Abstract
Abstract: :
Purpose:Aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in the corneal epithelium and is considered to play a significant role in cellular defense mechanisms against oxidative damage. We have recently shown that ALDH3A1 may be involved in the control of corneal epithelial cell growth by inhibiting proliferation (Invest Ophthalmol Vis Sci, 44:922, 2003). The aim of this study was to investigate the mechanisms by which ALDH3A1 exerts such an inhibitory effect on corneal epithelial cell proliferation. Methods: Kinase assays and Western blot analysis were used to determine cyclin–dependent kinase activities and the levels of cell cycle–regulatory proteins in human corneal epithelial (HCE) and human keratinocyte (NCTC 2544) cell lines stably transfected with human ALDH3A1 cDNA. The intracellular distribution of ALDH3A1 in these cell lines was assessed by confocal microscopy and Western blot analysis. Expression of ALDH3A1 was monitored in primary explant cultures of human corneal epithelium by RT–PCR, Western blotting and immunofluorescence. Results: We found a strong inhibition of cyclin A– and cyclin B–dependent kinase activities as well as inhibition of phosphorylation of the retinoblastoma protein (pRb) in ALDH3A1–transfected cells compared to control. This inhibition was associated with a down regulation of cyclins A, B and E, transcription factor E2F1, and the cell–regulatory protein p21. Interestingly, although ALDH3A1 is known to be cytosolic, a considerable amount of this protein was also localized in the nucleus. In human explant cultures, ALDH3A1 decreased with time and outgrowth of the epithelial cells and was not observed when cells reached confluence. Conclusions: These results provide evidence that ALDH3A1 may be involved in the control of cell growth by modulating levels and activities of cell cycle regulatory proteins. The nuclear localization of the protein raises the possibility for a nuclear function associated with cell proliferation. The loss of ALDH3A1 expression in human primary corneal epithelial cells maintained in culture further supports the involvement of ALDH3A1 in regulating proliferation. It is possible that ALDH3A1 by influencing cell proliferation may promote corneal epithelial cell repair and survival.
Keywords: cornea: basic science • proliferation • crystallins