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H.-S. Kwon, H.-S. Lee, J. Rubin, S. Tomarev; Myocilin May Regulate Actin Cytoskeleton Through Components of Wnt Signaling Pathway. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5111.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate molecular mechanisms of myocilin (Myoc) action.
Conditioned media was prepared from HEK293 cells that were transiently transfected with cDNAs encoding wild-type or mutated human Myoc and its N-terminal and C-terminal fragments. Flag-tagged Myoc was purified using Flag-agarose. Actin cytoskeleton was visualized by staining with Rhodamin-phalloidin. Flag- and alkaline phosphatase-tagged Myoc were used to study its interaction with frizzed receptors and secreted frizzled-related proteins (sFRPs).
Addition of conditioned media from Myoc expressing cells or purified Myoc protein to NIH3T3 or human trabecular meshwork cells induced changes in their actin cytoskeleton similar to those observed after addition of Wnt3a conditioned media. Conditioned media from cells expressing non-secreted mutated Myoc did not induce changes in the actin cytoskeleton. Pre-incubation of conditioned media from Myoc expressing cells (but not from Wnt-expressing cells) with monoclonal antibodies against Myoc blocked changes in actin cytoskeleton. Changes in the actin cytoskeleton were also blocked by addition of Wnt signaling antagonist, sFRP1, to conditioned media. Direct interaction of Myoc with sFRP1 was confirmed in co-immunoprecipitation experiments. The olfactomedin domain of Myoc was critical for this interaction. Addition of conditioned media from Myoc-expressing cells to NIH3T3 cells increased levels of activated Rac1 and JNK. Alkaline phosphatase-tagged human Myoc interacted with several frizzled receptors and sFRPs with frizzled-7 and sFRP3 giving the highest affinity of binding.
Myoc may be involved in the regulation of actin cytoskeleton in the trabecular meshwork through components of Wnt signaling pathway. Our data suggest that Myoc may work as a modulator of Wnt signaling.
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