HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion.

Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.

Rbm3 deficiency leads to transcriptome-wide splicing alterations.

Rbm3 (RNA-binding motif protein 3) is a stress responsive gene, which maintains cellular homeostasis and promotes survival upon various harmful cellular stimuli. Rbm3 protein shows conserved structural and molecular similarities to heterogeneous nuclear ribonucleoproteins (hnRNPs), which regulate all steps of the mRNA metabolism. Growing evidence is pointing towards a broader role of Rbm3 in various steps of gene expression. Here, we demonstrate that Rbm3 deficiency is linked to transcriptome-wide pre-mRNA splicing alterations, which can be reversed through Rbm3 co-expression from a cDNA. Using an MS2 tethering assay, we show that Rbm3 regulates splice site selection similar to other hnRNP proteins when recruited between two competing 5 ' splice sites. Furthermore, we show that the N-terminal part of Rbm3 encompassing the RNA recognition motif (RRM), is sufficient to elicit changes in splice site selection. On the basis of these findings, we propose a novel, undescribed function of Rbm3 in RNA splicing that contributes to the preservation of transcriptome integrity.

Sodium butyrate induces ferroptosis in endometrial cancer cells via the RBM3/SLC7A11 axis.

Ferroptosis is a form of programmed cell death with important biological functions in the progression of various diseases, and targeting ferroptosis is a new tumor treatment strategy. Studies have shown that sodium butyrate plays a tumor-suppressing role in the progression of various tumors, however, the mechanism of NaBu in endometrial cancer is unclear. Cell viability, clone formation, proliferation, migration, invasion abilities and cell cycle distribution were assessed by CCK8 assay, Clone formation ability assay, EdU incorporation, Transwell chambers and flow cytometry. The level of ferroptosis was assayed by the levels of ROS and lipid peroxidation, the ratio of GSH/GSSG and the morphology of mitochondria. Molecular mechanisms were explored by metabolome, transcriptome, RNA-pulldown and mass spectrometry. The in-vivo mechanism was validated using subcutaneous xenograft model. In this study, NaBu was identified to inhibit the progression of endometrial cancer in vitro and in vivo. Mechanistically, RBM3 has a binding relationship with SLC7A11 mRNA. NaBu indirectly downregulates the expression of SLC7A11 by promoting the expression of RBM3, thereby promoting ferroptosis in endometrial cancer cells. In conclusion, Sodium butyrate can promote the expression of RBM3 and indirectly downregulate the expression of SLC7A11 to stimulate ferroptosis, which may be a promising cancer treatment strategy.

RBM3 is associated with acute lung injury in septic mice and patients via the NF-κB/NLRP3 pathway.

Sepsis refers to host response disorders caused by infection, leading to life-threatening organ dysfunction. RNA-binding motif protein 3 (RBM3) is an important cold-shock protein that is upregulated in response to mild hypothermia or hypoxia. In this study, we aimed to investigate whether RBM3 is involved in sepsis-associated acute lung injury (ALI). Intraperitoneal injection of LPS (10 mg/kg) was performed in wild type (WT) and RBM3 knockout (KO, RBM3-/-) mice to establish an in vivo sepsis model. An NLRP3 inflammasome inhibitor, MCC950 (50 mg/kg), was injected intraperitoneally 30 min before LPS treatment. Serum, lung tissues, and BALF were collected 24 h later for further analysis. In addition, we also collected serum from sepsis patients and healthy volunteers to detect their RBM3 expression. The results showed that the expression of RBM3 in the lung tissues of LPS-induced sepsis mice and the serum of patients with sepsis was significantly increased and positively correlated with disease severity. In addition, RBM3 knockout (KO) mice had a low survival rate, and RBM3 KO mice had more severe lung damage, inflammation, lung cell apoptosis, and oxidative stress than WT mice. LPS treatment significantly increased the levels of nucleotide binding and oligomerization domain-like receptor family 3 (NLRP3) inflammasomes and mononuclear cell nuclear factor-κB (NF-κB) in the lung tissues of RBM3 KO mice. However, these levels were only slightly elevated in WT mice. Interestingly, MCC950 improved LPS-induced acute lung injury in WT and RBM3 KO mice but inhibited the expression of NLRP3, caspase-1, and IL-1ß. In conclusion, RBM3 was overexpressed in sepsis patients and LPS-induced mice. RBM3 gene deficiency aggravated sepsis-associated ALI through the NF-κB/NLRP3 pathway.

Study on the protective effect of RNA-binding motif protein 3 in mild hypothermia oxygen-glucose deprivation/reoxygenation cell model.

Mild hypothermia is proven neuroprotective in clinical practice. While hypothermia leads to the decrease of global protein synthesis rate, it upregulates a small subset of protein including RNA-binding motif protein 3 (RBM3). In this study, we treated mouse neuroblastoma cells (N2a) with mild hypothermia before oxygen-glucose deprivation/reoxygenation (OGD/R) and discovered the decrease of apoptosis rate, down-regulation of apoptosis-associated protein and enhancement of cell viability. Overexpression of RBM3 via plasmid exerted similar effect while silencing RBM3 by siRNAs partially reversed the protective effect exerted by mild hypothermia pretreatment. The protein level of Reticulon 3(RTN3), a downstream gene of RBM3, also increased after mild hypothermia pretreatment. Silencing RTN3 weakened the protective effect of mild hypothermia pretreatment or RBM3 overexpression. Also, the protein level of autophagy gene LC3B increased after OGD/R or RBM3 overexpression while silencing RTN3 decreased this trend. Furthermore, immunofluorescence observed enhanced fluorescence signal of LC3B and RTN3 as well as a large number of overlaps after RBM3 overexpressing. In conclusion, RBM3 plays a cellular protective role by regulating apoptosis and viability via its downstream gene RTN3 in the hypothermia OGD/R cell model and autophagy may participate in it.

The mRNA-Binding Protein RBM3 Regulates Activity Patterns and Local Synaptic Translation in Cultured Hippocampal Neurons.

The activity and the metabolism of the brain change rhythmically during the day/night cycle. Such rhythmicity is also observed in cultured neurons from the suprachiasmatic nucleus, which is a critical center in rhythm maintenance. However, this issue has not been extensively studied in cultures from areas less involved in timekeeping, as the hippocampus. Using neurons cultured from the hippocampi of newborn rats (both male and female), we observed significant time-dependent changes in global activity, in synaptic vesicle dynamics, in synapse size, and in synaptic mRNA amounts. A transcriptome analysis of the neurons, performed at different times over 24 h, revealed significant changes only for RNA-binding motif 3 (Rbm3). RBM3 amounts changed, especially in synapses. RBM3 knockdown altered synaptic vesicle dynamics and changed the neuronal activity patterns. This procedure also altered local translation in synapses, albeit it left the global cellular translation unaffected. We conclude that hippocampal cultured neurons can exhibit strong changes in their activity levels over 24 h, in an RBM3-dependent fashion.SIGNIFICANCE STATEMENT This work is important in several ways. First, the discovery of relatively regular activity patterns in hippocampal cultures implies that future studies using this common model will need to take the time parameter into account, to avoid misinterpretation. Second, our work links these changes in activity strongly to RBM3, in a fashion that is independent of the canonical clock mechanisms, which is a very surprising observation. Third, we describe here probably the first molecule (RBM3) whose manipulation affects translation specifically in synapses, and not at the whole-cell level. This is a key finding for the rapidly growing field of local synaptic translation.

RBM3 is an outstanding cold shock protein with multiple physiological functions beyond hypothermia.

RNA-binding motif protein 3 (RBM3), an outstanding cold shock protein, is rapidly upregulated to ensure homeostasis and survival in a cold environment, which is an important physiological mechanism in response to cold stress. Meanwhile, RBM3 has multiple physiological functions and participates in the regulation of various cellular physiological processes, such as antiapoptosis, circadian rhythm, cell cycle, reproduction, and tumogenesis. The structure, conservation, and tissue distribution of RBM3 in human are demonstrated in this review. Herein, the multiple physiological functions of RBM3 were summarized based on recent research advances. Meanwhile, the cytoprotective mechanism of RBM3 during stress under various adverse conditions and its regulation of transcription were discussed. In addition, the neuroprotection of RBM3 and its oncogenic role and controversy in various cancers were investigated in our review.

Mild hypothermia ameliorates hepatic ischemia reperfusion injury by inducing RBM3 expression.

Liver ischemia reperfusion injury (IRI) is a serious complication of certain liver surgeries, and it is difficult to prevent. As a potential drug-free treatment, mild hypothermia has been shown to promote positive outcomes in patients with IRI. However, the protective mechanism remains unclear. We established in vivo and in vitro models of hepatic ischemia reperfusion (IR) and mild hypothermia pretreatment. Hepatocytes were transfected with RNA-binding motif protein 3 (RBM3) overexpression plasmids, and IR was performed. Cell, culture medium, blood and tissue samples were collected to assess hepatic injury, oxidative stress, apoptosis and changes in RBM3 expression in the liver. Upregulation of RBM3 expression by mild hypothermia reduced the aminotransferase release, liver tissue injury and mitochondrial injury induced by liver IR. Hepatic IR-induced p38 and c-Jun N-terminal kinase (JNK) signaling pathway activation, oxidative stress injury and apoptosis could be greatly reversed by mild hypothermia. Overexpression of RBM3 mimicked the hepatoprotective effect of mild hypothermia. Mild hypothermia protects the liver from ischemia reperfusion-induced p38 and JNK signaling pathway activation, oxidative stress injury and apoptosis through the upregulation of RBM3 expression.

Pre-clinical and clinical studies on the role of RBM3 in muscle-invasive bladder cancer: longitudinal expression, transcriptome-level effects and modulation of chemosensitivity.

BACKGROUND: The response to neoadjuvant cisplatin-based chemotherapy (NAC) in muscle-invasive bladder cancer (MIBC) is impaired in up to 50% of patients due to chemoresistance, with no predictive biomarkers in clinical use. The proto-oncogene RNA-binding motif protein 3 (RBM3) has emerged as a putative modulator of chemotherapy response in several solid tumours but has a hitherto unrecognized role in MIBC. METHODS: RBM3 protein expression level in tumour cells was assessed via immunohistochemistry in paired transurethral resection of the bladder (TURB) specimens, cystectomy specimens and lymph node metastases from a consecutive cohort of 145 patients, 65 of whom were treated with NAC. Kaplan-Meier and Cox regression analyses were applied to estimate the impact of RBM3 expression on time to recurrence (TTR), cancer-specific survival (CSS), and overall survival (OS) in strata according to NAC treatment. The effect of siRNA-mediated silencing of RBM3 on chemosensitivity was examined in RT4 and T24 human bladder carcinoma cells in vitro. Cellular functions of RBM3 were assessed using RNA-sequencing and gene ontology analysis, followed by investigation of cell cycle distribution using flow cytometry. RESULTS: RBM3 protein expression was significantly higher in TURB compared to cystectomy specimens but showed consistency between primary tumours and lymph node metastases. Patients with high-tumour specific RBM3 expression treated with NAC had a significantly reduced risk of recurrence and a prolonged CSS and OS compared to NAC-untreated patients. In high-grade T24 carcinoma cells, which expressed higher RBM3 mRNA levels compared to RT4 cells, RBM3 silencing conferred a decreased sensitivity to cisplatin and gemcitabine. Transcriptomic analysis revealed potential involvement of RBM3 in facilitating cell cycle progression, in particular G1/S-phase transition, and initiation of DNA replication. Furthermore, siRBM3-transfected T24 cells displayed an accumulation of cells residing in the G1-phase as well as altered levels of recognised regulators of G1-phase progression, including Cyclin D1/CDK4 and CDK2. CONCLUSIONS: The presented data highlight the potential value of RBM3 as a predictive biomarker of chemotherapy response in MIBC, which could, if prospectively validated, improve treatment stratification of patients with this aggressive disease.

Time-resolved proteome and transcriptome of paraquat-induced pulmonary fibrosis.

BACKGROUNDS: Pulmonary fibrosis (PF) is a pathological state presenting at the progressive stage of heterogeneous interstitial lung disease (ILD). The current understanding of the molecular mechanisms involved is incomplete. This clinical toxicology study focused on the pulmonary fibrosis induced by paraquat (PQ), a widely-used herbicide. Using proteo-transcriptome analysis, we identified differentially expressed proteins (DEPs) derived from the initial development of fibrosis to the dissolved stage and provided further functional analysis. METHODS: We established a mouse model of progressive lung fibrosis via intratracheal instillation of paraquat. To acquire a comprehensive and unbiased understanding of the onset of pulmonary fibrosis, we performed time-series proteomics profiling (iTRAQ) and RNA sequencing (RNA-Seq) on lung samples from paraquat-treated mice and saline control. The biological functions and pathways involved were evaluated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Correlation tests were conducted on comparable groups 7 days and 28 days post-exposure. Differentially expressed proteins and genes following the same trend on the protein and mRNA levels were selected for validation. The functions of the selected molecules were identified in vitro. The protein level was overexpressed by transfecting gene-containing plasmid or suppressed by transfecting specific siRNA in A549 cells. The levels of endothlial-mesenchymal transition (EMT) markers, including E-cadherin, vimentin, FN1, and α-SMA, were determined via western blot to evaluate the fibrotic process. RESULTS: We quantified 1358 DEPs on day 7 and 426 DEPs on day 28 post exposure (Fold change >1.2; Q value < 0.05). The top 5 pathways - drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, protein digestion and absorption - were involved on both day 7 and day 28. Several pathways, including tight junction, focal adhesion, platelet activation, and ECM-receptor interaction, were more enriched on day 28 than on day 7. Integrative analysis of the proteome and transcriptome revealed a moderate correlation of quantitative protein abundance ratios with RNA abundance ratios (Spearman R = 0.3950 and 0.2477 on days 7 and 28, respectively), indicating that post-transcriptional regulation plays an important role in lung injury and repair. Western blot identified that the protein expressions of FN1, S100A4, and RBM3 were significantly upregulated while that of CYP1A1, FMO3, and PGDH were significantly downregulated on day 7. All proteins generally recovered to baseline on day 28. qPCR showed the mRNA levels of Fn1, S100a4, Rbm3, Cyp1a1, Fmo3, and Hpgd changed following the same trend as the levels of their respective proteins. Further, in vitro experiments showed that RBM3 was upregulated while PGDH was downregulated in an EMT model established in human lung epithelial A549 cells. RBM3 overexpression and PGDH knockout could both induce EMT in A549 cells. RBM3 knockout or PGDH overexpression had no reverse effect on EMT in A549 cells. CONCLUSIONS: Our proteo-transcriptomic study determined the proteins responsible for fibrogenesis and uncovers their dynamic regulation from lung injury to repair, providing new insights for the development of biomarkers for diagnosis and treatment of fibrotic diseases.

Amyloid pathology and synaptic loss in pathological aging.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory dysfunction and cognitive decline. Pathological aging (PA) describes patients who are amyloid-positive but cognitively unimpaired at time of death. Both AD and PA contain amyloid plaques dominated by amyloid ß (Aß) peptides. In this study, we investigated and compared synaptic protein levels, amyloid plaque load, and Aß peptide patterns between AD and PA. Two cohorts of post-mortem brain tissue were investigated. In the first, consisting of controls, PA, AD, and familial AD (FAD) individuals, synaptic proteins extracted with tris(hydroxymethyl)aminomethane-buffered saline (TBS) were analyzed. In the second, consisting of tissue from AD and PA patients from three different regions (occipital lobe, frontal lobe, and cerebellum), a two-step extraction was performed. Five synaptic proteins were extracted using TBS, and from the remaining portion Aß peptides were extracted using formic acid. Subsequently, immunoprecipitation with several antibodies targeting different proteins/peptides was performed for both fractions, which were subsequently analyzed by mass spectrometry. The levels of synaptic proteins were lower in AD (and FAD) compared with PA (and controls), confirming synaptic loss in AD patients. The amyloid plaque load was increased in AD compared with PA, and the relative amount of Aß40 was higher in AD while for Aß42 it was higher in PA. In AD loss of synaptic function was associated with increased plaque load and increased amounts of Aß40 compared with PA cases, suggesting that synaptic function is preserved in PA cases even in the presence of Aß.

Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

RBM3 Increases Cell Survival but Disrupts Tight Junction of Microvascular Endothelial Cells in Acute Lung Injury.

BACKGROUND: RNA-binding motif protein 3 (RBM3) is an important cold shock protein, which also responds to hypothermia or hypoxia. RBM3 is involved into multiple physiologic processes, such as promoting cell survival. However, its expression and function in acute lung injury (ALI) have not been reported. METHODS: A mouse ALI model was established by lipopolysaccharides (LPS) treatment. The RBM3 and cold inducible RNA-binding protein mRNA levels were examined by RT-qPCR, and MMP9 mRNA stability was determined by actinomycin D assay. RBM3 and MMP9 mRNA was tested by RNA immunoprecipitation (RIP assay). RBM3 overexpression or silent stable cell lines were established using recombinant lentivirus and subsequently used for cell survival and tight junction measurements. RESULTS: In this study, we found that RBM3, rather than cold inducible RNA-binding protein, was upregulated in lung tissue of ALI mice. RBM3 was increased in human pulmonary microvascular endothelial cells (HPMVECs) in response to LPS treatment, which is modulated by the NF-κB signaling pathway. Furthermore, RBM3 could reduce cell apoptosis induced by LPS, probably through suppressing p53 expression. Because increased permeability of HPMVECs leads to pulmonary edema in ALI, we subsequently examined the effect of RBM3 on cell tight junctions. Unexpectedly, RBM3 decreased the expression of tight junction protein zonula occludens-1 and increased cell permeability, and RBM3 overexpression increased MMP9 mRNA stability. Furthermore, RIP assay confirmed the interaction between RBM3 and MMP9 mRNA, possibly explaining the contribution of RBM3 to increase cell permeability. CONCLUSIONS: RBM3 seems to act as a "double-edged sword" in ALI, that RBM3 alleviates cell apoptosis but increases HPMVEC permeability in ALI.

RNA-binding proteins RBM-HuR, RBM3 and PODXL expression in urothelial carcinoma of the urinary bladder. Prognostic and clinical implications.

AIM OF THE STUDY: The clinical significance and predictive and prognostic value of HuR, RBM3, and PODXL expression in patients with urothelial bladder cancer (UBC) are not clear yet. The aim of this study was to assess HuR, RBM3 and PODXL expression in muscle invasive and non-muscle invasive UBC tissues, and to investigate the clinicopathological correlations and their predictive and prognostic impact in patients with such type of cancer. MATERIAL AND METHODS: RBM-HuR, RBM3 and PODXL expression levels were evaluated in 70 patients with urothelial carcinoma by immunohistochemistry. The relationships between their expression, clinicopathological findings and prognostic data were analyzed. RESULTS: High RBM-HuR expression was related to muscle invasion (p = 0.008), metastasis to lymph nodes (p = 0.007), and presence of blood spread (p = 0.049). High RBM3 expression was associated with lower grade (p = 0.044), absence of distant metastasis (p = 0.025), and absence of lymph node metastasis (p = 0.018). High PODXL expression was significantly associated with advanced tumor stage (p < 0.001), larger tumor size (p = 0.050), lymphovascular invasion (p = 0.006), lymph node metastasis (p = 0.008), higher grade (p = 0.043) and distant metastasis (p = 0.002).Three-year overall survival rate was negatively associated with high expression of both RBM-HuR and PODXL while it was directly correlated with high expression of RBM3 (p = 0.008, 0.009 and 0.015 respectively). High RBM-HuR and PODXL expression and low expression of RBM3 were related to tumor recurrence (p = 0.022, 0.011 and 0.015). CONCLUSIONS: RBM-HuR and PODXL expressions are markers of poor prognosis while RBM3 is a good prognostic marker for urothelial carcinoma of the bladder.

Role of RBM3 in the regulation of cell proliferation in hepatocellular carcinoma.

RNA binding motif protein 3 (RBM3) has been shown to be upregulated in several types of human tumors. Using tissue microarrays and immunohistochemistry, we showed here that both nuclear and cytoplasmic RBM3 expression levels were higher in hepatocellular carcinoma (HCC) tissues than in adjacent non-tumorous tissues. High nuclear RBM3 was found to be correlated with larger tumor size (P = 0.030), high serum AFP levels (P = 0.011), and advanced Edmonson grading (P = 0.006). Cytoplasmic RBM3 was associated with advanced Edmonson grading (P = 0.003). Kaplan-Meier survival analysis revealed that, although not statistically significant, there was a trend toward shortened overall survival in the subset of HCC patients with high RBM3 expression (both nuclear and cytoplasmic). In addition, we found that RBM3 could promote YAP1 expression in HCC cells. Moreover, we found that YAP1 played an essential part in RBM3-induced proliferation of HCC cells. Furthermore, we demonstrated that Verteporfin, a YAP1 inhibitor, could repress RBM3-induced proliferation of HCC cells. Our findings provide a new experimental basis for further understanding of the possible role of RBM3-YAP1 in the regulation of HCC proliferation.

Cold-inducible protein RBM3 mediates hypothermic neuroprotection against neurotoxin rotenone via inhibition on MAPK signalling.

Mild hypothermia and its key product, cold-inducible protein RBM3, possess robust neuroprotective effects against various neurotoxins. However, we previously showed that mild hypothermia fails to attenuate the neurotoxicity from MPP+ , one of typical neurotoxins related to the increasing risk of Parkinson disease (PD). To better understand the role of mild hypothermia and RBM3 in PD progression, another known PD-related neurotoxin, rotenone (ROT) was utilized in this study. Using immunoblotting, cell viability assays and TUNEL staining, we revealed that mild hypothermia (32°C) significantly reduced the apoptosis induced by ROT in human neuroblastoma SH-SY5Y cells, when compared to normothermia (37°C). Meanwhile, the overexpression of RBM3 in SH-SY5Y cells mimicked the neuroprotective effects of mild hypothermia on ROT-induced cytotoxicity. Upon ROT stimulation, MAPK signalling like p38, JNK and ERK, and AMPK and GSK-3ß signalling were activated. When RBM3 was overexpressed, only the activation of p38, JNK and ERK signalling was inhibited, leaving AMPK and GSK-3ß signalling unaffected. Similarly, mild hypothermia also inhibited the activation of MAPKs induced by ROT. Lastly, it was demonstrated that the MAPK (especially p38 and ERK) inhibition by their individual inhibitors significantly decreased the neurotoxicity of ROT in SH-SY5Y cells. In conclusion, these data demonstrate that RBM3 mediates mild hypothermia-related neuroprotection against ROT by inhibiting the MAPK signalling of p38, JNK and ERK.

Regulating glycolysis, the TLR4 signal pathway and expression of RBM3 in mouse liver in response to acute cold exposure.

At low temperatures, the liver increases glucose utilization and expresses RNA-binding motif 3 (RBM3) to cope with cold exposure. In this study, the expression of heat shock protein 70 (HSP70), Toll-like receptor 4 (TLR4), bone marrow differentiation factor 88 (MYD88), and phosphorylated nuclear factor-κB (NF-κB) was consistent with fluctuations in insulin in fasted cold-exposed mice. We also found up-regulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in acute cold exposure with a decrease in core body temperature. RBM3 transcription and translation were activated 2 h after cold exposure. The anti-apoptotic factor Bcl-2/Bax ratio also increased, while expression of apoptosis factors: cleaved caspase-3, cleaved poly(ADP-ribose)polymerase 1 (PARP-1) and cytochrome-c (Cyt-c) was unchanged. Liver glycogen was depleted after 2 h of cold exposure, and blood glucose decreased after 4 h. Glycogen synthase kinase 3ß (GSK3ß) phosphorylation continued to increase to promote hepatic glycogen synthesis. We found a high level of protein kinase B (AKT) phosphorylation after 6 h of cold exposure. In addition, we demonstrated that after cold exposure for 2 h, in the liver, continued phosphorylation of fructose-2,6-diphosphate (PFKFB2) and decreased accumulation of glycogen intermediates fructose-1,6-diphosphate (FDP) and pyruvic acid (PA). In summary, the liver responds to cold exposure through a number of different pathways, including activation of HSP70/TLR4 signaling pathways, up-regulation of RBM3 expression, and increased glycolysis and glycogen synthesis. We propose a possible signaling pathway in which regulation of RBM3 expression by the liver affects the AKT metabolic signaling pathway. Lay summary In response to changes in ambient temperature, mice regulate global metabolism and gene expression through hormones. This study focused on the effects of environmental hypothermia on molecular pathways of glucose metabolism in the liver, which is the important metabolic organ in mice. This provides a basis for further study of mice against cold exposure damage.

Mild hypothermia promotes the viability of in vitro-produced bovine blastocysts and their transcriptional expression of the cold-inducible transcription factor Rbm3 during in vitro culture.

In this study, we evaluated the effects of holding in vitro-produced bovine blastocysts under mild hypothermia (33°C or 35°C), by examining viability and hatching rates of day 7 blastocysts (day 0: in vitro fertilization) cultured for 6 days and transcriptional expression of cold-inducible transcription factors Cirp and Rbm3, implicated in mild hypothermia-induced cellular protection against various types of stress. In the normothermic control (38.5°C), viability of the embryos decreased rapidly after day 10, and most samples were degenerated on day 13. However, mild hypothermia, particularly at 33°C, resulted in maintenance of high embryonic survival rates until day 13 (77.1% on day 13) and significant increases in transcriptional expression of Rbm3 in day 11 embryos compared with those at 38.5°C. Thus, our results suggested that upregulation of Rbm3 may occur in response to mild hypothermia in many bovine embryos, providing insights into the effects of mild hypothermia on embryo quality.

RBM3 expression is upregulated by NF-κB p65 activity, protecting cells from apoptosis, during mild hypothermia.

The RNA-binding protein RBM3, a cold shock protein whose expression is elevated under hypothermic conditions, plays an important role in cell survival; however, little is known about the mechanism underlying the mild hypothermia-mediated regulation of RBM3 expression and apoptosis. Here we show that the transcription factor NF-κB p65 is phosphorylated at Ser276 and activates RBM3 gene transcription via binding to a particular element within the promoter region in response to induced hypothermia, elevating the protein expression, and suppressing apoptosis. Treatment with caffeic acid phenethyl ester (CAPE), a potent and specific inhibitor that suppresses the translocation of NF-κB p65 from the cytoplasm to the nucleus, resulted in decreased levels of RBM3 mRNA and protein and increased incidence of apoptosis despite cells were cultured under hypothermic conditions. Overexpression of RBM3 abolished the induction of apoptosis in cells treated with CAPE, indicating that NF-κB p65-upregulated RBM3 expression is necessary for the suppression of apoptosis. In addition, experiments with cells overexpressing RBM3 supported the finding demonstrating that the mild hypothermia-mediated higher expression of RBM3 suppressed the induction of apoptosis. Conversely, experiments with cells deficient in RBM3 supported the finding demonstrating that the CAPE-mediated loss of RBM3 induced apoptosis. These results suggest that NF-κB p65 is a critical mediator of mild hypothermia, to which cells are exposed as an extracellular environment, and a central inducer of RBM3 expression, which is responsible for preventing cells from apoptosis. Moreover, CAPE may have a potential for the application to a therapeutic agent for the treatment of cancers.

Hypothermia-induced RNA-binding motif protein 3 (RBM3) stimulates osteoblast differentiation via the ERK signaling pathway.

The RNA-binding motif protein 3 (RBM3) belongs to a small group of proteins whose synthesis increases during hypothermia while global protein production is slowed down. Bone homeostasis is maintained by a balance between bone resorption and bone formation. Osteoblasts are key components of the bone and have an important role in bone remodeling cycle. However, hypothermia-induced RBM3 between osteoblasts remains unclear. At 32°C, expression of RBM3 and Runx2 was increased in a time-dependent manner and mineralization was also increased. RBM3 was also increased in a time-dependent manner under osteogenic conditions. Overexpression of RBM3 increased the expression of osteogenic genes such as Runx2 and OC. The osteogenic condition-induced expressions of RBM3, Runx2 and OC gene were decreased by RBM3 siRNA. Moreover, RBM3 promoted ERK and p38 phosphorylation. The inhibitor of ERK decreased the expression of Runx2 but did not affect the expression of RBM3. Taken together, these results demonstrate that RBM3 stimulates osteoblast differentiation via the ERK signaling pathway.