title: Substratum stiffness and latrunculin B modulate the gene expression of the mechanotransducers YAP and TAZ in human trabecular meshwork cells.
abstract: The compliance of the human trabecular meshwork (HTM) has been shown to dramatically stiffen in glaucomatous patients. The purpose of this study was to determine the impact of substratum stiffness and latrunculin-B (Lat-B) on the expression and activity of the mechanotransducers, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ), in primary HTM cells as the cells start to recover from Lat-B treatment. Primary human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in DMSO or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of YAP, TAZ, 14-3-3σ, plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF). Immunoblotting was used to determine the expression of YAP and TAZ as well as the phosphorylation status of YAP. Immunofluorescence was used to determine YAP protein localization. YAP and TAZ mRNA expression were upregulated on the 75 kPa hydrogels in comparison to the 5 kPa hydrogels and TCP. Treatment with Lat-B resulted in a rapid and dramatic downregulation of YAP and TAZ on the 75 kPa hydrogels. On hydrogels, Lat-B treatment increased the phosphorylation of YAP at S127, while decreasing it on TCP. Similarly, Lat-B treatment resulted in markedly decreased nuclear localization of YAP on the hydrogels but elevated nuclear localization on TCP. Lat-B treatment of HTM cells on the 75 kPa hydrogels also increased 14-3-3σ mRNA, a protein important in YAP/TAZ degradation. In addition, Lat-B treatment decreased CTGF and PAI-1 mRNA on the 75 kPa hydrogels. In conclusion, substratum stiffness alters YAP/TAZ expression and YAP localization in primary HTM cells which then may modulate the expression of extracellular matrix proteins important in glaucoma. During the recovery period after Lat-B treatment, gene expression changes are more dramatic on substrates with stiffness similar to glaucomatous meshwork. Use of these hydrogels may more accurately reflect the alterations occurring in HTM cells in glaucoma after treatment with this drug.
authors.affiliation: Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, 1 Shields Ave., CA 95616, Davis, United States.
authors.forename: Sara M
authors.initials: SM
authors.lastname: Thomasy
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Morgan
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Wood
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Murphy
authors.forename: Paul
authors.initials: P
authors.lastname: Russell
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases; D005109:Extracellular Matrix / Q000187:drug effects* / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D005901:Glaucoma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053778:Thiazolidines / Q000494:pharmacology*; D014129:Trabecular Meshwork / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015534:Trans-Activators; D014157:Transcription Factors; D000091364:Transcriptional Coactivator with PDZ-Binding Motif Proteins; D014602:Uvea / Q000378:metabolism; D000091102:YAP-Signaling Proteins
chemical_list: D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D014408:Biomarkers, Tumor; D019086:Bridged Bicyclo Compounds, Heterocyclic; C526317:CCN2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C051321:SERPINE1 protein, human; D053778:Thiazolidines; D015534:Trans-Activators; D014157:Transcription Factors; D000091364:Transcriptional Coactivator with PDZ-Binding Motif Proteins; C551991:WWTR1 protein, human; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human; D055513:Connective Tissue Growth Factor; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; C037068:latrunculin B
keywords: biophysical cues; extracellular matrix; glaucoma; mechanotransduction; substratum compliance; trabecular meshwork