Abstract
Abstract: :
Purpose: The purpose of this study was to test the hypothesis that the adherence of conjunctival epithelial cells to those laminin isoforms resident in normal conjunctival basement membrane elicits differential gene expression. Methods: Human conjunctival epithelial cells (the HC0597 virally transformed cell line) were cultured on BSA–coated plastic tissue culture plates or plates coated with 10 µg/ml laminin–2 for 24 hours. Cells were cultured in serum–free keratinocyte growth medium to confluency. A TransSignal p53 Target Gene Array kit was purchased from Panomics (Redwood City, CA). Total RNA was isolated from the cells using the Qiagen RNeasy kit, and reverse–transcribed using the primer mix supplied with the array kit to created biotin–labeled cDNA probes. The probes were hybridized to the supplied array membranes, and binding was visualized by chemiluminescence. Relative expression of target genes was determined by densitometry and is reported as the average integrated density values (IDV). Results: By comparing average IDV we determined that the expression levels of a number of genes differed (increased or decreased) by over two–fold in HC0597 cells adherent to laminin–2 compared to BSA control. These genes included matrix remodeling factors (MMP–2 and PAI–1) and regulators of cell proliferation (PNUTL2, DD3, p53). Additionally, p53–induced genes involved in controlling apoptosis were regulated by laminin–2 (PPM1D, Bax). Conclusions: We have hypothesized that ocular surface epithelial cell adhesion to laminin isoforms may regulate cell proliferation, adhesion, and differentiation. Analysis of the expression of p53 target genes suggests that in human conjunctival epithelial cells in vitro, laminin–2 may play roles in regulating cell cycle dynamics, apoptosis, and matrix remodeling.
Keywords: conjunctiva • extracellular matrix • gene screening