Purchase this article with an account.
Joshua Morgan, Christopher Murphy, Paul Russell; Decrease of nuclear Yes-associated protein (YAP) with human trabecular meshwork (HTM) cell passage. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3543.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
YAP is a transcriptional coactivator with roles in proliferation and cell survival. YAP has recently been implicated in mechanotransduction, with cells grown on stiffer substrates exhibiting increased nuclear localization and transcriptional activity by this protein. Among YAP’s transcription targets are numerous genes associated with glaucoma such as transforming growth factor-β and connective tissue growth factor. The expression of these genes with disease would be consistent with more nuclear localization of YAP as a result of the elevated stiffness found in the glaucomatous HTM. We wished to quantify the nuclear localization of YAP as a function of passage number and presence of serum in cultured HTM cells as a baseline for future studies on the impact of substratum stiffness.
HTM cells from four donors were maintained in DMEM/F12 + 10% serum. For each passage, cells were plated at 100,000 cells/35 mm tissue culture plastic (TCPS) dish and allowed to attach overnight. The following day the media was replaced with media ± serum. After 3 days, the cells were fixed and permeabilized with 4% formaldehyde and 0.5% Triton. The cells were labeled with rabbit anti-YAP and counterstained with DAPI. The cells were imaged and the YAP nuclear contrast ratio (NCR), defined as the ratio of the YAP staining intensity in the nucleus or in the adjacent cytosol, was quantified.
In cells from all four donors there was substantial nuclear labeling of YAP and this was not influenced by the presence or absence of serum. The average nuclear contrast ratio decreased with passage from around 1.8 in earlier passages to ~1.2 - 1.4 at terminal passage (12-14), indicating that even in senescence YAP was not totally excluded from the nucleus of HTM cells. The decrease in nuclear labeling of YAP was mirrored by a decrease in cell density, consistent with the role of YAP in proliferation.
In culture, HTM cells cultured on TCPS express YAP and the localization is not influenced by the presence or absence of serum. A decrease in NCR is observed with increasing cell passage. With YAP targets including multiple genes believed to be important in glaucoma and the published observations of the elevated stiffness of the glaucomatous HTM and the stiffness-dependent nuclear localization of YAP, this protein represents a novel potential target for pharmacological intervention in glaucoma.
This PDF is available to Subscribers Only