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对表皮稳态有巨大影响，可导致内瑟顿综合征或纤维化等严重病理状况。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，我们认为meprins可能是增殖性和纤维化疾病发生和进展的关键调节因子。直到最近，唯一可用于体内研究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，观察到皮肤中胶原沉积受损，与患有Ehler - danlos综合征的患者相似。此外，我们最近证明了Mep1a-/-小鼠的伤口愈合和VII胶原成熟受损。其他分析Mep1a/b-/-单敲除和双敲除小鼠的研究揭示了疾病条件下细胞因子水平和免疫表型的变化。然而，迄今为止，没有一例人类纯合子功能缺失突变导致meprin α或meprin β活性完全丧失。因此，我们建议研究meprins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，正如在某些皮肤疾病中观察到的那样。为此，我们构建了他莫昔芬诱导小鼠，并将Mep1a或Mep1b cDNA（包括c端ha标签）插入野生型C57BL/6小鼠的Rosa26位点。将这些小鼠与cre驱动动物（Krt5-和Col1a2-CreERT2）杂交可以增加meprin在角质形成细胞或成纤维细胞中的表达。这些动物将显示meprin活性的增加是否足以诱导角化过度、促炎和纤维化疾病的发生。我们最近通过在角质形成细胞（Krt5-CreERT2）中诱导meprin β表达，导致细胞粘附减少和角化过度，证明了所提出概念的可行性。观察到的表型与Fra-2tg小鼠Mep1b表达增加完全一致，Fra-2tg小鼠是牛皮癣和系统性硬化症的模型。基于与Dr. Alexander Nyström （Dermatology, Freiburg）合作完成的成功的dfg资助项目（已删除符号），我们将使用这些小鼠在体内对假定的病理性meprin底物进行蛋白质组学鉴定，并为研究特异性meprin抑制剂用于角化过度、炎症和纤维化的潜在治疗的可行性提供基础。

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.