Cold-inducible RNA-binding Protein mRNA Research Articles

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

BackgroundRNA-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. MethodsA 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. ResultsIn 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. ConclusionsRBM3 seems to act as a "double-edged sword" in ALI, that RBM3 alleviates cell apoptosis but increases HPMVEC permeability in ALI.

Cold-inducible RNA binding protein promotes breast cancer cell malignancy by regulating Cystatin C levels.

Cold-inducible RNA binding protein (CIRBP) is a stress-responsive protein that promotes cancer development and inflammation. Critical to most CIRBP functions is its capacity to bind and posttranscriptionally modulate mRNA. However, a transcriptome-wide analysis of CIRBP mRNA targets in cancer has not yet been performed. Here, we use an ex vivo breast cancer model to identify CIRBP targets and mechanisms. We find that CIRBP transcript levels correlate with breast cancer subtype and are an indicator of luminal A/B prognosis. Accordingly, overexpression of CIRBP in nontumoral MCF-10A cells promotes cell growth and clonogenicity, while depletion of CIRBP from luminal A MCF-7 cells has opposite effects. We use RNA immunoprecipitation followed by high-throughput sequencing (RIP-seq) to identify a set of 204 high confident CIRBP targets in MCF-7 cells. About 10% of these showed complementary changes after CIRBP manipulation in MCF-10A and MCF-7 cells, and were highly interconnected with known breast cancer genes. To test the potential of CIRBP-mediated regulation of these targets in breast cancer development, we focused on Cystatin C (CST3), one of the most highly interconnected genes, encoding a protein that displays tumor suppressive capacities. CST3 depletion restored the effects of CIRBP depletion in MCF-7 cells, indicating that CIRBP functions, at least in part, by down-regulating CST3 levels. Our data provide a resource of CIRBP targets in breast cancer, and identify CST3 as a novel downstream mediator of CIRBP function.

The Expression of Cold-Inducible RNA-Binding Protein mRNA in Sow Genital Tract Is Modulated by Natural Mating, But Not by Seminal Plasma.

The RNA-binding proteins (RBPs), some of them induced by transient receptor potential (TRP) ion channels, are crucial regulators of RNA function that can contribute to reproductive pathogenesis, including inflammation and immune dysfunction. This study aimed to reveal the influence of spermatozoa, seminal plasma, or natural mating on mRNA expression of RBPs and TRP ion channels in different segments of the internal genital tract of oestrous, preovulatory sows. Particularly, we focused on mRNA expression changes of the cold-inducible proteins (CIPs) and related TRP channels. Pre-ovulatory sows were naturally mated (NM) or cervically infused with semen (Semen-AI) or sperm-free seminal plasma either from the entire ejaculate (SP-TOTAL) or the sperm-rich fraction (SP-AI). Samples (cervix to infundibulum) were collected by laparotomy under general anaesthesia for transcriptomic analysis (GeneChip® Porcine Gene 1.0 ST Array) 24 h after treatments. The NM treatment induced most of the mRNA expression changes, compared to Semen-AI, SP-AI, and SP-TOTAL treatments including unique significative changes in CIRBP, RBM11, RBM15B, RBMS1, TRPC1, TRPC4, TRPC7, and TRPM8. The findings on the differential mRNA expression on RBPs and TRP ion channels, especially to CIPs and related TRP ion channels, suggest that spermatozoa and seminal plasma differentially modulated both protein families during the preovulatory phase, probably related to a still unknown early signalling mechanism in the sow reproductive tract.

Luteolin Suppresses Sepsis-Induced Cold-Inducible RNA-Binding Protein Production and Lung Injury in Neonatal Mice.

Neonatal sepsis is a life-threatening inflammatory condition. Extracellular cold-inducible RNA-binding protein (CIRP), a proinflammatory mediator, plays a critical role in the pathogenesis of sepsis-induced lung injury in neonates. Luteolin, a polyphenolic flavonoid, has potent anti-inflammatory properties. However, the effects of luteolin on CIRP production and neonatal sepsis-induced lung injury remained unknown. We therefore hypothesize that treatment with luteolin suppresses CIRP production and attenuates lung injury in neonatal sepsis. To study this, sepsis was induced in C57BL/6J mouse pups (5-7 days) by intraperitoneal cecal slurry injection (CSI). One hour after CSI, luteolin (10 mg/kg body weight) or vehicle (normal saline) was administered through intraperitoneal injection. CIRP mRNA and protein were determined and lung injury was assessed at 10 h after CSI. Our results showed that administration of luteolin decreased CIRP mRNA and protein, improved lung architecture, reduced lung edema, and apoptosis after CSI. To examine the direct effect of luteolin on CIRP production, peritoneal macrophages were isolated from neonatal mice and stimulated with 100 ng/mL LPS with or without the presence of luteolin. The result indicates that luteolin directly inhibited LPS-induced CIRP production in neonatal macrophages. In addition, luteolin also downregulated hypoxia-inducible factor-1α (HIF-1α) and NOD-like receptor 3 (NLRP3) expression in septic neonates and in LPS-stimulated neonatal macrophages. In conclusion, administration of luteolin suppresses CIRP production and attenuates lung injury in neonatal sepsis. The beneficial effect of luteolin may be related to downregulation of HIF-1α and NLRP3 expression in neonatal macrophages. Luteolin may be developed as an adjunctive therapy for neonatal sepsis.

Cold-inducible RNA-binding protein contributes to intracerebral hemorrhage-induced brain injury via TLR4 signaling.

IntroductionExcessive neuroinflammation aggravates the brain injury caused by intracerebral hemorrhage (ICH), while the upstream mechanisms that initiate neuroinflammation remain unclear. Toll‐like receptor 4 (TLR4) signaling is important to trigger inflammatory responses in ICH, and cold‐inducible RNA‐binding protein (CIRP) has been shown as a novel ligand of TLR4 by recent studies. However, whether the CIRP could trigger the neuroinflammation via activating TLR4 signaling in ICH still needs to be investigated.MethodsHuman serum CIRP levels were measured using the ELISA kits. Western blot, FJB staining, brain water content, and neurological deficit scores were used to investigate the roles of CIRP in brain injury caused by ICH.ResultFirst, we found increased CIRP levels in the blood of patients with ICH when compared to the control individuals, and the ICH patients with mRS > 2 have higher serum CIRP levels in contrast to those with mRS ≤ 2. In the ICH mice, we also found that brain CIRP protein and mRNA levels were also increased after ICH. Furthermore, using the CIRP−/− mice, we found that CIRP−/− mice had less brain damages showing in less FJB+ cells, reduced brain water content (BWC) and lower neurological deficit scores (NDS) compared to that in WT mice after ICH. Cytokines including IL‐6, TNF‐α, and IL‐1β from CIRP−/− mice were attenuated after ICH. CIRP−/− mice also exhibited reduced TLR4 expression which was accompanied by the decreased activity of NF‐κB. This suggests that TLR4 signaling might be involved in CIRP‐mediated inflammatory injury possibly via NF‐κB activation after ICH.ConclusionOur findings suggest that CIRP may activate TLR4 signaling, and further inducing NF‐κB activation to increase the expression levels of cytokines and aggravate inflammatory injury in ICH. Targeting CIRP may be a promising strategy for ICH treatment.

RBM3 and CIRP expressions in targeted temperature management treated cardiac arrest patients-A prospective single center study.

BackgroundManagement of cardiac arrest patients includes active body temperature control and strict prevention of fever to avoid further neurological damage. Cold-shock proteins RNA-binding motif 3 (RBM3) and cold inducible RNA-binding protein (CIRP) expressions are induced in vitro in response to hypothermia and play a key role in hypothermia-induced neuroprotection.ObjectiveTo measure gene expressions of RBM3, CIRP, and inflammatory biomarkers in whole blood samples from targeted temperature management (TTM)-treated post-cardiac arrest patients for the potential application as clinical biomarkers for the efficacy of TTM treatment.MethodsA prospective single center trial with the inclusion of 22 cardiac arrest patients who were treated with TTM (33°C for 24 hours) after ROSC was performed. RBM3, CIRP, interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and inducible nitric oxide synthase (iNOS) mRNA expressions were quantified by RT-qPCR. Serum RBM3 protein concentration was quantified using an enzyme-linked immunosorbent assay (ELISA).ResultsRBM3 mRNA expression was significantly induced in post-cardiac arrest patients in response to TTM. RBM3 mRNA was increased 2.2-fold compared to before TTM. A similar expression kinetic of 1.4-fold increase was observed for CIRP mRNA, but did not reached significancy. Serum RBM3 protein was not increased in response to TTM. IL-6 and MCP-1 expression peaked after ROSC and then significantly decreased. iNOS expression was significantly increased 24h after return of spontaneous circulation (ROSC) and TTM.ConclusionsRBM3 is temperature regulated in patients treated with TTM after CA and ROSC. RBM3 is a possible biomarker candidate to ensure the efficacy of TTM treatment in post-cardiac arrest patients and its pharmacological induction could be a potential future intervention strategy that warrants further research.

Hypothermia induces changes in the alternative splicing pattern of cold-inducible RNA-binding protein transcripts in a non-hibernator, the mouse.

Cold-inducible RNA-binding protein (CIRBP) plays important roles in protection against harmful effects of cold temperature. We previously found that several splicing variants of CIRBP mRNA are constitutively expressed in the heart of non-hibernating euthermic hamsters and that one of the variants is predominantly expressed with remarkable reduction in the expression of other variants in hibernating hypothermic hamsters. The aim of this study was to determine whether the regulation of alternative splicing is a common function in a non-hibernator, the mouse. The expression of CIRBP mRNA was assessed by RT-PCR. In euthermic control mice, several splicing variants of CIRBP mRNA were detected in various organs. When hypothermia was induced in mice by using isoflurane anesthesia, the short form variant, which encodes full-length functional CIRBP, was predominantly detected. Keeping body temperature of anesthetized mice at 37°C prevented changes in the splicing pattern. Exposure of mice to a low temperature did not change the splicing pattern, suggesting that endogenous neuronal and/or humoral pathways activated in response to cold stimuli applied to the body surface play minor roles. In agreement with this, the shift in alternative splicing was reproduced in isolated leukocytes in vitro when they were incubated at 28°C. Since application of a TRPM8 or TRPA1 agonist at 37°C failed to promote the shift in the splicing pattern, it seems likely that cold-sensitive channels are not involved in the splicing regulation. Therefore, it is probable that a substantial reduction of temperature is a major cause of the regulation of alternative splicing of CIRBP transcripts. The regulatory system of CIRBP expression at the level of alternative splicing, which was originally discovered in the hibernating hamster, commonly exists in non-hibernators such as mice.

Mild hypothermia causes a shift in the alternative splicing of cold-inducible RNA-binding protein transcripts in Syrian hamsters

Cold-shock proteins are thought to participate in the cold-tolerant nature of hibernating animals. We previously demonstrated that an alternative splicing may allow rapid induction of functional cold-inducible RNA-binding protein (CIRBP) in the hamster heart. The purpose of the present study was to determine the major cause of the alternative splicing in Syrian hamsters. RT-PCR analysis revealed that CIRBP mRNA is constitutively expressed in the heart, brain, lung, liver, and kidney of nonhibernating euthermic hamsters with several alternative splicing variants. In contrast, the short variant containing an open-reading frame for functional CIRBP was dominantly found in the hibernating animals. Keeping the animals in a cold and dark environment did not cause a shift in the alternative splicing. Induction of hypothermia by central administration of an adenosine A1-receptor agonist reproduced the shift in the splicing pattern. However, the agonist failed to shift the pattern when body temperature was kept at 37°C, suggesting that central adenosine A1 receptors are not directly linked to the shift of the alternative splicing. Rapid reduction of body temperature to 10°C by isoflurane anesthesia combined with cooling did not alter the splicing pattern, but maintenance of mild hypothermia (~28°C) for 2 h elicited the shift in the pattern. The results suggest that animals need to be maintained at mild hypothermia for an adequate duration to induce the shift in the alternative splicing. This is applicable to natural hibernation because hamsters entering hibernation show a gradual decrease in body temperature, being maintained at mild hypothermia for several hours.

The regulation of CIRBP by transforming growth factor beta during heat shock-induced testicular injury.

Cold-inducible RNA-binding protein (CIRBP) is associated with cell stress. However, its upstream regulatory factors are still largely unknown. This study investigated whether CIRBP expression was regulated by transforming growth factor beta (TGF-β) during the process of heat-induced testicular damage. Ten male adult ICR mice were allocated to heat treatment (scrotal hyperthermia at 43°C for 30min, n=5) and control group (n=5); CIRBP and TGF-β1, TGF-β2, and TGF-β3 expression levels in the testis in mRNA and protein were analyzed. Then, we conducted invivo and invitro studies to investigate the regulatory effects of TGF-β on CIRBP. In the invivo study, male adult ICR mice were subjected to testicular hyperthermia followed by a local testicular injection of TGF-β antagonist (non-selective TGF-β I/II receptor inhibitor, 5μg or 10μg). In the invitro study, GC2-spd cells were cultured under 43°C for 30min or with different TGF-β isoforms (10ng/mL), and CIRBP expression levels in the testis and GC2-spd cells were analyzed 24 and 48h, respectively, after treatment. As a result, heat treatment significantly downregulated the relative CIRBP mRNA and protein expression (p=0.006 and 0.011), and significantly upregulated TGF-β2 and TGF-β3 expression levels (p=0.022 and 0.04, for mRNA, and p=0.001 for both protein levels). Local testicular injection of 10μg TGF-β antagonist significantly attenuated heat-induced histological damage to the testes and CIRBP downregulation (p=0.038). Furthermore, TGF-β2 and TGF-β3 significantly downregulated CIRBP mRNA and protein expression in GC2-spd cells (all p<0.01), exerting a similar effect to heat treatment. Our invivo and invitro experiments demonstrated that heat-induced CIRBP downregulation in the testes was mediated by the upregulation of TGF-β. Further studies are needed to clarify the molecular mechanisms underlying these processes.

The Mechanism Enabling Hibernation in Mammals.

Some rodents including squirrels and hamsters undergo hibernation. During hibernation, body temperature drops to only a few degrees above ambient temperature. The suppression of whole-body energy expenditure is associated with regulated, but not passive, reduction of cellular metabolism. The heart retains the ability to beat constantly, although body temperature drops to less than 10°C during hibernation. Cardiac myocytes of hibernating mammals are characterized by reduced Ca2+ entry into the cell membrane and a concomitant enhancement of Ca2+ release from and reuptake by the sarcoplasmic reticulum. These adaptive changes would help in preventing excessive Ca2+ entry and its overload and in maintaining the resting levels of intracellular Ca2+. Adaptive changes in gene expression in the heart prior to hibernation may be indispensable for acquiring cold resistance. In addition, protective effects of cold-shock proteins are thought to have an important role. We recently reported the unique expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters. The CIRP mRNA is constitutively expressed in the heart of a nonhibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP, while the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to nonhibernating hamsters, only the short product was found in hibernating animals. Thus, these results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation. We will summarize the current understanding of the cold-resistant property of the heart in hibernating animals.

Cold-inducible RNA-binding protein mediates airway inflammation and mucus hypersecretion through a post-transcriptional regulatory mechanism under cold stress

Acute or chronic cold exposure exacerbates chronic inflammatory airway diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. Cold-inducible RNA-binding protein (CIRP) is a cold-shock protein and is induced by various environmental stressors, such as hypothermia and hypoxia. In this study, we showed that CIRP gene and protein levels were significantly increased in patients with COPD and in rats with chronic airway inflammation compared with healthy subjects. Similarly, inflammatory cytokine production and MUC5AC secretion were up-regulated in rats following cigarette smoke inhalation. Cold temperature-induced CIRP overexpression and translocation were shown to be dependent on arginine methylation in vitro. CIRP overexpression promoted stress granule (SG) assembly. In the cytoplasm, the stability of pro-inflammatory cytokine mRNAs was increased through specific interactions between CIRP and mediator mRNA 3⿲-UTRs; these interactions increased the mRNA translation, resulting in MUC5AC overproduction in response to cold stress. Conversely, CIRP silencing and a methyltransferase inhibitor (adenosine dialdehyde) promoted cytokine mRNA degradation and inhibited the inflammatory response and mucus hypersecretion. These findings indicate that cold temperature can induce an airway inflammatory response and excess mucus production via a CIRP-mediated increase in mRNA stability and protein translation.

Therapeutic Hypothermia Enhances Cold-Inducible RNA-Binding Protein Expression and Inhibits Mitochondrial Apoptosis in a Rat Model of Cardiac Arrest.

Therapeutic hypothermia is well known for its protective effect against brain injury after cardiac arrest, but the exact mechanism remains unclear. Cold-inducible RNA-binding protein (CIRP), a member of cold shock protein, enables mammalian cells to withstand decreased temperature by regulating gene translation. However, the role of CIRP in global cerebral ischemia after therapeutic hypothermia has not been clearly elucidated. In the present study, rats resuscitated from 4min of cardiac arrest were separately treated with therapeutic hypothermia (immediately after return of spontaneous circulation (ROSC); targeted temperature at 33°C) and therapeutic normothermia (targeted temperature at 36.8°C) for 6h. The hippocampus was harvested at 0h (baseline), 6h, 12h, 1day, 3days, and 7days after ROSC. The expression of CIRP messenger RNA (mRNA) was assessed by real-time PCR. CIRP and mitochondrial apoptosis-associated proteins were tested by Western blot. The histological changes and neurological function were respectively evaluated by hematoxylin-eosin staining and neurological deficit score (NDS). Compared with baseline, rats resuscitated from cardiac arrest showed increased expression of CIRP, Bax, Caspase 9, and Caspase 3 and decreased expression of Bcl-2 in hippocampus (P < 0.05). However, therapeutic hypothermia after ROSC alleviated the alterations of Bax, Caspase 9, Caspase 3, and Bcl-2, while further increased the hippocampal expression of CIRP mRNA and protein, when compared with the normothermia rats (P < 0.05). In addition, compared with the therapeutic normothermia rats, histopathological damage in CA1 zone and NDS were respectively decreased and increased in the hypothermia rats (P < 0.05). Our findings suggest that 32°C therapeutic hypothermia exerts an important neuroprotective effects by up-regulating CIRP expression and inhibiting mitochondrial apoptosis factor production in the cardiac arrest rat model.

Effect of hypothermia on expression of cold-inducible RNA-binding protein after cardiopulmonary resuscitation in a rat model of cardiac arrest

Objective To evaluate the effect of hypothermia on the expression of cold-inducible RNA-binding protein (CIRP) after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest. Methods Ninety pathogen-free male Sprague-Dawley rats, weighing 280-320 g, were randomly divided into 3 groups (n=30 each) using a random number table: sham operation group (group Sham), cardiac arrest-CPR group (group CPR), and hypothermia group (group H). Group Sham underwent only cannulation of the artery and vein and tracheal intubation.In group CPR, cardiac arrest was induced by electric stimulation via the esophagus, and then CPR was performed.In group H, the rectal temperature was cooled down to 32-34 ℃ within 15 min by putting rats on the ice bags immediately after recovery of spontaneous circulation (ROSC), and maintained at this level for 6 h, while the rectal temperature was maintained at 36-37 ℃ in the other groups.Neurological deficit score (NDS) was evaluated at 24 and 72 h after ROSC, and the rats were then sacrificed.The hippocampus was removed for examination of the pathological changes of pyramidal cells in the hippocampal CA1 region (using hematoxylin and eosin staining), and for determination of the expression of CIRP mRNA and protein (using fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction or Western blot). Results Compared with group Sham, NDS score was significantly decreased, and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in CPR and H groups (P<0.05). Compared with group CPR, NDS score was significantly increased at 72 h after ROSC, and the expression of CIRP mRNA and protein was up-regulated at 24 and 72 h after ROSC in group H (P<0.05). Conclusion Hypothermia can effectively reduce cerebral ischemia-reperfusion injury and improve neurological function after CPR, and the mechanism is probably related to up-regulation of CIRP expression in a rat model of cardiac arrest. Key words: Hypothermia, induced; Heart arrest, induced; Cardiopulmonary resuscitation; Carrier proteins; Hippocampus

Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury.

Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury.

Changes in expression of cold-inducible RNA binding protein in hippocampus after cardiopulmonary resuscitation in a rat model of cardiac arrest

Objective To evaluate the changes in the expression of cold-inducible RNA binding protein (CIRP) in the hippocampus after cardiopulmonary resuscitation (CPR) in a rat model of cardiac arrest. Methods Eighty-five male Sprague-Dawley rats, weighing 280–320 g, aged 3 months, were randomly divided into 3 groups using a random number table: control (group C, n=15), sham operation group (group S, n=15) and CPR group (n=55). Cardiac arrest was induced through ventricular fibrillation caused by transoesophageal cardiac pacing, and then CPR was performed in group CPR.At 6 h, 12 h, and 1, 3 and 7 days after CPR, and the hippocampi were removed for detection of the expression of CIRP mRNA (using real-time reverse transcriptase polymerase chain reaction) and CIRP protein (using Western blot). The brain tissues were also obtained at 3 days after CPR for detection of CIRP expression in the hippocampal CA1 region by immunohistochemistry. Results Compared with group C, the expression of CIRP mRNA was significantly up-regulated at 1, 3, and 7 days after CPR, and the expression of CIRP was up-regulated at 1 and 3 days after CPR in group CRP (P 0.05). Compared with the values at 3 days after CPB, the expression of CIRP mRNA was significantly down-regulated at each time point after CPR, and the expression of CIRP was down-regulated at 6 h, 12 h, and 7 days after CPR in group CRP (P<0.05). CIRP was mainly expressed in the nucleus of pyramidal cells in the hippocampal CA1 region. Conclusion Up-regulated expression of hippocampal CIRP is one of the endogenous mechanisms that inhibit cell apoptosis in the brain tissues of rats. Key words: Heart arrest, induced; Cardiopulmonary resuscitation; RNA-binding proteins; Hippocampus

Hibernation-specific alternative splicing of the mRNA encoding cold-inducible RNA-binding protein in the hearts of hamsters

The hearts of hibernating animals are capable of maintaining constant beating despite a decrease in body temperature to less than 10 °C during hibernation, suggesting that the hearts of hibernators are highly tolerant to a cold temperature. In the present study, we examined the expression pattern of cold-inducible RNA-binding protein (CIRP) in the hearts of hibernating hamsters, since CIRP plays important roles in protection of various types of cells against harmful effects of cold temperature. RT-PCR analysis revealed that CIRP mRNA is constitutively expressed in the heart of a non-hibernating euthermic hamster with several different forms probably due to alternative splicing. The short product contained the complete open reading frame for full-length CIRP. On the other hand, the long product had inserted sequences containing a stop codon, suggesting production of a C-terminal deletion isoform of CIRP. In contrast to non-hibernating hamsters, only the short product was amplified in hibernating animals. Induction of artificial hypothermia in non-hibernating hamsters did not completely mimic the splicing patterns observed in hibernating animals, although a partial shift from long form mRNA to short form was observed. Our results indicate that CIRP expression in the hamster heart is regulated at the level of alternative splicing, which would permit a rapid increment of functional CIRP when entering hibernation.

Cold-Inducible RNA-Binding Protein Is an Important Mediator of Alcohol-Induced Brain Inflammation

Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP), a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT) mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP−/−) mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH) were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP−/− mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP−/− mice. In cultured BV2 cells (mouse microglia), ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

Construction of pECFP-C1- CIRP and its effect on cold-induced apoptosis in mouse NIH/3T3 cells

Cold-inducible RNA-binding protein (CIRP) is suggested to be involved in protecting cells from the damage caused by cold stress. In this study, the recombinant plasmid, pECFP-C1-CIRP, was constructed and transfected into NIH/3T3 cells by lipofectamineTM 2000. The fluorescence microscopy, real-time polymerase chain reaction (RT-PCR) and Western blot detection were done for transient and stable expression assays. CIRP gene was successfully subcloned into eukaryotic expression vector of pECFP-C1, confirmed by restrictive enzyme digestion analysis and DNA sequencing. Fluorescent microscopy study showed that green fluorescence was generated from NIH/3T3 cell. The results of RT-PCR indicated that the CIRP mRNA was expressed in NIH/3T3 cells, while the results of Western blot detection further indicated that the CIRP protein was overexpressed in NIH/3T3 cells. Stable expression in cells transfected with pECFP-C1-CIRP was achieved by selection in a G418-containing medium for 2 weeks. We performed experiments of cold-induced apoptosis under cold treatment of 4, 14 and 24°C. The results of the cell apoptosis detected by fluorescence microscopy indicated that the apoptosis level was reduced in pECFP-C1-CIRP stably expressed group due to overexpression of CIRP. This study suggested that CIRP played a role in protecting cells from cold-induced apoptosis. Key words : Cold inducible RNA-binding protein, NIH/3T3 cell, transfection, cold-induced apoptosis.

Effects of hypothermia and cerebral ischemia on cold-inducible RNA-binding protein mRNA expression in rat brain

CIRP (cold-inducible RNA-binding protein) mRNA is highly expressed in hypothermic conditions in mammalian cells, and the relationship between CIRP and neuroprotection for cerebral ischemia under hypothermia has been focused upon. At present, however, the expression characteristics of CIRP under hypothermia and cerebral ischemia in vivo are not clearly elucidated. In this study, CIRP mRNA expression in various regions of rat brain was examined by reverse transcriptase polymerase chain reaction (RT-PCR). CIRP expression levels were found to be similar in the hippocampus and cortex. Real-time quantitative PCR analysis revealed increasing CIRP mRNA expression in the cortex during the 24-h observation period following treatment with hypothermia or cerebral ischemia, with a greater increase in the hypothermia group. When cerebral ischemia was induced following hypothermia, CIRP mRNA expression in the cortex again showed a significant increasing tendency, but ischemia delayed the appearance of this increase. To reveal the relationship between CIRP and energy metabolism in the rat brain, lactate and pyruvate concentrations in the cortex of the rats treated with hypothermia, ischemia and ischemia after hypothermia were determined by spectrophotometric assay, and levels of phosphofructokinas-1 (PFK-1), the major regulatory enzyme of the glycolytic pathway, in the rat cortex in the three groups was also analyzed by Western blot. Using linear correlation, lactate and pyruvate concentrations, and PFK-1 levels, were each analyzed in the three groups in association with CIRP mRNA expression levels. The analysis did not reveal any correlation between the three metabolic parameters and CIRP mRNA expression induced by hypothermia, suggesting that while playing a role in neuroprotection under hypothermia, CIRP does not affect cerebral energy metabolism.

Cold-Inducible RNA-Binding Protein Bypasses Replicative Senescence in Primary Cells through Extracellular Signal-Regulated Kinase 1 and 2 Activation

Embryonic stem cells are immortalized cells whose proliferation rate is comparable to that of carcinogenic cells. To study the expression of embryonic stem cell genes in primary cells, genetic screening was performed by infecting mouse embryonic fibroblasts (MEFs) with a cDNA library from embryonic stem cells. Cold-inducible RNA-binding protein (CIRP) was identified due to its ability to bypass replicative senescence in primary cells. CIRP enhanced extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, and treatment with an MEK inhibitor decreased the proliferation caused by CIRP. In contrast to CIRP upregulation, CIRP downregulation decreased cell proliferation and resulted in inhibition of phosphorylated ERK1/2 inhibition. This is the first evidence that ERK1/2 activation, through the same mechanism as that described for a Val12 mutant K-ras to induce premature senescence, is able to bypass senescence in the absence of p16(INK4a), p21(WAF1), and p19(ARF) upregulation. Moreover, these results show that CIRP functions by stimulating general protein synthesis with the involvement of the S6 and 4E-BP1 proteins. The overall effect is an increase in kinase activity of the cyclin D1-CDK4 complex, which is in accordance with the proliferative capacity of CIRP MEFs. Interestingly, CIRP mRNA and protein were upregulated in a subgroup of cancer patients, a finding that may be of relevance for cancer research.