Stramer B M, Cook J R, Fini M E, Taylor A, Obin M
Vision Research Laboratories, New England Eye Center, Tufts University School of Medicine, Tufts University, Boston, MA 02111, USA.
Invest Ophthalmol Vis Sci. 2001 Jul;42(8):1698-706.
To examine dynamics and function of the ubiquitin (Ub)-proteasome pathway (UPP) during corneal stromal cell acquisition of the repair fibroblast phenotype.
An established cell culture model was used in which freshly isolated rabbit corneal stromal cells acquire a repair fibroblast phenotype, thereby mimicking injury-induced stromal cell activation.
Transition to the repair fibroblast phenotype during the 72 hours after initial plating was coincident with progressive UPP induction. Levels of Ub, Ub-conjugated proteins, ubiquitinylating enzymes E1 and E2-25K, and 26 S proteasome increased two- to fivefold in activated stromal cells. These increases were associated with enhanced (>10-fold) capacity for Ub-dependent proteolysis of (125)I-labeled H2A and with progressive (>6-fold) increases in the UPP substrate, inhibitor of kappaBalpha (IkappaBalpha). Because IkappaBalpha expression is induced by nuclear factor (NF)-kappaB, this finding suggests that rates of constitutive NF-kappaB activation, and thus IkappaBalpha degradation, are elevated in activated stromal cells. Both freshly isolated and activated stromal cells degraded IkappaBalpha in response to IL-1alpha; yet, only activated stromal cells maintained autocrine IL-1alpha expression after 24 hours. UPP induction was coincident with a more than 90% loss of tissue transketolase (TKT) and aldehyde dehydrogenase (ALDH) class 1. TKT was stabilized during the repair phenotype transition by proteasome inhibition and was degraded (>30%/h) by the UPP in cell-free assays.
Coordinate induction of the UPP during stromal cell activation alters levels of IkappaBalpha and TKT, two UPP substrates that are implicated in the loss of tissue stasis and corneal clarity after injury.
研究角膜基质细胞获得修复性成纤维细胞表型过程中泛素(Ub)-蛋白酶体途径(UPP)的动态变化及功能。
采用已建立的细胞培养模型,其中新鲜分离的兔角膜基质细胞获得修复性成纤维细胞表型,从而模拟损伤诱导的基质细胞活化。
初始接种后72小时内转变为修复性成纤维细胞表型与UPP的逐渐诱导相一致。在活化的基质细胞中,Ub、Ub缀合蛋白、泛素化酶E1和E2-25K以及26S蛋白酶体水平增加了2至5倍。这些增加与(125)I标记的H2A的Ub依赖性蛋白水解能力增强(>10倍)以及UPP底物κBα抑制因子(IkappaBalpha)的逐渐增加(>6倍)相关。由于IkappaBalpha表达由核因子(NF)-κB诱导,这一发现表明活化的基质细胞中组成型NF-κB活化率以及因此的IkappaBalpha降解率升高。新鲜分离的和活化的基质细胞均响应IL-1α而降解IkappaBalpha;然而,只有活化的基质细胞在24小时后维持自分泌IL-1α表达。UPP诱导与组织转酮醇酶(TKT)和1类醛脱氢酶(ALDH)超过90%的丧失同时发生。在修复表型转变过程中,TKT通过蛋白酶体抑制得以稳定,并且在无细胞试验中被UPP降解(>30%/小时)。
基质细胞活化过程中UPP的协同诱导改变了IkappaBalpha和TKT的水平,这两种UPP底物与损伤后组织稳态丧失和角膜透明度下降有关。