Knock-in mouse models for the characterization of meprin metalloproteases in hyperkeratosis, inflammation and systemic sclerosis

皮肤中蛋白水解酶的失调会极大地影响表皮的动态平衡，这可能会导致严重的病理情况，如Netherton综合征或纤维化。MEPRIN金属蛋白酶的表达和活性增加与增生性疾病、炎症和系统性硬化症有关。基于小鼠模型和患者数据，我们建议MEPRIPS可能是增生性和纤维性疾病发生和发展的关键调节因子。直到最近，唯一可用于研究体内meprin活性的转基因小鼠是Mep1a-/-和Mep1b-/-小鼠，它们是完全敲除的，因此完全缺乏蛋白酶。在这些动物中，观察到皮肤胶原沉积受损，类似于患有埃勒-丹洛斯综合征的患者。此外，我们最近在Mep1a-/-小鼠身上证明了伤口愈合和VII型胶原成熟受损。其他分析Mep1a/b-/-单基因敲除小鼠和双基因敲除小鼠的研究表明，在疾病条件下，细胞因子水平和免疫表型发生了变化。然而，到目前为止，还没有一个人类纯合子功能丧失突变的病例被记录下来，这会导致meprinα或meprinβ活性完全丧失。因此，我们建议研究meprint的病理影响的合适模型是允许这些酶的细胞类型和组织特异性诱导表达的小鼠，正如在某些皮肤病中观察到的那样。为了实现这一点，我们产生了他莫昔芬诱导的小鼠，并将Mep1a或Mep1b基因插入到野生型C57BL/6小鼠的rosa26基因座中，其中包括一个C端的HA-tag。将这些小鼠与CRE驱动动物(Krt5-和Col1a2-CreERT2)杂交，可以增加角质形成细胞或成纤维细胞中meprin的表达。这些动物将展示增加的meprin活性是否足以导致角化过度、促炎和纤维化疾病的发生。我们最近通过在角质形成细胞中诱导meprinβ的表达(Krt5-creert2)来证明所提出的概念的可行性，从而导致细胞黏附减少和角化过度。观察到的表型与Mep1b在Fra-2tg小鼠中的表达增加很好地一致，这是银屑病和系统性硬化症的模型。在DFG资助的与Alexander Nyström博士(皮肤科，弗赖堡)合作实施的成功项目(Sign Remote)的基础上，我们将使用这些小鼠在体内鉴定可能的病理性meprin底物的蛋白质组，并为研究特定meprin抑制剂潜在治疗角化过度、炎症和纤维化的可行性提供基础。

Dysregulation of proteolytic enzymes in skin has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as Netherton syndrome or fibrosis. Increased expression and activity of meprin metalloproteases has been associated with hyperproliferative diseases, inflammation and systemic sclerosis. Based on mouse models and patient data we suggest meprins as possible key regulators in the onset and progression of hyperproliferative and fibrotic disorders. Until recently, the only genetically altered mice available to study meprin activities in vivo were Mep1a-/- and Mep1b-/- mice, which are full knock-outs and thus completely lack the proteases. In these animals impaired collagen deposition in skin was observed, similar to patients suffering from Ehler’s-Danlos-syndrome. Additionally, we recently demonstrated impaired wound healing and collagen VII maturation in Mep1a-/- mice. Other studies analyzing Mep1a/b-/- single and double knock-out mice revealed alterations in cytokine levels and immunological phenotypes under diseases conditions. However, to date, not a single case of a homozygous loss-of-function mutation in humans is documented that leads to a complete loss of meprin α or meprin β activity. Therefore, we propose that suitable models to study the pathological impact of meprins are mice that allow for cell type and tissue-specific induced expression of these enzymes as observed for certain skin diseases. To achieve this, we generated tamoxifen-inducible mice and inserted the Mep1a or Mep1b cDNA, including a C-terminal HA-tag, into the Rosa26 locus of wild-type C57BL/6 mice. Crossing these mice to Cre-driver animals (Krt5- and Col1a2-CreERT2) allows for increased meprin expression in keratinocytes or fibroblast. These animals will show whether increased meprin activity will be sufficient to induce the onset of hyperkeratosis, pro-inflammatory and fibrotic diseases. We recently demonstrated the feasibility of the proposed concept by inducing meprin β expression in keratinocytes (Krt5-CreERT2) leading to decreased cell adhesion and hyperkeratosis. The observed phenotype was nicely in line with increased Mep1b expression in Fra-2tg mice, which serve as a model for psoriasis and systemic sclerosis. Based on the successful DFG-funded Project (sign removed), which was performed in collaboration with Dr. Alexander Nyström (Dermatology, Freiburg), we will use these mice for proteomic identification of putative pathological meprin substrates in vivo and provide the basis to investigate the feasibility of specific meprin inhibitors for potential therapeutic treatment of hyperkeratosis, inflammation and fibrosis.