Cold-inducible RNA-binding Protein Protein Research Articles

Intracellular CIRP promotes liver regeneration via STAT3 signaling pathway activation after partial hepatectomy in mice.

Cold‑inducible RNA‑binding protein (CIRP) is a cold shock protein implicated in the regulation of multiple biological processes depending on its cellular localization. However, to the best of our knowledge, the role of CIRP in liver regeneration and injury after hepatectomy has not been investigated. The present study was therefore designed to explore whether CIRP is involved in liver regeneration after hepatectomy and its specific role and underlying molecular mechanism. The overall involvement of CIRP in liver regeneration and injury after hepatectomy was evaluated in CIRP‑deficient mice. C23, an antagonist of extracellular CIRP, was used to assess the effect of extracellular CIRP on liver regeneration and injury after hepatectomy. CIRP overexpression and short hairpin RNA plasmids were transfected into HepG2 cells to study the effect of intracellular CIRP on cell proliferation. The effects of extracellular CIRP on cell proliferation and injury were determined via the use of recombinant CIRP protein to stimulate HepG2 cells in vitro. The results indicated that both hepatic and serum CIRP levels significantly increased after partial hepatectomy. Additionally, CIRP deficiency impaired liver regeneration but alleviated liver injury after partial hepatectomy in mice. C23 administration attenuated liver injury and suppressed endoplasmic reticulum (ER) stress and oxidative stress. Loss‑ and gain‑of‑function analyses in HepG2 cells indicated that an increase in intracellular CIRP promoted cell proliferation via signal transducers and activation of transcription 3 (STAT3) signaling pathway activation. Moreover, recombinant CIRP had no effect on cell proliferation or STAT3 phosphorylation but induced ER stress, which was blocked by TAK242, an inhibitor of Toll‑like receptor 4 (TLR4), in HepG2 cells. Taken together, the results of the present study demonstrated that intracellular CIRP promotes liver regeneration by activating the STAT3 pathway, whereas extracellular CIRP induces ER stress possibly via the TLR4 signaling pathway after hepatectomy.

AAV9-mediated CIRP gene transfer protects against cardiac dysfunction and remodelling in a rat model of myocardial infarction.

Cold-inducible RNA-binding protein (CIRP) is a stress-response protein that has been shown to protect cardiomyocytes under a variety of stress conditions from apoptosis. Our recent study showed that the expression of CIRP protein in the heart was downregulated in patients with heart failure and an animal model of ischaemia heart failure, but its role in heart failure is still unknown. The present study aimed at evaluating the potential role of CIRP on the heart in an animal model of myocardial infarction (MI). MI model of rats was induced by the ligation of the left coronary artery. CIRP overexpression was mediated by direct intracardiac injection of adeno-associated virus serotype 9 (AAV9) vectors carrying a CIRP coding sequence with a cardiac-specific promoter before the induction of the MI model. The effects of CIRP elevation on MI-induced heart were analysed through echocardiographic, pathological and molecular analysis. Our results showed that the intracardiac injection of AAV9 successfully mediated CIRP upregulation in cardiomyocytes. Upregulation of cardiac CIRP prevented MI-induced cardiac dysfunction and adverse remodelling, coupled with the reduced inflammatory response in the heart. Collectively, these results demonstrated the beneficial role of intracellular CIRP on the heart and suggest that CIRP may be a therapeutic target in ischaemic heart disease.

Regulation of CIRP by genetic factors of SP1 related to cold sensitivity.

Cold-inducible RNA-binding-protein (CIRP) is a cold shock protein that plays a protective role in genotoxic stress response. CIRP modulates inflammation in human diseases, inhibits cell proliferation, and protects cells from genotoxic damage during cellular stress. The mild cold responsive element and specificity protein 1 (SP1) play a role in Cirp expression at low temperatures. Although previous studies have provided insights into the immune functions of SP1 or CIRP, the mechanisms by which CIRP and SP1 me diate inflammatory responses remain largely unknown. Therefore, in the current study, we examined whether Cirp expression is affected by genetic factors related to temperature sensitivity as well as under low temperature. We performed a genome-wide association study on cold sensitivity in 2,000 participants. Fifty-six genome-wide significant trait-locus pairs were identified (p<1×10-5, false discovery rate < 0.05). Among these variants, rs1117050 and rs11170510 had a strong linkage disequilibrium (r2 > 0.8) relationship and expression quantitative trait locus-associated signals with the nearest Sp1 gene. We confirmed that the minor alleles of rs11170510 and rs58123204 were associated with increased Sp1 expression. Additionally, Sp1 overexpression led to CIRP translocation from the nucleus to the cytoplasm. CIRP protein levels increased in serum samples that had minor alleles of rs11170510 and rs58123204. Levels of various pro-inflammatory cytokines were also significantly increased in human peripheral blood mononuclear cells with minor alleles of rs11170510 and rs58123204. These results suggest that genetic factors related to cold sensitivity regulate CIRP expression and function and provide valuable insights into prediction of potential diseases through analysis of inherent genetic factors in humans.

What's New in Shock, February 2021?

What's New in Shock, February 2021?

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.

Cold‐Inducible RNA Binding Protein (CIRP) Decreases Inflammation and Breast Tumor Growth

RNA binding proteins (RBPs) post‐transcriptionally regulate gene expression by associating with the regulatory sequences in the untranslated regions of mRNAs. RBPs are often disrupted in cancer, but current knowledge of their role in regulating cancer‐associated genes is largely based on in vitro studies. Cold‐inducible RBP (CIRP) is a stress‐induced RBP that was recently shown to modulate inflammation in response to cellular stress. CIRP expression is altered in several cancers, including breast cancer. The effect of CIRP on inflammation in breast cancer is not known. This study tested the hypothesis, based on our preliminary data and published studies that CIRP changes the inflammatory profile of breast tumors from pro‐ to anti‐tumorigenic, and that CIRP expression and this altered inflammatory milieu are associated with lower tumorgenicity. To test this hypothesis a transgenic mouse overexpressing CIRP in the mammary epithelium was crossed with the PyMT mouse model of breast cancer. Effects on both early (7‐week‐old mice) and late (14‐week‐old mice) tumorigenesis and inflammation were assessed in CIRP/PyMT and PyMT mice. Mammary glands from 7‐week‐old CIRP/PyMT mice had 50% fewer mammary intraepithelial neoplasias than PyMT mice while 14 week‐old CIRP/PyMT mice had slower tumor growth kinetics, smaller tumors, decreased tumor burden and decreased pulmonary metastasis compared to PyMT mice. Importantly, CIRP protein level correlated with decreased tumorigenesis in CIRP/PyMT mice. To assess effects on the inflammatory environment, mammary glands from 7 and 14‐week‐old mice were incubated in medium overnight and Luminex cytokine assays were performed on the medium. Luminex assays were also performed on sera from 14‐wk mice to assess effects on systemic inflammation. There was a decrease in cytokines that promote inflammation, angiogenesis and metastasis at both timepoints for mammary glands, and in 14‐week sera. Overall, these data support a role for CIRP as a potent anti‐tumor molecule that suppresses local and systemic pro‐tumorigenic inflammation. To begin to probe whether CIRP's effects on cytokine levels were cell‐autonomous, we performed in silico analysis of CIRPs binding affinity for individual targets using the web‐based beRBP tool (Yu et al.). CIRP was not predicted to bind the cytokine mRNAs so may be acting in a non‐cell autonomous fashion.Support or Funding InformationNational Institutes of Health Grant/Award Number: F31‐CA213933American Association of Anatomists, Fellows Grant Award ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Cold-inducible RNA-binding Protein (CIRP) on Liver Glycolysis during Acute Cold Exposure in C57BL/6 Mice.

Cold-inducible RNA-binding protein (CIRP) is a stress-responsive protein involved in several signal transduction pathways required for cellular function, which are associated with apoptosis and proliferation. The present study aimed to investigate the possible effects of CIRP-mediated regulation of glucose metabolism in the liver following acute cold exposure. The livers and serum of male C57BL/6 mice were collected following cold exposure at 4 °C for 0 h, 2 h, 4 h, and 6 h. Glucose metabolic markers and the expression of glucose metabolic-related proteins were detected in the liver. Acute cold exposure was found to increase the consumption of glycogen in the liver. Fructose-1,6-diphosphate (FDP) and pyruvic acid (PA) were found to show a brief increase followed by a sharp decrease during cold exposure. Anti-apoptotic protein (Bcl-2) expression was upregulated. CIRP protein expression displayed a sequential increase with prolonged acute cold exposure time. Acute cold exposure also increased the level of protein kinase B (AKT) phosphorylation, and activated the AKT-signaling pathway. Taken together, these findings indicate that acute cold exposure increased the expression of CIRP protein, which regulates mouse hepatic glucose metabolism and maintains hepatocyte energy balance through the AKT signaling pathway, thereby slowing the liver cell apoptosis caused by cold exposure.

Cloning and expression of cold-inducible RNA binding protein in domestic yak (Bos grunniens).

Cold-inducible RNA binding protein (CIRP) is over-expressed during cold and many other stresses, and could regulate the adaptation to hypothermia. In the present investigation, the objective was to determine the expression of CIRP in adult yak heart, liver, spleen, lung, kidney, brain, ovary, testis and skin by relative quantitative real time polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry from mRNA and protein levels. The CIRP open reading frame encoding was cloned from the domestic yak brain. Results of RT-PCR and Western blot showed the high expression level of CIRP in the heart, kidney, brain, testis and skin, and the lower expression level of CIRP in the lung. Immunohistochemical staining showed CIRP was expressed in the nucleus of neuronal cells, spermatogonia, primary spermatocytes and epidermal cells, and in the cytoplasm of the residual tissues. These observations may provide new data to understand and further study the important role of CIRP protein in the plateau adaptation of the domestic yak on long-term evolution.

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.

Cold-inducible RNA-binding protein mediates cold air inducible airway mucin production through TLR4/NF-κB signaling pathway

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases and cold air stimulation has been shown to be associated with the severity of these diseases. However, the regulatory mechanisms that mediate excessive mucin production under cold stress remain elusive. Recently, the cold-inducible RNA-binding protein (CIRP) has been shown to be markedly induced after exposure to cold air. In this study, we sought to explore the expression of CIRP within bronchial biopsy specimens, the effect on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in cold air stimulation process. We found that CIRP protein expression was significantly increased in patients with COPD and in mice treated with cold air. Moreover, cold air stimulation induced MUC5AC expression in wild-type mice but not in CIRP−/− mice. In vitro, cold air stress significantly elevated the transcriptional and protein expression levels of MUC5AC in human bronchial epithelial cells. CIRP, toll-like receptor 4 (TLR4) and phosphorylated NF-κB p65 (p-p65) increased significantly in response to cold stress and CIRP siRNA, TLR4 - neutralizing Ab and a specific inhibitor of NF-κB could attenuated cold stress inducible MUC5AC expression. In addition, CIRP siRNA could hindered the expression levels of TLR4 and p-p65 both induced by cold stress. Taken together, these results suggest that airway epithelial cells constitutively express CIRP in vitro and in vivo. CIRP is responsible for cold-inducible MUC5AC expression by activating TLR4/NF-κB signaling pathway.

Cold-inducible proteins CIRP and RBM3, a unique couple with activities far beyond the cold.

Cold-inducible RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) are two evolutionarily conserved RNA-binding proteins that are transcriptionally upregulated in response to low temperature. Featuring an RNA-recognition motif (RRM) and an arginine–glycine-rich (RGG) domain, these proteins display many similarities and specific disparities in the regulation of numerous molecular and cellular events. The resistance to serum withdrawal, endoplasmic reticulum stress, or other harsh conditions conferred by RBM3 has led to its reputation as a survival gene. Once CIRP protein is released from cells, it appears to bolster inflammation, contributing to poor prognosis in septic patients. A variety of human tumor specimens have been analyzed for CIRP and RBM3 expression. Surprisingly, RBM3 expression was primarily found to be positively associated with the survival of chemotherapy-treated patients, while CIRP expression was inversely linked to patient survival. In this comprehensive review, we summarize the evolutionary conservation of CIRP and RBM3 across species as well as their molecular interactions, cellular functions, and roles in diverse physiological and pathological processes, including circadian rhythm, inflammation, neural plasticity, stem cell properties, and cancer development.

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

Cold-Inducible RNA-Binding Protein Regulates Cardiac Repolarization by Targeting Transient Outward Potassium Channels

Cold-inducible RNA-binding protein (CIRP) is constitutively expressed at low levels across various tissues. It is rapidly upregulated by multiple stresses, underlying a general role for CIRP in organic adaptations to pathophysiological conditions. However, the role of CIRP in the heart remains unclear. To examine the biofunctions of CIRP in the mammalian heart. Rats with targeted disruption of Cirp were generated using the TALEN (transcription activator-like effector nucleases)-based genome editing technique. The Cirp-knockout rats had structurally and functionally normal hearts. Resting ECG recordings revealed a short rate-corrected QT (QTc) interval in Cirp-null rats without any abnormalities in PR interval, RR interval or QRS waves as compared to wild-type animals. The shortened QTc interval from Cirp ablation was tightly linked to an abbreviated action potential duration in cardiac myocytes, which was attributable to increased transient outward potassium current (Ito). Furthermore, our findings uncovered that CIRP protein selectively bonded to KCND2 and KCND3 mRNAs encoding the functional α-subunits of Ito channel proteins. CIRP deficiency did not change the transcriptional activity of KCND2 or KCND3, but it facilitated their translation. Cirp knockout had no effect on the functional expression of ion channels other than Ito channels. CIRP modulates cardiac repolarization by negatively adjusting the expression and function of Ito channels. Our study may open a window to decipher the potential function of RNA-binding proteins in bioelectric activity.

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.

Cold-inducible RNA binding protein inhibits H2O2-induced apoptosis in rat cortical neurons

Objective The expression cold-inducible RNA-binding protein (CIRP) is significantly enhanced in neurons under hypothermia,but its roles remain unclear.This study aims to investigate whether the cerebral protection under hypothermia is mediated by the CIRP-mediated inhibition of neuronal apoptosis.Methods Primary rat cortical neurons were isolated,cultured,and transduced with lentiviral CIRP-RNAi.Apoptosis of the transduced neurons was induced with 100 μmol/L H2O2.The treated cells were divided into two groups,and cultured in 37℃ or 32℃ incubator respectively.Cell viability was detected by MTT colorimetric assay.Neuronal apoptosis was detected by flow cytometry after labeling the cells with Hoechst 33342 and Annexin V-FITC/PI.The protein expressions of CIRP,activated Caspase-3,and thioredoxin (TRX) were detected by Western blot.Results Under 32℃,CIRP protein was significantly induced in cortical neurons; the expression of activated Caspase-3 was decreased,while the TRX expression was increased.The rate of neuronal apoptosis was (4.5 ± 0.8) ％.Under 37 ℃,CIRP expression was evidently reduced in cortical neurons; the expression of activated caspase-3 was significantly enhanced,and TRX expression was reduced.The rate of neuronal apoptosis reaches (53.5 ± 1.7) ％ (P＜ 0.05,compared to that in 32 ℃ group).Conclusions The induction of CIRP protein in rat cortical neurons under hypothermia inhibits H2O2-induced neuronal apoptosis and thereby exerts neuroprotective effect,which forms one of the cerebral protective pathways under hypothermia. Key words: Cold-inducible RNA-binding protein; Neurons; Cerebral cortex ; Apoptosis; Hypothermia

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.