cAMP Response Element Modulator Research Articles

A liver immune rheostat regulates CD8 T cell immunity in chronic HBV infection.

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3-7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion.

(007) TESTOSTERONE POSITIVELY REGULATES ADENOSINE-INDUCED RELAXATION IN THE VAGINA: AN EXPERIMENTAL STUDY IN RATS

Abstract Introduction It has been previously demonstrated that testosterone (T) has a pivotal role in controlling vaginal relaxation through the related molecular machinery, i.e., improving the nitric oxide (NO)/protein kinase G (PKG)/cyclic guanosine monophosphate (cGMP) vaginal signaling and maintaining the integrity of the muscular wall. Adenosine (ADO) is a purinergic neuromodulator that, in the male, shows a positive effect on penile erection by controlling smooth muscle relaxation and local blood flow increase. However, the role, if any, of T on the ADO-induced relaxation signaling is yet to be investigated. Objective The aim of this study was to evaluate the effect of sex steroids on ADO-mediated vaginal relaxation using a validated animal model of ovariectomized (OVX) and sex steroid supplemented Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (E2; 10 mg/kg/die), testosterone (T; 30 mg/kg/week), or testosterone and letrozole (T+L, 2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were performed on noradrenaline (NA) pre-contracted distal vaginal strips, isolated from each experimental group, stimulated with increasing doses of ADO (1 μM - 1 mM), with or without NO-synthase inhibitor L-NAME (100 μM) pretreatment, or treated with ADORA2A selective agonist CGS21680 (10 Pm-100 μM). Messenger ribonucleic acid (mRNA) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results The results showed that OVX resulted in a decreased responsiveness to ADO, completely restored by T supplementation, with or without letrozole. In contrast, E2 did not affect the hyporesponsiveness to ADO in OVX rats. Likewise, ADO induced dose-dependent relaxation was significantly reduced by in vitro L-NAME (100 μM) only in the OVX animals treated with T or T+L. Accordingly, vagina expressed all ADO receptors (ADORA1, ADORA2A, ADORA2B, ADORA3) mRNAs, with pro-relaxant ADORA2A (ADO receptor coupled to adenylyl cyclase activation) being significantly modulated by hormonal treatments: OVX significantly reduced ADORA2A mRNA expression, an effect counteracted by either T or T+L administration, but not by E2. These data are supported by the observed T-induced upregulation of cyclic adenosine monophosphate (cAMP)-dependent genes, where T, but not E2, treatment upregulated the mRNA expression of the ubiquitous isoform of ADCY (ADCY4), protein kinase A (PKA) catalytic subunit b (PKAcb), PKA regulatory subunit 2b (PKAr2b) and cAMP responsive element modulator (CREM). Finally, as further demonstration of the functional activity of ADO signaling in distal vagina, stimulation by increasing doses of CGS21680, a selective agonist of ADORA2A receptor, enhanced the relaxant effect induced by T and T+L treatments but not by E2. Conclusions Our data demonstrated for the first time a novel ADO-mediated relaxant mechanism in vagina and suggested that T positively modulates the responsiveness to ADO stimulation, thus confirming this process as a major player in the relaxant mechanism regulation of muscle contractility in vagina. Disclosure No.

Panax ginseng Ameliorates Pituitary-Ovarian Dysfunction Induced by Radiofrequency Electromagnetic Radiation from Cell Phones via Upregulation of the CREM Signaling Pathway.

Panax ginseng (PG) is a plant that contains ginsenosides, which are considered adaptogens that confer cellular protection. However, the impact of PG on pituitary-ovarian dysfunction and subsequent infertility is unknown. This study investigated the hypothesis that PG would attenuate pituitary-ovarian dysfunction associated with mobile phone's Radiofrequency Electromagnetic Radiation (RF-EMR) in experimental rat models and the possible involvement of a cAMP Response Element Modulator (CREM)-dependent pathway. Twenty adult female Wistar rats were divided randomly into four groups, each consisting of five rats. The control group was administered a vehicle (distilled water) orally, while the P. ginseng group received 200 mg/kg of P. ginseng extract orally. The RF-EMR group was exposed to 900MHz radiation, and the RF-EMR + PG group was exposed to the same radiation while also being treated with 200 mg/kg of P. ginseng orally. These treatments were administered daily for a period of 56 days. The RF-EMR group exhibited significant reductions in serum levels of LH, FSH, estradiol, and progesterone compared to the control group. Moreover, levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were significantly lower in the RF-EMR group compared to the control. Additionally, there was a notable decrease in the expression of the CREM gene, accompanied by disrupted pituitary/ovarian morphology in the RF-EMR group compared to the control. However, the administration of PG mitigated these changes. The findings of this study indicate that P. ginseng extract shields against pituitary-ovarian impairment linked to RF-EMR exposure from cell phones by boosting antioxidant capacity and promoting the CREM-dependent pathway.

CAMP-Mediated CREM-MITF-TYR Axis Regulates Melanin Synthesis in Pacific Oysters.

Colorful shells in bivalves are mostly caused by the presence of biological pigments, among which melanin is a key component in the formation of shell colours. Cyclic adenosine monophosphate (cAMP) is an important messenger in the regulation of pigmentation in some species. However, the role of cAMP in bivalve melanogenesis has not yet been reported. In this study, we performed in vitro and in vivo experiments to determine the role of cAMP in regulating melanogenesis in Pacific oysters. Besides, the function of cAMP-responsive element modulator (CREM) and the interactions between CREM and melanogenic genes were investigated. Our results showed that a high level of cAMP promotes the expression of melanogenic genes in Pacific oysters. CREM controls the expression of the MITF gene under cAMP regulation. In addition, CREM can regulate melanogenic gene expression, tyrosine metabolism, and melanin synthesis. These results indicate that cAMP plays an important role in the regulation of melanogenesis in Pacific oysters. CREM is a key transcription factor in the oyster melanin synthesis pathway, which plays a crucial role in oyster melanin synthesis through a cAMP-mediated CREM-MITF-TYR axis.

Antileishmanial activity of cathelicidin and its modulation by Leishmania donovani in a CREM-dependent manner for establishing infection.

Concerns regarding toxicity and resistance of current drugs have been reported in visceral leishmaniasis. Anti-microbial peptides are considered as new promising candidates and amongst them, human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, coupled with its apoptosis-inducing role. Administration of hCAP18/LL-37 in infected macrophages also decreased parasite survival and increased the host favorable cytokine IL-12. However, 1,25-dihydroxyvitamin D3 (VitD3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the VitD3-receptor (VDR), transcription factor of hCAP18/LL-37. cAMP response element modulator (CREM), the repressor of VDR, was induced in infection resulting in suppression of both VDR and cathelicidin expression. PGE2/cAMP/PKA axis was found to regulate CREM induction during infection and silencing CREM in infected cells and BALB/c mice led to decreased parasite survival. Present study thus documents the anti-leishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.

Methylation Status of cAMP-responsive Element Modulator (CREM) Gene in Infertile Men and Its Association with Sperm Parameters.

The methylation pattern of non-imprinting genes was little studied, although it is widely known that the abnormal methylation levels of imprinting genes are associated with different forms of male infertility. The purpose of this research was to assess the CREM gene's methylation status and seminal characteristics in infertile individuals who were potential intracytoplasmic sperm injection (ICSI) candidates. A total of 45 semen samples (15 normospermia, 15 asthenospermia, and 15 oligoasthenoteratospermia) were examined. Using aniline blue (AB) staining, we carried out conventional semen analysis, chromatin quality, and sperm maturity testing. DNA was taken from semen samples, and all isolated DNA was assessed using Nanodrop and gel electrophoresis. A quantitative methylation-specific polymerase chain reaction (Q-MSP) approach was used to quantify the methylation at the DMRs of the CREM gene. According to our findings, sperm count (P=0.012), concentration (P= 0.019), motility (P=0.006), progression (P=0.006), and normal morphology (P=0.004) were all inversely correlated with abnormal sperm chromatin condensation. Additionally, we noted that the methylation level of the CREM gene was considerably more significant in the oligoasthenoteratospermia group compared to the asthenospermia and normospermia groups (P<0.05). Additionally, sperm count (P=0.043), progression (P=0.026), and normal morphology (P=0.024) were all inversely linked with CREM methylation. Overall, the abnormal CREM methylation patterns have a negative impact on sperm parameters. Additionally, the CREM gene's DNA methylation status may serve as an epigenetic indicator of male infertility.

CaMK4 controls follicular helper T cell expansion and function during normal and autoimmune T-dependent B cell responses

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated B cell compartment responsible for the production of autoantibodies. Here, we show that T cell-specific expression of calcium/calmodulin-dependent protein kinase IV (CaMK4) leads to T follicular helper (Tfh) cells expansion in models of T-dependent immunization and autoimmunity. Mechanistically, CaMK4 controls the Tfh-specific transcription factor B cell lymphoma 6 (Bcl6) at the transcriptional level through the cAMP responsive element modulator α (CREMα). In the absence of CaMK4 in T cells, germinal center formation and humoral immunity is impaired in immunized mice, resulting in reduced anti-dsDNA titres, as well as IgG and complement kidney deposition in the lupus-prone B6.lpr mouse. In human Tfh cells, CaMK4 inhibition reduced BCL6 expression and IL-21 secretion ex vivo, resulting in impaired plasmablast formation and IgG production. In patients with SLE, CAMK4 mRNA levels in Tfh cells correlated with those of BCL6. In conclusion, we identify CaMK4/CREMα as a driver of T cell-dependent B cell dysregulation in autoimmunity.

Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20mg/kg·bw) once every 5days via intratracheal instillation for 35days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20μg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

Prognostic value and potential mechanism of cellular senescence and tumor microenvironment in hepatocellular carcinoma: Insights from bulk transcriptomics and single-cell sequencing analysis.

The high mortality rate and postoperative recurrence of hepatocellular carcinoma (HCC) contribute to the burden on society and healthcare. The prognostic value and underlying mechanisms of cellular senescence and tumor microenvironment (TME) in HCC remain unclear. Bulk transcriptomic data were obtained from 368 HCC samples in The Cancer Genome Atlas-liver hepatocellular carcinoma cohort and 64 samples from the GSE116174 dataset. Single-cell RNA sequencing (scRNA-seq) data of HCC were obtained from the GSE149614 dataset, including 18 tumor samples from 10 patients. Prognosis-related cellular senescence genes and immune cells were identified through univariate analysis. Least absolute shrinkage and selection operator regression analysis was performed to construct the CellAge score and TME score, both of which were identified as independent prognostic factors for HCC based on multivariate Cox analysis. The combined CellAge and TME scores showed improved prognostic stratification for HCC patients, as confirmed by multivariate Cox analysis (p < .001). The gene set enrichment analysis (GSEA) revealed enrichment of the extracellular matrix receptor interaction signaling pathway in the group with high CellAge scores and low TME scores, which exhibited a worse prognosis. Single-cell sequencing results revealed higher expression activity of the cAMP response element modulator (CREM) extended transcription factor in HCC cells and most immune cells, indicating its involvement in TME remodeling. Finally, the tumor immune dysfunction and exclusion (TIDE) analysis demonstrated that the combined scores could predict the outcomes of immune therapy in patients with HCC. In conclusion, cellular senescence contributes to TME remodeling in HCC, and the developed CellAge and TME scores serve as independent prognostic factors and predictors of immune therapy in HCC.

Expanding the Spectrum of EWSR1::CREM Fusion Tumors: An Unusual Pediatric Intranasal Myxoid Tumor.

EWSR1::CREM gene fusions are increasingly being recognized in a diverse number of soft tissue tumors, including well-defined entities such as angiomatoid fibrous histiocytoma or clear cell sarcoma, and other unclassifiable tumors. As a group, EWSR1::CREM fused tumors often demonstrate primitive spindle or epithelioid cells, myxoid stroma, and a broad immunophenotype. Herein we present an unusual case of a child diagnosed with an intranasal malignant myxoid tumor harboring an EWSR1::CREM gene fusion. To the best of our knowledge, this is the first case of intranasal myxoid tumor with this particular fusion. Diagnosis and management of the case is discussed.

Mild dehydration effects on the murine kidney single-nucleus transcriptome and chromatin accessibility.

Daily, we may experience mild dehydration with a rise in plasma osmolality that triggers the release of vasopressin. Although the effect of dehydration is well characterized in collecting duct principal cells (CDPCs), we hypothesized that mild dehydration (<12 h) results in many kidney cell-specific changes in transcriptomes and chromatin accessibility. Single-nucleus (sn) multiome (RNA-assay for transposase-accessible chromatin) sequencing and bulk RNA sequencing of kidneys from male and female mice that were mildly water deprived or not were compared. Water-deprived mice had a significant increase in plasma osmolality. sn-multiome-seq resulted in 19,837 nuclei that were annotated into 33 clusters. In CDPCs, aquaporin 2 (Aqp2) and aquaporin 3 (Apq3) were greater in dehydrated mice, but there were novel genes like gremlin 2 (Grem2; a cytokine) that were increased compared with ad libitum mice. The transcription factor cAMP-responsive element modulator (Crem) was greater in CDPCs of dehydrated mice, and the Crem DNA motif was more accessible. There were hundreds of sex- and dehydration-specific differentially expressed genes (DEGs) throughout the kidney, especially in the proximal tubules and thin limbs. In male mice, DEGs were enriched in pathways related to lipid metabolism, whereas female DEGs were enriched in organic acid metabolism. Many highly expressed genes had a positive correlation with increased chromatin accessibility, and mild dehydration exerted many transcriptional changes that we detected at the chromatin level. Even with a rise in plasma osmolality, male and female kidneys have distinct transcriptomes suggesting that there may be diverse mechanisms used to remain in fluid balance.NEW & NOTEWORTHY The kidney consists of >30 cell types that work collectively to maintain fluid-electrolyte balance. Kidney single-nucleus transcriptomes and chromatin accessibility profiles from male and female control (ad libitum water and food) or mildly dehydrated mice (ad libitum food, water deprivation) were determined. Mild dehydration caused hundreds of cell- and sex-specific transcriptomic changes, even though the kidney function to conserve water was the same.

Analyzation of the Peripheral Blood Mononuclear Cells Atlas and Cell Communication of Rheumatoid Arthritis Patients Based on Single-Cell RNA-Seq.

Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease with a multifactorial etiology. Peripheral blood is the main channel of the immune system, and peripheral blood mononuclear cells (PBMCs) are the immune cells that initiate the autoimmune inflammatory process. However, there are few reports on the mechanisms of peripheral blood immunity in RA. ScRNA-seq was performed on four RA samples and integrated with single-cell transcriptome data from four healthy control samples downloaded from publicly available databases for analysis. A total of 52,073 cells were used for descending clustering analysis to map RA peripheral blood immune cells at single-cell resolution. Redimensional clustering analysis of four major immune cells (T cells, monocytes, B cells, and natural killer cells) revealed that double-negative T (DNT) cells were significantly altered in abundance and function. And a number of genes (including SOCS3, cAMP-responsive element modulator (CREM), B2M, MTFP1, RSRP1, and YWHAB) were specifically downregulated in DNT cells. RA T cells, especially DNT cells, exhibit significant metabolic defects and dysfunction, mainly in the form of inhibition of oxidative phosphorylation, ATP synthesis, and major histocompatibility complex (MHC)-I-mediated antigen presentation. In addition, cellular communication networks were established, and it was evident that RA is significantly attenuated in the number and intensity of cellular communication. Monocytes and T cells play key roles in the process of the immune inflammatory response through CCL and MHC-related pathways. This study describes the landscape of the peripheral blood immune system and cell communication in RA, characterizes the abundance of PBMCs, gene expression profiles, and changes in signaling pathways in RA patients, and identifies several key cell subpopulations (DNT and classic monocytes) and specific genes (SOCS3, CREM, B2M, MTFP1, RSRP1, and YWHAB). Meanwhile, we propose that classic monocytes in peripheral blood may migrate to sites of inflammation in synovial tissue under the chemotaxis of the chemokines CCL3 and CCL3L1, differentiate into macrophages, secrete proinflammatory cytokines, and thus participate in the inflammatory response. These findings provide new insights for the future elucidation of the peripheral blood immune mechanisms of RA and the search for new clinical therapeutic targets.

Novel Aspects of cAMP-Response Element Modulator (CREM) Role in Spermatogenesis and Male Fertility.

Spermatogenesis is a very complex process with an intricate transcriptional regulation. The transition from the diploid to the haploid state requires the involvement of specialized genes in meiosis, among other specific functions for the formation of the spermatozoon. The transcription factor cAMP-response element modulator (CREM) is a key modulator that triggers the differentiation of the germ cell into the spermatozoon through the modification of gene expression. CREM has multiple repressor and activator isoforms whose expression is tissue-cell-type specific and tightly regulated by various factors at the transcriptional, post-transcriptional and post-translational level. The activator isoform CREMτ controls the expression of several relevant genes in post-meiotic stages of spermatogenesis. In addition, exposure to xenobiotics negatively affects CREMτ expression, which is linked to male infertility. On the other hand, antioxidants could have a positive effect on CREMτ expression and improve sperm parameters in idiopathically infertile men. Therefore, CREM expression could be used as a biomarker to detect and even counteract male infertility. This review examines the importance of CREM as a transcription factor for sperm production and its relevance in male fertility, infertility and the response to environmental xenobiotics that may affect CREMτ expression and the downstream regulation that alters male fertility. Also, some health disorders in which CREM expression is altered are discussed.

MITF::CREM-rearranged tumor: a novel group of cutaneous tumors with melanocytic differentiation.

Cutaneous tumors with melanocytic differentiation represent a broad group of neoplasms of both melanocytic and non-melanocytic origin. Besides traditional members such as clear-cell sarcoma (CCS) and PEComa, the latter group has recently expanded to also include MITF::CREM fusion-associated tumors, but the available data are limited. Herein, we present a third case of this rare neoplasm which occurred in the temporal region in a 1-year-old girl. It was an infiltratively growing polypoid dermal-based lesion lacking an intraepidermal component. It consisted of cellular solid sheets or small nests of epithelioid to spindled cells with a predominantly eosinophilic and much less commonly clear cytoplasm. The nuclei had round to ovoid shape and exhibited moderate to high-grade atypia and prominent nucleoli. The mitotic activity was 11 mitoses per 10 high-power fields, and atypical mitotic figures were present. Immunohistochemically, the tumor was strongly positive with S100 protein, SOX10, and MITF, while HMB45, tyrosinase, and Melan A were negative. Extensive molecular analysis revealed only MITF::CREM gene fusion. There had no evidence of disease 9 months after the diagnosis. These tumors need to be distinguished from malignant tumors with melanocytic differentiation, primarily from melanoma. However, additional cases still need to be studied to precisely define their biological potential and establish their nosologic status.

The Mechanisms Underlying the Pharmacological Effects of GuiPi Decoction on Major Depressive Disorder based on Network Pharmacology and Molecular Docking

Major Depressive Disorder (MDD) is a common affective disorder. GuiPi decoction (GPD) is used to treat depression in China, Japan, and Korea. However, its effective ingredients and antidepressant mechanisms remain unclear. We attempted to reveal the potential mechanisms of GPD in the treatment of MDD by network pharmacology and molecular docking. In addition, we conducted an enzymatic activity assay to validate the results of molecular docking. GPD-related compounds and targets, and MDD-related targets were retrieved from databases and literature. The herb-compound-target network was constructed by Cytoscape. The protein- protein interaction network was built using the STRING database to find key targets of GPD on MDD. Enrichment analysis of shared targets was analyzed by MetaCore database to obtain the potential pathway and biological process of GPD on MDD. The main active compounds treating MDD were screened by molecular docking. The PDE4s inhibitors were screened and verified by an enzyme activity assay. GPD contained 1222 ingredients and 190 potential targets for anti-MDD. Possible biological processes regulated by GPD were neurophysiological processes, blood vessel morphogenesis, Camp Responsive Element Modulator (CREM) pathway, and Androgen Receptor (AR) signaling crosstalk in MDD. Potential pathways in MDD associated with GPD include neurotransmission, cell differentiation, androgen signaling, and estrogen signaling. Fumarine, m-cresol, quercetin, betasitosterol, fumarine, taraxasterol, and lupeol in GPD may be the targets of SLC6A4, monoamine oxidase A (MAOA), DRD2, OPRM1, HTR3A, Albumin (ALB), and NTRK1, respectively. The IC50 values of trifolin targeting Phosphodiesterase (PDE) 4A and girinimbine targeting PDE4B1 were 73.79 μM and 31.86 μM, respectively. The IC50 values of girinimbine and benzo[a]carbazole on PDE4B2 were 51.62 μM and 94.61 μM, respectively. Different compounds in GPD may target the same protein, and the same component in GPD can target multiple targets. These results suggest that the effects of GPD on MDD are holistic and systematic, unlike the pattern of one drug-one target.

CAMP responsive element modulator α promotes effector T cells in systemic autoimmune diseases.

T lymphocytes play a crucial role in adaptive immunity. Dysregulation of T cell-derived inflammatory cytokine expression and loss of self-tolerance promote inflammation and tissue damage in several autoimmune/inflammatory diseases, including systemic lupus erythematosus (SLE) and psoriasis. The transcription factor cAMP responsive element modulator α (CREMα) plays a key role in the regulation of T cell homeostasis. Increased expression of CREMα is a hallmark of the T cell-mediated inflammatory diseases SLE and psoriasis. Notably, CREMα regulates the expression of effector molecules through trans-regulation and/or the co-recruitment of epigenetic modifiers, including DNA methyltransferases (DNMT3a), histone-methyltransferases (G9a) and histone acetyltransferases (p300). Thus, CREMα may be used as a biomarker for disease activity and/or target for future targeted therapeutic interventions.

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.