Abstract
Purpose :
Prior studies have suggested that expression of TGFβ2, αvβ3 integrin and alternatively spliced fibronectin (FN) isoforms (EDA+FN and EDB+FN) are upregulated as we age and that expression of TGFβ2 and β3 integrin mRNA is controlled by the transcription factor NFATc1. In this study, we examined if age-related increases in TGFβ2, β3 integrin and FN expression could be detected in HTM cells and whether activation of NFATc1 affected expression.
Methods :
TGFβ2, β3 integrin and FN levels were analyzed in confluent cultures of HTM cells isolated from young (17-35 yrs) and old (71-96 yrs) donor eyes. Changes in TGFβ2, and β3 integrin levels were also analyzed over time in confluent cultures of young HTM cells grown continuously for 21 days. An adenovirus vector expressing a constitutively active GFP-NFATc1 was used to determine if NFATc1 could drive expression of these genes in young HTM cells over a 4-5 day period. Western blotting (WB), TGFβ2 ELISAs and qPCR analyses were used to detect TGFβ2, β3 integrin and FN mRNA and/or protein levels. An on-cell western was used to determine FN protein levels.
Results :
TGFβ2, β3 integrin and EDB+FN mRNA levels were significantly increased in cells from older eyes compared to young eyes while total FN and EDA+FN mRNA levels were unchanged. Older cells also incorporated more EDA+FN and EDB+FN into the extracellular matrix compared to young cells. Prolonged culturing of young cells for 21 days, significantly increased TGFβ2 mRNA levels over time. β3 integrin and EDB+FN mRNA levels were initially upregulated and then remained unchanged or slowly declined. β3 integrin protein levels mirrored the mRNA levels. Finally, levels of TGFβ2 and β3 integrin mRNA and protein levels could be increased in young cells by activation of NFATc1.
Conclusions :
These studies show that TGFβ2, β3 integrin and EDA+/EDB+FN levels are higher in older HTM cells and that TGFβ2 and β3 integrin levels can be increased in young cells by prolonging the culturing time or by activating NFATc1. These studies also suggest that the activity of NFATc1 could be playing a role in the age-related expression of these proteins.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.