Abstract
Purpose :
Recently we showed that the inactivation of cathepsin K (CTSK), a secretory lysosomal protease, increased the ECM deposition and actin-bundling in the trabecular meshwork (TM) and elevated the intraocular pressure. In this study, we explored the loss-of-function effect of CTSK on the human TM proteome.
Methods :
Normal primary human TM (HTM) cells (n=4) in vitro were transfected with either siRNA against CTSK (siCTSK) or scrambled siRNA (siScr) as control. 72h post-transfection, the protein was collected, and mass spectrometry-based quantitative proteomics was performed. Changes in protein levels were screened using criteria- p ≤ 0.05 and mean ± 2σ of log2 as confidence fold change limits. MetaCoreTM was used for the functional pathway enrichment analysis.
Results :
We found an increase and decrease in 398 and 132 proteins in TM respectively due to siCTSK. The upregulated proteins were related to– a) TM cell contractility and cell adhesion proteins- actin scaffolding protein tensin 1 (p=0.03), paxillin (p=0.01), docking protein involved in tyrosine kinase-based signaling related to cell adhesion BCAR1/p130cas (p=0.04), tyrosine-phosphorylated growth factor receptor adaptor protein NCK1 (0.04) and NCK2 (p=0.03), serine/threonine-protein kinase D1 (PRKD1) (p=0.001), and Guanine nucleotide exchange factor Vav2 (p=0.04), intracellular signal transducer and transcriptional modulator activated by TGFβ SMAD3 (p=0.04); b) sphingosine kinase 2 (SPHK2) (p=0.0001), which is involved in phosphorylation of the lysosphingolipid sphingosine-1-phosphate; and c) apoptosis-related proteins including caspase 1 (p=0.03), which cleaves and activates caspase 3 (p=0.02), caspase 7 (p=0.01), nuclear factorκB (NFkB) p105 subunit (p=0.05), and a critical apoptosis regulator receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (p=0.03). Interestingly there was a significant decrease in the levels of actin depolymerizing protein phosphatase SSH1 (p=0.03), cell survival or anti-apoptotic protein tank binding kinase 1 (TBK1) (p=0.03), TBK1 adaptor protein 5-azacytidine-induced protein 2 (AZI2) (p=0.01), and as expected CTSK (p=0.04).
Conclusions :
These unbiased observations establish the functionality of CTSK in regulating the TM actin cytoskeleton and the maintenance of TM cellularity. We propose that dysregulation of CTSK in TM can cause increased contractility and cell loss leading to elevated IOP and glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.