Versican splice variants in human trabecular meshwork and ciliary muscle.
OBJECTIVE
Versican, chondroitin sulfate glycoprotein 2, is thought to play a role in regulating aqueous humor outflow and intraocular pressure via the human trabecular meshwork (HTM) of the eye. This protein was upregulated in HTM cells when they were treated with TGF-beta. There are four splice variant forms of versican (V0-V3) with different numbers of glycoaminoglycan (GAG) attachment domains. In this study, we investigated the various isoforms of versican from ocular tissues and cultured cells.


METHODS
HTM and human ciliary muscle (HCM) tissues were dissected from three pairs of donors eyes with no histories of eye disease. Cultures of HTM and HCM cells were established from five donor eyes, and HTM cells were treated for 72 h with 1 ng/ml of either with hrTGF-beta1 or hrTGF-beta2. Total RNA was isolated from cells and tissues from each of the samples. Relative quantitation of gene expression of each variant was detected by real-time PCR with SYBR Green dye.


RESULTS
All four variants of versican existed in each sample. In cultured HTM cells, the two variants with the largest number of GAG domains predominate. The V1 form is about 20% greater than the V0 form. There is an upregulation, particularly in the V0 form, when the cells are cultured. In tissue, the V1 form is about five fold greater than the other three. In the ocular ciliary muscle, the V1 form is the most prominent, but with this tissue, the relative amount of the V0 form did not change when cells were cultured. There was an upregulation of all splice variants in HTM cells when they were cultured with either TGF-beta1 or TGF-beta2. The increase in expression in the HTM with TGF-beta treatments were greatest with the V0 and V2 forms.


CONCLUSIONS
This is the first report about the presence of various forms of versican in the anterior segment of the eye and the alterations in the mRNA patterns of these forms when cells are placed in culture. The results indicate the variants with the largest numbers of GAG attachment domains are the most prominent in the HTM and HCM. The increases in these forms that have the most GAG domains would be consistent with the increases in chondroitin sulfate reported in glaucoma.
Zhao
Xiujun
X
Section on Aging and Ocular Disease, National Eye Institute, National Institutes of Health, Bethesda, MD, USA. xiujun.zhao@mssm.edu
Russell
Paul
P
D018929:Cell Culture Techniques; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism*; D002924:Ciliary Body / Q000187:drug effects / Q000378:metabolism*; D004274:DNA, Recombinant; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D015854:Up-Regulation; D053675:Versicans
D011508:Chondroitin Sulfate Proteoglycans; D004274:DNA, Recombinant; D037181:Lectins, C-Type; D020033:Protein Isoforms; D012333:RNA, Messenger; C508960:TGFB1 protein, human; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; C508322:VCAN protein, human; D053675:Versicans