Epigenetic control of the angiotensin-converting enzyme in endothelial cells during inflammation.

The angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system, which is involved in the regulation of blood pressure. Alterations in ACE expression or activity are associated with various pathological phenotypes, particularly cardiovascular diseases. In human endothelial cells, ACE was shown to be negatively regulated by tumor necrosis factor (TNF) α. To examine, whether or not, epigenetic factors were involved in ACE expression regulation, methylated DNA immunoprecipitation and RNA interference experiments directed against regulators of DNA methylation homeostasis i.e., DNA methyltransferases (DNMTs) and ten-eleven translocation methylcytosine dioxygenases (TETs), were performed. TNFα stimulation enhanced DNA methylation in two distinct regions within the ACE promoter via a mechanism linked to DNMT3a and DNMT3b, but not to DNMT1. At the same time, TET1 protein expression was downregulated. In addition, DNA methylation decreased the binding affinity of the transcription factor MYC associated factor X to the ACE promoter. In conclusion, DNA methylation determines the TNFα-dependent regulation of ACE gene transcription and thus protein expression in human endothelial cells.

Role of the angiotensin-converting enzyme in the G-CSF-induced mobilization of progenitor cells.

In addition to being a peptidase, the angiotensin-converting enzyme (ACE) can be phosphorylated and involved in signal transduction. We evaluated the role of ACE in granulocyte-colony-stimulating factor (G-CSF)-induced hematopoietic progenitor cell (HPC) mobilization and detected a significant increase in mice-lacking ACE. Transplantation experiments revealed that the loss of ACE in the HPC microenvironment rather than in the HPCs increased mobilization. Indeed, although ACE was expressed by a small population of bone-marrow cells, it was more strongly expressed by endosteal bone. Interestingly, there was a physical association of ACE with the G-CSF receptor (CD114), and G-CSF elicited ACE phosphorylation on Ser1270 in vivo and in vitro. A transgenic mouse expressing a non-phosphorylatable ACE (ACES/A) mutant demonstrated increased G-CSF-induced HPC mobilization and decreased G-CSF-induced phosphorylation of STAT3 and STAT5. These results indicate that ACE expression/phosphorylation in the bone-marrow niche interface negatively regulates G-CSF-induced signaling and HPC mobilization.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.