Purchase this article with an account.
Paul Russell, Sara Thomasy, Christopher Murphy; Dynamic Re-establishment of the Actin Cytoskeleton Impacts the Mechanotranducing Pathway in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3529. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
After treatment of human trabecular meshwork (HTM) cells with Latrunculin B (Lat B), an actin cytoskeleton disruptor, there is a transient increase in the Young’s modulus (YM) or stiffness of HTM cells. This increase in YM was reported on hydrogels with moduli similar to normal (5 kPa) and glaucomatous (75 kPa) meshwork as well as on hard surfaces (>1 GPa). The increase was approximately 4 fold over the base line YM on the hard surfaces at 90 minutes post recovery. The increases were less for the hydrogels. Recently, the proteins Yes-associated protein (YAP) and transcription coactivator with PDZ-binding motif (TAZ) were reported to be the mechanotranducers of extracellular alterations. The purpose of this study was to investigate gene expression changes in YAP and TAZ and other proteins in the mechanotranducing pathway during the dynamic changes occurring with the rearrangement of the actin cytoskeleton after Lat B treatment.
HTM cells were treated with 2 μM Lat B or vehicle for 30 minutes. The cells were washed and placed in serum containing medium for 1 hour. RNA was then harvested and quantitative PCR was done to determine mRNA expression of several genes.
There was a significant increase in YAP/TAZ mRNA in HTM cells on the 75 kPa hydrogels compared to cells on the 5 kPa hydrogels or the tissue culture plastic (TCP). Addition of Lat B resulted in a significant decrease of YAP mRNA on both hydrogels, but no change on the TCP. TAZ mRNA was also significantly decreased on the 75 kPa hydrogels, but was not altered on either the 5 kPa hydrogels or the TCP. The protein 14-3-3σ associates with YAP/TAZ and sequesters them in the cytoplasm. The mRNA for 14-3-3σ was increased on both hydrogels compared to TCP and was significantly increased with Lat B treatment on the hydrogels but not altered on TCP. Connective tissue growth factor, whose mRNA expression is directed by YAP, was down regulated on the hydrogels with Lat B treatment but not altered on TCP.
During the dynamic reassembly of the actin cytoskeleton after Lat B treatment, there were substantial alterations in the signaling pathway involving the mechanotransducers YAP/TAZ. If the HTM cells are merely grown on TCP, this alteration was not observed. These data highlight the need to conduct experiments on substrates that mimic the compliances measured in normal or glaucomatous HTM.
This PDF is available to Subscribers Only