scholarly journals Effects of Netarsudil on Actin-Driven Cellular Functions in Normal and Glaucomatous Trabecular Meshwork Cells: A Live Imaging Study

2020 ◽  
Vol 9 (11) ◽  
pp. 3524
Author(s):  
Kate E. Keller ◽  
Casey Kopczynski
Keyword(s):  
Trabecular Meshwork ◽  
Kinase Inhibitor ◽  
Imaging Study ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Cellular Functions ◽  
Tunneling Nanotubes ◽  
Trabecular Meshwork Cells ◽  
Lateral Fusion ◽  
Actin Stress Fibers

The actin cytoskeleton of trabecular meshwork (TM) cells is a therapeutic target for lowering intraocular pressure (IOP) in glaucoma patients. Netarsudil (the active ingredient in RhopressaTM) is a Rho-associated protein kinase inhibitor that induces disassembly of actin stress fibers. Here, we used live cell imaging of SiR-actin-labeled normal (NTM) and glaucomatous TM (GTM) cells to investigate actin dynamics during actin-driven biological processes with and without netarsudil treatment. Actin stress fibers were thicker in GTM than NTM cells and took longer (>120 min) to disassemble following addition of 1 µM netarsudil. Actin-rich extracellular vesicles (EVs) were derived by two mechanisms: exocytosis of intracellular-derived vesicles, and cleavage of filopodial tips, which detached the filopodia from the substratum, allowing them to retract to the cell body. While some phagocytosis was noted in untreated TM cells, netarsudil potently stimulated phagocytic uptake of EVs. Netarsudil treatment induced lateral fusion of tunneling nanotubes (TNTs) that connected adjacent TM cells; TNTs are important for TM cellular communication. Together, our results suggest that netarsudil may clear outflow channels in TM tissue by inducing phagocytosis and/or by modulating TM communication via EVs and TNTs. These cellular functions likely work together to regulate IOP in normal and glaucomatous TM.

scholarly journals WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization

2012 ◽  
Vol 302 (1) ◽  
pp. F103-F115 ◽  
Author(s):  
Jane H. Kim ◽  
Amitava Mukherjee ◽  
Sethu M. Madhavan ◽  
Martha Konieczkowski ◽  
John R. Sedor
Keyword(s):  
Kinase Inhibitor ◽  
Adherens Junctions ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Transcriptional Responses ◽  
Interacting Protein ◽  
Cell Matrix ◽  
Actin Stress Fibers ◽  
Cell Cell

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

scholarly journals Tunneling Nanotubes are Novel Cellular Structures That Communicate Signals Between Trabecular Meshwork Cells

2017 ◽  
Vol 58 (12) ◽  
pp. 5298 ◽  
Author(s):  
Kate E. Keller ◽  
John M. Bradley ◽  
Ying Ying Sun ◽  
Yong-Feng Yang ◽  
Ted S. Acott
Keyword(s):  
Trabecular Meshwork ◽  
Cellular Structures ◽  
Tunneling Nanotubes ◽  
Trabecular Meshwork Cells

scholarly journals p38 MAP Kinase Inhibitor Suppresses Transforming Growth Factor-β2–Induced Type 1 Collagen Production in Trabecular Meshwork Cells

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120774 ◽  
Author(s):  
Miyuki Inoue-Mochita ◽  
Toshihiro Inoue ◽  
Tomokazu Fujimoto ◽  
Takanori Kameda ◽  
Nanako Awai-Kasaoka ◽  
...  
Keyword(s):  
Growth Factor ◽  
Map Kinase ◽  
Trabecular Meshwork ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
P38 Map Kinase ◽  
Collagen Production ◽  
Trabecular Meshwork Cells ◽  
Transforming Growth Factor Β2

Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells

2006 ◽  
Vol 82 (3) ◽  
pp. 362-370 ◽  
Author(s):  
Tomoyo Koga ◽  
Takahisa Koga ◽  
Maiko Awai ◽  
Jun-ichiro Tsutsui ◽  
Beatrice Y.J.T. Yue ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Trabecular Meshwork ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Trabecular Meshwork Cells

scholarly journals Hyperosmolarity regulates SOX9 mRNA posttranscriptionally in human articular chondrocytes

2009 ◽  
Vol 297 (4) ◽  
pp. C898-C906 ◽  
Author(s):  
Simon R. Tew ◽  
Mandy J. Peffers ◽  
Tristan R. McKay ◽  
Emma T. Lowe ◽  
Wasim S. Khan ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Half Life ◽  
Articular Chondrocytes ◽  
Kinase Inhibitor ◽  
Stress Fibers ◽  
Human Articular Chondrocytes ◽  
Luciferase Assay ◽  
Mrna Levels ◽  
Matrix Gene ◽  
Actin Stress Fibers

The transcription factor SOX9 regulates cartilage extracellular matrix gene expression and is essential for chondrocyte differentiation. We previously showed that activation of p38 MAPK by cycloheximide in human chondrocytes leads to stabilization of SOX9 mRNA (Tew SR and Hardingham TE. J Biol Chem 281: 39471–39479, 2006). In this study we investigated whether regulation of p38 MAPK caused by changes in osmotic pressure could control SOX9 mRNA levels expression by a similar mechanism. Primary human articular chondrocytes isolated from osteoarthritic cartilage at passage 2- 4 showed significantly raised SOX9 mRNA levels when exposed to hyperosmotic conditions for 5 h. The effect was strongest and most reproducible when actin stress fibers were disrupted by the Rho effector kinase inhibitor Y27632, or by culturing the cells within alginate beads. Freshly isolated chondrocytes, used within 24–48 h of isolation, did not contain actin stress fibers and upregulated SOX9 mRNA in response to hyperosmolarity in the presence and absence of Y27632. In these freshly isolated chondrocytes, hyperosmolarity led to an increase in the half-life of SOX9 mRNA, which was sensitive to the p38 MAPK inhibitor SB202190. SOX9 protein levels were increased by hyperosmotic culture over 24 h, and, in passaged chondrocytes, the activity of a COL2A1 enhancer driven luciferase assay was upregulated. However, in freshly isolated chondrocytes, COL2A1 mRNA levels were reduced by hyperosmotic conditions and the half-life was decreased. The results showed that the osmotic environment regulated both SOX9 and COL2A1 mRNA posttranscriptionally, but in fresh cells resulted in increased SOX9, but decreased COL2A1.

scholarly journals The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

2007 ◽  
Vol 292 (5) ◽  
pp. C1830-C1836 ◽  
Author(s):  
Amanda M. D. Malone ◽  
Nikhil N. Batra ◽  
Giri Shivaram ◽  
Ron Y. Kwon ◽  
Lidan You ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluid Flow ◽  
Actin Polymerization ◽  
Bone Adaptation ◽  
Stress Fibers ◽  
Calcium Flux ◽  
Actin Dynamics ◽  
Osteoblastic Cells ◽  
Cox 2 ◽  
Actin Stress Fibers

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE2 release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE2 release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE2 release. In fact, PGE2 release was increased threefold in the polymerization inhibited cells and this PGE2 release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE2 release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE2 release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells

2016 ◽  
Vol 145 ◽  
pp. 393-400 ◽  
Author(s):  
Tomokazu Fujimoto ◽  
Toshihiro Inoue ◽  
Miyuki Inoue-Mochita ◽  
Hidenobu Tanihara
Keyword(s):  
Trabecular Meshwork ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Actin Dynamics ◽  
Trabecular Meshwork Cells
Sign in / Sign up

Export Citation Format

Share Document