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K. Keller, M.J. Kelley, Y. Chen, T.S. Acott; Alternative Splicing by Trabecular Meshwork Cells Alters Glycosaminoglycan Chains in Response to Mechanical Stretch, TNF and IL–1 Treatment . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1857.
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Glycosaminoglycans (GAGs) are thought to be a source of resistance to aqueous humor outflow in the trabecular meshwork (TM). Elevated intraocular pressure (IOP), sensed as mechanical stretching by TM cells, triggers extracellular matrix (ECM) remodeling to achieve IOP homeostasis. Laser trabeculoplasty treatment alleviates IOP elevation due in part to the release of tumor necrosis factor (TNF) and interleukin–1 (IL–1), which induce ECM remodeling and also decrease outflow resistance. Versican and CD44, two ECM molecules modified during remodeling, each contain GAG attachment sites in alternatively spliced domains. The mRNA splice forms of versican and CD44 expressed by TM cells in response to mechanical stretch, TNFα, or IL–1α treatment were therefore investigated.
Cultured porcine TM cells were mechanically stretched or treated with either 10 ng/ml TNFα or IL–1α for 12, 24 or 48 hrs. RNA was isolated and RT–PCR was performed using primers that flanked previously reported splice sites. DNA sequencing determined the identity of each product. Quantitative RT–PCR with a real–time thermocycler was used to quantitate changes in splicing of particular isoforms using primers anchored within the spliced exon. These results were normalized against the total expression level of each gene.
The V1 isoform of versican lacks the αGAG domain that contains 5–8 chondroitin sulfate attachment sites. qRT–PCR indicated that mechanical stretch decreases the total number of GAG chains contributed by versican in two ways: (a) by reducing mRNA expression of versican and (b) by increasing the proportion of the V1 splice form. These effects were most apparent at 12 hr. Treatment with TNFα reduced total versican mRNA expression at 12 hr and 48 hr, but did not significantly affect V1 splicing. Conversely, IL–1α increased total versican but decreased the V1 isoform at 12 hr. CD44, a cell surface receptor for hyaluronan, has up to 9 additional exons (v2–v10) included due to alternative splicing. Treatment with either TNFα or IL–1α, but not mechanical stretch, increased the expression of exons v3–v7 as compared to the control at all three time points. Inclusion of these exons allows CD44 to bind multiple GAG chains in addition to hyaluronate.
Mechanical stretch, TNFα and IL–1α induce TM cells to express alternate isoforms of versican and CD44 that contain differing GAG attachment sites. These modulations may be one mechanism by which TM cells regulate outflow resistance and IOP.
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