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Chengrong Yu, Waynekid Kam, Ivy Dambuza, Bernadette Marrero, Rashid Mahdi, Charles E. Egwuagu, ; Osteopontin is Expressed by Microglia and T Cells and Regulated by STAT3. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2050.
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© ARVO (1962-2015); The Authors (2016-present)
Osteopontin (OPN) is a SIBLING family protein that binds to integrin receptors including α4β1, α9β1, and α9β4 and it promotes chemotactic properties of leukocytes. In previous studies, we showed that mice with STAT3 deletion in T cells do not develop EAU or EAE because their uveitogenic or encephalotigenic T cells are defective in the expression of OPN and α4β1 and could not traffic into the retina or brain during CNS inflammation (J Immunol. 180:6070-6; J Immunol. 187:3338-46). Furthermore, OPN and α4β1, along with αΒ crystalline, have been implicated in mechanism that mediate relapsing/remitting CNS autoimmune diseases. In this study, we have investigated whether OPN is expressed in the retina and whether its expression is directly regulated by STAT3.
Expression of OPN in T cells, microglia, and retinal tissues was determined by FACS, confocal microscopy (using anti-IBa 1 and anti-OPN abs), and RT-PCR. Naïve CD4 T cells were polarized under Th0, Th1 and Th17 conditions and their lineage makers and intracellular cytokine expression were analyzed by FACS. Experimental autoimmune uveitis (EAU) was induced in C57BL/6 mice by immunization with IRBP/CFA. Bioinformatic tools were used to analyze whether there are potential STAT binding sites within distal and proximal core OPN promoter regions of the OPN gene. Chromatin immunoprecipitation (CHIP) analysis was performed using STAT3 and STAT1 Abs to determine potential STAT binding to OPN promoter.
We detected constitutive OPN expression by the microglia cell line, BV-2 and analysis of the various T-helper subsets revealed that OPN is preferentially expressed in Th17 and Th1 but barely detectable in Th0 cells. By CHIP assays, we identified 2 potential STAT binding sites and have demonstrated here that STAT3 preferentially binds the TTCagaGAA (-1162). By immunohistochemistry we detected increased OPN expression in perivascular cells of retinal blood vesicles and retinal microglial cells of mice with EAU.
We provide for the first time direct evidence that STAT3 binds to the OPN promoter and is a positive regulator of OPN gene expression. Increased OPN expression by microglia and perivascular cells during uveitis, taken together with our previous finding that defective OPN expression correlates with resistance to EAU, suggest that OPN is a potential therapeutic target in uveitis.
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