Ribonuclease 1 (RNase1) is a circulating extracellular endonuclease that regulates the vascular homeostasis of extracellular RNA and acts as a vessel‐ and tissue‐protective enzyme. Upon long‐term inflammation, high amounts of proinflammatory cytokines affect endothelial cell (EC) function by down‐regulation of RNase1. Here, we investigated the transcriptional regulation of RNase1 upon inflammation in HUVECs. TNF‐α or IL‐1β stimulation reduced the expression of RNase1 relative to the acetylation state of histone 3 at lysine 27 and histone 4 of the RNASE1 promoter. Inhibition of histone deacetylase (HDAC) 1, 2, and 3 by the specific class I HDAC inhibitor MS275 abolished the TNF‐α‐ or IL‐1β–mediated effect on the mRNA and chromatin levels of RNase1. Moreover, chromatin immunoprecipitation kinetics revealed that HDAC2 accumulates at the RNASE1 promoter upon TNF‐β stimulation, indicating an essential role for HDAC2 in regulating RNase1 expression. Thus, proinflammatory stimulation induced recruitment of HDAC2 to attenuate histone acetylation at the RNASE1 promoter site. Consequently, treatment with HDAC inhibitors may provide a new therapeutic strategy to stabilize vascular homeostasis in the context of inflammation by preventing RNase1 down‐regulation in ECs.—Bedenbender, K., Scheller, N., Fischer, S., Leiting, S., Preissner, K. T., Schmeck, B. T., Vollmeister, E. Inflammation‐mediated deacetylation of the ribonuclease 1 promoter via histone deacetylase 2 in endothelial cells. FASEB J. 33, 9017–9029 (2019). http://www.fasebj.org

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.

SLC10A7 represents an orphan member of the Solute Carrier Family SLC10. Recently, mutations in the human SLC10A7 gene were associated with skeletal dysplasia, amelogenesis imperfecta, and decreased bone mineral density. However, the exact molecular function of SLC10A7 and the mechanisms underlying these pathologies are still unknown. For this reason, the role of SLC10A7 on intracellular calcium signaling was investigated. SLC10A7 protein expression was negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. Whereas SLC10A7 knockout HAP1 cells showed significantly increased calcium influx after thapsigargin, ionomycin and ATP/carbachol treatment, SLC10A7 overexpression reduced this calcium influx. Intracellular Ca 2+ levels were higher in the SLC10A7 knockout cells and lower in the SLC10A7-overexpressing cells. The SLC10A7 protein colocalized with STIM1, Orai1, and SERCA2. Most of the previously described human SLC10A7 mutations had no effect on the calcium influx and thus were confirmed to be functionally inactive. In the present study, SLC10A7 was established as a novel negative regulator of intracellular calcium signaling that most likely acts via STIM1, Orai1 and/or SERCA2 inhibition. Based on this, SLC10A7 is suggested to be named as negative regulator of intracellular calcium signaling (in short: RCAS). Ca 2+ is one of the most versatile second messengers in eukaryotic cells. It is involved in many cellular processes such as muscle contraction, vesicle exocytosis, cell proliferation and growth, and gene expression 1. Whereas calcium influx into excitable cells is mainly mediated by voltage-gated calcium channels, the major entry pathway of calcium into non-excitable cells involves calcium release-activated calcium channels in the plasma membrane that allow store-operated calcium entry (SOCE) 2,3. Orail and the canonical transient receptor potential protein (TRPC) are two well-recognized store-operated Ca 2+ channels 4. While SOCE through Orail is dependent on activation and translocation of the stromal interaction molecule 1 (STIM1), SOCE through TRPC can function in a STIM1-dependent or-independent manner 4. These store-operated calcium channels are opened in response to calcium depletion of the endoplasmic reticulum (ER). Release of Ca 2+ from the intracellular stores leads to activation of the ER Ca 2+ sensor STIM1, which then interacts with Orai1 subunits to form a STIM-Orai complex that supports SOCE at a typically high selectivity for Ca 2+ 5-7. A local increase in Ca 2+ levels or gradual refilling of the calcium stores then inactivates the channels by negative feedback regulation 8,9. The sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) transports Ca 2+ from the cytosol into the SR/ER lumen to maintain the cytosolic Ca 2+ at its low resting level 10. The activity of SERCA is regulated according to the cellular requirements and extracellular signals of two different small proteins, phospholamban and sarcolipin. Both regulator proteins are only expressed in muscl...

Self-extracellular RNA (eRNA), released from stressed or injured cells upon various pathological situations such as ischemia-reperfusion-injury, has been shown to act as an alarmin by inducing procoagulatory and proinflammatory responses. In particular, M1-polarization of macrophages by eRNA resulted in the expression and release of a variety of cytokines, including tumor necrosis factor (TNF)-α or interleukin-6 (IL-6). The present study now investigates in which way self-eRNA may influence the response of macrophages towards various Toll-like receptor (TLR)-agonists. Isolated agonists of TLR2 (Pam2CSK4), TLR3 (PolyIC), TLR4 (LPS), or TLR7 (R848) induced the release of TNF-α in a concentration-dependent manner in murine macrophages, differentiated from bone marrow-derived stem cells by mouse colony stimulating factor. Here, the presence of eRNA shifted the dose-response curve for Pam2CSK4 (Pam) considerably to the left, indicating that eRNA synergistically enhanced the cytokine liberation from macrophages even at very low Pam-levels. The synergistic activation of TLR2 by eRNA/Pam was duplicated by other TLR2-agonists such as FSL-1 or Pam3CSK4. In contrast, for TLR4-agonists such as LPS a synergistic effect of eRNA was much weaker, and was not existent for TLR3-, or TLR7-agonists. The synergistic eRNA/Pam action was dependent on the NFκB-signaling pathway as well as on p38MAP- and MEK1/ERK-kinases and was prevented by predigestion of eRNA with RNase1 or by antibodies against TLR2. Thus, the presence of self-eRNA as alarming molecule sensitizes innate immune responses towards pathogen-associated molecular patterns (PAMPs) in a synergistic way and may thereby contribute to the differentiated outcome of inflammatory responses.

Deletion of the Habp2 gene encoding Factor VII-activating protease (FSAP) increases liver fibrosis in mice. A single nucleotide polymorphism (G534E) in HABP2 leads to lower enzymatic activity and is associated with enhanced liver fibrosis in humans. Liver fibrosis is associated with a decrease in FSAP expression but, to date, nothing is known about how this might be regulated. Primary mouse hepatocytes or the hepatocyte cell line, AML12, were treated with different factors, and expression of FSAP was determined. Of the various regulatory factors tested, only transforming growth factor-β (TGF-β) demonstrated a concentration- and time-dependent inhibition of FSAP expression at the mRNA and protein level. The TGF-β-Type I receptor (ALK-5) antagonist SB431542 and Smad2 siRNA, but neither SIS3, which inhibits SMAD3, nor siRNA against Smad3 could block this effect. Various regions of the HABP2 promoter region were cloned into reporter constructs, and the promoter activity was determined. Accordingly, the promoter activity, which could phenocopy changes in Habp2 mRNA in response to TGF-β, was found to be located in the 177-bp region upstream of the transcription start site, and this region did not contain any SMAD binding sites. Mutation analysis of the promoter and chromatin immunoprecipitation assays were performed to identify an important role for the ATF3 binding element. Thus, TGF-β is the most likely mediator responsible for the decrease in FSAP expression in liver fibrosis.