Increased CpG methylation at the CDH1 locus in inflamed ileal mucosa of patients with Crohn disease.

BACKGROUND: E-cadherin, a major actor of cell adhesion in the intestinal barrier, is encoded by the CDH1 gene associated with susceptibility to Crohn Disease (CD) and colorectal cancer. Since epigenetic mechanisms are suspected to contribute to the multifactorial pathogenesis of CD, we studied CpG methylation at the CDH1 locus. The methylation of the CpG island (CGI) and of the 1st enhancer, two critical regulatory positions, was quantified in surgical specimens of inflamed ileal mucosa and in peripheral blood mononuclear cells (PBMC) of 21 CD patients. Sixteen patients operated on for a non-inflammatory bowel disease, although not normal controls, provided a macroscopically normal ileal mucosa and PBMC for comparison. RESULTS: In ileal mucosa, 19/21 (90%) CD patients vs 8/16 control patients (50%) (p < 0.01) had a methylated CDH1 promoter CGI. In PBMC, CD patients with methylated CGI were 11/21 (52%) vs 7/16 controls (44%), respectively. Methylation in the 1st enhancer of CDH1 was also higher in the CD group for each of the studied CpGs and for their average value (45 ± 17% in CD patients vs 36 ± 17% in controls; p < 0.001). Again, methylation was comparable in PBMC. Methylation of CGI and 1st enhancer were not correlated in mucosa or PBMC. CONCLUSIONS: Methylation of several CpGs at the CDH1 locus was increased in the inflamed ileal mucosa, not in the PBMC, of CD patients, suggesting the association of CDH1 methylation with ileal inflammation. Longitudinal studies will explore if this increased methylation is a risk marker for colorectal cancer.

Correction of a knock-in mouse model of acrodysostosis with gene therapy using a rAAV9-CAG-human PRKAR1A vector.

Acrodysostosis is a rare skeletal dysplasia caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). In a knock-in mouse model (PRKAR1Awt/mut) expressing one copy of the recurrent R368X mutation, we tested the effects of a rAAV9-CAG-human PRKR1A (hPRKAR1A) vector intravenously administered at 4 weeks of age. Caudal vertebrae and tibial diaphyses contained 0.52 ± 0.7 and 0.13 ± 0.3 vector genome per cell (VGC), respectively, at 10 weeks of age and 0.22 ± 0.04 and 0.020 ± 0.04 at 16 weeks while renal cortex contained 0.57 ± 0.14 and 0.26 ± 0.05 VGC. Vector-mediated hPRKAR1A expression was found in growth plate chondrocytes, osteoclasts, osteoblasts, and kidney tubular cells. Chondrocyte architecture was restored in the growth plates. Body length, tail length, and body weight were improved in vector treated PRKAR1Awt/mut mice, not the bone length of their limbs. These results provide one of the few proofs for gene therapy efficacy in a mouse model of chondrodysplasia. In addition, the increased urinary cAMP of PRKAR1Awt/mut mice was corrected almost to normal. In conclusion, gene therapy with hPRKAR1A improved skeletal growth and kidney dysfunction, the hallmarks of acrodysostosis in R368X mutated mice and humans.

Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression.

IL2RA, a subunit of the high affinity receptor for interleukin-2 (IL2), plays a crucial role in immune homeostasis. Notably, IL2RA expression is induced in CD4+ T cells in response to various stimuli and is constitutive in regulatory T cells (Tregs). We selected for our study 18 CpGs located within cognate regulatory regions of the IL2RA locus and characterized their methylation in naive, regulatory, and memory CD4+ T cells. We found that 5/18 CpGs (notably CpG + 3502) show dynamic, active demethylation during the in vitro activation of naive CD4+ T cells. Demethylation of these CpGs correlates with appearance of IL2RA protein at the cell surface. We found no influence of cis located SNP alleles upon CpG methylation. Treg cells show constitutive demethylation at all studied CpGs. Methylation of 9/18 CpGs, including CpG +3502, decreases with age. Our data thus identify CpG +3502 and a few other CpGs at the IL2RA locus as coordinated epigenetic regulators of IL2RA expression in CD4+ T cells. This may contribute to unravel how the IL2RA locus can be involved in immune physiology and pathology.

Correction to: Fetal growth is associated with CpG methylation in the P2 promoter of the IGF1 gene.

[This corrects the article DOI: 10.1186/s13148-018-0489-9.].

Fetal growth is associated with CpG methylation in the P2 promoter of the IGF1 gene.

Background: There are many reasons to think that epigenetics is a key determinant of fetal growth variability across the normal population. Since IGF1 and INS genes are major determinants of intrauterine growth, we examined the methylation of selected CpGs located in the regulatory region of these two genes. Methods: Cord blood was sampled in 159 newborns born to mothers prospectively followed during their pregnancy. A 142-item questionnaire was filled by mothers at inclusion, during the last trimester of the pregnancy and at the delivery. The methylation of selected CpGs located in the promoters of the IGF1 and INS genes was measured in cord blood mononuclear cells collected at birth using bisulfite-PCR-pyrosequencing. Results: Methylation at IGF1 CpG-137 correlated negatively with birth length (r = 0.27, P = 3.5 × 10-4). The same effect size was found after adjustment for maternal age, parity, and smoking: a 10% increase in CpG-137 methylation was associated with a decrease of length by 0.23 SDS. Conclusion: The current results suggest that the methylation of IGF1 CpG-137 contributes to the individual variation of fetal growth by regulating IGF1 expression in fetal tissues.

Genome-Wide Methylation Analysis Identifies Specific Epigenetic Marks In Severely Obese Children.

Obesity is a heterogeneous disease with many different subtypes. Epigenetics could contribute to these differences. The aim of this study was to investigate genome-wide DNA methylation searching for methylation marks associated with obesity in children and adolescents. We studied DNA methylation profiles in whole blood cells from 40 obese children and controls using Illumina Infinium HumanMethylation450 BeadChips. After correction for cell heterogeneity and multiple tests, we found that compared to lean controls, 31 CpGs are differentially methylated in obese patients. A greatest proportion of these CpGs is hypermethylated in obesity and located in CpG shores regions. We next focused on severely obese children and identified 151 differentially methylated CpGs among which 10 with a difference in methylation greater than 10%. The top pathways enriched among the identified CpGs included the "IRS1 target genes" and several pathways in cancer diseases. This study represents the first effort to search for differences in methylation in obesity and severe obesity, which may help understanding these different forms of obesity and their complications.

Role of DNA methylation at the placental RTL1 gene locus in type 1 diabetes.

Genome-wide association studies (GWAS) have identified more than 40 T1D loci associated with type 1 diabetes (T1D). How these polymorphisms interact with environmental factors to trigger T1D is unknown, but recent evidence suggests that epigenetic mechanisms could play a role. To begin to explore the contribution of epigenetics to T1D, we have examined DNA methylation in a pilot study of whole blood cells DNA from 10 young T1D patients and 10 young controls. Through the study of >900 000 CG loci across a diverse set of functionally relevant genomic regions using a custom DNA methylation array, we identified 250 T1D-differentially methylated region (DMR) at p < 0.05 and 1 DMR using next a permutation-based multiple testing correction method. This DMR is located in an imprinted region previously associated with T1D on the chromosome 14 that encompasses RTL1 gene and 2 miRNAs (miR136 and miR432). Using pyrosequencing-based bisulfite PCR, we replicated this association in a different and larger set of T1D patients and controls. DNA methylation at this DMR was inversely correlated with RTL1 gene expression and positively correlated with miR136 expression in human placentas. The DMR identified in this study presents suggestive evidence for altered methylation site in T1D and provide a promising new candidate gene. RTL1 is essential for placental permeability function in the mid-to-late fetal stages. We suggest that hypo-methylation could increase the fetal exposure to environmental factors in T1D susceptibility.

The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth.

BACKGROUND: Even if genetics play an important role, individual variation in stature remains unexplained at the molecular level. Indeed, genome-wide association study (GWAS) have revealed hundreds of variants that contribute to the variability of height but could explain only a limited part of it, and no single variant accounts for more than 0.3% of height variance. At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits, an emerging challenge in humans, has not been attempted for height. Since insulin-like growth factor 1 (IGF1) controls postnatal growth, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene is a potential epigenetic contributor to the individual variation in circulating IGF1 and stature in growing children. RESULTS: Child height was closely correlated with serum IGF1. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. The highest association was for CG-137 methylation, which contributed 13% to the variance of height and 10% to serum IGF1. CG methylation (studied in children undergoing surgery) was approximately 50% lower in liver and growth plates, indicating that the IGF1 promoters are tissue-differentially methylated regions (t-DMR). CG methylation was inversely correlated with the transcriptional activity of the P2 promoter in mononuclear blood cells and in transfection experiments, suggesting that the observed association of methylation with the studied traits reflects true biological causality. CONCLUSIONS: Our observations introduce epigenetics among the individual determinants of child growth and serum IGF1. The P2 promoter of the IGF1 gene is the first epigenetic quantitative trait locus (QTL(epi)) reported in humans. The CG methylation of the P2 promoter takes place among the multifactorial factors explaining the variation in human stature.

CpG methylation changes within the IL2RA promoter in type 1 diabetes of childhood onset.

None of the polymorphic variants of the IL2RA gene found associated with Type 1 Diabetes (T1D) was shown to have a functional effect. To test if the epigenetic variation could play a role at this locus, we studied the methylation of 6 CpGs located within the proximal promoter of IL2RA gene in 252 T1D patients compared with 286 age-matched controls. We found that DNA methylation at CpGs -373 and -456 was slightly but significantly higher in patients than in controls (40.4 ± 4.6 vs 38.3 ± 5.4, p=1.4E4; 91.4 ± 2.8 vs 89.5 ± 5.3, p=1.8E-6), while other CpG showed a strictly comparable methylation. Among 106 single nucleotide polymorphisms (SNPs) located in the neighboring 180 kb region, we found that 28 SNPs were associated with DNA methylation at CpG -373. Sixteen of these SNPs were known to be associated with T1D. Our findings suggest that the effect of IL2RA risk alleles on T1D may be partially mediated through epigenetic changes.

Beneficial paracrine effects of cannabinoid receptor 2 on liver injury and regeneration.

UNLABELLED: The cannabinoid receptor 2 (CB2) plays a pleiotropic role in innate immunity and is a crucial mediator of liver disease. In this study, we investigated the impact of CB2 receptors on the regenerative process associated with liver injury. Following acute hepatitis induced by carbon tetrachloride (CCl(4)), CB2 was induced in the nonparenchymal cell fraction and remained undetectable in hepatocytes. Administration of CCl(4) to CB2(-/-) mice accelerated liver injury, as shown by increased alanine/aspartate aminotransferase levels and hepatocyte apoptosis, and delayed liver regeneration, as reflected by a retarded induction of hepatocyte proliferating cell nuclear antigen expression; proliferating cell nuclear antigen induction was also delayed in CB2(-/-) mice undergoing partial hepatectomy. Conversely, following treatment with the CB2 agonist JWH-133, CCl(4)-treated WT mice displayed reduced liver injury and accelerated liver regeneration. The CCl(4)-treated CB2(-/-) mice showed a decrease in inducible nitric oxide synthase and tumor necrosis factor-alpha expression, and administration of the nitric oxide donor moldomine (SIN-1) to these animals reduced hepatocyte apoptosis, without affecting liver regeneration. Impaired liver regeneration was consecutive to an interleukin-6 (IL-6)-mediated decrease in matrix metalloproteinase 2 (MMP-2) activity. Indeed, CCl(4)-treated CB2(-/-) mice displayed lower levels of hepatic IL-6 messenger RNA and increased MMP-2 activity. Administration of IL-6 to these mice decreased MMP-2 activity and improved liver regeneration, without affecting hepatocyte apoptosis. Accordingly, administration of the MMP inhibitor CTTHWGFTLC to CCl(4)-treated CB2(-/-) mice improved liver regeneration. Finally, in vitro studies demonstrated that incubation of hepatic myofibroblasts with JWH-133 increased tumor necrosis factor-alpha and IL-6 and decreased MMP-2 expressions. CONCLUSION: CB2 receptors reduce liver injury and promote liver regeneration following acute insult, via distinct paracrine mechanisms involving hepatic myofibroblasts. These results suggest that CB2 agonists display potent hepatoprotective properties, in addition to their antifibrogenic effects.

Cannabinoid CB2 receptor potentiates obesity-associated inflammation, insulin resistance and hepatic steatosis.

BACKGROUND: Obesity-associated inflammation is of critical importance in the development of insulin resistance and non-alcoholic fatty liver disease. Since the cannabinoid receptor CB2 regulates innate immunity, the aim of the present study was to investigate its role in obesity-induced inflammation, insulin resistance and fatty liver. METHODOLOGY: Murine obesity models included genetically leptin-deficient ob/ob mice and wild type (WT) mice fed a high fat diet (HFD), that were compared to their lean counterparts. Animals were treated with pharmacological modulators of CB2 receptors. Experiments were also performed in mice knock-out for CB2 receptors (Cnr2 -/-). PRINCIPAL FINDINGS: In both HFD-fed WT mice and ob/ob mice, Cnr2 expression underwent a marked induction in the stromal vascular fraction of epididymal adipose tissue that correlated with increased fat inflammation. Treatment with the CB2 agonist JWH-133 potentiated adipose tissue inflammation in HFD-fed WT mice. Moreover, cultured fat pads isolated from ob/ob mice displayed increased Tnf and Ccl2 expression upon exposure to JWH-133. In keeping, genetic or pharmacological inactivation of CB2 receptors decreased adipose tissue macrophage infiltration associated with obesity, and reduced inductions of Tnf and Ccl2 expressions. In the liver of obese mice, Cnr2 mRNA was only weakly induced, and CB2 receptors moderately contributed to liver inflammation. HFD-induced insulin resistance increased in response to JWH-133 and reduced in Cnr2 -/- mice. Finally, HFD-induced hepatic steatosis was enhanced in WT mice treated with JWH-133 and blunted in Cnr2 -/- mice. CONCLUSION/SIGNIFICANCE: These data unravel a previously unrecognized contribution of CB2 receptors to obesity-associated inflammation, insulin resistance and non-alcoholic fatty liver disease, and suggest that CB2 receptor antagonists may open a new therapeutic approach for the management of obesity-associated metabolic disorders.

Progesterone reduces the migration of mast cells toward the chemokine stromal cell-derived factor-1/CXCL12 with an accompanying decrease in CXCR4 receptors.

Mast cell recruitment is implicated in many physiological functions and several diseases. It depends on microenvironmental factors, including hormones. We have investigated the effect of progesterone on the migration of HMC-1(560) mast cells toward CXCL12, a chemokine that controls the migration of mast cells into tissues. HMC-1(560) mast cells were incubated with 1 nM to 1 microM progesterone for 24 h. Controls were run without progesterone. Cell migration toward CXCL12 was monitored with an in vitro assay, and statistical analysis of repeated experiments revealed that progesterone significantly reduced cell migration without increasing the number of apoptotic cells (P = 0.0084, n = 7). Differences between progesterone-treated and untreated cells were significant at 1 microM (P < 0.01, n = 7). Cells incubated with 1 microM progesterone showed no rearrangment of actin filaments in response to CXCL12. Progesterone also reduced the calcium response to CXCL12 and Akt phosphorylation. Cells incubated with progesterone had one-half the control concentrations of CXCR4 (mRNA, total protein, and membrane-bound protein). Progesterone also inhibited the migration of HMC-1(560) cells transfected with hPR-B-pSG5 plasmid, which contained 2.5 times as much PR-B as the control. These transfected cells responded differently (P < 0.05, n = 5) from untreated cells to 1 nM progesterone. We conclude that progesterone reduces mast cell migration toward CXCL12 and that CXCR4 may be a progesterone target in mast cells.

Progesterone increases csk homologous kinase in HMC-1560 human mast cells and reduces cell proliferation.

Mast cells proliferate in vivo in areas of active fibrosis, during parasite infestations, in response to repeated immediate hypersensitivity reactions and in patients with mastocytosis. We investigated how progesterone reduces the proliferation of HMC-1(560) mast cells that proliferate spontaneously in culture. Cells were incubated with 1 microM to 1 nM progesterone for 24-48 h. Progesterone (1 microM) reduced the spontaneous proliferation of HMC-1(560) mast cells to half that of cells cultured without hormone. [(3)H] thymidine incorporation was only 50% of control; there were fewer cells in G2/M and more cells in G0/G1. The amounts of phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK proteins were also reduced. In contrast progesterone had no effect on MAP kinase-phosphatase-1. The Raf/MAPK pathway, which depends on Src kinase activity, is implicated in the control of cell proliferation. HMC-1(560) cells incubated with the tyrosine kinase inhibitor PP1 proliferated more slowly than controls and had less phospho-Raf-1 (Tyr 340-341) and phospho-p42/p44 MAPK. The Csk homologous kinase (CHK), an endogenous inhibitor of Src protein tyrosine kinases, was also enhanced in progesterone-treated cells. In contrast, progesterone had no effect on the growth of cells transfected with siRNA CHK. We conclude that progesterone increases the amount of csk homologous kinase, which in turn reduces HMC-1(560) mast cell proliferation. This effect parallels decreases in the phosphorylated forms of Raf-1 and p42/44 MAPK, as their production depends on Src kinase activity.