Zinc finger protein ZBTB17 controls cellular senescence via interacting with nuclear receptor RXRA and its downstream calcium signaling.

Cellular senescence is broadly known as a stable cell cycle arrest accompanied by a senescence-associated secretory phenotype (SASP). In the past decades, calcium signaling has emerged as a key mediator of cellular senescence. However, the transcriptional regulation of calcium signaling during cellular senescence remains partially understood. We have previously identified the nuclear receptor RXRA as a key senescence repressor through inhibiting the endoplasmic reticulum (ER) calcium release channel inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) mediated intracellular calcium signaling. Nevertheless, as a transcriptional recruiter, the mechanism by which RXRA inhibits ITPR2 during cellular senescence remains unclear. Here we identified the zinc finger protein ZBTB17 can interact with RXRA. Interestingly, knockdown of ZBTB17 induces a cascade of RXRA-dependent intracellular calcium signaling, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) accumulation, DNA damages, and ultimately cellular senescence. Moreover, the signaling and senescence phenotype induced by knocking down of ZBTB17 can also be abolished after silencing ITPR2. Altogether, our work provides a new mechanism controlling intracellular calcium signaling and cellular senescence and unveils novel insight toward the role of zinc finger proteins.

Proteomic analysis of ITPR2 as a new therapeutic target for curcumin protection against AFB1-induced pyroptosis.

Aflatoxin B1 (AFB1) is extremely carcinogenic and can cause liver cancer in humans and animals with continued ingestion. As a natural compound, curcumin (Cur) exhibits excellent anti-inflammatory, and anti-cancer properties with few side effects. In this study, a total of 60 male mice (6-week-olds, 15 per group). After one week of acclimatization feeding, the mice were divided into control group (Con), AFB1 group, curcumin group (Cur), and AF+Cur group. The mice were gavaged with curcumin (Cur, 100 mg/kg) and/or AFB1 (0.75 mg/kg). To identify a new therapeutic target for AFB1-induced pyroptosis, we performed proteomic profiling for curcumin alleviating liver injury caused by AFB1 to further validate the targets through volcano plot analysis, Venn analysis, heatmap analysis, correlation, cluster analysis, GO and KEGG enrichment. AFB1 exposure resulted in the loss of hepatocyte membrane, swelling of the endoplasmic reticulum, and a significant increase in transaminase (ALT and AST) contents, while curcumin greatly improved these changes. We found that differentially expressed proteins are enriched in the endoplasmic reticulum membrane and identified ITPR2 as a target of curcumin that alleviates AFB1-induced liver injury by proteomics. Furthermore, ITPR2 expression was detected by immunofluorescence, and qRT-PCR for mRNA expression of genes downstream of ITPR2 (calpain1, calpain2, caspase-12, caspase-3). ITPR2-activated endoplasmic reticulum stress-related proteins (calpain1, calpaini2, bcl-2, BAX, cl-caspase-12, cl-caspase-3), apoptosis (PARP) and pyroptosis (DFNA5) related proteins were examined by western blotting. The analysis showed that it effectively prevents AFB1-induced pyroptosis by lowering endoplasmic reticulum stress via interfering with ITPR2 and its downstream proteins (calpain1, calpain2, bcl-2, Bax) and inhibiting caspase-12/caspase-3 pathway. Conclusively, this study applied proteomic profiling to elucidate ITPR2 as a new target, which might give a new perspective on the mechanism of curcumin alleviating AFB1-induced pyroptosis.

Rearing-environment-dependent hippocampal local field potential differences in wild-type and inositol trisphosphate receptor type 2 knockout mice.

KEY POINTS: Mice reared in an enriched environment are demonstrated to have larger hippocampal gamma oscillations than those reared in isolation, thereby confirming previous observations in rats. To test whether astrocytic Ca2+ surges are involved in this experience-dependent LFP pattern modulation, we used inositol trisphosphate receptor type 2 (IP3 R2)-knockout (KO) mice, in which IP3 /Ca2+ signalling in astrocytes is largely diminished. We found that this experience-dependent gamma power alteration persists in the KO mice. Interestingly, hippocampal ripple events, the synchronized events critical for memory consolidation, are reduced in magnitude and frequency by both isolated rearing and IP3 R2 deficiency. ABSTRACT: Rearing in an enriched environment (ENR) is known to enhance cognitive and memory abilities in rodents, whereas social isolation (ISO) induces depression-like behaviour. The hippocampus has been documented to undergo morphological and functional changes depending on these rearing environments. For example, rearing condition during juvenility alters CA1 stratum radiatum gamma oscillation power in rats. In the present study, hippocampal CA1 local field potentials (LFP) were recorded from bilateral CA1 in urethane-anaesthetized mice that were reared in either an ENR or ISO condition. Similar to previous findings in rats, gamma oscillation power during theta states was higher in the ENR group. Ripple events that occur during non-theta periods in the CA1 stratum pyramidale also had longer intervals in ISO mice. Because astrocytic Ca2+ elevations play a key role in synaptic plasticity, we next tested whether these changes in LFP are also expressed in inositol trisphosphate receptor type 2 (IP3 R2)-knockout (KO) mice, in which astrocytic Ca2+ elevations are largely diminished. We found that the gamma power was also higher in IP3 R2-KO-ENR mice compared to IP3 R2-KO-ISO mice, suggesting that the rearing-environment-dependent gamma power alteration does not necessarily require the astrocytic IP3 /Ca2+ pathway. By contrast, ripple events showed genotype-dependent changes, as well as rearing condition-dependent changes: ISO housing and IP3 R2 deficiency both lead to longer inter-ripple intervals. Moreover, we found that ripple magnitude in the right CA1 tended to be smaller in IP3 R2-KO. Because IP3 R2-KO mice have been reported to have depression phenotypes, our results suggest that ripple events and the mood of animals may be broadly correlated.

Germline genetic variations at 11q13 and 12p11 locus modulate age at onset for renal cell carcinoma.

PURPOSE: Few risk factors have been identified for renal cell carcinoma. We performed a validation study in a population with a European background to identify the most significant variants previously identified in association with renal cell carcinoma risk. MATERIALS AND METHODS: We performed a case-control validation study after recruiting 463 controls and 463 patients with a histologically confirmed diagnosis of clear cell renal cell carcinoma. For each patient and matched control we genotyped 8 single nucleotide polymorphisms selected from previous studies to evaluate the association between candidate single nucleotide polymorphisms and renal cell carcinoma susceptibility. RESULTS: After adjusting for patient age, gender, smoking status and body mass index the AG + AA genotypes from rs7105934 (11q13) were associated with a decreased risk of renal cell carcinoma (OR 0.50, 95% CI 0.33-0.75, p = 0.001) and the AC + CC genotypes from rs1049380 (ITPR2) were associated with an increased risk (OR 1.66, 95% CI 1.28-2.16, p <0.001). Kidney cancer developed at an older age in patients carrying the dominant risk allele A for rs7105934 (mean age at diagnosis 73.1 vs 68.9 years, p = 0.002) and at a younger age in those carrying the dominant allele C for rs1049380 (mean 68.1 vs 70.8 years, p = 0.005). CONCLUSIONS: In what is to our knowledge the first validation study of the main 8 single nucleotide polymorphism variants associated with renal cell carcinoma susceptibility we confirmed the association of 2 single nucleotide polymorphisms with the risk of renal cell carcinoma. Each variant influenced patient age at disease diagnosis.

ITPR2 Mediated Calcium Homeostasis in Oligodendrocytes is Essential for Myelination and Involved in Depressive-Like Behavior in Adolescent Mice.

Ca2+ signaling is essential for oligodendrocyte (OL) development and myelin formation. Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) is an endoplasmic reticulum calcium channel and shows stage-dependent high levels in postmitotic oligodendrocyte precursor cells (OPCs). The role and potential mechanism of ITPR2 in OLs remain unclear. In this study, it is revealed that loss of Itpr2 in OLs disturbs Ca2+ homeostasis and inhibits myelination in adolescent mice. Animals with OL-specific deletion of Itpr2 exhibit anxiety/depressive-like behaviors and manifest with interrupted OPC proliferation, leading to fewer mature OLs in the brain. Detailed transcriptome profiling and signal pathway analysis suggest that MAPK/ERK-CDK6/cyclin D1 axis underlies the interfered cell cycle progression in Itpr2 ablated OPCs. Besides, blocking MAPK/ERK pathway significantly improves the delayed OPC differentiation and myelination in Itpr2 mutant. Notably, the resting [Ca2+]i is increased in Itpr2 ablated OPCs, with the elevation of several plasma calcium channels. Antagonists against these plasma calcium channels can normalize the resting [Ca2+]i level and enhance lineage progression in Itpr2-ablated OPCs. Together, the findings reveal novel insights for calcium homeostasis in manipulating developmental transition from OPCs to pre-OLs; additionally, the involvement of OLs-originated ITPR2 in depressive behaviors provides new therapeutic strategies to alleviate myelin-associated psychiatric disorders.

Calcium transferring from ER to mitochondria via miR-129/ITPR2 axis controls cellular senescence in vitro and in vivo.

Senescent cells are known to be accumulated in aged organisms. Although the two main characteristics, cell cycle arrest (for dividing cells) and secretion of senescence-associated secretory phenotype (SASP) factors, have been well described, the lack of sufficient senescent markers and incomplete understanding of mechanisms have limited the progress of the anti-senescence field. Calcium transferred from the endoplasmic reticulum (ER) via inositol 1, 4, 5-trisphosphate receptor type 2 (ITPR2) to mitochondria has emerged as a key player during cellular senescence and aging. However, the internal regulatory mechanisms, particularly those of endogenous molecules, remain only partially understood. Here we identified miRNA-129 (miR-129) as a direct repressor of ITPR2. Interestingly, miR-129 controlled a cascade of intracellular calcium signaling, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA damage, and consequently cellular senescence through ITPR2 and mitochondrial calcium uniporter (MCU). In addition, miR-129 was repressed in different senescence models and delayed bleomycin-induced cellular senescence. Importantly, intraperitoneal injection of miR-129 partly postponed bleomycin-accelerated lung aging and natural aging markers as well as reduced immunosenescence markers in mice. Altogether, these findings demonstrated that miR-129 regulated cellular senescence and aging markers via intracellular calcium signaling by directly targeting ITPR2.

Expression of ITPR2 regulated by lncRNA-NONMMUT020270.2 in LPS-stimulated HT22 cells.

Background: Long non-coding RNA (lncRNA)-NONMMUT020270.2 is downregulated and co-expressed with inositol 1,4,5-trisphosphate receptor type 2 (ITPR2) in the hippocampus of Alzheimer's disease (AD) mice. However, whether the expression of ITPR2 was regulated by lncRNA-NONMMUT020270.2 remains unclear. we aimed to investigate regulating relationship of lncRNA-NONMMUT020270.2 and ITPR2. Methods: HT22 cells were firstly transfected with the pcDNA3.1-lncRNA-NONMMUT020270.2 overexpression plasmid or with the lncRNA-NONMMUT020270.2 smart silencer, and then were stimulated with lipopolysaccharide (LPS) for 24h. The mRNA expression levels of lncRNA-NONMMUT020270.2 and ITPR2 were measured by reverse transcription-quantitative PCR. Cell viability was assessed using a Cell Counting Kit 8 assay. The expression of Aß1-42 was detected by ELISA. The expression levels of p-tau, caspase-1, and inositol trisphosphate receptor (IP3R) proteins were detected by western-blotting. Nuclear morphological changes were detected by Hoechst staining. Flow cytometry and Fluo-3/AM were carried out to determine cell apoptosis and the intracellular Ca2+. Results: LPS significantly decreased cell viability, and ITPR2 mRNA and IP3R protein expression levels. While it markedly enhanced the expression levels of p-tau and Aß1-42, cell apoptosis rate, as well as intracellular Ca2+ concentration (P < 0.05). In addition, lncRNA-NONMMUT020270.2 overexpression significantly increased the expressions levels of ITPR2 mRNA and IP3R protein (P < 0.05), and inhibited expression of p-tau and Aß1-42, cell apoptosis rate, and reduced intracellular Ca2+ concentration (P < 0.05). By contrast, lncRNA-NONMMUT020270.2 silencing notably downregulated expressions levels of ITPR2 mRNA and IP3R protein (P < 0.05), and elevated expression levels of p-tau and Aß1-42, cell apoptosis rate, and intracellular Ca2+ concentration (P < 0.05). Conclusion: lncRNA-NONMMUT020270.2 was positively correlated with ITPR2 expression in LPS-induced cell. Downregulating the lncRNA-NONMMUT020270.2 and ITPR2 may promote cell apoptosis and increase intracellular Ca2+ concentration.

Genetic ablation of phospholipase C delta 1 increases survival in SOD1(G93A) mice.

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, resulting in selective motor neuron degeneration and paralysis. Patients die approximately 3-5 years after diagnosis. Disease pathophysiology is multifactorial, including excitotoxicity, but is not yet fully understood. Genetic analysis has proven fruitful in the past to further understand genes modulating the disease and increase knowledge of disease mechanisms. Here, we revisit a previously performed microsatellite analysis in ALS and focus on another hit, PLCD1, encoding phospholipase C delta 1 (PLCδ1), to investigate its role in ALS. PLCδ1 may contribute to excitotoxicity as it increases inositol 1,4,5-trisphosphate (IP3) formation, which releases calcium from the endoplasmic reticulum through IP3 receptors. We find that expression of PLCδ1 is increased in ALS mouse spinal cord and in neurons from ALS mice. Furthermore, genetic ablation of this protein in ALS mice significantly increases survival, but does not affect astrogliosis, microgliosis, aggregation or the amount of motor neurons at end stage compared to ALS mice with PLCδ1. Interestingly, genetic ablation of PLCδ1 prevents nuclear shrinkage of motor neurons in ALS mice at end stage. These results indicate that PLCD1 contributes to ALS and that PLCδ1 may be a new target for future studies.

Genetic polymorphisms of Ca2+ transport proteins and molecular chaperones in mitochondria-associated endoplasmic reticulum membrane and non-alcoholic fatty liver disease.

Background: Non-alcoholic fatty liver disease (NAFLD) is recognized to be closely associated with endoplasmic reticulum stress and mitochondrial dysfunction, while previous studies have emphasized the important role of calcium homeostasis from the mitochondria-associated endoplasmic reticulum membrane (MAM) in the endoplasmic reticulum and mitochondria. This article will assess the association between genetic polymorphisms of Ca2+ transport proteins and molecular chaperones in MAM and NAFLD risk. Methods: A case-control study was conducted in a community of Nanjing, China during April to December 2020. 2701 subjects were enrolled and genotyped for 6 genetic variants in HSPA5 and ITPR2 genes. Logistic regression analysis was used to assess impact of these variants on NAFLD risk. Results: After adjusting for age, gender, total cholesterol and glucose, we identified that HSPA5 rs12009 variant genotypes (recessive model: OR= 0.801, 95% CI= 0.652-0.986, P= 0.036), rs430397 variant genotypes (recessive model: OR= 0.546, 95% CI= 0.314-0.950, P= 0.032), and ITPR2 rs11048570 variant genotypes (recessive model: OR= 0.673, 95% CI= 0.453-0.999, P= 0.049) were associated with a reduced risk of NAFLD. Multivariate stepwise regression analysis indicated that gender, glucose, body mass index, triglycerides and favorable alleles were independent influencers of NAFLD (all P< 0.05). The area under the receiver operating characteristic curve was 0.764 (95% CI= 0.745-0.783, P< 0.001). Conclusion: The variant genotypes of Ca2+ transport-associated genes HSPA5 (rs12009 and rs430397) and ITPR2 (rs11048570) might contribute to the reduction of the NAFLD risk in Chinese Han population, which can provide new insight into NAFLD pathogenesis.

Cardio-omentopexy requires a cardioprotective innate immune response to promote myocardial angiogenesis in mice.

Objective: The pedicled greater omentum, when applied onto stressed hearts using omentopexy, has been shown to be protective in humans and animals. The mechanisms underlying cardioprotection using omentopexy remain elusive. This study examined whether macrophage-mediated angiogenesis accounts for the cardioprotective effect of omentopexy in mice. Methods: C57BL/6 mice were subjected to minimally invasive transverse aortic constriction for 6 weeks and subsequent cardio-omentopexy for 8 weeks. Control mice underwent the same surgical procedures without aortic constriction or cardio-omentopexy. Results: Transverse aortic constriction led to left ventricular concentric hypertrophy, reduced mitral E/A ratio, increased cardiomyocyte size, and myocardial fibrosis in the mice that underwent sham cardio-omentopexy surgery. The negative effects of transverse aortic constriction were prevented by cardio-omentopexy. Myocardial microvessel density was elevated in the mice that underwent aortic constriction and sham cardio-omentopexy surgery, and cardio-omentopexy further enhanced angiogenesis. Nanostring gene array analysis uncovered the activation of angiogenesis gene networks by cardio-omentopexy. Flow cytometric analysis revealed that cardio-omentopexy triggered the accumulation of cardiac MHCIIloLyve1+TimD4+ (Major histocompatibility complex class IIlow lymphatic vessel endothelial hyaluronan receptor 1+ T cell immunoglobulin and mucin domain conataining 4+) resident macrophages at the omental-cardiac interface. Intriguingly, the depletion of macrophages with clodronate-liposome resulted in the failure of cardio-omentopexy to protect the heart and promote angiogenesis. Conclusions: Cardio-omentopexy protects the heart from pressure overload-elicited left ventricular hypertrophy and dysfunction by promoting myocardial angiogenesis. Cardiac MHCIIloLyve1+TimD4+ resident macrophages play a critical role in the cardioprotective effect and angiogenesis of cardio-omentopexy.

Evidence That ITPR2-Mediated Intracellular Calcium Release in Oligodendrocytes Regulates the Development of Carbonic Anhydrase II + Type I/II Oligodendrocytes and the Sizes of Myelin Fibers.

Myelination of neuronal axons in the central nervous system (CNS) by oligodendrocytes (OLs) enables rapid saltatory conductance and axonal integrity, which are crucial for normal brain functioning. Previous studies suggested that different subtypes of oligodendrocytes in the CNS form different types of myelin determined by the diameter of axons in the unit. However, the molecular mechanisms underlying the developmental association of different types of oligodendrocytes with different fiber sizes remain elusive. In the present study, we present the evidence that the intracellular Ca2+ release channel associated receptor (Itpr2) contributes to this developmental process. During early development, Itpr2 is selectively up-regulated in oligodendrocytes coinciding with the initiation of myelination. Functional analyses in both conventional and conditional Itpr2 mutant mice revealed that Itpr2 deficiency causes a developmental delay of OL differentiation, resulting in an increased percentage of CAII+ type I/II OLs which prefer to myelinate small-diameter axons in the CNS. The increased percentage of small caliber myelinated axons leads to an abnormal compound action potentials (CAP) in the optic nerves. Together, these findings revealed a previously unrecognized role for Itpr2-mediated calcium signaling in regulating the development of different types of oligodendrocytes.

[Changes in three-dimensional arterial spin labeling perfusion imaging of the hippocampus in depressive Itpr2-/- mice].

OBJECTIVE: To study the behavioral changes of inositol 1, 4, 5-trisphosphate receptor type 2 knockout (Itpr2-/- mice) and investigate the blood perfusion changes in the hippocampus using three-dimensional arterial spin labeling (3D-ASL). METHODS: 28 Itpr2-/- mice and 20 wild-type mice were assessed for depressive phenotype using behavioral tests (including sucrose consumption test, tail suspension test, forced swimming test and open field test). 15 Itpr2-/- mice and 14 wild-type mice were randomly selected for 3D-T2WI imaging of the whole brain and 3D-ASL imaging of the middle hippocampal layer, and cerebral blood flow (CBF) of the middle hippocampal layer was calculated. ITK-SNAP was used to delineate the bilateral hippocampal area and measure the average CBF value. RESULTS: Compared with the wild-type mice, Itpr2-/- mice exhibited a distinct depressive phenotype with significantly decreased sucrose preference (P < 0.05) and increased immobile time in tail suspension test (P < 0.05) and forced swimming test (P < 0.01), without obvious changes in the performance in open field test (P > 0.05). Significantly decreased mean CBF values were found in the left and right hippocampus of Itpr2-/- mice as compared with the wild-type mice (left: 73.30 ±5.609 vs 95.77±5.095; right: 73.53±5.700 vs 100.5±4.696; bilateral means: 73.42±5.607 vs98.12±4.754; P < 0.01). CONCLUSIONS: Itpr2 deficiency can cause depressive phenotype and affect the cerebral blood flow in the hippocampus of mice.

Anakinra Reduces Epileptogenesis, Provides Neuroprotection, and Attenuates Behavioral Impairments in Rats in the Lithium-Pilocarpine Model of Epilepsy.

Temporal lobe epilepsy is a widespread chronic disorder that manifests as spontaneous seizures and is often characterized by refractoriness to drug treatment. Temporal lobe epilepsy can be caused by a primary brain injury; therefore, the prevention of epileptogenesis after a primary event is considered one of the best treatment options. However, a preventive treatment for epilepsy still does not exist. Neuroinflammation is directly involved in epileptogenesis and neurodegeneration, leading to the epileptic condition and cognitive decline. In the present study, we aimed to clarify the effect of treatment with a recombinant form of the Interleukin-1 receptor antagonist (anakinra) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that anakinra administration during the latent phase of the model significantly suppressed the duration and frequency of spontaneous recurrent seizures in the chronic phase. Moreover, anakinra administration prevented some behavioral impairments, including motor hyperactivity and disturbances in social interactions, during both the latent and chronic periods. Histological analysis revealed that anakinra administration decreased neuronal loss in the CA1 and CA3 areas of the hippocampus but did not prevent astro- and microgliosis. The treatment increased the expression level of the solute carrier family 1 member 2 gene (Slc1a2, encoding excitatory amino acid transporter 2 (EAAT2)) in the hippocampus, potentially leading to a neuroprotective effect. However, the increased gene expression of proinflammatory cytokine genes (Interleukin-1ß (Il1b) and tumor necrosis factor α (Tnfa)) and astroglial marker genes (glial fibrillary acidic protein (Gfap) and inositol 1,4,5-trisphosphate receptor type 2 (Itpr2)) in experimental rats was not affected by anakinra treatment. Thus, our data demonstrate that the administration of anakinra during epileptogenesis has some beneficial disease-modifying effects.

Genetic variants in the ITPR2 gene are associated with Kashin-Beck Disease in Tibetan.

BACKGROUND: Kashin-Beck Disease (KBD) is a chronic, endemic osteoarthropathy. Inositol 1,4,5-triphosphate receptor type 2 (ITPR2) gene is involved in chondrocytes. We speculated that single-nucleotide polymorphisms (SNPs) in ITPR2 gene may have an association with KBD in Tibetan. METHODS: To prove this hypothesis, a total of eight SNPs (rs1049376, rs11048526, rs11048556, rs11048585, rs16931011, rs10842759, rs2230372, and rs7134213) were selected, and genotyped in 316 KBD patients and 320 controls. The association between ITPR2 variants and KBD risk was assessed by logistic regression analysis. RESULTS: The study identified significant association (p = 0.019) between KBD and a novel locus, ITPR2 SNP rs11048526 (OR = 1.49, 95% CI = 1.07-2.08). The variant A/G genotype frequency in rs11048526 had a significantly increased risk of KBD in co-dominant model (OR = 3.70, 95% CI = 1.26-10.89, p = 0.018), dominant model (OR = 3.56, 95% CI = 1.22-10.40, p = 0.020), log-additive model (OR = 3.00, 95% CI = 1.12-8.00, p = 0.029) after adjusted for age and gender. CONCLUSION: The results indicate a potential association between ITPR2 and KBD risk in Tibetan. Further work is required to confirm these results and explore the mechanisms of these effects.

Development of a high efficient promoter finding method based on transient transfection.

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.

The nuclear receptor RXRA controls cellular senescence by regulating calcium signaling.

Calcium signaling is emerging as a key pathway controlling cellular senescence, a stable cell proliferation arrest playing a fundamental role in pathophysiological conditions, such as embryonic development, wound healing, cancer, and aging. However, how calcium signaling is regulated is still only partially understood. The inositol 1, 4, 5-trisphosphate receptor type 2 (ITPR2), an endoplasmic reticulum calcium release channel, was recently shown to critically contribute to the implementation of senescence, but how ITPR2 expression is controlled is unclear. To gain insights into the regulation of ITPR2 expression, we performed an siRNA screen targeting 160 transcription factors and epigenetic regulators. Interestingly, we discovered that the retinoid X receptor alpha (RXRA), which belongs to the nuclear receptor family, represses ITPR2 expression and regulates calcium signaling though ITPR2 and the mitochondrial calcium uniporter (MCU). Knockdown of RXRA induces the production of reactive oxygen species (ROS) and DNA damage via the ITPR2-MCU calcium signaling axis and consequently triggers cellular senescence by activating p53, whereas RXRA overexpression decreases DNA damage accumulation and then delays replicative senescence. Altogether, our work sheds light on a novel mechanism controlling calcium signaling and cellular senescence and provides new insights into the role of nuclear receptors.

High expression of inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) as a novel biomarker for worse prognosis in cytogenetically normal acute myeloid leukemia.

Inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) is a key regulator for the activity of calcium ion transmembrane transportation, which plays a critical role in cell cycle and proliferation. However, the clinical impact of ITPR2 in cytogenetically normal acute myeloid leukemia (CN-AML) remained unknown. Several microarray datasets were used to evaluate the association between ITPR2 expression and clinical and molecular characteristics. ITPR2 showed a higher expression in CN-AML patients than normal persons. In a cohort of 157 CN-AML patients, high ITPR2 expression (ITPR2high) was associated with dramatically shorter overall survival (OS; P = 0.004) and event-free survival (EFS; P = 0.01), which were also shown in the European Leukemia Net (ELN) intermediate-I genetic category (OS: P = 0.0066; EFS: P = 0.009). Multivariable analyses adjusting for known prognostic factors confirmed ITPR2high to be associated with shorter OS (P = 0.0019) and EFS (P = 0.012). The prognostic value of ITPR2 was further validated in another cohort of 162 CN-AML patients (P = 0.007). In addition, first gene/microRNA expression signatures were derived that associated with ITPR2high on the genome-wide scale, which provided many indications to illustrate the possible mechanisms why ITPR2 could function. These results could aid to identify new targets and design novel therapeutic strategies for CN-AML patients.