Abstract
Purpose :
We recently showed that cathepsin k (CTSK) is negatively regulated by pathological stressors known to affect extracellular matrix (ECM) remodeling in trabecular meshwork (TM) and intraocular pressure (IOP). Using loss of CTSK function by siRNA mediated knockdown (siCTSK) and induced expression of CTSK using adenovirus expression system (AdCTSK) in human TM (HTM) cells, this study unravels the mechanism of CTSK action on the regulation of ECM processing and maturation.
Methods :
Evaluate the effects of– a) siCTSK, dexamethasone (Dex), transforming growth factor β2 (TGFβ2) - on ECM associated proteins- Procollagen C-Endopeptidase Enhancer 1 (PCOLCE1), procollagen (pCOL1), collagen (COL1), and fibronectin (FN), and procollagen C-proteinase (PCP/BMP1) activity assay. b) siCTSK and AdCTSK on adrenomedullin (AM) and its receptors- calcitonin gene-related peptide (CLR), receptor activity-modifying proteins- RAMP1, and RAMP2. c) Relationship between CTSK and AM on ECM and intercellular adhesion molecule (ICAM1). qPCR and immunoblotting experiments were done in biologically different HTM lines (n≥3) with p≤0.05 indicating significance.
Results :
siCTSK significantly induced ECM protein fibronectin (FN) (p=0.007), ICAM (p=0.02), and PCP activity (p=0.04) but reduced intracellular (p=0.009) and secretory PCOLCE (p=0.0003), intracellular pCOL1 (p=0.0007), COL1 (p=0.04) and secretory COL1 (p=0.008). Similarly, Dex decreased Intracellular PCOLCE (p=0.008). Intracellular pCOL1 was increased in TGFβ2 (p=0.03) and Dex (p=0.05) whereas secretory pCOL1 was increased in TGFβ2 (p=0.04) and Dex (p=0.005). Based on an unbiased proteomic analysis, we found siCTSK increased AM levels (log2-0.66; p=0.02) and qPCR analysis showed increased AM (p=0.05) and its receptor- CLR (p=0.05). Contrarily, AdCTSK significantly decreased the AM (p=0.0009) and its receptors- RAMP1 (p=0.0003), RAMP2 (p=0.02). Interestingly, AdCTSK significantly attenuated the levels of secretory COL1 (p=0.02), total FN (p=0.001), and ICAM (p=0.0005), which were significantly induced upon AM treatment -secretory COL1 (p=0.02), FN (p=0.04) and ICAM (p=0.01).
Conclusions :
We show that CTSK via AM and its receptors participates in processing and maturation of ECM like COL and FN and modulates cell adhesive interactions in TM. Further elucidation of the relationship between CTSK and AM on ECM remodeling will provide novel mechanism in IOP regulation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.