Novel insight into the role of clusterin on intraocular pressure regulation by modifying actin polymerization and extracellular matrix remodeling in the trabecular meshwork

Abstract

This study provides comprehensive mechanistic evidence for the role of clusterin, a stress‐response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress‐related clusterin function loss. Small interfering RNA‐mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine‐protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and β5. A complete loss of clusterin as seen in clusterin knockout mice (Clu‐/‐) led to significant IOP elevation at postnatal Day 70. Contrarily, constitutive clusterin expression using adenovirus (AdCLU) in HTM cells resulted in the loss of actin polymerization via decreased PRK2, and LIMK1 and negative regulation of integrin αV and β5. Furthermore, we found that AdCLU treatment in HTM cells significantly decreased the ECM protein expression and distribution by significantly increasing matrix metalloprotease 2 (MMP2) activity and lowering the levels of pro‐fibrotic proteins such as transforming growth factor‐β2 (TGFβ2), thrombospondin‐1 (TSP‐1), and plasminogen activator inhibitor‐1 (PAI‐1). Finally, we found that HTM cells supplemented with recombinant human clusterin attenuated the pro‐fibrotic effects of TGFβ2. For the first time this study demonstrates the importance of clusterin in the regulation of TM actin cytoskeleton ‐ ECM interactions and the maintenance of IOP, thus making clusterin an interesting target to reverse elevated IOP.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation

“…Elevated IOP was modeled using porcine ex vivo perfusion system as described earlier (20, 54). Post perfusion the TM tissue was carefully isolated and frozen at -80°C until further processing.…”

“…Total RNA was extracted from HTM cells using the Trizol method following the manufacturer’s protocol. The RNA amounts measurement, cDNA conversion, and qPCR reaction were conducted as published before (54). Sequence-specific forward and reverse oligonucleotide primers for the indicated genes are provided in Table 2 .…”

“…q PCR reaction was performed and calculated by the delta-delta Ct method as described earlier (54, 61). The normalization was performed using 18S ribosomal RNA (18S rRNA) for HTM.…”

“…After determining the protein concentration and the immunoblotting process was performed as published earlier (38, 54). Blots were stripped using mild stripping buffer if required to reprobe for the loading control and multiple proteins within the same molecular weight range.…”

“…IF staining was done on HTM cells grown on 2% gelatin-coated glass coverslip. The methodologies used for IHC and IF has been published from the lab earlier (20, 54).…”