Abstract
Abstract: :
Purpose: The membrane-spanning mucin MUC4 is a major mucin of the ocular surface, primarily expressed in conjunctival epithelia in humans. In animal models, vitamin A deficiency downregulates the expression of mucins, but little is known regarding regulation of the MUC4 gene in human ocular surface epithelium. The purpose of this study was to identify potential regulators of MUC4 in ocular surface epithelium using a TERT-immortalized human conjunctival epithelial cell line. Methods: The TERT-immortalized human conjunctival epithelial cell line was derived as previously described (Gipson et al. IOVS 42:S484, 2001). For this study, the immortalized cells were cultured in GIBCO Keratinocyte Serum Free Medium (K-sfm), followed by culture in a 1:1 mixture of K-sfm and low calcium DMEM/F12 to confluence. At confluence, the cells were cultured in DMEM/F12 with 10% calf serum and 10 ng/ml EGF for 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, and 7 days. In other experiments, the cells were cultured in DMEM/F12 without serum and EGF but with all trans retinoic acid (RA) (0, 1 nM, 100 nM, 1000 nM) for 96 h. In a third set of experiments, the cells were cultured with TNF-alpha (20 ng/ml), or dexamethasone (10-6M) for 24 h following 24 h serum starvation. Total RNA was isolated from the cells, and after DNase treatment, reverse transcription (RT) and polymerase chain reaction (PCR) were performed. The relative expression level of MUC4 mRNA was determined using real-time RT-PCR. Results: MUC4 mRNA was undetectable in cells cultured without serum or up to 3 hours after addition of serum to the culture medium. Furthermore, real-time RT-PCR revealed that treatment with serum resulted in a continued increase in the level of MUC4 mRNA in cultures from 6 h to 72 h with a decrease to the 48 h level by day 7. Treatment with 100 nM RA in the absence of serum induced MUC4 mRNA expression. No MUC4 mRNA was detected with 1 and 1000 nM RA, and at the concentration of 1000 nM, RA was found to be toxic to the cells. Culture with TNF-alpha and dexamethasone did not affect the expression of MUC4 mRNA. Conclusion: Serum and vitamin A induce upregulation of MUC4 mRNA expression in the TERT-immortalized human conjunctival epithelial cell line. Since there is lack of MUC4 expression in the keratinized epithelium of vitamin A deficient rats (Tei et al. IOVS 41:82-8, 2000), and since RA induces MUC4 expression, we infer that MUC4 is an important hydrating molecule of the ocular surface. The clinical efficacy of applying autologous serum, containing vitamin A, to treat severe dry eye may be related to the upregulation of expression of MUC4.
Keywords: 365 conjunctiva • 375 cornea: surface mucins • 376 cornea: tears/tear film/dry eye