Li F, Goncalves J, Faughnan K, Steiner M G, Pagan-Charry I, Esposito D, Chin B, Providence K M, Higgins P J, Staiano-Coico L
Department of Surgery, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, USA.
Exp Cell Res. 2000 Aug 1;258(2):245-53. doi: 10.1006/excr.2000.4918.
In the adult epidermis, keratinocytes do not normally express the type-1 inhibitor of plasminogen activator (PAI-1). Basal epithelial cell-specific PAI-1 synthesis, however, accompanies epidermal wound repair in vivo in which PAI-1 transcripts and immunoreactive protein are confined to epithelial cells in the migrating tongue and the hyperproliferative zone. A model system using human keratinocytes (HaCaT cells) was developed to assess functional relationships between epithelial growth state transitions and PAI-1 expression. PAI-1 synthesis was maximal in low population density, exponentially growing HaCaT cultures; relative PAI-1 mRNA and protein levels progressively declined as cells attained, and were maintained in, a postconfluent condition. While the fraction of PAI-1(+) keratinocytes remained stable (at approximately 85-90% of the population) throughout the culture period, both PAI-1 mRNA abundance and mean cell-associated PAI-1 protein declined by >90% during prolonged (i.e., 8-day) growth arrest. Similar to epidermal trauma in vivo, scrape wounding of HaCaT monolayers resulted in the rapid and location-specific induction of PAI-1 protein (an increase of 11- to 16-fold relative to unwounded cultures) in cells immediately bordering the injury site. PAI-1 expression was evident in keratinocytes that comprised the opposed migrating fronts and remained elevated until wound closure. Down-regulation of PAI-1 synthesis in HaCaT cells transfected with an inducible LacSwitch-based antisense vector system markedly impaired both the rate and the extent of wound closure. All injuries created in antisense PAI-1 monolayers remained unhealed at day 8 postinjury compared to the 3-day complete repair typical of control cultures. Vector-driven modulation of PAI-1 synthesis was also associated with an increase in the percentage of suprabasal-type keratinocytes within the wound field. PAI-1 expression by migrating HaCaT cells appears necessary to maintain the basal epidermal phenotype and/or appropriate cell-to-substrate adhesion during injury repair.
在成人表皮中，角质形成细胞通常不表达纤溶酶原激活物-1型抑制剂（PAI-1）。然而，在体内表皮伤口修复过程中会出现基底上皮细胞特异性PAI-1合成，其中PAI-1转录本和免疫反应性蛋白局限于迁移舌部和增殖活跃区的上皮细胞。开发了一种使用人角质形成细胞（HaCaT细胞）的模型系统，以评估上皮生长状态转变与PAI-1表达之间的功能关系。在低细胞密度、呈指数生长的HaCaT培养物中，PAI-1合成最高；随着细胞达到汇合后状态并维持该状态，PAI-1相对mRNA和蛋白质水平逐渐下降。虽然在整个培养期间，PAI-1(+)角质形成细胞的比例保持稳定（约占细胞群体的85-90%），但在长时间（即8天）生长停滞期间，PAI-1 mRNA丰度和平均细胞相关PAI-1蛋白均下降了>90%。与体内表皮创伤相似，刮伤HaCaT单层细胞会导致紧邻损伤部位的细胞中PAI-1蛋白迅速且在特定位置诱导表达（相对于未受伤培养物增加11至16倍）。PAI-1表达在构成相对迁移前沿的角质形成细胞中很明显，并一直升高直到伤口闭合。用基于可诱导LacSwitch的反义载体系统转染的HaCaT细胞中PAI-1合成的下调显著损害了伤口闭合的速度和程度。与对照培养物典型的3天完全修复相比，反义PAI-1单层细胞在损伤后第8天所有造成的损伤仍未愈合。载体驱动的PAI-1合成调节还与伤口区域内基底上层型角质形成细胞百分比的增加有关。迁移的HaCaT细胞表达PAI-1似乎是在损伤修复过程中维持基底表皮表型和/或适当的细胞与底物粘附所必需的。
