Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.

Dysregulated autophagy and ER stress are involved in the etiology of human IBD. Aden et al. show that loss of ATG16L1 function renders intestinal epithelial cells vulnerable to IL-22–induced ER stress and necroptosis via STING signaling, which induces ileal inflammation in vivo.

Cellular senescence, a state of persistent cell cycle arrest, has emerged as a potent tumor suppressor mechanism by restricting proliferation of cells at risk for neoplastic transformation. Senescent cells secrete various growth factors, cytokines, and other proteins that can either elicit the clearance of tumor cells or potentially promote tumor progression. In addition, this senescence-associated secretory phenotype (SASP) includes various factors that are synthesized as transmembrane precursors and subsequently converted into their soluble counterparts. Despite the importance of the SASP to tumor biology, it is virtually unknown how transmembrane proteins are released from senescent cancer cells. Here we show in different models of senescence that the metalloprotease A disintegrin and metalloproteinase 17 (ADAM17) is activated and releases the epidermal growth factor receptor ligand amphiregulin and tumor necrosis factor receptor I (TNFRI) from the surface of senescent cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail by mitogen-activated protein kinase (MAPK) p38. Interestingly, unlike amphiregulin and TNFRI, full-length intercellular adhesion molecule 1 (ICAM1) is released from senescent cells by microvesicles independently of ADAM17. Thus, our results suggest that transmembrane proteins can be released by two distinct mechanisms and point to a crucial role for ADAM17 in shaping the secretory profile of senescent cells.

Background & AimsRNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis.MethodsWe generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data.ResultsThe H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times.ConclusionsIn analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.

BackgroundChemokines and their receptors play a decisive role in tumor progression and metastasis. We recently found a new signaling mechanism in malignant glioma cells mediated by transmembrane chemokines that we termed “inverse signaling”. According to this hypothesis, soluble (s)-CXCL16 binds to the surface-expressed transmembrane (tm) -CXCL16, and induces signaling and different biological effects in the stimulated cells, so that the transmembrane ligand itself acts as a receptor for its soluble counterpart. Now, we hypothesized that “inverse signaling” via tm-CXCL16 might also take place in meningiomas, a completely different, benign tumor entity.MethodsWe used quantitative reverse-transcription polymerase chain reaction, immunocytochemistry and western blot to detect CXCL16 and CXCR6 in human meningioma cells isolated from 28 human meningiomas. Subsequently, we stimulated cultured human tm-CXCL16-positive, CXCR6-negative meningioma cells with recombinant s-CXCL16 and analyzed binding, signaling and biological effects using RNAi silencing to verify specificity.ResultsIn fact, cultured human meningioma cells considerably express CXCL16, but substantially lack CXCR6, the only known CXCL16 receptor. These receptor-negative cells could bind s-CXCL16, and responded to s-CXCL16 application with activation of the intracellular kinases ERK1/2 und Akt. As a consequence, we observed increased proliferation and rescue of apoptosis of cultured meningioma cells. Since binding and signaling were abolished by siRNA silencing, we concluded that tm-CXCL16 specifically acts as a receptor for s-CXCL16 also in human meningioma cells.ConclusionThese findings underline our recent report on the mechanism of inverse signaling as a broad biological process also observable in more benign tumor cells and contributing to tumor progression.