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Dong-Jin Oh, Min Kang, Douglas J Rhee; Trafficking of SPARC. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5686.
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© ARVO (1962-2015); The Authors (2016-present)
SPARC, a matricellular protein, is secreted into the extracellular space but does not serve a structural function, and modulates interactions between cells and the surrounding extracellular matrix. Fibrosis is characterized by excessive deposition of extracellular matrix that interferes with normal tissue architecture and function, resulting in hypertensive ocular diseases. Myocilin, which has matricellular like properties, and considered a peripheral member of the matricellular family, has altered intracellular trafficking as part of its pathophysiology. Increased expression of SPARC raises IOP in an ex vivo perfusion chamber system and the absence of SPARC results in a lower IOP in mice. We hypothesized that SPARC could be altered trafficking.
A normal state, incubation with TGF-β2 of 3 ng for 24 hrs, 25 multiplicity of infection of Ad5.hSPARC for SPARC overexpression for 18 hrs with 2% serum media and then for 24 hrs with 6% serum media, and 2 x 10^7 TU/mL of lenti.shRNA.hSPARC with 4 µg/mL of polybrene for SPARC knockdown for 4 days with 10% serum media were used for SPARC secretion in trabecular meshwork cell cultures. PDI antibody was used as an ER marker, EEA1 antibody as an early endosomal marker, and GM130 antibody as a golgi complex marker, and also SPARC antibodies were used for secretion markers using immunofluorescence staining.
In a normal state weak GM130 stains showed SPARC secretion pattern with golgi complex in perinuclear distribution, EEA1 stains displayed in small-dots distribution of SPARC in perinucleus region, and PDI stains produced a perinuclear and dotted distribution of SPARC in endoplasmc reticulum lumen. TGF-β2 displayed 7 times more SPARC expression in GM130, EEA1, and PDI stain distributions than the normal state. Ad5.hSPARC exhibited 10 times more SPARC expression in GM130, EEA1, and PDI stain distributions than the normal state. Lenti.shRNA.hSPARC demonstrated little SPARC secretion levels in GM130, EEA1, and PDI stain distributions.
TGF-β2 and Ad5.hSPARC produced 7 times and 10 times more SPARC expressions in GM130, EEA1, and PDI stain distributions than the normal state, respectively. Lenti.shRNA.hSPARC demonstrated little SPARC secretion levels. Secretory proteins are mostly glycoproteins. These results can be used for SPARC trafficking as preliminary data. SPARC mutations or treatments to regulate SPARC secretion need to be found.
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