It is well documented that mutations in the MYOCILIN gene may lead to juvenile- and adult-onset primary open-angle glaucoma. However, the functions of wild-type myocilin are still not well understood. To study functions of human myocilin and its two proteolytic fragments, these proteins were expressed in HEK293 cells. Conditioned medium from myocilin-expressing cells as well as purified myocilin induced formation of stress fibers in primary cultures of human trabecular meshwork or NIH3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin as well as secreted inhibitors of Wnt-signaling, sFRP1 or sFRP3, and beta-catenin siRNA. Interaction of myocilin with sFRP1, sFRP3 and several frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different frizzled and sFRPs. Treatment of NIH3T3 cells with myocilin and its fragments induced intracellular re-distribution of beta-catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of beta-catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of beta-catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, as myocilin modulates Wnt signaling by interacting with components of this signaling pathway.

Protein transduction (PT) is a method for delivering proteins into mammalian cells. PT is accomplished by linking a small peptide tag-called a PT domain (PTD)-to a protein of interest, which generates a functional fusion protein that can penetrate efficiently into mammalian cells. In order to study the functions of a transcription factor (TF) of interest, expression plasmids that encode the TF often are transfected into mammalian cells. However, the efficiency of DNA transfection is highly variable among different cell types and is usually very low in primary cells, stem cells and tumor cells. Zinc-finger transcription factors (ZF-TFs) can be tailor-made to target almost any gene in the human genome. However, the extremely low efficiency of DNA transfection into cancer cells, both in vivo and in vitro, limits the utility of ZF-TFs. Here, we report on an artificial ZF-TF that has been fused to a well-characterized PTD from the human immunodeficiency virus-1 (HIV-1) transcriptional activator protein, Tat. This ZF-TF targeted the endogenous promoter of the human VEGF-A gene. The PTD-attached ZF-TF was delivered efficiently into human cells in vitro. In addition, the VEGF-A-specific transcriptional repressor retarded the growth rate of tumor cells in a mouse xenograft experiment.

Gene expression is regulated at the transcriptional and post-transcriptional levels. Therefore, in order to achieve a high level of silencing, which includes minimizing any residual expression of a target gene, suppression at both the transcriptional and post-transcriptional levels is required. In this study, we describe a new method for highly efficient gene silencing that combines zinc finger protein-mediated transcriptional repression and small interfering RNA (siRNA)-mediated inhibition of post-transcriptional events. To measure the amount of gene expression under various conditions, we used a luciferase reporter gene that was driven by a variety of promoters, including that of the human vascular endothelial growth factor-A (VEGF-A) gene. We also measured expression of the endogenous VEGF-A gene. Inhibition of gene expression by each of the two individual technologies was effective, but in-depth analyses revealed residual expression of the target gene. The combination of specific zinc finger transcription factors and siRNAs greatly enhanced the silencing of the human VEGF-A gene, not only when cells were grown in the presence of normal amounts of oxygen but also under conditions of hypoxic stimulation. These results suggest that a bi-level approach to the silencing of VEGF-A expression may be clinically beneficial as part of a cancer treatment protocol.