Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1ß release by mononuclear phagocytes.

Objective: The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in host defense against infections. High systemic IL-1ß levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1ß release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1ß release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Methods: Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1ß was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. Results: The inhibitory effect of nAChR agonists on the BzATP-induced IL-1ß release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1ß release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1ß release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. Conclusion: We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1ß release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis.

The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.

Genomic Location of PRMT6-Dependent H3R2 Methylation Is Linked to the Transcriptional Outcome of Associated Genes.

Protein arginine methyltransferase 6 (PRMT6) catalyzes asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a). This mark has been reported to associate with silent genes. Here, we use a cell model of neural differentiation, which upon PRMT6 knockout exhibits proliferation and differentiation defects. Strikingly, we detect PRMT6-dependent H3R2me2a at active genes, both at promoter and enhancer sites. Loss of H3R2me2a from promoter sites leads to enhanced KMT2A binding and H3K4me3 deposition together with increased target gene transcription, supporting a repressive nature of H3R2me2a. At enhancers, H3R2me2a peaks co-localize with the active enhancer marks H3K4me1 and H3K27ac. Here, loss of H3R2me2a results in reduced KMT2D binding and H3K4me1/H3K27ac deposition together with decreased transcription of associated genes, indicating that H3R2me2a also exerts activation functions. Our work suggests that PRMT6 via H3R2me2a interferes with the deposition of adjacent histone marks and modulates the activity of important differentiation-associated genes by opposing transcriptional effects.

Identification and in silico structural analysis of Gallus gallus protein arginine methyltransferase 4 (PRMT4).

Protein arginine methyltransferase 4 (PRMT4) is an essential epigenetic regulator of fundamental and conserved processes during vertebrate development, such as pluripotency and differentiation. Surprisingly, PRMT4 homologs have been identified in nearly all vertebrate classes except the avian genome. This raises the possibility that in birds PRMT4 functions are taken over by other PRMT family members. Here, we reveal the existence of a bona fide PRMT4 homolog in the chicken, Gallus gallus. Using a biochemical approach, we initially purified a putative chicken PRMT4 protein and thus provided the first evidence for the presence of an endogenous PRMT4-specific enzymatic activity toward histone H3 arginine 17 (H3R17) in avian cells. We then isolated a G. gallus PRMT4 (ggPRMT4) transcript encompassing the complete open reading frame. Recombinant ggPRMT4 possesses intrinsic methyltransferase activity toward H3R17. CRISPR/Cas9-mediated deletion of ggPRMT4 demonstrated that the transcript identified here encodes avian PRMT4. Combining protein-protein docking and homology modeling based on published crystal structures of murine PRMT4, we found a strong structural similarity of the catalytic core domain between chicken and mammalian PRMT4. Strikingly, in silico structural comparison of the N-terminal Pleckstrin homology (PH) domain of avian and murine PRMT4 identified strictly conserved amino acids that are involved in an interaction interface toward the catalytic core domain, facilitating for the first time a prediction of the relative spatial arrangement of these two domains. Our novel findings are particularly exciting in light of the essential function of the PH domain in substrate recognition and methylation by PRMT4.

Expression of acetylcholine receptors by experimental rat renal allografts.

Chronic allograft injury (CAI) is a major cause for renal allograft dysfunction and characterized by vasculopathies, tubular atrophy, and fibrosis. We demonstrated that numerous leukocytes interact with vascular endothelial cells of allografts and produce acetylcholine, which contributes to vascular remodeling. The cholinergic system might be a promising target for the development of novel therapies. However, neither the cellular mechanisms nor the acetylcholine receptors involved in CAI are known. Kidney transplantation was performed in the Lewis to Lewis and in the Fischer-334 to Lewis rat strain combination, which is an established experimental model for CAI. Expression of nicotinic and muscarinic acetylcholine receptors mRNA was quantified in renal tissue by real-time RT-PCR on days 9 and 42 after surgery. We detected CHRNA2-7, CHRNA10, CHRNB2, CHRNB4, and CHRM1-3 mRNA in normal kidneys and in renal transplants. In contrast, CHRNA9, CHRM4, and CHRM5 mRNA remained below the threshold of detection. In renal allografts, CHRNA3 and CHRNB4 mRNA expression were dramatically reduced compared to isografts. In conclusion, we demonstrated that most acetylcholine receptor subtypes are expressed by normal and transplanted kidneys. Allograft rejection downmodulates CHRNA3 and CHRNB4 mRNA. The role of different acetylcholine receptor subtypes in the development of CAI remains to be established.

Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands.

2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.

Myelination in the absence of UDP-galactose:ceramide galactosyl-transferase and fatty acid 2 -hydroxylase.

BACKGROUND: The sphingolipids galactosylceramide (GalCer) and sulfatide are major myelin components and are thought to play important roles in myelin function. The importance of GalCer and sulfatide has been validated using UDP-galactose:ceramide galactosyltransferase-deficient (Cgt-/-) mice, which are impaired in myelin maintenance. These mice, however, are still able to form compact myelin. Loss of GalCer and sulfatide in these mice is accompanied by up-regulation of 2-hydroxylated fatty acid containing (HFA)-glucosylceramide in myelin. This was interpreted as a partial compensation of the loss of HFA-GalCer, which may prevent a more severe myelin phenotype. In order to test this hypothesis, we have generated Cgt-/- mice with an additional deletion of the fatty acid 2-hydroxylase (Fa2h) gene. RESULTS: Fa2h-/-/Cgt-/- double-deficient mice lack sulfatide, GalCer, and in addition HFA-GlcCer and sphingomyelin. Interestingly, compared to Cgt-/- mice the amount of GlcCer in CNS myelin was strongly reduced in Fa2h-/-/Cgt-/- mice by more than 80%. This was accompanied by a significant increase in sphingomyelin, which was the predominant sphingolipid in Fa2h-/-/Cgt-/- mice. Despite these significant changes in myelin sphingolipids, compact myelin was formed in Fa2h-/-/Cgt-/- mice, and g-ratios of myelinated axons in the spinal cord of 4-week-old Fa2h-/-/Cgt-/- mice did not differ significantly from that of Cgt-/- mice, and there was no obvious phenotypic difference between Fa2h-/-/Cgt-/- and Cgt-/- mice CONCLUSIONS: These data show that compact myelin can be formed with non-hydroxylated sphingomyelin as the predominant sphingolipid and suggest that the presence of HFA-GlcCer and HFA-sphingomyelin in Cgt-/- mice does not functionally compensate the loss of HFA-GalCer.

Absence of 2-hydroxylated sphingolipids is compatible with normal neural development but causes late-onset axon and myelin sheath degeneration.

Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H(-/-)) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H(-/-) mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGFalpha receptor and the time course of myelin formation were not altered in FA2H(-/-) mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H(-/-) and wild-type mice. Moreover, myelin of FA2H(-/-) mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H(-/-) mice. Aged (18-month-old) FA2H(-/-) mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.