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William Foster, Joshua Davis; Connective Tissue Growth Factor Protein Expression by Müller Cells is Bipartite as a Function of Substrate Stiffness. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4689.
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© ARVO (1962-2015); The Authors (2016-present)
Müller cells have been previously found to change their expression of connective tissue growth factor (Ctgf) mRNA by more than 100-fold when cultured on substrates of different elastic moduli. To better understand the mechanosensitivity of Müller cells, we quantified the expression of CTGF protein utilizing a Sandwich ELISA with streptavidin-biotin and utilized immunohistochemistry to determine the localization of the mechanotransduction markers YAP/TAZ in Müller cells as a function of substrate stiffness.
A conditionally immortalized mouse Müller cell line (ImM10) was cultured on polyacrylamide substrates with a calibrated Young’s modulus of 500 Pa, 1000 Pa, and 5000 Pa with glass as a control. A uniform coating of laminin was cross-linked to the substrates. For the ELISA studies, Müller cells were cultured for to 24 days and the media was collected, stored at -80C until analysis, and changed every 3 days. All substrates were studied in triplicate. For immunohistochemistry, Müller cells were cultured for either 15 or 21 days, and stained with antibodies to YAP/TAZ, utilizing a standard protocol.
The ELISA demonstrated unexpected, bipartite behavior, as a function of substrate stiffness. Cells grown on substrates firmer than 1000 Pa had a peak in their protein expression at 15 days while substrates of 1000 Pa and less had a peak in their protein expression at day 21. Both the differences between the different substrates and as a function of time were significant (p=0.006, p=0.022, respectively; two-way ANOVA). Immunohistochemistry at day 15 demonstrated YAP/TAZ localization to the cytoplasm only in cells grown on firmer substrates while studies at day 21 demonstrated localization to the cytoplasm only in cells grown on softer substrates.
There appear to be two regimes in stiffness that influence CTGF protein production in Müller, 1000 Pa and under and over 1000 Pa. Consistent with this finding, cytoplasmic localization of the mechanotransduction markers YAP/TAZ corresponded with increased production of protein. It is interesting to note that the transition point in stiffness occurs at the point at which the Müller cells are normally found in vivo (1000 Pa).
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