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.

Antileishmanial Activity of Cathelicidin and its Modulation by Leishmania donovani in a cAMP Response Element Modulator-Dependent Manner in Infection.

Concerns regarding toxicity and resistance of current drugs in visceral leishmaniasis have been reported. Antimicrobial peptides are considered to be promising candidates and among them human cathelicidin hCAP18/LL-37 showed significant parasite killing on drug-sensitive and resistant Leishmania promastigotes, in addition to its apoptosis-inducing role. Administration of hCAP18/LL-37 to infected macrophages also decreased parasite survival and increased the host favorable cytokine interleukin 12. However, 1,25-dihydroxyvitamin D3 (vitamin D3)-induced endogenous hCAP18/LL-37 production was hampered in infected THP-1 cells. Infection also suppressed the vitamin D3 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. This study documents the antileishmanial potential of cathelicidin and further identifies CREM as a repressor of cathelicidin in Leishmania infection.

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.

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.

Atrial proteomic profiling reveals a switch towards profibrotic gene expression program in CREM-IbΔC-X mice with persistent atrial fibrillation.

BACKGROUND: Overexpression of the CREM (cAMP response element-binding modulator) isoform CREM-IbΔC-X in transgenic mice (CREM-Tg) causes the age-dependent development of spontaneous AF. PURPOSE: To identify key proteome signatures and biological processes accompanying the development of persistent AF through integrated proteomics and bioinformatics analysis. METHODS: Atrial tissue samples from three CREM-Tg mice and three wild-type littermates were subjected to unbiased mass spectrometry-based quantitative proteomics, differential expression and pathway enrichment analysis, and protein-protein interaction (PPI) network analysis. RESULTS: A total of 98 differentially expressed proteins were identified. Gene ontology analysis revealed enrichment for biological processes regulating actin cytoskeleton organization and extracellular matrix (ECM) dynamics. Changes in ITGAV, FBLN5, and LCP1 were identified as being relevant to atrial fibrosis and structural based on expression changes, co-expression patterns, and PPI network analysis. Comparative analysis with previously published datasets revealed a shift in protein expression patterns from ion-channel and metabolic regulators in young CREM-Tg mice to profibrotic remodeling factors in older CREM-Tg mice. Furthermore, older CREM-Tg mice exhibited protein expression patterns reminiscent of those seen in humans with persistent AF. CONCLUSIONS: This study uncovered distinct temporal changes in atrial protein expression patterns with age in CREM-Tg mice consistent with the progressive evolution of AF. Future studies into the role of the key differentially abundant proteins identified in this study in AF progression may open new therapeutic avenues to control atrial fibrosis and substrate development in AF.

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 20 mg/kg·bw) once every 5 days via intratracheal instillation for 35 days. 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 20 µg/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 15 days 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.

Cardiac-specific overexpression of CREM-IbΔC-X via CRISPR/Cas9 in mice presents a new model of atrial cardiomyopathy with spontaneous atrial fibrillation.

Atrial cardiomyopathy (ACM) forms the substrate for atrial fibrillation (AF) and underlies the potential for atrial thrombus formation and subsequent stroke. However, generating stable animal models that accurately replicate the entire progression of atrial lesions, particularly the onset of AF, presents significant challenges. In the present study, we found that the isoform of CRE-binding protein modulator (CREM-IbΔC-X), which is involved in the regulation of cardiac development and atrial rhythm, was highly expressed in atrial biopsies from patients with AF. Building upon this finding, we employed CRISPR/Cas9 technology to create a mouse model with cardiac-specific overexpression of CREM-IbΔC-X (referred to as CS-CREM mice). This animal model effectively illustrated the development of ACM through electrophysiological and structural remodelings over time. Proteomics and Chip-qPCR analysis of atrial samples revealed significant upregulation of cell-matrix adhesion and extracellular matrix structural components, alongside significant downregulation of genes related to atrial functions in the CS-CREM mice. Furthermore, the corresponding responses to anti-arrhythmia drugs, i.e., amiodarone and propafenone, suggested that CS-CREM mice could serve as an ideal in vivo model for drug testing. Our study introduced a novel ACM model with spontaneous AF by cardiac-specifically overexpressing CREM-IbΔC-X in mice, providing valuable insights into the mechanisms and therapeutic targets of ACM.

Effects of cadmium exposure during pregnancy on genome-wide DNA methylation and the CREB/CREM pathway in the testes of male offspring rats.

This experimental study explored the multigenerational and transgenerational effects of cadmium (Cd) exposure during pregnancy on the testicular tissue and spermatogenesis of male offspring rats. CdCl2 at different doses (0, 0.5, 1, 2 mg/kg/day) were dispensed to pregnant SD rats, thus producing generation F1. Adult females in F1 (PND 56) were mated with untreated fertile males so as to produce generation F2. Likewise, adult females in F2 were mated to produce generation F3. Damages to testicular tissue were observed in all the three generations, with serum testosterone (T) increased in F2 and F3. Notably, the genome-wide DNA methylation level in the testicular tissue of F1 was altered, as was the expression of F1-F3 methyltransferases. In addition, the expression of Creb/Crem pathway, a pathway critical for the metamorphosis from postmeiotic round spermatocytes to spermatozoa, was also remarkably altered in the three generations. In concludion, prenatal Cd exposure might bring multigenerational and transgenerational toxic effects to testes via genome-wide DNA methylation and the regulation of CREB/CREM pathway.

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.

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.

Epithelioid mesenchymal neoplasm with FUS::CREM gene fusion in the tongue: Report of a rare and challenging diagnosis.

FET (encompassing both EWSR1 and FUS) fusions with genes from the CREB family (CREB1, ATF1, and CREM) are involved in a variety of neoplasms. Recently, FET::CREB fusions were recognized in a group of malignant epithelioid neoplasm with a striking predilection to mesothelial-lined cavities and frequent cytokeratin immunoexpression. Herein, we report a rare mesenchymal neoplasm with epithelioid morphology and nonspecific immunoprofile harboring a FUS::CREM fusion arising in the oral tongue of a 53-year-old man. Histology showed a well-circumscribed tumor composed of epithelioid cells with eosinopohilic or clear cytoplasm with sparse stroma, accompanied by peripheral lymphoplasmacytic infiltrates. Immunohistochemically, an extensive panel revealed only patchy expression of synaptophysin and weak-to-moderate nuclear expression of TFE3, and negativity for other markers including cytokeratins, epithelial membrane antigen, p63/p40, vimentin, S100, smooth muscle actin, CD34, desmin, SOX10, glial fibrillary acidic protein, melan-A, HMB45, and CD68. A FUS::CREM gene fusion was detected by next generation sequencing at an outside institution, and subsequent fluorescence in situ hybridization analysis confirmed the presence of FUS gene rearrangement. The identification and analysis of additional cases should help to clarify the nosologic status and the biologic potential of this tumor.

A novel SMARCA2-CREM fusion expending the molecular spectrum of salivary gland hyalinazing clear cell carcinoma beyond the FET genes.

Hyalinizing clear cell carcinoma (HCCC) is a rare salivary gland carcinoma with a generally indolent behavior, characterized by recurrent chromosomal translocation involving EWSR1 (22q12.2) leading to two fusion genes EWSR1::ATF1 or EWSR1::CREM. We report one case of HCCC with a novel SMARCA2::CREM fusion, identified by targeted RNA next generation sequencing by LD-RT-PCR, which has until now never been described in salivary glands. The exon 4 of SMARCA2 is fused to exon 5 of CREM. This fusion has been described previously in only one tumor, a central nervous system tumor (intracranial mesenchymal tumor) but not in other FET::CREB fused tumors. This fusion was confirmed by CREM break-apart FISH and reverse transcriptase polymerase chain reaction (RT-PCR). The tumor cells showed retained expression of INI1, SMARCA2, and SMARCA4 by immunohistochemistry. We compare its clinical, histopathological, immunophenotypic, genetic features with those previously described in HCCC, FET::CREB fusion-positive. Our results added data suggesting that different histomolecular tumor subtypes seem to be included within the terminology "HCCC, FET::CREB fusion-positive," and that further series of cases are needed to better characterize them.

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

BACKGROUND: Overproduction 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. RESULTS: CREMα 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. CONCLUSIONS: Decreased 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.

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

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 ß-cell regulators in check.

Evidence that the transcriptional repressor ICER is regulated via the N-end rule for ubiquitination.

ICER is a transcriptional repressor that is mono- or poly-ubiquitinated. This either causes ICER to be translocated from the nucleus, or degraded via the proteasome, respectively. In order to further studies the proteins involved in ICER regulation mass spectrometry analysis was performed to identify potential candidates. We identified twenty eight ICER-interacting proteins in human melanoma cells, Sk-Mel-24. In this study we focus on two proteins with potential roles in ICER proteasomal degradation in response to the N-end rule for ubiquitination: the N-alpha-acetyltransferase 15 (NAA15) and the E3 ubiquitin-protein ligase UBR4. Using an HA-tag on the N- or C-terminus of ICER (NHAICER or ICERCHA) it was found that the N-terminus of ICER is important for its interaction to UBR4, whereas NARG1 interaction is independent of HA-tag position. Silencing RNA experiments show that both NAA15 and UBR4 up-regulates ICER levels and that ICER's N-terminus is important for this regulation. The N-terminus of ICER was found to have dire consequences on its regulation by ubiquitination and cellular functions. The half-life of NHAICER was found to be about twice as long as ICERCHA. Polyubiquitination of ICER was found to be dependent on its N-terminus and mediated by UBR4. This data strongly suggests that ICER is ubiquitinated as a response to the N-end rule that governs protein degradation rate through recognition of the N-terminal residue of proteins. Furthermore, we found that NHAICER inhibits transcription two times more efficiently than ICERCHA, and causes apoptosis 5 times more efficiently than ICERCHA. As forced expression of ICER has been shown before to block cells in mitosis, our data represent a potentially novel mechanism for apoptosis of cells in mitotic arrest.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Genetic inhibition of nuclear factor of activated T-cell c2 prevents atrial fibrillation in CREM transgenic mice.

AIMS: Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. METHODS AND RESULTS: Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription. CONCLUSION: Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.

Glutathione peroxidase 4-regulated neutrophil ferroptosis induces systemic autoimmunity.

The linkage between neutrophil death and the development of autoimmunity has not been thoroughly explored. Here, we show that neutrophils from either lupus-prone mice or patients with systemic lupus erythematosus (SLE) undergo ferroptosis. Mechanistically, autoantibodies and interferon-α present in the serum induce neutrophil ferroptosis through enhanced binding of the transcriptional repressor CREMα to the glutathione peroxidase 4 (Gpx4, the key ferroptosis regulator) promoter, which leads to suppressed expression of Gpx4 and subsequent elevation of lipid-reactive oxygen species. Moreover, the findings that mice with neutrophil-specific Gpx4 haploinsufficiency recapitulate key clinical features of human SLE, including autoantibodies, neutropenia, skin lesions and proteinuria, and that the treatment with a specific ferroptosis inhibitor significantly ameliorates disease severity in lupus-prone mice reveal the role of neutrophil ferroptosis in lupus pathogenesis. Together, our data demonstrate that neutrophil ferroptosis is an important driver of neutropenia in SLE and heavily contributes to disease manifestations.

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.

Clear cell tumor with melanocytic differentiation and MITF-CREM translocation: a novel entity similar to clear cell sarcoma.

The presence of melanocytic differentiation in tumors of non-melanocyte origin is uncommon and is typically associated with the overexpression of MITF, the master regulator of melanin synthesis, or another member of the MIT/TFE3 family. In clear cell sarcoma, the presence of either an EWSR1-ATF1 or EWSR1-CREB1 translocation-derived fusion protein is thought to drive melanocytic differentiation by directly stimulating the expression of MITF. Here, we describe a clear cell neoplasm with melanocytic differentiation that is characterized by a novel MITF-CREM gene fusion. CREM is the third member of the ATF1/CREB1/CREM family, and the nature of the MITF-CREM fusion appears analogous to the EWSR1-ATF1 and EWSR1-CREB1 fusions. Thus, this MITF-CREM-rearranged clear cell tumor represents a novel entity with morphologic, immunohistochemical, and molecular similarity to clear cell sarcoma.