Epigenetic regulation of chemokine CXCL12 gene transcription influences its prosurvival effect on pancreatic beta cells

Abstract

Background and aims: The CXC chemokine ligand 12 (CXCL12/SDF-1) promotes the expression of the proliferative phenotype that improves the resistance of pancreatic beta cells (b-cells) to diabetogenic stimuli. Our aim was to demonstrate the positive impact of chemokine CXCL12 expression on increased survival of b-cells, to provide a novel insight into the epigenetic regulation of CXCL12 gene ( Cxcl12 ) transcription and to initiate a screening study designed to test whether certain compounds present in the Epigenetic Compound Library (ECL-COST-TD0905) possess a DNMT1 inhibitory po- tential. Materials and methods: The RIN-5F rat pancreatic b-cell line (wt), its coun- terpart that possesses a stably integrated human gene for CXCL12 (#1) and MIN6 cells were exposed to increasing concentrations of streptozotocin. The prosurvival potential of CXCL12 was assessed by the viability assay (MTT). For the DNA methylation studies, DNA was isolated from: rat RIN-5F wt and #1 cells, rat Langerhans islets and mouse MIN6, NIH 3T3 wt and PARP-1 knockout cells. DNA methylation of the rat and mouse Cxcl12 was assessed using real-time methylation-specific PCR (MSP) with primers designed for each CpG island predicted within the promoter, the first exon and intron of Cxcl12 . Each component from ECL-COST-TD0905 was used at 15 μM con- centration for demethylation studies (5-aza-2’-deoxycytidine, was used as positive control). Results: Our results confirmed that the ιncreased presence of CXCL12 im- proves pancreatic b-cell survival during oxidative stress induced by a dia- betogenic stimulus. The CpG island analysis of the rat and mouse Cxcl12 promoter, first exon and intron revealed the same number and very similar distribution of CpG islands in both species. MSP showed that the CpG-rich regions within the Cxcl12 promoter, first exon and intron are semi-methyl- ated in the rat Rin-5F cells. In the rat Langerhans islets, the core promoter is unmethylated, while the first exon exhibited methylation of both alleles. In mouse cells, large differences in methylation patterns of the core promoter were observed: wt cells possess a unmethylated and PARP-1 knockout cells a fully methylated core promoter. One of the eight analysed compounds from the ECL-COST-TD0905 possesses potential to inhibit DNMT1 in vitro . Conclusion: We confirmed that CXCL12 exerts a prosurvival effect on pancreatic b-cells. The differences observed in the methylation status of the Cxcl12 gene, points to decreased gene responsiveness to external stimuli. The clear differences in the methylation status of the promoter and the first exon in the rat insulinoma cell line and ex vivo isolated Langerhans islets have to be underlined. Furthermore, observed hypermethylation of mouse Cxcl12 in PARP-1 knockout cells, points to the involvement of PARP-1 in the inhibition of the methylation in vivo.