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Frances Fan, Anna Maria Maraschi, Annalisa Montemari, Chaunte Stampley, Craig Crosson, Manuela Bartoli; TGF Beta Stimulates Lysosomal Activity Through Translocation of TFEB in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4649.
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Increased intraocular pressure by lysosomal dysfunction within trabecular meshwork (TM) cells has been shown to play a role in the pathogenesis of glaucoma. Transforming growth factor beta (TGF-beta) is known to alter TM cell homeostasis and activity, also affecting the lysosomal proteolytic system of these cells. Recently, it has been discovered that lysosome-mediated degradative pathways are under the control of a specific gene network, CLEAR (Coordinated Lysosomal Expression and Regulation), that is regulated by the transcription factor EB (TFEB) in HeLa cells. Here we examined TFEB expression and localization in human TM cells (hTM) treated with TGF-beta, to determine whether the lysosomal functionality in TM cells is under TFEB regulation.
Human TM cells were stimulated with TGF-beta (2ng/ml) for different times (6, 12, 24 and 48 hours). Stimulation with sucrose (100mM) for the same time points was performed in hTM as a control for increased lysosomal activity. Control cells were treated with vehicle only. Western analysis was used to assess the expression levels of TFEB. Intracellular localization of TFEB was determined by immunocytochemical analysis and by nuclear fractionation studies.
Western blotting analysis revealed that TFEB is expressed in hTM and is actively translocated in the nuclear compartment of these cells in response to sucrose treatment (an activator of lysosomal activity). In addition, stimulation of hTM cells with TGF-beta induced translocation of TFEB to the nuclear compartment at all the tested times with maximal nuclear accumulation at 24 and 48 hours, as shown by immunocytochemical and nuclear fractionation studies.
The results of this study show that TFEB activation in hTM is stimulated by TGF-beta thus implicating this molecular player in TGF-beta-mediated dysfunction of hTM homeostasis. Based on the emerging role of TFEB as a critical regulator of lysosmal activity and biogenesis, our results suggest a possible role for this transcription factor in the regulation of lysosomal activity in hTM and potentially implicate it in the pathogenesis of glaucoma.
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