Nitrogen availability is important for preventing catastrophic mitosis in fission yeast.

Mitosis is a critical stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe may demonstrate catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from e.g. mutations in acetyl-CoA/biotin carboxylase (cut6), in fatty acid synthase (fas2/lsd1), or in the transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It was previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride. In this study we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.

Critical importance of DNA binding for CSL protein functions in fission yeast.

CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central ß-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.

Targeted deletion of von-Hippel-Lindau in the proximal tubule conditions the kidney against early diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.

Dupilumab but not cyclosporine treatment shifts the microbiome toward a healthy skin flora in patients with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) patients display an altered skin microbiome which may not only be an indicator but also a driver of inflammation. We aimed to investigate associations among AD patients' skin microbiome, clinical data, and response to systemic therapy in patients of the TREATgermany registry. METHODS: Skin swabs of 157 patients were profiled with 16S rRNA gene amplicon sequencing before and after 3 months of treatment with dupilumab or cyclosporine. For comparison, 16s microbiome data from 258 population-based healthy controls were used. Disease severity was assessed using established instruments such as the Eczema Area and Severity Index (EASI). RESULTS: We confirmed the previously shown correlation of Staphylococcus aureus abundance and bacterial alpha diversity with AD severity as measured by EASI. Therapy with Dupilumab shifted the bacterial community toward the pattern seen in healthy controls. The relative abundance of Staphylococci and in particular S. aureus significantly decreased on both lesional and non-lesional skin, whereas the abundance of Staphylococcus hominis increased. These changes were largely independent from the degree of clinical improvement and were not observed for cyclosporine. CONCLUSIONS: Systemic treatment with dupilumab but not cyclosporine tends to restore a healthy skin microbiome largely independent of the clinical response indicating potential effects of IL-4RA blockade on the microbiome.

Crosstalk between microbiome, regulatory T cells and HCA2 orchestrates the inflammatory response in a murine psoriasis model.

The organ-specific microbiome plays a crucial role in tissue homeostasis, among other things by inducing regulatory T cells (Treg). This applies also to the skin and in this setting short chain fatty acids (SCFA) are relevant. It was demonstrated that topical application of SCFA controls the inflammatory response in the psoriasis-like imiquimod (IMQ)-induced murine skin inflammation model. Since SCFA signal via HCA2, a G-protein coupled receptor, and HCA2 expression is reduced in human lesional psoriatic skin, we studied the effect of HCA2 in this model. HCA2 knock-out (HCA2-KO) mice reacted to IMQ with stronger inflammation, presumably due to an impaired function of Treg. Surprisingly, injection of Treg from HCA2-KO mice even enhanced the IMQ reaction, suggesting that in the absence of HCA2 Treg switch from a suppressive into a proinflammatory type. HCA2-KO mice differed in the composition of the skin microbiome from wild type mice. Co-housing reversed the exaggerated response to IMQ and prevented the alteration of Treg, implying that the microbiome dictates the outcome of the inflammatory reaction. The switch of Treg into a proinflammatory type in HCA2-KO mice could be a downstream phenomenon. This opens the opportunity to reduce the inflammatory tendency in psoriasis by altering the skin microbiome.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Tubular IKKß Deletion Alleviates Acute Ischemic Kidney Injury and Facilitates Tissue Regeneration.

Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate immune response converging to NF-ĸB pathway induction. Despite the clear role of NF-ĸB in inflammation, it has recently been acknowledged that NF-ĸB may impact other cell functions. To identify NF-ĸB function with respect to metabolism, vascular function and oxidative stress after I/R injury and to decipher in detail the underlying mechanism, we generated a transgenic mouse model with targeted deletion of IKKß along the tubule and applied I/R injury followed by its analysis after 2 and 14 days after I/R injury. Tubular IKKß deletion ameliorated renal function and reduced tissue damage. RNAseq data together with immunohistochemical, biochemical and morphometric analysis demonstrated an ameliorated vascular organization and mRNA expression profile for increased angiogenesis in mice with tubular IKKß deletion at 2 days after I/R injury. RNAseq and protein analysis indicate an ameliorated metabolism, oxidative species handling and timely-adapted cell proliferation and apoptosis as well as reduced fibrosis in mice with tubular IKKß deletion at 14 days after I/R injury. In conclusion, mice with tubular IKKß deletion upon I/R injury display improved renal function and reduced tissue damage and fibrosis in association with improved vascularization, metabolism, reactive species disposal and fine-tuned cell proliferation.

Microbial regulation of hexokinase 2 links mitochondrial metabolism and cell death in colitis.

Hexokinases (HK) catalyze the first step of glycolysis limiting its pace. HK2 is highly expressed in gut epithelium, contributes to immune responses, and is upregulated during inflammation. We examined the microbial regulation of HK2 and its impact on inflammation using mice lacking HK2 in intestinal epithelial cells (Hk2ΔIEC). Hk2ΔIEC mice were less susceptible to acute colitis. Analyzing the epithelial transcriptome from Hk2ΔIEC mice during colitis and using HK2-deficient intestinal organoids and Caco-2 cells revealed reduced mitochondrial respiration and epithelial cell death in the absence of HK2. The microbiota strongly regulated HK2 expression and activity. The microbially derived short-chain fatty acid (SCFA) butyrate repressed HK2 expression via histone deacetylase 8 (HDAC8) and reduced mitochondrial respiration in wild-type but not in HK2-deficient Caco-2 cells. Butyrate supplementation protected wild-type but not Hk2ΔIEC mice from colitis. Our findings define a mechanism how butyrate promotes intestinal homeostasis and suggest targeted HK2-inhibition as therapeutic avenue for inflammation.

CD79a promotes CNS-infiltration and leukemia engraftment in pediatric B-cell precursor acute lymphoblastic leukemia.

Central nervous system (CNS) involvement remains a challenge in the diagnosis and treatment of acute lymphoblastic leukemia (ALL). In this study, we identify CD79a (also known as Igα), a signaling component of the preB cell receptor (preBCR), to be associated with CNS-infiltration and -relapse in B-cell precursor (BCP)-ALL patients. Furthermore, we show that downregulation of CD79a hampers the engraftment of leukemia cells in different murine xenograft models, particularly in the CNS.

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice.

BACKGROUND & AIMS: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. RESULTS: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. LAY SUMMARY: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

SETDB1 is required for intestinal epithelial differentiation and the prevention of intestinal inflammation.

OBJECTIVE: The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. DESIGN: We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. RESULTS: Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. CONCLUSION: SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

Neutrophil Extracellular Trap Induced Dendritic Cell Activation Leads to Th1 Polarization in Type 1 Diabetes.

Neutrophils releasing neutrophil extracellular traps (NETs) infiltrate the pancreas prior to type 1 diabetes (T1D) onset; however, the precise nature of their contribution to disease remains poorly defined. To examine how NETs affect immune functions in T1D, we investigated NET composition and their effect on dendritic cells (DCs) and T lymphocytes in T1D children. We showed that T1D patient NET composition differs substantially from that of healthy donors and that the presence of T1D-NETs in a mixed peripheral blood mononuclear cell culture caused a strong shift toward IFNγ-producing T lymphocytes, mediated through activation of innate immunity cells in T1D samples. Importantly, in a monocyte-derived DC (moDC) culture, NETs induced cytokine production, phenotypic change and IFNγ-producing T cells only in samples from T1D patients but not in those from healthy donors. RNA-seq analysis revealed that T1D-NETs presence causes TGFß downregulation and IFNα upregulation and creates pro-T1D signature in healthy moDCs.

NOD2 Influences Trajectories of Intestinal Microbiota Recovery After Antibiotic Perturbation.

BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. METHODS: Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. RESULTS: Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. CONCLUSIONS: Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn's disease.

Experimental Parasite Infection Causes Genome-Wide Changes in DNA Methylation.

Parasites are arguably among the strongest drivers of natural selection, constraining hosts to evolve resistance and tolerance mechanisms. Although, the genetic basis of adaptation to parasite infection has been widely studied, little is known about how epigenetic changes contribute to parasite resistance and eventually, adaptation. Here, we investigated the role of host DNA methylation modifications to respond to parasite infections. In a controlled infection experiment, we used the three-spined stickleback fish, a model species for host-parasite studies, and their nematode parasite Camallanus lacustris. We showed that the levels of DNA methylation are higher in infected fish. Results furthermore suggest correlations between DNA methylation and shifts in key fitness and immune traits between infected and control fish, including respiratory burst and functional trans-generational traits such as the concentration of motile sperm. We revealed that genes associated with metabolic, developmental, and regulatory processes (cell death and apoptosis) were differentially methylated between infected and control fish. Interestingly, genes such as the neuropeptide FF receptor 2 and the integrin alpha 1 as well as molecular pathways including the Th1 and Th2 cell differentiation were hypermethylated in infected fish, suggesting parasite-mediated repression mechanisms of immune responses. Altogether, we demonstrate that parasite infection contributes to genome-wide DNA methylation modifications. Our study brings novel insights into the evolution of vertebrate immunity and suggests that epigenetic mechanisms are complementary to genetic responses against parasite-mediated selection.

DNA methylation QTL analysis identifies new regulators of human longevity.

Human longevity is a complex trait influenced by both genetic and environmental factors, whose interaction is mediated by epigenetic mechanisms like DNA methylation. Here, we generated genome-wide whole-blood methylome data from 267 individuals, of which 71 were long-lived (90-104 years), by applying reduced representation bisulfite sequencing. We followed a stringent two-stage analysis procedure using discovery and replication samples to detect differentially methylated sites (DMSs) between young and long-lived study participants. Additionally, we performed a DNA methylation quantitative trait loci analysis to identify DMSs that underlie the longevity phenotype. We combined the DMSs results with gene expression data as an indicator of functional relevance. This approach yielded 21 new candidate genes, the majority of which are involved in neurophysiological processes or cancer. Notably, two candidates (PVRL2, ERCC1) are located on chromosome 19q, in close proximity to the well-known longevity- and Alzheimer's disease-associated loci APOE and TOMM40. We propose this region as a longevity hub, operating on both a genetic (APOE, TOMM40) and an epigenetic (PVRL2, ERCC1) level. We hypothesize that the heritable methylation and associated gene expression changes reported here are overall advantageous for the LLI and may prevent/postpone age-related diseases and facilitate survival into very old age.

Two different epigenetic information channels in wild three-spined sticklebacks are involved in salinity adaptation.

Epigenetic inheritance has been proposed to contribute to adaptation and acclimation via two information channels: (i) inducible epigenetic marks that enable transgenerational plasticity and (ii) noninducible epigenetic marks resulting from random epimutations shaped by selection. We studied both postulated channels by sequencing methylomes and genomes of Baltic three-spined sticklebacks (Gasterosteus aculeatus) along a salinity cline. Wild populations differing in salinity tolerance revealed differential methylation (pop-DMS) at genes enriched for osmoregulatory processes. A two-generation experiment demonstrated that 62% of these pop-DMS were noninducible by salinity manipulation, suggesting that they are the result of either direct selection or associated genomic divergence at cis- or trans-regulatory sites. Two-thirds of the remaining inducible pop-DMS increased in similarity to patterns detected in wild populations from corresponding salinities. The level of similarity accentuated over consecutive generations, indicating a mechanism of transgenerational plasticity. While we can attribute natural DNA methylation patterns to the two information channels, their interplay with genomic variation in salinity adaptation is still unresolved.

Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis.

The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.IMPORTANCE All animals are associated with microorganisms; hence, host-microbe interactions are of fundamental importance for life on earth. However, we know little about the molecular basis of these interactions. Therefore, we studied the deep-sea Riftia pachyptila symbiosis, a model association in which the tubeworm host is associated with only one phylotype of endosymbiotic bacteria and completely depends on this sulfur-oxidizing symbiont for nutrition. Using a metaproteomics approach, we identified both metabolic interaction processes, such as substrate transfer between the two partners, and interactions that serve to maintain the symbiotic balance, e.g., host efforts to control the symbiont population or symbiont strategies to modulate these host efforts. We suggest that these interactions are essential principles of mutualistic animal-microbe associations.

A combined epigenome- and transcriptome-wide association study of the oral masticatory mucosa assigns CYP1B1 a central role for epithelial health in smokers.

BACKGROUND: The oral mucosa has an important role in maintaining barrier integrity at the gateway to the gastrointestinal and respiratory tracts. Smoking is a strong environmental risk factor for the common oral inflammatory disease periodontitis and oral cancer. Cigarette smoke affects gene methylation and expression in various tissues. This is the first epigenome-wide association study (EWAS) that aimed to identify biologically active methylation marks of the oral masticatory mucosa that are associated with smoking. RESULTS: Ex vivo biopsies of 18 current smokers and 21 never smokers were analysed with the Infinium Methylation EPICBeadChip and combined with whole transcriptome RNA sequencing (RNA-Seq; 16 mio reads per sample) of the same samples. We analysed the associations of CpG methylation values with cigarette smoking and smoke pack year (SPY) levels in an analysis of covariance (ANCOVA). Nine CpGs were significantly associated with smoking status, with three CpGs mapping to the genetic region of CYP1B1 (cytochrome P450 family 1 subfamily B member 1; best p = 5.5 × 10-8) and two mapping to AHRR (aryl-hydrocarbon receptor repressor; best p = 5.9 × 10-9). In the SPY analysis, 61 CpG sites at 52 loci showed significant associations of the quantity of smoking with changes in methylation values. Here, the most significant association located to the gene CYP1B1, with p = 4.0 × 10-10. RNA-Seq data showed significantly increased expression of CYP1B1 in smokers compared to non-smokers (p = 2.2 × 10-14), together with 13 significantly upregulated transcripts. Six transcripts were significantly downregulated. No differential expression was observed for AHRR. In vitro studies with gingival fibroblasts showed that cigarette smoke extract directly upregulated the expression of CYP1B1. CONCLUSION: This study validated the established role of CYP1B1 and AHRR in xenobiotic metabolism of tobacco smoke and highlights the importance of epigenetic regulation for these genes. For the first time, we give evidence of this role for the oral masticatory mucosa.

Metastasis of pancreatic cancer: An uninflamed liver micromilieu controls cell growth and cancer stem cell properties by oxidative phosphorylation in pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed when liver metastases already emerged. We recently demonstrated that hepatic stromal cells determine the dormancy status along with cancer stem cell (CSC) properties of pancreatic ductal epithelial cells (PDECs) during metastasis. This study investigated the influence of the hepatic microenvironment - and its inflammatory status - on metabolic alterations and how these impact cell growth and CSC-characteristics of PDECs. Coculture with hepatic stellate cells (HSCs), simulating a physiological liver stroma, but not with hepatic myofibroblasts (HMFs) representing liver inflammation promoted expression of Succinate Dehydrogenase subunit B (SDHB) and an oxidative metabolism along with a quiescent phenotype in PDECs. SiRNA-mediated SDHB knockdown increased cell growth and CSC-properties. Moreover, liver micrometastases of tumor bearing KPC mice strongly expressed SDHB while expression of the CSC-marker Nestin was exclusively found in macrometastases. Consistently, RNA-sequencing and in silico modeling revealed significantly altered metabolic fluxes and enhanced SDH activity predominantly in premalignant PDECs in the presence of HSC compared to HMF. Overall, these data emphasize that the hepatic microenvironment determines the metabolism of disseminated PDECs thereby controlling cell growth and CSC-properties during liver metastasis.

Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes.

Biologic activity of proteases is mainly characterized by the substrate specificity, tissue distribution, and cellular localization. The human metalloproteases meprin α and meprin ß share 41% sequence identity and exhibit a similar cleavage specificity with a preference for negatively charged amino acids. However, shedding of meprin α by furin on the secretory pathway makes it a secreted enzyme in comparison with the membrane-bound meprin ß. In this study, we identified human meprin α and meprin ß as forming covalently linked membrane-tethered heterodimers in the early endoplasmic reticulum, thereby preventing furin-mediated secretion of meprin α. Within this newly formed enzyme complex, meprin α was able to be activated on the cell surface and detected by cleavage of a novel specific fluorogenic peptide substrate. However, the known meprin ß substrates amyloid precursor protein and CD99 were not shed by membrane-tethered meprin α. On the other hand, being linked to meprin α, activation of or substrate cleavage by meprin ß on the cell surface was not altered. Interestingly, proteolytic activity of both proteases was increased in the heteromeric complex, indicating an increased proteolytic potential at the plasma membrane. Because meprins are susceptibility genes for inflammatory bowel disease (IBD), and to investigate the physiologic impact of the enzyme complex, we performed transcriptome analyses of intestinal mucosa from meprin-knockout mice. Comparison of the transcriptional gene analysis data with gene analyses of IBD patients revealed that different gene subsets were dysregulated if meprin α was expressed alone or in the enzyme complex, demonstrating the physiologic and pathophysiological relevance of the meprin heterodimer formation.-Peters, F., Scharfenberg, F., Colmorgen, C., Armbrust, F., Wichert, R., Arnold, P., Potempa, B., Potempa, J., Pietrzik, C. U., Häsler, R., Rosenstiel, P., Becker-Pauly, C. Tethering soluble meprin α in an enzyme complex to the cell surface affects IBD-associated genes.