Ries Christian, Popp Tanja, Egea Virginia, Kehe Kai, Jochum Marianne
Division of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University of Munich, Nussbaumstrasse 20, 80336 Munich, Germany.
Toxicology. 2009 Sep 1;263(1):26-31. doi: 10.1016/j.tox.2008.08.011. Epub 2008 Sep 3.
Matrix metalloproteinases (MMPs), especially MMP-9 and MMP-2, degrade various proteins of the extracellular matrix, including collagen type IV the major component of basement membranes which also separate the epidermis from the dermis. Although previous work indicates the contribution of MMPs and their inhibitors (TIMPs) to the pathophysiology of skin lesions induced by the toxic chemical warefare agent sulphur mustard (SM), little is known about the underlying molecular and cellular mechanisms. In this study we demonstrate in a 3D-skin model that topical application of SM significantly upregulated basal MMP-9 mRNA expression and release from the cells as shown by qRT-PCR and zymography, whereas that of MMP-2, membrane-type 1 (MT1)-MMP, TIMP-1 and TIMP-2 remained almost unaffected by SM. Further studies in neonatal human dermal fibroblasts (NHDF) and HaCaT keratinocytes revealed that MMP-9 was not secreted from these cells, neither with or without exposure to SM. However, when NHDF and HaCaT were cocultivated, MMP-9 was expressed and released from the cell mixture, suggesting that interaction between both cell types is essential for MMP-9 production. Moreover, SM-treatment of NHDF/HaCaT cocultures further upregulated MMP-9 biosynthesis and secretion, which was consistent with our findings obtained in the 3D-skin model. Addition of conditioned medium derived from SM-exposed HaCaT cells to NHDF was able to stimulate MMP-9 secretion and also increased the migratory potential of NHDF as shown in a scratch-wound healing assay and a fluorescent cell invasion assay. In contrast, culture supernatants of SM-treated NHDF had not such an effect on HaCaT cells. Taken together, our findings provide first evidence that SM exposure of skin stimulates keratinocytes to release soluble factors which in turn induce enhanced MMP-9 secretion and invasiveness of fibroblasts in vitro. This provides a potential mechanism probably contributing to SM-evoked tissue injury in vivo.
基质金属蛋白酶（MMPs），尤其是MMP-9和MMP-2，可降解细胞外基质的各种蛋白质，包括IV型胶原蛋白，它是基底膜的主要成分，基底膜也将表皮与真皮分隔开来。尽管先前的研究表明MMPs及其抑制剂（TIMPs）在由有毒化学战剂硫芥（SM）引起的皮肤损伤的病理生理学中发挥了作用，但对于其潜在的分子和细胞机制知之甚少。在本研究中，我们在三维皮肤模型中证明，如qRT-PCR和酶谱分析所示，局部应用SM可显著上调基底MMP-9 mRNA的表达并促进其从细胞中的释放，而MMP-2、膜型1（MT1）-MMP、TIMP-1和TIMP-2的表达和释放几乎不受SM的影响。对新生儿人真皮成纤维细胞（NHDF）和HaCaT角质形成细胞的进一步研究表明，无论是否暴露于SM，这些细胞均不分泌MMP-9。然而，当NHDF和HaCaT共培养时，MMP-9从细胞混合物中表达并释放，这表明两种细胞类型之间的相互作用对于MMP-9的产生至关重要。此外，对NHDF/HaCaT共培养物进行SM处理可进一步上调MMP-9的生物合成和分泌，这与我们在三维皮肤模型中获得的结果一致。将来自暴露于SM的HaCaT细胞的条件培养基添加到NHDF中，能够刺激MMP-9的分泌，并且如划痕愈合试验和荧光细胞侵袭试验所示，还增加了NHDF的迁移潜力。相比之下，经SM处理的NHDF的培养上清液对HaCaT细胞没有这种作用。综上所述，我们的研究结果首次证明，皮肤暴露于SM会刺激角质形成细胞释放可溶性因子，进而诱导成纤维细胞在体外增强MMP-9的分泌和侵袭能力。这提供了一种可能导致体内SM诱发组织损伤的潜在机制。
