Loss of Cathepsin K Impairs Collagen Biogenesis and Enhances Actin Polymerization in Trabecular Meshwork

Compromise in trabecular meshwork (TM) function due to extracellular matrix (ECM) accumulation contributes to increased intraocular pressure (IOP) in primary open-angle glaucoma (POAG). Our previous study demonstrated the role of cathepsin K (CTSK), a potent collagenase, on ECM homeostasis, actin bundling in TM, and IOP regulation. This study was designed to understand the response of TM cells to the loss of CTSK function.

Normal primary human TM (HTM) cells transfected with either small interfering RNA (siRNA) against CTSK (siCTSK) or scrambled siRNA (siScr) as a control were screened using mass spectrometry-based quantitative proteomics for changes in protein levels. Immunofluorescence imaging was used to identify changes in the distribution of differentially expressed proteins. Flow cytometry analysis provided evidence for the cell death mechanism resulting from the loss of CTSK function. Biochemical analysis was performed to quantify filamentous actin, assess BMP1 activity, and measure calcium levels.

CTSK loss significantly disrupted collagen biogenesis and ECM remodeling and increased intracellular calcium levels and the expression of calcium-regulatory proteins. Actin polymerization was increased due to protein kinase D1 (PRKD1) activation through the slingshot phosphatase 1 (SSH1)/cofilin pathway, promoting focal adhesion maturation. Despite increased apoptotic markers (CASP3, CASP7, TRADD, PPM1F), caspase-3/-7 activation was not induced, suggesting apoptosis-independent cellular remodeling. Notably, RhoQ and myosin motor proteins were significantly downregulated, indicating altered mechanotransduction.

Our findings underscore the multifaceted role of CTSK in maintaining critical cellular processes within the TM. Specifically, we have shown that CTSK is closely involved in regulating ECM homeostasis, influencing calcium signaling, and governing cytoskeletal dynamics and TM cellularity.