ObjectiveTo study how CD73 is shed from the retinal pigment epithelium (RPE) surface.MethodsCD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared.ResultsLPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73.ConclusionMMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.