Abstract
Purpose :
In our previous study, we demonstrated that the clusterin knockout mice showed increased intraocular pressure (IOP) as a result of enhanced actin polymerization. We aimed to elucidate the impact of clusterin gain-of-function on the actin cytoskeletal dynamics in human TM (HTM) cells.
Methods :
We performed-A) tandem mass spectrometry-based global proteomic analysis on HTM cells constitutively expressing clusterin (AdCLU) compared to control (AdMT). Screening criteria for protein level changes included p≤0.05 and mean±2σ of log2≥0.2 as the confidence fold change (FC). B) Label-free proteomic analysis of co-immunoprecipitated (Co-IP) samples with tensin 3 (TNS3) antibody to determine its interactors. C) Immunofluorescence (IF) to determine - the distribution of TNS3 in the human ocular hypertensive (OHT) outflow pathway, the effect of siRNA-mediated loss of TNS3 (siTNS3), and the effect of pcDNA-EGFP-TNS3 (eTNS3)-mediated overexpression on actin fibers. D) Immunoblotting (IB) to analyze the effect of the siTNS3 on actin-associated proteins. Statistical analyses were performed using a student’s t-test with significance at p≤0.05 (n=4).
Results :
Proteomic analysis showed AdCLU reduced actin alpha 1 (p=0.046, log2FC=-0.5), TNS1 (p= 0.08, log2FC=-0.23), TNS3 (p=0.02, log2FC=-0.44), and TNS4 (p=0.04, log2FC=-0.220) levels. Confirmatory analysis by IB showed that AdCLU significantly reduced TNS3 (p=0.01). IF demonstrated higher TNS3 expression in the TM outflow pathway of the OHT tissues compared to normotensive tissues. Proteomics of Co-IP samples using TNS3 antibody revealed interactions with major cytoskeletal and extracellular matrix proteins, including vimentin, actins- actin gamma 1, actin alpha cardiac muscle 1, filamin-A, twinfilin-2, and tenascin-c. siTNS3 reduced F-actin distribution compared to siScrambled (siScr). Further, IB analysis showed a significant downregulation of fibronectin (p=0.04) and vimentin (p=0.03) in siTNS3 compared to siScr. Additionally, IF demonstrated that induction of TNS3 using eTNS3 expression plasmid induced F-actin and increased the distribution of active integrin β1 and β3 compared to control.
Conclusions :
This novel study presents the firsthand report on how clusterin regulates actin and integrin adhesome complex regulators like the tensins that can modify fibrillogenesis via integrins, defining players in effecting TM biomechanics and IOP.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.