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M Elizabeth Fini, Shinwu Jeong; Trabecular Meshwork Cell Expression of Two Novel Mucin Genes Located in the MHC Class I Locus. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3562.
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Mucins, characterized by tandem repeats that serve as sites for O-glycosylation, are important to ocular surface health. Two novel mucin genes, PBMUCL1 (MUC22) and PBMUCL2 (HCG22), were recently identified in a mucin gene cluster within the MHC Class I locus on chromosome 6 (Hu Genet 129:117, 2011). We investigated expression in cells of the anterior segment.
We used two immortalized human cell lines: HCLE from corneal limbal epithelium and TM-1 from trabecular meshwork (TM). We also used primary TM cell lines isolated from human corneal rims. Cells were untreated or treated with agents linked to ocular hypertension: IL-1beta (10 ng/ml), the glucocorticoid (GC) triamcinolone acetonide (TA; 100 ng/ml), and TGF-beta (10 ng/ml). RT-PCR was used to determine mRNA expression. Statistical significance was determined by use of Student’s t-test. FLAG or GFP-tagged cDNAs were cloned in the pcDNA3.1+ vector and expressed in TM-1 cells followed by fluorescence microscopy, subcellular fractionation and/or western blotting.
HCLE cells express PBMUCL1 but not PBMUCL2; TM cells express both. IL-1 stimulates expression of both genes in TM cells, while TA inhibits basal and IL-1-stimulated expression. TGF-beta also inhibits expression. Conceptual translation indicates that PBMUCL1 mRNA encodes a plasma membrane-associated protein of 174 kDa and PBMUCL2 mRNA encodes a secreted protein of 26kDa. A cloned variant of PBMUCL1 cDNA with a truncated mucin repeat region (57 kDa), tagged with GFP (27 kDa) and expressed in TM-1 cells, enriches in the plasma membrane, migrating at ~ 200 kDa on westerns. FLAG-tagged PBMUCL2 cDNA is expressed as a ~40 kDa cytoplasmic form and a ~67 kDa secreted form. O-glycosylation predicted by sequence analysis explains the larger than expected sizes.
The expression of mucins by TM cells has not previously been considered to our knowledge. These experiments confirm that the novel mucin genes, PBMUCL1 (MUC22) and PBMUCL2 (HCG22), are regulated by agents associated with ocular hypertension and encode proteins translated in TM cells. Interestingly, an association between the MHC class I locus and ocular hypertension was reported more than 35 years ago (Science 194:1427, 1976). The presence of a mucin layer that collapses with tissue fixation would help reconcile the discrepancy between measured outflow resistance and observed size of outflow pathway spaces.
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