes from environment stress, was down regulated by P11S. However, caspase-14 bound to neither MVK WT nor P11S. MVK WT and P11S shared the same location in cytoplasm, which indicated neither of them could translocate into nucleus to affect the expression of caspase-14. Meanwhile, we found that Lamin A/C especially bound to MVK WT but not P11S detected by Mass Spectrometry. This project will involve an in depth analysis of the molecular mechanism of how MVK regulated the expression of caspase-14, and whether the regulation was associated with Lamin A/C. Further investigation into the pathogenesis of porokeratosis caused by MVK mutation will help to improve treatment strategies and may lead to a better understanding of the disease mechanisms that are essential to provoke the development of target therapeutic strategies.

汗孔角化症是一种遗传性皮肤角质化异常疾病，影响人的外貌，且有癌变可能，目前没有特别有效的治疗办法。MVK是汗孔角化症的致病基因，但是致病原因仍然未知。前期工作中我们发现了导致汗孔角化症的MVK致病突变P11S，其稳定性比野生型WT显著性降低；进一步发现MVK可以调控caspase-14的表达；质谱分析MVK与核纤层蛋白Lamin A/C存在相互作用，但MVK致病突变体则丧失这一功能。本项目将在前期工作的基础上，深入分析MVK是否通过Lamin A/C调节caspase-14的表达，从而影响对filaggrin的水解以及对细胞的保护。该研究将阐明MVK P11S突变导致汗孔角化症发生的分子机制，并为汗孔角化症提供可能的靶向治疗方案。

MVK是汗孔角化症的致病基因。前期工作中，本人在汗孔角化症患者的MVK基因中发现了新的致病突变P11S，本项目旨在研究突变对MVK功能造成的影响以及导致汗孔角化症发生的分子机制。本研究的主要研究内容有三项：（1）在人角质形成细胞HaCaT中，分别转染MVK WT野生型与MVK P11S突变型质粒，观察HaCaT中caspase-14的本底表达水平，检测MVK突变后是否可以影响caspase-14的表达；（2）分析MVK在转录水平对caspase-14的调控机制；（3）检测MVK对caspase-14的转录水平进行调节，在HaCaT细胞中分别转染MVK WT及P11S质粒后，利用qPCR技术检测caspase-14的mRNA表达是否受到影响。研究结果发现：（1）MVK在正常人及汗孔角化症患者的表皮层中皆有表达，但汗孔角化症患者的病灶处表达位置异常，真皮层中也有大量聚集性表达，提示皮肤结构异常:；（2）HaCaT细胞转染了MVK WT/P11S质粒后，显微镜明场观察发现细胞更容易出现凋亡情况，提示MVK突变后促进角质形成细胞发生异常凋亡；（3）通过Western blotting检测MVK突变后会降低caspase-14的表达；（4）通过qPCR检测MVK突变后不影响caspase-14的转录水平；（5）caspase-14表达的降低可能通过溶酶体降解途径；（6）通过Western blotting检测MVK突变后下调caspase-14的表达，同时也抑制了caspase-14对底物filaggrin的降解；（7）通过qPCR检测MVK突变后不影响filaggrin的转录水平。本研究推测，MVK突变后会降低caspase-14的蛋白水平表达，从而降低对filaggrin的水解作用，可能导致表皮屏障功能受损，引发了汗孔角化症。该研究拓宽了对汗孔角化症发病机制的理解，有可能对后续治疗或者药物研发有启示作用。