Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES)
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The human 5-lipoxygenase (5-LO), encoded by the ALOX5 gene, is the key enzyme in the formation of pro-inflammatory leukotrienes. ALOX5 gene transcription is strongly stimulated by calcitriol (1α, 25-dihydroxyvitamin D3) and TGFβ (transforming growth factor-β). Here, we investigated the influence of MLL (activator of transcript initiation), AF4 (activator of transcriptional elongation) as well as of the leukemogenic fusion proteins MLL-AF4 (ectopic activator of transcript initiation) and AF4-MLL (ectopic activator of transcriptional elongation) on calcitriol/TGFβ-dependent 5-LO transcript elongation. We present evidence that the AF4 complex directly interacts with the vitamin D receptor (VDR) and promotes calcitriol-dependent ALOX5 transcript elongation. Activation of transcript elongation was strongly enhanced by the AF4-MLL fusion protein but was sensitive to Flavopiridol. By contrast, MLL-AF4 displayed no effect on transcriptional elongation. Furthermore, HDAC class I inhibitors inhibited the ectopic effects caused by AF4-MLL on transcriptional elongation, suggesting that HDAC class I inhibitors are potential therapeutics for the treatment of t(4;11)(q21;q23) leukemia.
Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation.
R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance–associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
The G2A receptor (GPR132) contributes to oxaliplatin-induced mechanical pain hypersensitivity
(2017)
Chemotherapy-induced peripheral neuropathic pain (CIPN) is a common and severe debilitating side effect of many widely used cytostatics. However, there is no approved pharmacological treatment for CIPN available. Among other substances, oxaliplatin causes CIPN in up to 80% of treated patients. Here, we report the involvement of the G-protein coupled receptor G2A (GPR132) in oxaliplatin-induced neuropathic pain in mice. We found that mice deficient in the G2A-receptor show decreased mechanical hypersensitivity after oxaliplatin treatment. Lipid ligands of G2A were found in increased concentrations in the sciatic nerve and dorsal root ganglia of oxaliplatin treated mice. Calcium imaging and patch-clamp experiments show that G2A activation sensitizes the ligand-gated ion channel TRPV1 in sensory neurons via activation of PKC. Based on these findings, we conclude that targeting G2A may be a promising approach to reduce oxaliplatin-induced TRPV1-sensitization and the hyperexcitability of sensory neurons and thereby to reduce pain in patients treated with this chemotherapeutic agent.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
Chemotherapy, nerve injuries, or diseases like multiple sclerosis can cause pathophysiological processes of persistent and neuropathic pain. Thereby, the activation threshold of ion channels is reduced in peripheral sensory neurons to normally noxious stimuli like heat, cold, acid, or mechanical due to sensitization processes. This leads to enhanced neuronal activity, which can result in mechanical allodynia, cold allodynia, thermal hyperalgesia, spontaneous pain, and may initiate persistent and neuropathic pain. The treatment options for persistent and neuropathic pain patients are limited; for about 50% of them, current medication is not efficient due to severe side effects or low response to the treatment. Therefore, it is of special interest to find additional treatment strategies. One approach is the control of neuronal sensitization processes. Herein, signaling lipids are crucial mediators and play an important role during the onset and maintenance of pain. As preclinical studies demonstrate, lipids may act as endogenous ligands or may sensitize transient receptor potential (TRP)-channels. Likewise, they can cause enhanced activity of sensory neurons by mechanisms involving G-protein coupled receptors and activation of intracellular protein kinases. In this regard, oxidized metabolites of the essential fatty acid linoleic acid, 9- and 13-hydroxyoctadecadienoic acid (HODE), their dihydroxy-metabolites (DiHOMEs), as well as epoxides of linoleic acid (EpOMEs) and of arachidonic acid (EETs), as well as lysophospholipids, sphingolipids, and specialized pro-resolving mediators (SPMs) have been reported to play distinct roles in pain transmission or inhibition. Here, we discuss the underlying molecular mechanisms of the oxidized linoleic acid metabolites and eicosanoids. Furthermore, we critically evaluate their role as potential targets for the development of novel analgesics and for the treatment of persistent or neuropathic pain.
Fibrogenesis is a progressive scarring event resulting from disrupted regular wound healing due to repeated tissue injury and can end in organ failure, like in liver cirrhosis. The protagonists in this process, either liver-resident cells or patrolling leukocytes attracted to the site of tissue damage, interact with each other by soluble factors but also by direct cell-cell contact mediated by cell adhesion molecules. Since cell adhesion molecules also support binding to the extracellular matrix, they represent excellent biosensors, which allow cells to modulate their behavior based on changes in the surrounding microenvironment. In this review, we focus on selectins, cadherins, integrins and members of the immunoglobulin superfamily of adhesion molecules as well as some non-classical cell adhesion molecules in the context of hepatic fibrosis. We describe their liver-specific contributions to leukocyte recruitment, cell differentiation and survival, matrix remodeling or angiogenesis and touch on their suitability as targets in antifibrotic therapies.
Background: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-β and microRNA 218.
Objective: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue.
Methods: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis.
Results: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro.
Conclusions: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.
Patient therapy is based mainly on a combination of diagnosis, suitable monitoring or support devices and drug treatment and is usually employed for a pre-existing disease condition. Therapy remains predominantly symptom-based, although it is increasingly clear that individual treatment is possible and beneficial. However, reasonable precision medicine can only be realized with the coordinated use of diagnostics, devices and drugs in combination with extensive databases (4Ds), an approach that has not yet found sufficient implementation. The practical combination of 4Ds in health care is progressing, but several obstacles still hamper their extended use in precision medicine.
Inflammation is a highly regulated biological response of the immune system that is triggered by assaulting pathogens or endogenous alarmins. It is now well established that some soluble extracellular matrix constituents, such as small leucine-rich proteoglycans (SLRPs), can act as danger signals and trigger aseptic inflammation by interacting with innate immune receptors. SLRP inflammatory signaling cascade goes far beyond its canonical function. By choosing specific innate immune receptors, coreceptors, and adaptor molecules, SLRPs promote a switch between pro- and anti-inflammatory signaling, thereby determining disease resolution or chronification. Moreover, by orchestrating signaling through various receptors, SLRPs fine-tune inflammation and, despite their structural homology, regulate inflammatory processes in a molecule-specific manner. Hence, the overarching theme of this review is to highlight the molecular and functional specificity of biglycan-, decorin-, lumican-, and fibromodulin-mediated signaling in inflammatory and autoimmune diseases.
Inhibitor-kappaB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1) are non-canonical IκB kinases, both described as contributors to tumor growth and metastasis in different cancer types. Several hints indicate that they are also involved in the pathogenesis of melanoma; however, the impact of their inhibition as a potential therapeutic measure in this “difficult-to-treat” cancer type has not been investigated so far. We assessed IKKε and TBK1 expression in human malignant melanoma cells, primary tumors and the metastasis of melanoma patients. Both kinases were expressed in the primary tumor and in metastasis and showed a significant overexpression in tumor cells in comparison to melanocytes. The pharmacological inhibition of IKKε/TBK1 by the approved drug amlexanox reduced cell proliferation, migration and invasion. Amlexanox did not affect the cell cycle progression nor apoptosis induction but significantly suppressed autophagy in melanoma cells. The analysis of potential functional downstream targets revealed that NF-кB and ERK pathways might be involved in kinase-mediated effects. In an in vivo xenograft model in nude mice, amlexanox treatment significantly reduced tumor growth. In conclusion, amlexanox was able to suppress tumor progression potentially by the inhibition of autophagy as well as NF-кB and MAP kinase pathways and might therefore constitute a promising candidate for melanoma therapy.
Pain is the most frequent cause triggering patients to visit a physician. The worldwide incidence of chronic pain is in the range of 20% of adults, and chronic pain conditions are frequently associated with several comorbidities and a drastic decrease in patients’ quality of life. Although several approved analgesics are available, such therapy is often not satisfying due to insufficient efficacy and/or severe side effects. Therefore, novel strategies for the development of safe and highly efficacious pain killers are urgently needed. To reach this goal, it is necessary to clarify the causes and signal transduction cascades underlying the onset and progression of the different types of chronic pain. The papers in this Special Issue cover a wide variety of mechanisms involved in different pain types such as inflammatory, neuropathic or cancer pain. Therefore, the results summarized here might contribute to a better understanding of the mechanisms in chronic pain and thereby to the development of novel therapeutic strategies for pain patients.
Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.
5‐Lipoxygenase (5‐LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5‐LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening of ALOX5 promoter‐interacting proteins, obtained by DNA pulldown and label‐free quantitative mass spectrometry. Protein preparations from myeloid and B‐lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specific ALOX5 promotor binding proteins. Among those, the 15 most likely candidates for a prominent role in ALOX5 gene regulation are the known ALOX5 interactors Sp1 and Sp3, the related factor Sp2, two Krüppel‐like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G‐quadruplex structures. As G‐quadruplexes are implicated in gene regulation, spectroscopic and antibody‐based methods were used to confirm their presence within the GC‐rich sequence of the ALOX5 promoter. In summary, we have systematically characterized the interactome of the ALOX5 promoter, identifying several zinc finger proteins as novel potential ALOX5 gene regulators. Further, we have shown that the ALOX5 promoter can form DNA G‐quadruplex structures, which may play a functional role in ALOX5 gene regulation.
Type 1 diabetes (T1D) is mainly precipitated by the destruction of insulin-producing β-cells in the pancreatic islets of Langerhans by autoaggressive T cells. The etiology of the disease is still not clear, but besides genetic predisposition the exposure to environmental triggers seems to play a major role. Virus infection of islets has been demonstrated in biopsies of T1D patients, but there is still no firm proof that such an infection indeed results in islet-specific autoimmunity. However, virus infection results in a local inflammation with expression of inflammatory factors, such as cytokines and chemokines that attract and activate immune cells, including potential autoreactive T cells. Many chemokines have been found to be elevated in the serum and expressed by islet cells of T1D patients. In mouse models, it has been demonstrated that β-cells express chemokines involved in the initial recruitment of immune cells to the islets. The bulk load of chemokines is however released by the infiltrating immune cells that also express multiple chemokine receptors. The result is a mutual attraction of antigen-presenting cells and effector immune cells in the local islet microenvironment. Although there is a considerable redundancy within the chemokine ligand-receptor network, a few chemokines, such as CXCL10, seem to play a key role in the T1D pathogenesis. Studies with neutralizing antibodies and investigations in chemokine-deficient mice demonstrated that interfering with certain chemokine ligand-receptor axes might also ameliorate human T1D. However, one important aspect of such a treatment is the time of administration. Blockade of the recruitment of immune cells to the site of autoimmune destruction might not be effective when the disease process is already ongoing. By that time, autoaggressive cells have already arrived in the islet microenvironment and a blockade of migration might even hold them in place leading to accelerated destruction. Thus, an anti-chemokine therapy makes most sense in situations where the cells have not yet migrated to the islets. Such situations include treatment of patients at risk already carrying islet-antigen autoantibodies but are not yet diabetic, islet transplantation recipients, and patients that have undergone a T cell reset as occurring after anti-CD3 antibody treatment.
The widely varying therapeutic response of patients with inflammatory bowel disease (IBD) continues to raise questions regarding the unclarified heterogeneity of pathological mechanisms promoting disease progression. While biomarkers for the differentiation of Crohn’s disease (CD) versus ulcerative colitis (UC) have been suggested, specific markers for a CD subclassification in ileal CD versus colonic CD are still rare. Since an altered signature of the tryptophan metabolism is associated with chronic inflammatory disease, we sought to characterize potential biomarkers by focusing on the downstream enzymes and metabolites of kynurenine metabolism. Using immunohistochemical stainings, we analyzed and compared the mucosal tryptophan immune metabolism in bioptic samples from patients with active inflammation due to UC or CD versus healthy controls. Localization-specific quantification of immune cell infiltration, tryptophan-metabolizing enzyme expression and mucosal tryptophan downstream metabolite levels was performed. We found generally increased immune cell infiltrates in the tissue of all patients with IBD. However, in patients with CD, significant differences were found between regulatory T cell and neutrophil granulocyte infiltration in the ileum compared with the colon. Furthermore, we observed decreased kynurenine levels as well as strong kynureninase (KYNU) expression specifically in patients with ileal CD. Correspondingly, significantly elevated levels of the kynurenine metabolite 3-hydroxyanthranilic acid were detected in the ileal CD samples. Highlighting the heterogeneity of the different phenotypes of CD, we identified KYNU as a potential mucosal biomarker allowing the localization-specific differentiation of ileal CD versus colonic CD.
Sphingosine‐1‐phosphate lyase 1 (S1P lyase or SGPL1) is an essential sphingosine‐1‐phosphate‐degrading enzyme. Its manipulation favors onset and progression of colorectal cancer and others in vivo. Thus, SGPL1 is an important modulator of cancer initiation. However, in established cancer, the impact of retrospective SGPL1 modulation is elusive. Herein, we analyzed how SGPL1 siRNA affects malignancy of the human colorectal cancer cells DLD‐1 and found that in parallel to the reduction of SGPL1 expression levels, migration, invasion, and differentiation status changed. Diminished SGPL1 expression was accompanied with reduced cell migration and cell invasion in scratch assays and transwell assays, whereas metabolic activity and proliferation was not altered. Decreased migration was attended by increased cell–cell‐adhesion through upregulation of E‐cadherin and formation of cadherin‐actin complexes. Spreading cell islets showed lower vimentin abundance in border cells. Furthermore, SGPL1 siRNA treatment induced expression of epithelial cell differentiation markers, such as intestinal alkaline phosphatase and cytokeratin 20. Hence, interference with SGPL1 expression augmented a partial redifferentiation of colorectal cancer cells toward normal colon epithelial cells. Our investigation showed that SGPL1 siRNA influenced tumorigenic activity of established colorectal cancer cells. We therefore suggest SGPL1 as a target for lowering malignant potential of already existing cancer.
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.