cAMP Response Element Modulator Research Articles

Identification of the promoter region regulating the transcription of the REV7 gene

REV7 is involved in various biological processes including DNA repair and mutagenesis, cell cycle regulation, gene transcription, and carcinogenesis. REV7 is highly expressed in adult testicular germ cells as well as several malignant tumors. REV7 expression levels are associated with prognosis in several human cancers, however, the mechanism of REV7 transcriptional regulation has not been elucidated. In this study, we characterized the promoter region of the REV7 gene. A luciferase reporter assay using the human germ cell tumor cell line NEC8 was utilized to examine the upstream genomic region of REV7 for transcriptional activity, and two transcriptional activation regions were identified. We determined a small genomic region important for transcriptional activation using site-directed mutagenesis; this region is shared by several putative binding motifs for transcription factors, including the cAMP-responsive element modulator (CREM), cAMP-response element binding protein (CREB), and B-lymphocyte-induced maturation protein-1 (BLIMP-1). Exogenous CREM and CREB expression had no effect on the transcriptional activity in NEC8 cells or the human embryonic kidney cell line HEK293T. In contrast, exogenous BLIMP-1 expression increased luciferase reporter activity in HEK293T cells but unexpectedly decreased activity in NEC8 cells. Chromatin immunoprecipitation analysis demonstrated that BLIMP-1 binds to the genomic region near the binding motif in the REV7 promoter. Additionally, BLIMP-1 overexpression promoted endogenous REV7 expression in HEK293T cells. These findings suggest that BLIMP-1 may be a putative transcriptional regulator of REV7 in mammalian cells.

Neonatal sevoflurane exposure induces long-term changes in dendritic morphology in juvenile rats and mice.

General anesthetics are potent neurotoxins when given during early development, causing apoptotic deletion of substantial number of neurons and persistent neurocognitive and behavioral deficits in animals and humans. The period of intense synaptogenesis coincides with the peak of susceptibility to deleterious effects of anesthetics, a phenomenon particularly pronounced in vulnerable brain regions such as subiculum. With steadily accumulating evidence confirming that clinical doses and durations of anesthetics may permanently alter the physiological trajectory of brain development, we set out to investigate the long-term consequences on dendritic morphology of subicular pyramidal neurons and expression on genes regulating the complex neural processes such as neuronal connectivity, learning, and memory. Using a well-established model of anesthetic neurotoxicity in rats and mice neonatally exposed to sevoflurane, a volatile general anesthetic commonly used in pediatric anesthesia, we report that a single 6 h of continuous anesthesia administered at postnatal day (PND) 7 resulted in lasting dysregulation in subicular mRNA levels of cAMP responsive element modulator (Crem), cAMP responsive element-binding protein 1 (Creb1), and Protein phosphatase 3 catalytic subunit alpha, a subunit of calcineurin (Ppp3ca) (calcineurin) when examined during juvenile period at PND28. Given the critical role of these genes in synaptic development and neuronal plasticity, we deployed a set of histological measurements to investigate the implications of anesthesia-induced dysregulation of gene expression on morphology and complexity of surviving subicular pyramidal neurons. Our results indicate that neonatal exposure to sevoflurane induced lasting rearrangement of subicular dendrites, resulting in higher orders of complexity and increased branching with no significant effects on the soma of pyramidal neurons. Correspondingly, changes in dendritic complexity were paralleled by the increased spine density on apical dendrites, further highlighting the scope of anesthesia-induced dysregulation of synaptic development. We conclude that neonatal sevoflurane induced persistent genetic and morphological dysregulation in juvenile rodents, which could indicate heightened susceptibility toward cognitive and behavioral disorders we are beginning to recognize as sequelae of early-in-life anesthesia.

Proton-Pump Inhibitors Suppress T Cell Response by Shifting Intracellular Zinc Distribution.

Proton-pump inhibitors (PPI), e.g., omeprazole or pantoprazole, are the most widely used drugs for various gastrointestinal diseases. However, more and more side effects, especially an increased risk of infections, have been reported in recent years. The underlying mechanism has still not yet been fully uncovered. Hence, in this study, we analyzed the T cell response after treatment with pantoprazole in vitro. Pantoprazole preincubation reduced the production and secretion of interferon (IFN)-γ and interleukin (IL)-2 after the T cells were activated with phytohemagglutinin (PHA)-L or toxic shock syndrome toxin-1 (TSST-1). Moreover, a lower zinc concentration in the cytoplasm and a higher concentration in the lysosomes were observed in the pantoprazole-treated group compared to the untreated group. We also tested the expression of the zinc transporter Zrt- and Irt-like protein (Zip)8, which is located in the lysosomal membrane and plays a key role in regulating intracellular zinc distribution after T cell activation. Pantoprazole reduced the expression of Zip8. Furthermore, we measured the expression of cAMP-responsive element modulator (CREM) α, which directly suppresses the expression of IL-2, and the expression of the phosphorylated cAMP response element-binding protein (pCREB), which can promote the expression of IFN-γ. The expression of CREMα was dramatically increased, and different isoforms appeared, whereas the expression of pCREB was downregulated after the T cells were treated with pantoprazole. In conclusion, pantoprazole downregulates IFN-γ and IL-2 expression by regulating the expression of Zip8 and pCREB or CREMα, respectively.

Transcriptional regulatory network during axonal regeneration of dorsal root ganglion neurons: laser-capture microdissection and deep sequencing.

The key regulators and regeneration-associated genes involved in axonal regeneration of neurons after injury have not been clarified. In high-throughput sequencing, various factors influence the final sequencing results, including the number and size of cells, the depth of sequencing, and the method of cell separation. There is still a lack of research on the detailed molecular expression profile during the regeneration of dorsal root ganglion neuron axon. In this study, we performed laser-capture microdissection coupled with RNA sequencing on dorsal root ganglion neurons at 0, 3, 6, and 12 hours and 1, 3, and 7 days after sciatic nerve crush in rats. We identified three stages after dorsal root ganglion injury: early (3-12 hours), pre-regeneration (1 day), and regeneration (3-7 days). Gene expression patterns and related function enrichment results showed that one module of genes was highly related to axonal regeneration. We verified the up-regulation of activating transcription factor 3 (Atf3), Kruppel like factor 6 (Klf6), AT-rich interaction domain 5A (Arid5a), CAMP responsive element modulator (Crem), and FOS like 1, AP-1 transcription factor Subunit (Fosl1) in dorsal root ganglion neurons after injury. Suppressing these transcription factors (Crem, Arid5a, Fosl1 and Klf6) reduced axonal regrowth in vitro. As the hub transcription factor, Atf3 showed higher expression and activity at the pre-regeneration and regeneration stages. G protein-coupled estrogen receptor 1 (Gper1), interleukin 12a (Il12a), estrogen receptor 1 (ESR1), and interleukin 6 (IL6) may be upstream factors that trigger the activation of Atf3 during the repair of axon injury in the early stage. Our study presents the detailed molecular expression profile during axonal regeneration of dorsal root ganglion neurons after peripheral nerve injury. These findings may provide reference for the clinical screening of molecular targets for the treatment of peripheral nerve injury.

Corticosterone stage-dependently inhibits progesterone production presumably via impeding the cAMP-StAR cascade in granulosa cells of chicken preovulatory follicles

Stress can suppress reproduction capacity in either wild or domestic animals, but the exact mechanism behind it, especially in terms of steroidogenesis, remains under-investigated so far. Considering the important roles of progesterone in avian breeding, we investigated the modulation of corticosterone on progesterone production in cultured granulosa cells of chicken follicles at different developmental stages. Using enzyme immunoassays, our study showed that corticosterone could only inhibit progesterone synthesis in granulosa cells from F5-6, F4, and F3 follicles, but not F2 and F1 follicles. Coincidentally, both quantitative real-time PCR and western blotting revealed that corticosterone could downregulate steroidogenic acute regulatory protein (StAR) expression, suggesting the importance of StAR in corticosterone-related actions. Using the dual-luciferase reporter system, we found that corticosterone can potentially enhance, rather than inhibit, the activity of StAR promoter. Of note, combining high-throughput transcriptomic analysis and quantitative real-time PCR, phosphodiesterase 10A (PDE10A), protein kinase cAMP-dependent type II regulatory subunit alpha (PRKAR2A) and cAMP responsive element modulator (CREM) were identified to exhibit the differential expression patterns consistent with cAMP blocking in granulosa cells from F5-6, F4, and F3, but not F2 and F1 follicles. Afterward, the expression profiles of these genes in granulosa cells of distinct developmental-stage follicles were examined by quantitative real-time PCR, in which all of them expressed correspondingly with progesterone levels of granulosa cells during development. Collectively, these findings indicate that corticosterone can stage-dependently inhibit progesterone production in granulosa cells of chicken preovulatory follicles, through impeding cAMP-induced StAR activity presumptively.

Decreased SUV39H1 at the promoter region leads to increased CREMα and accelerates autoimmune response in CD4+ T cells from patients with systemic lupus erythematosus

BackgroundOverproduction of cAMP-responsive element modulator α (CREMα) in total T cells from patients with systemic lupus erythematosus (SLE) can inhibit IL-2 and increase IL-17A. These ultimately promote progression of SLE. This study aims to investigate the expression of CREMα in SLE CD4+ T cells and find out the mechanisms for the regulation of CREMα in SLE CD4+ T cells.ResultsCREMα mRNA was overexpressed in CD4+ T cells from SLE patients. The levels of histone H3 lysine 9 trimethylation (H3K9me3) and suppressor of variation 3–9 homolog 1 (SUV39H1) at the CREMα promoter of SLE CD4+ T cells were markedly decreased. Down-regulating SUV39H1 in normal CD4+ T cells elevated the levels of CREMα, IL-17A, and histone H3 lysine 4 trimethylation (H3K4me3) in the CREMα promoter region, and lowered IL-2, H3K9me3, DNA methylation, and DNA methyltransferase 3a (DNMT3a) enrichments within the CREMα promoter, while no sharp change in SET domain containing 1 (Set1) at the CREMα promoter. Up-regulating SUV39H1 in SLE CD4+ T cells had the opposite effects. The DNA methylation and DNMT3a levels were obviously reduced, and H3K4me3 enrichment was greatly increased at the CREMα promoter of CD4+ T cells from SLE patients. The Set1 binding in the CREMα promoter region upgraded significantly, and knocking down Set1 in SLE CD4+ T cells alleviated the H3K4me3 enrichment within this region, suppressed CREMα and IL-17A productions, and promoted the levels of IL-2, CREMα promoter DNA methylation, and DNMT3a. But there were no obviously alterations in H3K9me3 and SUV39H1 amounts in the region after transfection.ConclusionsDecreased SUV39H1 in the CREMα promoter region of CD4+ T cells from SLE patients contributes to under-expression of H3K9me3 at this region. In the meantime, the Set1 binding at the CREMα promoter of SLE CD4+ T cells is up-regulated. As a result, DNMT3a and DNA methylation levels alleviate, and H3K4me3 binding increases. All these lead to overproduction of CREMα. Thus, the secretion of IL-2 down-regulates and the concentration of IL-17A up-regulates, ultimately promoting SLE.

Impact of Long Non-Coding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 on the Susceptibility of High Glucose-Treated Cardiomyocytes to Hypoxia/Reoxygenation

To estimate the effect of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on susceptibility of high glucose (HG)-treated cardiomyocytes (CMs) to hypoxia/reoxygenation (H/R). Forty healthy rats were assigned to the control, diabetes mellitus (DM), DM + ischemia/reperfusion injury (IRI) control, and DM IRI groups (n = 10 for each group). Rat CMs (H9C2) were subjected to HG and H/R treatments. LncRNA MALAT1 and cyclic-AMP responsive element modulator (CREM) mRNA levels were measured using quantitative polymerase chain reaction, and protein levels of CREM, myeloid differentiation primary response protein 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor associated factor 6 (TRAF6), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were quantified using western blotting. Flow cytometry was applied for detection of apoptosis, and a cell viability analyzer determined the number of living cells. IRI induced myocardial apoptosis and elevated lncRNA MALAT1, CREM, and MyD88/IRAK1/TRAF6 levels in DM group mice. In H9C2 cells, HG treatment downregulated CREM expression, which resulted in the downregulation of lncRNA MALAT1 expression, reducing the susceptibility to H/R, increasing cell viability and apoptosis, decreasing the activity of the MyD88/IRAK1/TRAF6 signaling pathway, and suppressing TNF-α and IL-6 expression. LncRNA MALAT1 regulates the susceptibility of HG-treated CMs to H/R through the MyD88 signaling pathway.

An extracranial CNS presentation of the emerging "intracranial" mesenchymal tumor, FET: CREB-fusion positive.

A novel histomolecular tumor, the "intracranial mesenchymal tumor (IMT), FET::CREB fusion-positive", has recently been identified and added to the 2021 World Health Organization Classification of Tumors of the Central Nervous System. One of the essential diagnostic criteria defined in this classification is the intracranial location of the tumor. Herein, we report a spinal case of IMT with a classical EWSR1::CREM fusion. We compare its clinical, histopathological, immunophenotypical, genetic and epigenetic features with those previously described in IMT, FET::CREB fusion-positive. The current case presented histopathological (epithelioid morphology with mucin-rich stroma, and expression of EMA and desmin), radiological (an extraparenchymal lobulated mass without dural tail), genetic (fusion implicating the EWSR1 and CREM genes), and epigenetic (DNA-methylation profiling) similarities to previously reported cases. This case constitutes the third "extracranial" observation of an IMT. Our results added data suggesting that the terminology "IMT, FET::CREB fusion-positive" is provisional and that further series of cases are needed to better characterize them.

Short-term zinc supplementation of zinc-deficient seniors counteracts CREMα - mediated IL-2 suppression

BackgroundAging is accompanied by a dramatic decline in the interleukin (IL)-2 production capacity of human immune cells, thus making seniors more susceptible to a variety of age-related diseases. A common cause of impaired cytokine production in advanced age is a deficiency of the essential micronutrient zinc. Nevertheless, the molecular mechanisms underlying a zinc deficiency-induced decrease in IL-2 production have not yet been satisfactorily elucidated. Recent animal and in vitro data suggested that the transcription factor cAMP-responsive element modulator (CREM) alpha plays a critical role in T cells´ disturbed IL-2 production in suboptimal zinc conditions. However, its role in the human aging process and the possibility of influencing this detrimental process by short-term zinc supplementation have not yet been evaluated.ResultsComparing peripheral lymphocytes of 23 young and 31 elderly subjects with either high, intermediate, or deficient zinc status, we observed zinc-dependent regulation of the IL-2 production mediated by the transcription factor CREM alpha. For the first time in humans, we report a mutual relationship between low zinc levels, high CREM alpha expression, subsequent impaired IL-2 production, and vice versa. Remarkably, an average of only 6 days of in vivo zinc supplementation to zinc-deficient seniors was sufficient to rapidly improve zinc status, reverse CREM alpha overexpression, and counteract subsequent low IL-2 production rates.ConclusionsOur ex vivo and in vivo data identify zinc deficiency-mediated CREM alpha overexpression as a key cellular mechanism underlying impaired IL-2 production in the elderly and point toward the use of zinc as a rapidly immune-enhancing add-on nutraceutical in geriatric therapy.Graphical abstractDuring the aging process, there is a progressive decrease in zinc status, which in turn leads to overexpression of the transcription factor CREMmathrm{alpha } in peripheral lymphocytes. CREMα is a negative regulator of the IL-2 gene, the overexpression of which dramatically limits adequate IL-2 production. This deleterious mechanism can be counteracted by short-term oral zinc administration, which can adjust IL-2 production in old, zinc-deficient individuals to a level similar to that of young adults.

Dual regulation of miR-375 and CREM genes in pancreatic beta cells

ABSTRACT MicroRNA-375 (miR-375) is upregulated in the islets of some diabetics and is correlated with poor outcome. Previous work in our laboratory showed that cyclic adenosine monophosphate (cAMP) reduces miR-375 expression and could provide a way to restore normal miR-375 levels, however the transcription repression mechanism is unknown. Using a chromatin immunoprecipitation assay we show that cAMP response element modulator (CREM) binds to the miR-375 promoter 3-fold above background and we find that CREM represses transcription from the miR-375 promoter 1.8-fold. While investigating miR-375 target genes we discovered that several microRNA:mRNA target prediction algorithms listed human CREM as a target gene of miR-375. The predicted binding site is conserved in primates but not in other species. We found that indeed miR-375 binds to the predicted site on human CREM and represses translation of a green fluorescent protein reporter gene by 30%. These findings suggest a primate-specific double-negative feedback loop, a mechanism that would keep these important β-cell regulators in check.

Genetic and epigenetic alterations of cyclic AMP response element modulator in rheumatoid arthritis.

Genetic and epigenetic factors are strongly associated with the autoimmune disease rheumatoid arthritis (RA). Cyclic AMP response element modulator (CREM), a gene related to immune system regulation, has been implicated in various immune-mediated inflammatory processes, although it remains unknown whether CREM is involved in RA. This study enrolled 278 RA patients and 262 controls. Three variants [rs12765063, rs17499247, rs1213386] were identified through linkage disequilibrium and expression quantitative trait locus analysis, and CREM transcript abundance was determined by quantitative real-time polymerase chain reaction. The identified variants were genotyped using the TaqMan Allelic Discrimination assay, and CREM promoter methylation was assessed by bisulphite sequencing. Differences between groups and correlations between variables were assessed with Student's t-tests and Pearson's correlation coefficients. Associations between phenotypes and genotypes were evaluated with logistic regression. Rheumatoid arthritis patients exhibited increased CREM expression (p<.0001), which was decreased by methotrexate (p=.0223) and biologics (p=.0001), but could not be attributed to CREM variants. Interestingly, rs17499247 displayed a significant association with serositis (p=.0377), and rs1213386 increased the risk of lymphadenopathy (p=.0398). Furthermore, seven CpG sites showed decreased methylation in RA (p=.0477~ p<.0001). Collectively, our results indicate that CREM hypomethylation and CREM upregulation occur in RA and that CREM variants are involved in the development of serositis and lymphadenopathy in RA. This study highlights the novel roles of CREM in RA pathophysiology.

CREM Is Correlated With Immune-Suppressive Microenvironment and Predicts Poor Prognosis in Gastric Adenocarcinoma.

The transcriptional repressor cAMP response element modulator (CREM) has an important role in T-cell development. In this study, we used the integrated Bioinformatics Methods to explore the role of CREM in gastric adenocarcinoma (GAC). Our results showed that high CREM expression was closely related with poorer overall survival in GAC. By GSEA cluster analysis, we found that the high expression of CREM was associated with the cancer-associated pathway in GAC. Moreover, single-cell sequencing data showed that CREM is mainly localized in exhausted CD8+ T cells. Its prognostic value and the potential function lead to T-cell exhaustion in the tumor microenvironment (TME). Similar results were also obtained in glioma and lung cancer. High expression of CREM, correlated with clinical relevance of GAC, was associated with T-cell exhaustion and M2 polarization in GAC. These findings suggest that CREM can be used as a prognostic biomarker in GAC, which might provide a novel direction to explore the pathogenesis of GAC.

Dynamic Expression and Chromatin Incorporation of ACT and CREM Transcription Factors in Testis Tissues of Infertile Men.

ObjectiveActivator of CREM in the testis (ACT) is a tissue specific transcription factor which activates cAMP responsive element modulator (CREM), a key transcription factor in differentiation of round spermatids into mature spermatozoa. They bind to CRE region in the promoters of transition protein genes (TNP1, TNP2) and protamine genes (PRM1 and PRM2), which are essential for sperm chromatin compaction, and regulates their transcription. This study was conducted to consider the expression of ACT and CREM and their regulatory roles on the expression of PRM1, PRM2, TNP1 and TNP2 genes in testis tissues of infertile men.Materials and MethodsIn this case-control study, testicular biopsies were collected from 40 infertile men and classified into three groups: obstructive azoospermia (OA, n=10, positive control), round spermatid maturation arrest (SMA, n=20), Sertoli cell-only syndrome (SCOS, n=10, negative control group). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of ACT, CREM, TNP1, TNP2, PRM1 and PRM2 genes were assessed in testicular samples and incorporation of ACT and CREM proteins on the promoters of PRM1, PRM2, TNP1 and TNP2 genes were also evaluated by ChIP-real time PCR. ResultsOur results demonstrated significant decrease in the expression levels of ACT, CREM and in their incorporations on their target genes in SMA group in comparison to control groups (P<0.05).ConclusionThese data confirm that there is low expression and incorporation of ACT and CREM and of their target genes in infertilities which are associated with post-meiotic arrest.

A novel SMARCA2-CREM fusion: expanding the molecular spectrum of intracranial mesenchymal tumors beyond the FET genes

A novel histomolecular tumor of the central nervous system, the “intracranial mesenchymal tumor (IMT), FET-CREB fusion-positive” has recently been identified in the literature and will be added to the 2021 World Health Organization Classification of Tumors of the Central Nervous System. However, our latest study using DNA-methylation analyses has revealed that intracranial FET-CREB fused tumors do not represent a single molecular tumor entity. Among them, the main subgroup presented classical features of angiomatoid fibrous histiocytoma, having ultrastructural features of arachnoidal cells, for. Another tumor type with clear cell component and histopathological signs of aggressivity clustered in close vicinity with clear cell sarcoma of soft tissue. Herein, we report one case of IMT with a novel SMARCA2-CREM fusion which has until now never been described in soft tissue or the central nervous system. We compare its clinical, histopathological, immunophenotypic, genetic and epigenetic features with those previously described in IMT, FET-CREB fusion-positive. Interestingly, the current case did not cluster with IMT, FET-CREB fusion-positive but rather presented histopathological (clear cell morphology with signs of malignancy), clinical (with a dismal course with several recurrences, metastases and finally the patient’s death), genetic (fusion implicating the CREM gene), and epigenetic (DNA-methylation profiling) similarities with our previously reported clear cell sarcoma-like tumor of the central nervous system. Our results added data suggesting that different clinical and histomolecular tumor subtypes or grades seem to be included within the terminology “IMT, FET-CREB fusion-positive”, and that further series of cases are needed to better characterize them.

The cAMP responsive element modulator (CREM) is a regulator of CD4+ Tcell function.

The cAMP responsive element modulator (CREM) is a transcriptional regulator of different effector cytokines in CD4+ Tcells including IL-2, IL-17, IL-21 but also IL-4 and IL-13 and thus an important determinant of central T helper cell functions. Our review gives an overview over the regulation of CREM in Tcells and the pleiotropic effects of CREM on CD4+ Tcells in health and autoimmune diseases with a particular focus on systemic lupus erythematosus.

Expression of Transcription Factor CREM in Human Tissues.

Cyclic AMP element modulator (CREM) is a transcription factor best known for its intricate involvement in spermatogenesis. The CREM gene encodes for multiple protein isoforms, which can enhance or repress transcription of target genes. Recent studies have identified fusion genes, with CREM as a partner gene in many neoplastic diseases. EWSR1-CREM fusion genes have been found in several mesenchymal tumors and in salivary gland carcinoma. These genes encode fusion proteins that include the C-terminal DNA-binding domain of CREM. We used a transcriptomic approach and immunohistochemistry to study the expression of CREM isoforms that include DNA-binding domains across human tissues. We found that CREM protein is widely expressed in almost all normal human tissues. A transcriptomic analysis of normal tissues and cancer showed that transcription of CREM can be altered in tumors, suggesting that also wild-type CREM may be involved in cancer biology. The wide expression of CREM protein in normal human tissues and cancer may limit the utility of immunohistochemistry for identification of tumors with CREM fusions:

Integrative DNA Methylation and Gene Expression Analysis in the Prefrontal Cortex of Mexicans Who Died by Suicide.

BackgroundSuicide represents a major health concern, especially in developing countries. While many demographic risk factors have been proposed, the underlying molecular pathology of suicide remains poorly understood. A body of evidence suggests that aberrant DNA methylation and expression is involved. In this study, we examined DNA methylation profiles and concordant gene expression changes in the prefrontal cortex of Mexicans who died by suicide.MethodsIn collaboration with the coroner’s office in Mexico City, brain samples of males who died by suicide (n = 35) and age-matched sudden death controls (n = 13) were collected. DNA and RNA were extracted from prefrontal cortex tissue and analyzed with the Infinium Methylation480k and the HumanHT-12 v4 Expression Beadchips, respectively.ResultsWe report evidence of altered DNA methylation profiles at 4430 genomic regions together with 622 genes characterized by differential expression in cases vs controls. Seventy genes were found to have concordant methylation and expression changes. Metacore-enriched analysis identified 10 genes with biological relevance to psychiatric phenotypes and suicide (ADCY9, CRH, NFATC4, ABCC8, HMGA1, KAT2A, EPHA2, TRRAP, CD22, and CBLN1) and highlighted the association that ADCY9 has with various pathways, including signal transduction regulated by the cAMP-responsive element modulator, neurophysiological process regulated by the corticotrophin-releasing hormone, and synaptic plasticity. We therefore went on to validate the observed hypomethylation of ADCY9 in cases vs control through targeted bisulfite sequencing.ConclusionOur study represents the first, to our knowledge, analysis of DNA methylation and gene expression associated with suicide in a Mexican population using postmortem brain, providing novel insights for convergent molecular alterations associated with suicide.

Abstract 1631: GPR65 is a critical mediator of low pH induced immunosuppressive signalling in tumor associated macrophages: Human target validation of GPR65 as a novel innate immune checkpoint and discovery of potent, selective GPR65 antagonists

Abstract Tumor-associated macrophages (TAMs) are the major innate immune component in the microenvironment of solid tumors. These cells are highly heterogeneous and plastic but often display a pronounced immunosuppressive phenotype that supports primary tumor growth and metastasis. A recently identified determinant of the immunosuppressive properties of TAMs is the activation of the pH-sensing G protein-coupled receptor, GPR65, on these cells by the acidic microenvironment that is inherent to many advanced solid tumours1. Previous work in mouse macrophages has shown that GPR65 activation leads to an elevation of inducible cAMP early repressor (ICER), an isoform of the CREM gene, which in turn suppresses the expression of a host of proinflammatory mediators1. Consistent with a high expression of GPR65 and CREM in human myeloid cells, and particularly in human TAMs, we now show that equivalent immunosuppressive signaling is also present in human macrophages in response to low pH. Further substantiating GPR65 as an innate immune checkpoint in human cancers, we also show that subjects that are homozygous for a hypomorphic coding variant in GPR65 (I231L) have a significantly improved survival across a range of cancers compared to other genotypes. This survival advantage is maintained in patients with highly glycolytic tumors that would otherwise be predicted to respond poorly to immunotherapy, and is consistent with studies showing that genetic deletion of ICER attenuates the growth of anti-PD-1-resistant B16.F10 melanoma tumors in mice1. With a view to developing a therapeutic agent able to reverse low pH-dependent immunosuppression in TAMs, we identified potent and selective small molecule inhibitors of GPR65 from a screening campaign. These molecules were able to fully ablate the acidic pH-induced expression of ICER in human macrophages with downstream implications for pro-inflammatory cytokines known to support T cell anti-tumor responses. Additional medicinal chemistry optimization led to molecules exhibiting excellent oral bioavailability in preclinical species and systemic exposure that completely suppressed GPR65 signaling following oral dosing in mice. Profiling of these inhibitors in relevant mouse tumor models is currently ongoing. In conclusion, we have (i) demonstrated that GPR65 is a key determinant of low pH-induced immunosuppression in human macrophages and thus an important innate immune checkpoint in cancer, and (ii) identified selective small molecule inhibitors of GPR65 with potent in vivo activity. This work provides the basis for developing novel “macrophage conditioning” therapeutic agents that may have utility across a range of cancers, either as single agents or in combination with other approved immunoncology drugs. 1Nat Immunol 19:1319. Citation Format: Barbara Cipriani, Alan Naylor, Gavin Milne, Barbara Young, Rupert Satchell, Sourav Sarkar, Zoe Smith, Louise Healy, John Unitt, Jessica Holien, Rhoanne McPherson, Anastasia Nika, Jessy Cartier, Tozska Bohn, Tobias Bopp, Tom McCarthy, Stuart Hughes. GPR65 is a critical mediator of low pH induced immunosuppressive signalling in tumor associated macrophages: Human target validation of GPR65 as a novel innate immune checkpoint and discovery of potent, selective GPR65 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1631.

Quercetin upregulates CREM gene expression in cyanide-induced endocrine dysfunction

Cyanide is among the ubiquitous chemicals that humans are usually exposed to and it is well documented that cyanide induces infertility in humans and experimental rodents. However, the pathogenesis remains unknown. Likewise, quercetin is an important nutraceutical that detoxifies reactive oxygen species, but its effects on testicular damage is not clear. The present study investigated the role of nutraceutical, quercetin on cyanide-induced testicular toxicity and probable involvement of cAMP-response-element modulator (CREM) which is a transcription factor necessary for the process of spermatogenesis. Thus, this work hypothesized that quercetin will mitigate endocrine dysfunction induced by cyanide. Seventy-two adult male Wistar rats were divided into seven groups (A to G). Groups A, B, C, F and G comprised of eight (8) rats per group while groups D and E comprised of sixteen (16) rats per group. Group A was designated as control while Groups B and C were given 0.5 and 1 mg/kg of cyanide respectively for 56 days. Group D and E received 0.5 and 1 mg/kg body weight cyanide respectively for 30 days. At day 30, eight animals were sacrificed from Group D and E and the remaining eight (8) rats were subdivided into sub-groups (D1 and E1) and were given 20 and 40 mg/kg of quercetin respectively for twenty-six (26) days. Group F and G were given concurrent administration of cyanide and quercetin at a dose of 0.5 + 20 mg/kg and 1 + 40 mg/kg respectively for 56 days. Body and testicular weight were significantly reduced in cyanide treated groups while quercetin modulates the reduction. Significant down-regulation of CREM gene and reduction in serum level of follicle stimulating hormone (FSH), Luteinizing hormone (LH), testosterone, glutathione peroxidase (GPx) and zinc in cyanide-treated groups, whereas administration of quercetin concomitantly with cyanide exposure or post-treated significantly reversed the alterations.

Modified MYOMI-14 Korean herbal formulations have protective effects against cyclophosphamide-induced male infertility in mice.

Korean herbal formulation, MYOMI-14, has been reported to improve the idiopathic male infertility condition with poor semen. In this study, four MYOMI formulations were modified from MYOMI-14 by reducing the number of constituents. We investigated the therapeutic effect of MYOMI formulations on cyclophosphamide-induced male infertility using mice model. Cyclophosphamide treatment significantly decreased body weight, testicular weight, sperm count, normal-shaped sperm rate and sperm motility compared to untreated control group, whereas MYOMI formulations restore the cyclophosphamide-induced dysfunction, as determined by increased sperm count and motility, and decreased abnormally shaped spermatozoa. In addition, treatment with MYOMI formulations reduced cyclophosphamide-induced apoptosis of germ cells and oxidative stress. MYOMI-treated mice also showed improved spermatogenesis as shown by the increased expression of spermatogenesis-related genes, as cAMP-responsive element modulator (CREM) and cAMP response element-binding (CREB) protein. Among the MYOMI formulations, MYOMI-7 showed better results in terms of recovering CP-induced damages to testis and improving the fertility. Taken together, this study is expected to make significant contribution to the literature by exploring various formulations that reduced constituents of MYOMI-14, a Korean herbal medicine, in treating CP-induced male infertility.