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CC Zimmerman, D Vollrath, U Weitgasser, JR Polansky; Studies Of Differentially Regulated Genes In GC-treated Confluent HTM Cells Show Evidence For Parallel Regulation Of Alzheimer's-related Gene Products And TIGR/MYOC . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3957.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To explore the regulation of selected genes associated with Alzheimer's disease compared to TIGR/MYOC in our steroid-induced HTM cell model using semi-quantitative RT-PCR. The model was previously used to define TIGR/MYOC as a candidate gene for glaucoma based on its induction characteristics. Methods: Differential effects on gene expression were examined using confluent HTM cultures exposed to 1-2 day and more prolonged 7-14 day DEX (100 to 500 nM) treatments with and without bFGF (100 to 1000 pM). Highly induced genes were identified in an initial screening using microarray methods, with particular attention paid to those showing characteristics similar to TIGR/MYOC (i.e. substantial delayed, progressive inductions by dexamethasone (DEX) and blocking effects of bFGF). Specific primers were designed for RT-PCR for several of these genes, focusing on potential Alzheimer's-related products. Two products associated with the extracellular matrix (ECM) that showed putative reductions in the microarray studies were evaluated in parallel. Semi-quantitative methods using several different numbers of cycles (20, 25, 30) were used to obtain reproducible results in different experiments. Real time PCR was used to define estimated induction levels for some experiments. Results: A number of Alzheimer's-related products were confirmed as having substantial, delayed DEX inductions similar to TIGR/MYOC, including AACT (alpha-1 antichymotrypsin), APOD (apolipoprotein D), and SAA1 and SAA4 (serum amyloid A precursor proteins). A partial reversal of these inductions was seen with bFGF. In comparison, MMP1 (matrix metalloproteinase-1) THBS1 (thrombospondin-1), were confirmed as being substantially reduced in the DEX-treated cultures, without a significant bFGF reversal being observed. Real time PCR studies for TIGR/MYOC, AACT, and APOD show induction levels of greater than 500-fold. Conclusion: The finding that prolonged DEX treatment of HTM cells results in the substantial induction of mRNA for proteins associated with Alzheimer's disease may provide new clues to glaucoma pathogenic mechanisms. Further studies are needed to determine the relationship between changes in mRNA levels and protein levels for these genes, and to investigate potential interactions with both native and mutant TIGR/MYOC proteins.
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