Cold-inducible RNA-binding Protein Expression Research Articles

Cold-inducible RNA-binding protein mediates neuroinflammation in cerebral ischemia

BackgroundNeuroinflammation is a key cascade after cerebral ischemia. Excessive production of proinflammatory mediators in ischemia exacerbates brain injury. Cold-inducible RNA-binding protein (CIRP) is a newly discovered proinflammatory mediator that can be released into the circulation during hemorrhage or septic shock. Here, we examine the involvement of CIRP in brain injury during ischemic stroke. MethodsStroke was induced by middle cerebral artery occlusion (MCAO). In vitro hypoxia was conducted in a hypoxia chamber containing 1% oxygen. CIRP and tumor necrosis factor-α (TNF-α) levels were assessed by RT-PCR and Western blot analysis. ResultsCIRP is elevated along with an upregulation of TNF-α expression in mouse brain after MCAO. In CIRP-deficient mice, the brain infarct volume, induction of TNF-α, and activation of microglia are markedly reduced after MCAO. Using microglial BV2 cells, we demonstrate that hypoxia induces the expression, translocation, and release of CIRP, which is associated with an increase of TNF-α levels. Addition of recombinant murine (rm) CIRP directly induces TNF-α release from BV2 cells and such induction is inhibited by neutralizing antisera to CIRP. Moreover, rmCIRP activates the NF-κB signaling pathway in BV2 cells. The conditioned medium from BV2 cells exposed to hypoxia triggers the apoptotic cascade by increasing caspase activity and decreasing Bcl-2 expression in neural SH-SY5Y cells, which is inhibited by antisera to CIRP. ConclusionExtracellular CIRP is a detrimental factor in stimulating inflammation to cause neuronal damage in cerebral ischemia. General significanceDevelopment of an anti-CIRP therapy may benefit patients with brain ischemia.

Expression of Cold-Inducible RNA-Binding Protein in Normal Skin, Actinic Keratosis and Squamous Cell Carcinoma.

Dear Editor: Cold-inducible RNA-binding proteins (CIRP) are a kind of RNA binding protein associated with diverse cellular responses including cell growth, proliferation, and apoptosis1. CIRP are induced by various cellular stresses in addition to cold exposure such as ultraviolet (UV) radiation and hypoxia1,2. Recently, their proto-oncogenic functions have been suggested, but a detailed expression pattern in skin tumor has not been reported3,4,5. Therefore, we aimed to simply observe the expression pattern of CIRP in photo-damaged epidermis of actinic keratosis (AK), squamous cell carcinoma (SCC) and normal skin by immunohistochemical staining. Five samples of normal skin were obtained from 5 healthy individuals undergoing cosmetic surgery. Among them, 3 were taken from the back and 2 from the face. Specimens of AK and SCC were obtained from the faces of 5 patients who underwent excisional surgery. SCC samples were confined to that which developed after long-standing AK. Clinically and pathologically active lesions were taken as the samples. In all cases, informed consent was obtained from patients according to the ethics committee of the Chonnam National University Hospital. Serial paraffin sections of each specimen were stained with monoclonal antibodies specific for CIRP (Proteintech Group, Chicago, IL, USA) at a dilution of 1 : 100 according to the manufacturers' protocols. The expression of CIRP in epidermal keratinocytes was scored semi-quantitatively by two dermatologists. Nuclear and cytoplasmic staining were assessed separately. We considered both staining intensity and the ratio of positively stained cells in comparison with adjacent stromal cells, lymphocytes, and sebaceous and eccrine glands. No staining was cited as 0, weaker staining than stromal cells as 1, similar to stromal cells as 2, and stronger as 3. Statistical analysis was performed using the chi-square test and Wilcoxon's rank sum test to compare the expression pattern (SPSS ver. 17.0; SPSS Inc., Chicago, IL, USA). In normal skin specimens, CIRP expression was more evident in nuclei than in cytoplasm throughout the epidermal keratinocytes (Fig. 1). However, in specimens from the face, the most sun exposed area, cytoplasmic CIRP staining intensity was increased compared to that in specimens from the back, a less-sun exposed area. In the case of AK, nuclear CIRP expression was decreased while cytoplasmic expression was maintained or rather increased. And in the most pathologic spots of SCC, CIRP expression was significantly decreased both in the nuclei and cytoplasm. Statistical analysis revealed significantly decreased expression of CIRP in nuclei of AK and consequent SCC compared with normal skin. There was no statistically significant difference in cytoplasmic staining intensity among them (Fig. 2). Fig. 1 Cold-inducible RNA-binding protein (CIRP) expression is evident in the nuclei of epidermal keratinocytes in normal skin from back (CIRP immunohistochemical stain, ×200). Fig. 2 Expression patterns of Cold-inducible RNA-binding protein (CIRP). Representative pictures of five cases in each normal skin from face (A), actinic keratosis (AK) (B), and squamous cell carcinoma (SCC) (C) (A~C: CIRP immunohistochemical stain, ×200). ... In previous studies of human cancer, the majority of endometrial carcinoma showed decreased staining intensity4. However, staining intensity was increased in several other human tumors, such as colon and prostate cancer5. These conflicting results may come from the early inducing mechanism of CIRP. CIRP regulates gene expression at the level of translation1,3. Therefore, the exact cellular function of CIRP remains unknown at the moment and the expression pattern in cancer cells could vary according to the state of the tumor. The increased cytoplasmic CIRP expression we observed in sun-exposed areas might be explained by relocalization of CIRP that was triggered by UV exposure2. We observed a significant decrease of nuclear CIRP expression in AK and SCC compared with normal skin. Further studies are needed to elucidate the relationship between CIRP and UV radiation and consequent tumorigenesis in the skin.

Hypothermia Protects against Fulminant Hepatitis in Mice by Reducing Reactive Oxygen Species Production

Objective: Mild hypothermia (32-33°C) shows protective effects in patients with brain damage and cardiac arrest. Although cold-inducible RNA-binding protein (CIRP) contributes to the protective effects of hypothermia through extracellular signal-regulated kinase activation in fibroblasts, the effects of hypothermia in the liver remain unclear. Methods: We analysed the effects of cold temperature on fulminant hepatitis, a potentially fatal disease, using the <smlcap>D</smlcap>-galactosamine (GalN)/lipopolysaccharide (LPS) and concanavalin (con) A-induced hepatitis models in mice. After GalN/LPS administration and anaesthesia, mice in the hypothermia group were kept at 25°C and those in control group were kept at 35°C. After concanavalin A (con A) administration, the mice in the hypothermia group were placed in a chamber with an ambient temperature of 6°C for 1.5 h. Results: Hypothermia attenuated liver injury and prolonged survival. Activation of c-Jun N-terminal kinase and Akt, which are involved in reactive oxygen species (ROS) accumulation, was suppressed by low temperature. Hypothermia significantly decreased oxidized protein levels, and treatment with N-acetyl-<smlcap>L</smlcap>-cysteine, an antioxidant, attenuated GalN/LPS-induced liver injury. In con A-induced hepatitis, CIRP expression was upregulated and Bid expression was downregulated, resulting in decreased apoptosis of hepatocytes in the hypothermia group. Conclusions: These data suggest that hypothermia directly protects hepatocytes from cell death via reduction of ROS production in fulminant hepatitis.

The protective effect of Cold-inducible RNA-binding protein (CIRP) on testicular torsion/detorsion: An experimental study in mice

PurposeTo evaluate the expression of Cold-inducible RNA-binding protein (CIRP) in torsion/detorsion of the testes in different phases and demonstrate the protective effect of CIRP on testicular injury after torsion/detorsion (T/D) in an experimental mouse model. MethodsTwenty-four male BALB/c mice were divided randomly into 8 groups: normal control group (N), sham-operated group (S), torsion 2h group (T2h), torsion/detorsion 12h group (T/D12h), and T/D24h, T/D48h, T/D72h, and T/D96h groups. The testes were examined for the expression levels of CIRP. Another 32 male BALB/c mice were divided randomly in to 4 groups: normal control group (N), T/D group, T/D+pcDNA3.1 group, and T/D+pcDNA3.1-CIRP group. The plasmids were transfected into testes with in vivo-jetPEI. After 3days, morphological changes, mean seminiferous tubule diameter (MSTD), and the number of the germ cell layers were observed. Superoxide dismutase (SOD) activity, the levels of malondialdehyde (MDA), and Bcl-2/Bax ratios were studied in the different groups. ResultsCompared with the N and S groups, the expression of CIRP in the T2h group was down-regulated. In T/D groups, the levels of CIRP were reduced in a time dependent manner. Compared to T/D and T/D+pcDNA3.1 group, the MSTD, number of the germ cell layers, SOD activity, and Bcl-2/Bax ratio increased in T/D+pcDNA3.1-CIRP group, while the level of MDA decreased. ConclusionsThe results of our study have shown that down-regulated CIRP is involved in testicular injury after testicular torsion/detorsion. Up-regulation of the expression of CIRP may reduce the damage caused by torsion/detorsion, possibly by preventing germ cell oxidative stress and apoptosis.

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

Difference in the expression of CIRBP, RBM3 and HSP70 in the myocardium and cerebellum after death by hypothermia and carbon monoxide poisoning

We studied the expression of hypoxia-related antigens (e.g., cold-inducible antigens and apoptotic antigens) in the myocardium and the cerebellumthat were obtained from individuals after death by carbon monoxide or hypothermia. The immunohistochemistry results revealed that expression of cold-inducible RNA binding protein (CIRBP) and RNA-binding protein 3 (RBM3) may be associated with hpyothermic and the hypoxic conditions. The expression of CIRBP and RBM3 in the myocardium was different from their expression in the cerebellum, especially in the Purkinje cells. The results indicate that agonal duration influences antigen expression. In the hypothermic condition, the myocardium uses more ATP since the force of the excitation-contraction coupling of the myocardium increases by more than 400% when the experimental temperature is reduced from 35°C to 25°C. The results obtained in this study indicate that physicians should pay attention to the myocardium when cooling the patient's body to protect the brain. Key W ords: carbon monoxide death, cerebellum, CIRBP, hypothermic death, myocardium, RBM3.

Ketogenic diet and fasting induce the expression of cold-inducible RNA-binding protein with time-dependent hypothermia in the mouse liver

Cold-inducible RNA-binding protein (CIRBP) induced by cold stress modulates the molecular circadian clock in vitro. The present study examines the effect of a ketogenic diet (KD) and fasting on Cirbp expression in the mouse liver. Chronic KD administration induced time-dependent Cirbp expression with hypothermia in mice. The circadian expression of clock genes such as Bmal1 and Clock was phase-advanced and augmented in the liver of mice fed with a KD. Transient food deprivation also induced time-dependent Cirbp expression with hypothermia in mice. These findings suggest that hypothermia is involved in the increased expression of Cirbp under ketogenic or fasting conditions.

Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells

BackgroundThere are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5′ flanking region of the mouse cirp gene.ResultsBy transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5′ flanking region octanucleotide 5′-TCCCCGCC-3′ is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5′ flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5′ flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C.ConclusionsCold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.

Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatogenic function in mouse testes

Cold-inducible RNA-binding protein (CIRP) is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling (TUNEL) assay, and mitogen-activated protein kinase (MAPK) signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter (MSTD) and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly via the activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

Mild hypothermia facilitates the expression of cold-inducible RNA-binding protein and heat shock protein 70.1 in mouse brain

An appropriate thermal control system is essential for maintaining brain homeostasis. Hypothermia is a decrease in core body temperature that occurs when the thermoregulatory responses of homeothermic animals are impaired by environmental and situational influences, such as cold ambience and anesthesia. In recent years, hypothermia has been used for medical treatment, i.e., therapeutic hypothermia, for patients with stroke, traumatic brain injury, and heart surgery. However, the target molecules acting during hypothermia have not been identified. To understand the molecular mechanisms, we generated a mouse model of mild hypothermia (1°C–2°C below normal), and analyzed the expression of several genes. After mice were exposed to cold for 24 and 48h, their rectal temperature reached 33°C–35°C. Then, using real-time quantitative PCR, we analyzed the mRNA expression levels of c-fos, cold-inducible RNA-binding protein (CIRP), heat shock protein (hsp) 70.1, oxytocin, and representative inflammatory cytokines, i.e., tumor necrosis factor (TNF)-α and interleukin (IL)-6 in target organs. Importantly, we found that the expression levels of CIRP and hsp70.1 were elevated in the olfactory bulb within 48h. In the hypothalamus, CIRP expression levels increased and were followed by an increase in hsp70.1 expression. Meanwhile, TNF-α and IL-6 expression decreased gradually over 24 and 48h in the olfactory bulb and hypothalamus. These specific expression profiles, i.e., enhanced CIRP and hsp70.1 expression and depressed cytokine expression, suggest that they could regulate apoptosis related to the cytokine signaling.

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 100μmol/L H2O2, the treated cells were divided into two groups, and cultured in 37°C or 32°C 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°C, CIRP protein is significantly induced in cortical neurons; the expression of activated caspase-3 decreases, while the TRX expression increases. The rate of neuronal apoptosis is 4.5±0.8%. Under 37°C, CIRP expression is evidently reduced in cortical neurons; the expression of activated caspase-3 is significantly enhanced with reduced level of TRX expression. The rate of neuronal apoptosis reaches 53.5±1.7% (P<0.05, compared to that in 32°C 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.

Effects of Cold Stimulation with Different Intensities on Expression of CIRP in Heart,Brain,Testis and Lung Tissues of Wistar Rats

In order to investegate the changes of cold inducible RNA-binding protein(CIRP) expression in heart,lung,brain and testis,healthy Wistar rats were treated by cold stimulation with different intensities.Totally 100 Wistar rats were divided into control group,acute cold stimulation group and chronic cold stimulation group.The control group was raised under normal ambient temperature(20 ℃ ± 1 ℃),acute cold stimulation group was subject to cold stress under different temperature(-10 ℃±1 ℃,-4 ℃±1 ℃,0 ℃±1 ℃,4 ℃±1 ℃) for different time(3 h,6 h,12 h and 24 h),respectively,and the chronic cold stimulation group was subject to cold stress under 10 ℃ ± 1 ℃ for 1 weak,2 weak and 3 weak.Methods of SYBR green I real time PCR and Western-blot were used to qualify the levels of CIRP expression.The results indicated that CIRP in four tissues of rats existed the basic expression level under normal condition;CIRP was expressed at all time after cold stimulation,but the expression levels were different in different tissues;after acute cold stimulation,the expression level of CIRP mRNA in hearts,brains and testes of rats increased in 0~6 h and peaked at 6 h then started to decline,while in lungs the change tendency was opposite to the former;after chronic cold stimulation,the expression levels of CIRP increased in hearts,brains and testes,while decreased in lungs,but the difference was not significant among different groups.Therefore,it can be concluded that the expression of CIRP in hearts,lungs,brains and testes of healthy Wistar rats is regular,although there is tissue specificity.

Moderate low temperature preserves the stemness of neural stem cells and suppresses apoptosis of the cells via activation of the cold-inducible RNA binding protein

We hypothesized that one of the mechanisms underlying the protection of brain injury by therapeutic hypothermia is associated with preservation of neural stem cells. We investigated effects of moderate low temperature and the contribution of a cold-inducible molecule for the stemness of neural stem cells. The MEB5 mouse neural stem cell line was cultured in the presence or absence of EGF, and apoptosis, mRNA expression, and immunocytochemistry of the differentiation markers nestin and GFAP were evaluated at 37 or 32°C. We investigated the contribution of the cold-inducible RNA binding protein (CIRP) on apoptosis and differentiation of MEB5 cells at 32°C. EGF deprivation increased the number of apoptotic cells, decreased expression of nestin, and increased expression of GFAP. The moderate low temperature prevented apoptosis and decreases in expression of GFAP in MEB5 by EGF deprivation. The moderate low temperature significantly increased expression of CIRP. siRNA against CIRP significantly increased the apoptotic cell population of MEB5 cells via EGF deprivation at 32°C. These findings suggest that moderate low temperature preserved stemness of neural stem cells and prevented cell apoptosis via the stimulation of CIRP, and one of the mechanisms of rescue of brain injury by the moderate hypothermia is associated with preservation of neural stem cells.

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 modulates proliferation in the mouse mammary epithelium

Cold‐inducible RNA binding protein (CIRP) is upregulated in response to cellular stress, either facilitating or repressing translation of its mRNA targets. We showed that CIRP is upregulated in all breast cancer cell lines examined, while others have shown CIRP overexpression in primary breast tumors. In the MCF‐7 breast cancer cell line, CIRP upregulates the expression of another stress‐induced RNA binding protein, HuR, as well as the cell cycle regulator cyclin E1. Both HuR and cyclin E1 are also overexpressed in breast cancer, with higher expression correlating with a poor prognosis. In order to determine the in vivo function of CIRP in the breast epithelium, we produced a transgenic mouse expressing CIRP in the mammary epithelium under control of the mouse mammary tumor virus (MMTV) regulatory sequences. Real‐time RT‐PCR showed maximal expression of the transgene in the pregnant mouse at gestation day 16, and a second peak of expression after birth, at lactation day 14. Immunohistochemistry for the proliferation marker Ki‐67 showed decreased proliferation in the mammary epithelium of CIRP transgenic mice compared to wild type littermates at gestation day 16 (45% CIRP vs. 56% wild type, p=0.05) and lactation day 1 (7% CIRP vs. 25% wild type, p=0.0068). These results suggest that CIRP negatively regulates proliferation in the intact mammary gland. We are currently examining effects of CIRP expression on apoptosis in the involuting mammary gland. This work was supported by a grant from the National Cancer Institute‐National Institutes of Health to RSH (R01CA095898).

Cold‐inducible RNA‐binding protein contributes to human antigen R and cyclin E1 deregulation in breast cancer

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress-induced RNA-binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF-7 breast cancer cells by stabilizing its mRNA. We show here that cold-inducible RNA-binding protein (CIRP) co-regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co-regulate cyclin E1, HuR and CIRP levels were altered in MCF-7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co-precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co-localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR-containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets.

Cold-inducible RNA binding protein (CIRP) expression is modulated by alternative mRNAs

Cold-inducible RNA binding protein (CIRP) is a mammalian protein whose expression is up-regulated in response to mild hypothermia. Although the exact function of this protein is currently unknown, it is thought to function as an RNA chaperone, facilitating mRNA translation upon the perception of cold stress. In this study we have identified and characterized the major CIRP 5'-untranslated region (5'-UTR) transcripts in mouse embryonic fibroblast NIH-3T3 cells. We show that the 5'-UTR of CIRP, a protein highly homologous to the cold-shock protein Rbm3, is much shorter than the previously published 5' leader sequence of Rbm3. In addition, three major CIRP transcripts with different transcription start sites are generated, with the levels of each of these transcripts being regulated in response to time and temperature. The major transcript generated at 37 degrees C does not encode for the full-length CIRP open reading frame, while the two major transcripts at 32 degrees C do. Further, the longest transcript detected at 32 degrees C shows a discrete expression and stability profile under mild hypothermic conditions and exhibits internal ribosome entry segment (IRES)-like activity. The IRES-like activity is not responsive to conditions of mild hypothermia or hypoxia, but the levels and stability of the transcript harboring the putative IRES are increased at 32 degrees C. We discuss the emerging transcriptional and translational mechanisms by which CIRP expression appears to be controlled and the role that the 5'-UTR plays in the modulation of CIRP expression.

Overexpression of CIRP may reduce testicular damage induced by cryptorchidism

To investigate the protective effect of overexpression of cold-inducible RNA-binding protein (CIRP) on testicular damage induced by cryptorchidism. Male BALB/c mice were made surgically cryptorchid and CIRP gene was transferred into the cryptorchid testis by in vivo electroporation. Seven or ten days after electroporation, the expression of CIRP, p53 and Fas mRNA and protein were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Meanwhile, Histopathological changes were observed by light microscope, and flow cytometry was used to detect testicular cell apoptosis. Testicular weights after transfection with pVAX1-CIRP or pVAX1 were 0.083+/-0.005 g and 0.065+/-0.004 g, respectively, on day 7(P < 0.05) and 0.078+/-0.004 g and 0.052+/-0.007 g, on day 10 (P < 0.05). Testicular cell apoptosis after transfection with pVAX1-CIRP or pVAX1 were 9.8+/-1.1 % and 20.7+/-1.3 %, respectively, on day 7 (P < 0.01) and 10.4+/-0.9 % and 27.5+/-1.2 %, on day 10 (P < 0.01). In addition, the expression of CIRP mRNA and protein in the testes transfected with pVAX1-CIRP were both increased (P < 0.05) at each indicated time point. Meanwhile, the expression of p53 was decreased on day 7 (P < 0.05) and Fas was decreased on day 10(P < 0.05). Overexpression of CIRP may reduce testicular damage induced by cryptorchidism by down-regulating the levels of p53 and Fas.

Artificial cold exposure induced stroke in renovascular hypertensive rats and its association with cold-inducible RNA binding protein mRNA expression in brain tissue and blood pressure

Background High incidence of stroke at interchange period of autumn and winter was demonstrated by epidemiological survey, and the specific causes should be further investigated. Objective To investigate the influence of artificial cold exposure on the incidence of stroke in renovascular hypertensive rats (RHR), and analyze the association with blood pressure and cold-inducible RNA binding protein (CIRP) mRNA expression in brain tissue. Design A completely randomized grouping design, a randomized control animal trial. Settings Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University; Department of Chemistry, Open laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong. Materials Male SD rats ( n = 460), weighing 80–100 g were obtained from Guangdong Province Health Animal Unit. A modified RXZ-300A intelligent artificial climate cabinet (Ningbo Jiangnan Instrument Co., Ltd., China). Methods The experiment were processed in the Lab of Neurology, the First Affiliated Hospital of Sun Yat-sen University and the Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, University of Hong Kong from October 2004 to November 2005. Rats ( n = 400) were operated to establish 2-kidney 2-clip RHR model as described previously. The sham-operated rats ( n = 60) served as normotensive controls. Eight weeks later, 300 of RHR were randomly selected according to their systolic blood pressure (SBP) and divided into 3 sub-groups ( n = 100 per group): mild hypertensive group (SBP of 160–200 mm Hg), moderate hypertensive group (SBP of 200–220 mm Hg) and severe hypertensive group (SBP > 220 mm Hg). Each group was further divided into two groups ( n = 50) under ACE and non-ACE. Normal sham-operated SD rats ( n = 60), SBP < 140 mm Hg, were randomly divided into two groups: Sham-operated control group ( n = 30) under ACE and non-ACE. To establish the ACE and non-ACE treatment, rats were housed individually in artificial climate cabinet, and ACE was designed as three cycles of 12-hour light of 22 °C (7:00–19:00) and 12-hour dark of 4 °C(19:00–7:00). The non-ACE group was kept at 22 °C throughout the experiment. Main outcome measures Blood Pressure changes were measured and stroke symptom were observed; Expression of the CIRP were examined by reverse transcription-polymerase chain reaction. Results Finally 360 rats were involved in the analysis of results. ▪ Incidence of stroke: The incidence of stroke in 2k2c RHR was significantly higher after a three-day intermittent (12-hour) ACE (29.3%) as compared with that in non-ACE (17.3%) ( P < 0.05). Furthermore, the severe hypertensive 2k2c RHR (BP > 220 mm Hg) was found to have much higher incidence of stroke (66%, 33/50) than the mild (8%, 4/50) and moderate (18%) hypertensive 2k2c RHR. ▪ CIRP mRNA in brain tissue: ACE treatment stimulated the mRNA expression of CIRP in non-stroke 2k2c RHR but not in stroke 2k2c RHR ( P < 0.05). Conclusion High blood pressure and low expression of CIRP are associated with ACE induced stroke.

Down-regulation of cold-inducible RNA-binding protein does not improve hypothermic growth of Chinese hamster ovary cells producing erythropoietin

Discovery of the cold-inducible RNA-binding protein (CIRP) in mouse fibroblasts suggests that growth suppression at hypothermic conditions is due to an active response by the cell rather than due to passive thermal effects. To determine the effect of down-regulated CIRP expression on cell growth and erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells at low culture temperature, stable CHO cell clones with reduced CIRP expression level were established by transfecting (rCHO) cells with the CIRP siRNA vector with a target sequence of TCGTCCTTCCATGGCTGTA. For comparison of the degree of specific growth rate ( μ) reduction at low culture temperature, three CIRP-reduced clones with different μ and three control clones transfected with null vector were cultivated at two different temperatures, 32 °C and 37 °C. Unlike mouse fibroblasts, alleviation of hypothermic growth arrest of rCHO cells by CIRP down-regulation was insignificant, as shown by statistical analysis using the t-test ( P<0.18, n=3). The ratios of μ at 32 °C to μ at 37 °C of CIRP-reduced clones and control clones were 0.29±0.03 and 0.25±0.03 on an average, respectively. Furthermore, it was also found that overexpression of CIRP did not inhibit rCHO cell growth significantly at 37 °C. Taken together, the data obtained show that down-regulation of only CIRP in rCHO cells, unlike mouse fibroblasts, is not sufficient to recover growth arrest at low-temperature culture (32 °C).