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Ayan Chatterjee, Sarah Oh, Guadalupe Villarreal, Douglas Rhee; Characterization of Sirtuin family protein expression in human trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3552.
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© ARVO (1962-2015); The Authors (2016-present)
The Sirtuin (Sirt) family of proteins is a group of NAD+-dependent deacetylases that have been associated with the regulation of aging in mammals. Their expression profile in human trabecular meshwork (TM) cells at baseline and under conditions associated with glaucoma pathogenesis have not yet been characterized. We examined whether the Sirt family of proteins would be expressed at detectable levels in primary cultured human TM cells and whether their expression profile may be altered in response to treatment with TGF-β2 or dexamethasone.
Human TM tissues were isolated and primary TM cells were established from cadaveric donors ranging in age from 42 to 69. TM cell cultures were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 20% fetal bovine serum, 1% L-glutamine (2 mM), and gentamicin (10 µg/mL) at 37°C in 10% CO2 atmosphere. All experiments used TM cells in the fourth to fifth passage. TM cells were treated with either 3 ng/mL TGF-β2 or equal volumes of 4 mM HCl vehicle, added directly to the culture media. TM cells were treated with 100 nM dexamethasone or equal volumes of 0.004% ethanol vehicle, added directly to the culture media. Integrated band intensities were calculated from immunoblots using LiCor Image Studio software and normalized using β-actin. Student t-tests were used for statistical analyses.
All seven Sirt family proteins were detectable by immunoblot in human TM tissues (n=4) and also in cultured TM cells (n=4). Sirt family protein expression levels did not change significantly in 24-hour TGF-β2-treated TM cells versus control cells (n=4). However, Sirt3 protein levels were increased by 20.02 ± 4.68% (n=4; p=0.0052) in 24-hour dexamethasone-treated cells versus control cells. No other Sirt proteins showed statistically significant changes due to treatment with the specified conditions.
Our findings suggest that all seven Sirt family proteins are expressed in human TM. The baseline Sirt family expression profile does not change in response to 24-hour TGF-β2 treatment but does change in response to dexamethasone treatment. In cultured human TM cells, 24-hour dexamethasone treatment increases Sirt3 protein levels.
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