Elevated amounts of myocilin in the aqueous humor of transgenic mice cause significant changes in ocular gene expression

Abstract

Myocilin is a 55-57kDa secreted glycoprotein and member of the olfactomedin family, which is mutated in some forms of primary open-angle glaucoma. To assess the effects of elevated amounts of myocilin on aqueous humor outflow dynamics in an in vivo system, transgenic betaB1-crystallin-MYOC mice have been developed that strongly overexpress myocilin in their eyes. The transgenic overexpression of myocilin results in an almost five-fold increase of secreted normal myocilin in the aqueous humor of betaB1-crystallin-MYOC mice. In the present study, we wanted to use betaB1-crystallin-MYOC as a tool to identify the response of ocular tissues to the presence of higher than normal amounts of myocilin, and to identify changes in gene expression that could help to shed light on the functional in vivo properties of myocilin. RNA was isolated from ocular tissues of betaB1-crystallin-MYOC mice and wild-type littermates. Changes in gene expression were determined by hybridization of gene microarrays and confirmed by real time RT-PCR and Western blotting. The expression of genes that had been found to be differentially regulated in betaB1-crystallin-MYOC mice was further analyzed in cultured human trabecular meshwork (HTM) cells treated with recombinant myocilin. Although betaB1-crystallin-MYOC mice do not have an obvious phenotype, a statistically significant up- and downregulation of several distinct genes was found when compared to gene expression in wild-type littermates. Among the genes that were found to be differentially regulated were Wasl, Ceacam1, and Spon2, which are involved in cell adhesion and cell-matrix interactions. Differences in expression were also found for Six1 which encodes for a transcription factor, and for Pftk1 whose gene product is a cdc2-related protein kinase. The expression of these genes was also found to be regulated in vitro in HTM cells treated with recombinant myocilin. Substantially higher amounts in ocular tissues of betaB1-crystallin-MYOC mice were found for connexin 46 and alphaB-crystallin. In addition, several genes that encode for olfactomedin proteins showed distinct changes in expression. Olfml3 was significantly downregulated, while Lphn1, Lphn2, and Lphn3 were significantly upregulated. Our findings support a role for myocilin in modulating cellular adhesion, and suggest functional processes that involve other proteins of the olfactomedin family.

In Vivo Gene Expression Profiling of Retina Postintravitreal Injections of Dexamethasone and Triamcinolone at Clinically Relevant Time Points for Patient Care

“…Studies on steroid-induced glaucoma have shown that TAA and Dex induce dose dependent, differential gene profiles in human trabecular meshwork (TM) cells, revealed by microarray technology 18-21 . Today, the TM epithelium is considered to be the target tissue and the myocilin gene, expressed mainly in TM, the top candidate gene for the development of steroidinduced glaucoma, through the proposed mechanism of increased intraocular pressure (IOP) 22 . Although an elevated IOP is a significant risk factor for the development of glaucoma, it is important to note that sight-threatening effects of glaucoma are not necessarily coming from increased IOP but from retinal and optic nerve neurodegeneration.…”

Quantitative Real-Time RT-PCR of ITGA7, SVEP1, TNS1, LPHN3, SEMA3G, KLB and MMP13 mRNA Expression in Breast Cancer

“…LPHN3 (the latrophilin 3 gene) is a member of the G-protein coupled receptor (GPCR) family; it has large extracellular and intracellular domains, containing several cell adhesion modules, such as cadherin, IgG, laminin A, thrombospondin type 1, galactose lectin, EGF, and transmembrane segments that may be involved in intracellular signalling during cell-to-cell adhesion (Martinez et al, 2011). LPHN3 was up-regulated significantly in a transgenic mice model that over-expressed myocilin (Paper et al, 2008). Altered LPHN3 expression levels have been seen after brain ischemia (Bin et al, 2002), and during the period of adrenal gland development (Wing et al, 2009).…”

The Effects of Myocilin Expression on Functionally Relevant Trabecular Meshwork Genes: A Mini-Review

“…19,20 Subjecting the mouse eye to elevated levels of myocilin protein in the aqueous humor also caused changes in the entire eye gene expression. 21 Transgenic mice carrying myocilin cDNA under the control of the bB1-crystallin promoter expressed myocilin in the lens and secreted the protein into the aqueous humor. Transcriptome of the whole mouse eye (minus the lens) was compared to that of the wild-type littermate.…”