Abstract
Purpose :
Our earlier work showed that human TM (HTM) cells undergoing cyclic mechanical stretch (CMS) induced SREBP2, a cholesterogenic transcription factor, with alterations in TM lipid profile. This prompted us to mechanistically investigate the mechanosensing action of SREBPs in TM and the consequence of SREBPs activation on actin assembly and lipid biogenesis in the TM.
Methods :
a) Investigated the mechano-responsiveness of SREBPs activation or nuclear SREBPs (N-SREBPs) under elevated pressure (2x) in porcine anterior segments (PAS) and CMS in HTM cells; b) Evaluated the outcome of constitutively active SREBPs in HTM cells using adenovirus expressing the nuclear SREBP isoforms - Ad5-N-SREBP1a, Ad5-N-SREBP1c, and Ad5-N-SREBP2 in comparison to adenovirus control (AdMT) – on cell shape changes, actin organization, and responsive lipogenic genes and enzymes. These were performed using qPCR, semi-quantitative immunoblotting (IB), and immunofluorescence (IF) based image analysis. Student’s t-test was used for statistics and results were significant if p<0.05 with a sample size of N=3.
Results :
Under 2x pressure stress, we found SREBP activation since N-SREBP1 (p = 0.04) and N-SREBP2 (p = 0.03) protein levels were induced significantly, and CMS increased the nuclear distribution of SREBP1 and 2. Upon significantly inducing the N-SREBPs individually, we found pronounced cell shape changes, like highly contractile morphology with cell retraction. When N-SREBPs were overexpressed, we observed actin remodeling with strong induction in cortical actin as well as stress fibers and redistribution of focal adhesions including paxillin and vinculin. Interestingly, N-SREBP2 overexpression showed greater F-actin stress fibers compared to others. Acetyl CoA carboxylase (ACC) and peroxisome proliferator-activated receptor gamma (PPARG) genes were commonly upregulated to significant levels upon N-SREBPs overexpression. N-SREBP1a and 2 significantly upregulated both fatty acid and cholesterol biogenic genes- fatty acid synthase (FAS), HMG CoA synthase (HMGCS), and HMG CoA reductase (HMGCR).
Conclusions :
This is the first report where we mechanistically elucidate the relationship between mechanical stress-mediated SREBPs activation and the resultant actin polymerization. We propose that SREBPs-mediated TM contractile changes are modulated via lipogenic enzymes including ACC and PPARG.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.