Abstract
Purpose: :
Our goal was to characterize the expression of matrix metalloproteinases (MMPs), their physiological inhibitors (TIMPs) and other extracellular matrix (ECM) genes during lens fiber cell differentiation in a model of non–immortalized human lens epithelial cells cultured on ECM. MMPs and TIMPS have been studied in adult postmorten human lens, in post–cataract lens capsular bags and in immortalized human lens cells exposed to cytokines and other stressors. MMPs have also been studied in rat lens epithelial and fiber cells and TIMPs in whole lenses. Significant differences exist in the expression patterns reported with these various model systems.
Methods: :
A cultured human lens cell model (Blakely et al., IOVS 41:3808, 2000) was used for these studies. Lens cells were grown on matrix–coated substrate. RNA was harvested at different stages of fiber cell differentiation, as well as after 55 MeV proton irradiation. For gene array analysis, total RNA were reverse transcribed, PCR amplified and processed per directions provided by the manufacturer (Superarray Biosciences, Corp, MD). The biotin–labeled PCR amplicons were hybridized to human ECM gene family profiles, and applied to ECL film for fluorescent signal detection. Background noise signals were subtracted from the signal intensities for each gene spot and normalized to the positive housekeeping controls in each filter. In addition, RNA was characterized using the Human MultiGene–12TM Matrix Metaloproteinase RT–PCR Profiling Kit (Superarray), and the PCR products were analyzed by gel electrophoresis. Completely independent replicate experiments were completed.
Results: :
Differentiating lens epithelial cells demonstrated differences in ECM gene expression. High levels of MMP 1, 2, 3, 11, 14, 15, 16, 17, and TIMP 1, 2, 4, were expressed in epithelial cells, whereas MMP 1, 2, 9, 11, 13, 14, 15, 16, 17, 24, 26, and TIMP 1, 2, 4, were expressed in fiber cells. Low levels of MMP 7, 9, 10, 13, 24 and 26 were expressed in epithelial cells, whereas MMP 3, 7, 10 were expressed in fiber cells. Both cell types did not express MMP 8, 12, and 20 and TIMP 3. Irradiated epithelial cells showed upregulation of MT1–MMP, while irradiation fiber cells showed downregulation of MT1–MMP, MMP–16 and MMP–2 and upregulation of MMP–1
Conclusions: :
We have evidence for cell–specific expression of MMPs, TIMPs and other ECM genes during lens fiber differentiation on ECM, and for proton radiation induced alteration in the regulation of ECM gene expression.