RNA-binding Motif 3 Research Articles

Characterization of Circulating Cold Shock Proteins FGF21 and RBM3 in a Multi-Center Study of Pediatric Cardiac Arrest.

Fibroblast Growth Factor 21 (FGF21) is a neuroprotective hormone induced by cold exposure that targets the β-klotho co-receptor. β-klotho is abundant in the newborn brain but decreases rapidly with age. RNA-Binding Motif 3 (RBM3) is a potent neuroprotectant upregulated by FGF21 in hypothermic conditions. We characterized serum FGF21 and RBM3 levels in patients enrolled in a prospective multi-center study of pediatric cardiac arrest (CA) via a secondary analysis of samples collected to evaluate brain injury biomarkers. Patients (n = 111) with remnant serum samples available from at least two of three available timepoints (0-24, 24-48 or 48-72 hours post-resuscitation) were included. Serum samples from 20 healthy controls were used for comparison. FGF21 was measured by Luminex and internally validated enzyme-linked immunoassay (ELISA). RBM3 was measured by internally validated ELISA. Of postarrest patients, 98 were managed with normothermia, while 13 were treated with therapeutic hypothermia (TH). FGF21 increased >20-fold in the first 24 hours postarrest versus controls (681 pg/mL [200-1864] vs. 29 pg/mL [15-51], n = 99 vs. 19, respectively, p < 0.0001, median [interquartile range]) with no difference in RBM3. FGF21 did not differ by sex, while RBM3 was increased in females versus males at 48-72 hours postarrest (1866 pg/mL [873-5176] vs. 1045 pg/mL [535-2728], n = 40 vs. 54, respectively, p < 0.05). Patients requiring extracorporeal membrane oxygenation (ECMO) postresuscitation had increased FGF21 versus those who did not at 48-72 hours (6550 pg/mL [1455-66,781] vs. 1213 pg/mL [480-3117], n = 7 vs 74, respectively, p < 0.05). FGF21 and RBM3 did not correlate (Spearman's rho = 0.004, p = 0.97). We conclude that in a multi-center study of pediatric CA patients where normothermic targeted temperature management was largely used, FGF21 was markedly increased postarrest versus control and highest in patients requiring ECMO postresuscitation. RBM3 was sex-dependent. We provide a framework for future studies examining the effect of TH on FGF21 or use of FGF21 therapy after pediatric CA.

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

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

FGF21 modulates hippocampal cold-shock proteins and CA2-subregion proteins in neonatal mice with hypoxia-ischemia.

Fibroblast growth factor 21 (FGF21) is a neuroprotectant with cognitive enhancing effects but with poorly characterized mechanism(s) of action, particularly in females. Prior studies suggest that FGF21 may regulate cold-shock proteins (CSPs) and CA2-marker proteins in the hippocampus but empirical evidence is lacking. We assessed in normothermic postnatal day (PND) 10 female mice, if hypoxic-ischemic (HI) brain injury (25 min 8% O2/92% N2) altered endogenous levels of FGF21 in serum or in the hippocampus, or its receptor β-klotho. We also tested if systemic administration of FGF21 (1.5 mg/kg) modulated hippocampal CSPs or CA2 proteins. Finally, we measured if FGF21 therapy altered markers of acute hippocampal injury. HI increased endogenous serum FGF21 (24 h), hippocampal tissue FGF21 (4d), and decreased hippocampal β-klotho levels (4d). Exogenous FGF21 therapy modulated hippocampal CSP levels, and dynamically altered hippocampal CA2 marker expression (24 h and 4d). Finally, FGF21 ameliorated neuronal damage markers at 24 h but did not affect GFAP (astrogliosis) or Iba1 (microgliosis) levels at 4d. FGF21 therapy modulates CSP and CA2 protein levels in the injured hippocampus. These proteins serve different biological functions, but our findings suggest that FGF21 administration modulates them in a homeostatic manner after HI. Hypoxic-ischemic (HI) injury in female post-natal day (PND) 10 mice decreases hippocampal RNA binding motif 3 (RBM3) levels in the normothermic newborn brain. HI injury in normothermic newborn female mice alters serum and hippocampal fibroblast growth factor 21 (FGF21) levels 24 h post-injury. HI injury in normothermic newborn female mice alters hippocampal levels of N-terminal EF-hand calcium binding protein 2 (NECAB2) in a time-dependent manner. Exogenous FGF21 therapy ameliorates the HI-mediated loss of hippocampal cold-induced RNA-binding protein (CIRBP). Exogenous FGF21 therapy modulates hippocampal levels of CA2-marker proteins after HI.

Hypothermia increases cold-inducible protein expression and improves cerebellar-dependent learning after hypoxia ischemia in the neonatal rat.

Hypoxic ischemic encephalopathy remains a significant cause of developmental disability.1,2 The standard of care for term infants is hypothermia, which has multifactorial effects.3-5 Therapeutic hypothermia upregulates the cold-inducible protein RNA binding motif 3 (RBM3) that is highly expressed in developing and proliferative regions of the brain.6,7 The neuroprotective effects of RBM3 in adults are mediated by its ability to promote the translation of mRNAs such as reticulon 3 (RTN3).8 METHODS: Hypoxia ischemia or control procedure was conducted in Sprague Dawley rat pups on postnatal day 10 (PND10). Pups were immediately assigned to normothermia or hypothermia at the end of the hypoxia. In adulthood, cerebellum-dependent learning was tested using the conditioned eyeblink reflex. The volume of the cerebellum and the magnitude of cerebral injury were measured. A second study quantified RBM3 and RTN3 protein levels in the cerebellum and hippocampus collected during hypothermia. Hypothermia reduced cerebral tissue loss and protected cerebellar volume. Hypothermia also improved learning of the conditioned eyeblink response. RBM3 and RTN3 protein expression were increased in the cerebellum and hippocampus of rat pups subjected to hypothermia on PND10. Hypothermia was neuroprotective in male and female pups and reversed subtle changes in the cerebellum after hypoxic ischemic. Hypoxic ischemic produced tissue loss and a learning deficit in the cerebellum. Hypothermia reversed both the tissue loss and learning deficit. Hypothermia increased cold-responsive protein expression in the cerebellum and hippocampus. Our results confirm cerebellar volume loss contralateral to the carotid artery ligation and injured cerebral hemisphere, suggesting crossed-cerebellar diaschisis in this model. Understanding the endogenous response to hypothermia might improve adjuvant interventions and expand the clinical utility of this intervention.

RBM3 suppresses stemness remodeling of prostate cancer in bone microenvironment by modulating N6-methyladenosine on CTNNB1 mRNA

Bone metastasis is the most happened metastatic event in prostate cancer (PCa) and needs a large effort in treatment. When PCa metastasizes to the bone, the new microenvironment can induce the epigenome reprogramming and stemness remodeling of cancer cells, thereby increasing the adaptability of cancer cells to the bone microenvironment, and this even leads to the occurrence of secondary tumor metastasis. Our group has previously found that RNA binding motif 3 (RBM3) affects the stem cell-like properties of PCa by interfering with alternative splicing of CD44. However, whether RBM3, as a stress-response protein, can resist microenvironmental remodeling of PCa particularly in bone metastasis remains unknown. By co-culturing PCa cells with osteoblasts to mimic PCa bone metastases, we found that RBM3 upregulates the N6-methyladenosine (m6A) methylation on the mRNA of catenin beta 1 (CTNNB1) in a manner dependent on methyltransferase 3 (METTL3), an N6-adenosine-methyltransferase complex catalytic subunit. Consequently, this modification results in a decreased stability of CTNNB1 mRNA and a followed inactivation of Wnt signaling, which ultimately inhibits the stemness remodeling of PCa cells by osteoblasts. Thus, the present study may extend our understanding of the inhibitory role of RBM3 on particularly bone metastasis of PCa.

16: FGF21 THERAPY ATTENUATES ISCHEMIA-MEDIATED DOWNREGULATION OF NEUROPROTECTIVE RBM3 IN NEWBORN MICE

Introduction: Fibroblast Growth Factor 21 (FGF21) is a neuroprotective cold stress hormone – i.e., a hormone upregulated by cold exposure. The mechanisms underlying FGF21-mediated neuroprotection remain to be fully elucidated. A key clue comes from studies in cultured immature neurons which revealed that FGF21 administration augments the ability of cold-stress to increase the potent neuroprotective cold-shock protein (CSP) RNA-Binding Motif 3 (RBM3). Furthermore, Beta-klotho, the obligatory FGF21 co-receptor, is uniquely abundant in the infant brain suggesting that newborns may benefit most from FGF21 therapy. Here we tested in a mouse model of neonatal encephalopathy (NE) if FGF21 bolus increased RBM3 levels in the normothermic (non-cooled) brain. We also tested if it affected the levels of a related CSP termed cold-inducible RNA-binding protein (CIRBP). Methods: Post-natal day 10 C57BL/6 female pups (n = 8/group) underwent right common carotid artery ligation followed by 25 min of 8% hypoxia (the Rice-Vannucci model of NE). Post-injury, mice were randomized to FGF21 (1.5mg/kg) or vehicle via subcutaneous bolus. Shams were also studied. RBM3 and CIRBP levels were measured (western blot) 24h later in the ipsilateral hippocampus. Levels were analyzed by Kruskal-Wallis (KW) with post-hoc Dunn’s test. Results: Median RBM3 densitometry differed between sham (0.309, IQR: 0.269-0.356), vehicle (0.133, IQR: 0.077-0.191) and FGF21 (0.160, IQR: 0.125-0.341) treated mice (KW, p=0.0135). Post-hoc Dunn’s test revealed significant decreases in RBM3 in vehicle treated NE vs. sham mice (p=0.010), but not in FGF21 treated NE vs. sham mice (p=0.385). Median CIRBP densitometry differed between sham (0.672, IQR: 0.608-0.695), vehicle (0.470, IQR: 0.418-0.591) and FGF21 (0.659, IQR: 0.511-0.731) treated mice (KW, p=0.031). There were no significant differences in CIRBP for individual group comparisons. Conclusions: Bolus administration of FGF21 attenuates the loss of the powerful neuroprotectant RBM3 in the normothermic hippocampus at 24h post-insult. Future studies of FGF21 treatment in RBM3 knockout mice are warranted to determine the contribution of this pathway to FGF21-mediated neuroprotection in NE.

549: ANALYSIS OF NEUROPROTECTIVE COLD SHOCK PROTEINS IN A MULTICENTER STUDY OF PEDIATRIC CARDIAC ARREST

Introduction: The efficacy of hypothermic neuroprotection varies by age. Cold exposure stimulates production of cold stress hormones (CSHs) which trigger thermogenesis and may be neuroprotective. FGF21 is a neuroprotective CSH that requires the Beta-klotho (KLB) co-receptor to signal. KLB is abundant in the newborn brain, decreases with age, and is virtually absent in adults. Thus, infants and young children may uniquely benefit from FGF21/KLB mediated neuroprotection. In vitro studies revealed that FGF21 upregulates the powerful neuroprotectant RNA-Binding Motif-3 (RBM3) in developmentally immature neurons (but not mature neurons). We sought to characterize FGF21 and RBM3 serum levels in children after cardiac arrest (CA) and evaluate their interdependence. Methods: Secondary analysis of serum samples collected in a multi-institutional prospective observational study evaluating biomarkers after pediatric CA. We measured FGF21 (Luminex) and RBM3 (ELISA) levels. All patients with serum from at least two timepoints (0-24h, 24-48h, 48-72h) were included. We also included serum from healthy controls. FGF21 and RBM3 levels at each timepoint were compared to controls (Wilcoxon-Ranksum); the relationship between FGF21/RBM3 at each timepoint was also evaluated (Spearman). Results: A total of 296 samples from 111 post-arrest patients were analyzed, and 20 samples from controls. Median age of the post-arrest cohort was 31.3 months (IQR: 6.9-129.9); 37.3 months (IQR: 18.6-63.6) for controls. Median FGF21 levels in the controls was 282pg/mL (IQR: 64-736), and did not differ significantly at 0-24h, 24-48h, and 48-72h (383 [IQR: 72-867], 723 [IQR: 167-1,688] and 552 [IQR: 210-1,225], respectively, P>0.05). Median control RBM3 was 2,944pg/mL (IQR: 969-8,241), and did not differ significantly at 0-24h, 24-48h, and 48-72h (1,538 [IQR: 757-4,146], 1,454 [625-3,543] and 1,526 [IQR: 613-3,591], respectively, P>0.05). FGF21 and RBM3 were correlated at 0-24h and 48-72h (Spearman’s coefficient of 0.384 and 0.223, respectively; P< 0.05 for both). Conclusions: After pediatric CA, serum FGF21 levels trend upward and RBM3 levels downward and correlate at early and late timepoints. Ongoing studies are examining their relationship to age, demographics, CA endophenotype, use of hypothermia, other serum biomarkers, and outcomes.

RNA-binding protein RBM3 intrinsically suppresses lung innate lymphoid cell activation and inflammation partially through CysLT1R

Innate lymphoid cells (ILC) promote lung inflammation in asthma through cytokine production. RNA-binding proteins (RBPs) are critical post-transcriptional regulators, although less is known about RBPs in ILC biology. Here, we demonstrate that RNA-binding motif 3 (RBM3) is highly expressed in lung ILCs and is further induced by alarmins TSLP and IL-33. Rbm3−/− and Rbm3−/−Rag2−/− mice exposed to asthma-associated Alternaria allergen develop enhanced eosinophilic lung inflammation and ILC activation. IL-33 stimulation studies in vivo and in vitro show that RBM3 suppressed lung ILC responses. Further, Rbm3−/− ILCs from bone marrow chimeric mice display increased ILC cytokine production suggesting an ILC-intrinsic suppressive function of RBM3. RNA-sequencing of Rbm3−/− lung ILCs demonstrates increased expression of type 2/17 cytokines and cysteinyl leukotriene 1 receptor (CysLT1R). Finally, Rbm3−/−Cyslt1r−/− mice show dependence on CysLT1R for accumulation of ST2+IL-17+ ILCs. Thus, RBM3 intrinsically regulates lung ILCs during allergen-induced type 2 inflammation that is partially dependent on CysLT1R.

Hypoxia-ischemia-mediated effects on neurodevelopmentally regulated cold-shock proteins in neonatal mice under strict temperature control.

Neonates have high levels of cold-shock proteins (CSPs) in the normothermic brain for a limited period following birth. Hypoxic-ischemic (HI) insults in term infants produce neonatal encephalopathy (NE), and it remains unclear whether HI-induced pathology alters baseline CSP expression in the normothermic brain. Here we established a version of the Rice-Vannucci model in PND 10 mice that incorporates rigorous temperature control. Common carotid artery (CCA)-ligation plus 25 min hypoxia (8% O2) in pups with targeted normothermia resulted in classic histopathological changes including increased hippocampal degeneration, astrogliosis, microgliosis, white matter changes, and cell signaling perturbations. Serial assessment of cortical, thalamic, and hippocampal RNA-binding motif 3 (RBM3), cold-inducible RNA binding protein (CIRBP), and reticulon-3 (RTN3) revealed a rapid age-dependent decrease in levels in sham and injured pups. CSPs were minimally affected by HI and the age point of lowest expression (PND 18) coincided with the timing at which heat-generating mechanisms mature in mice. The findings suggest the need to determine whether optimized therapeutic hypothermia (depth and duration) can prevent the age-related decline in neuroprotective CSPs like RBM3 in the brain, and improve outcomes during critical phases of secondary injury and recovery after NE. The rapid decrease in endogenous neuroprotective cold-shock proteins (CSPs) in the normothermic cortex, thalamus, and hippocampus from postnatal day (PND) 11-18, coincides with the timing of thermogenesis maturation in neonatal mice. Hypoxia-ischemia (HI) has a minor impact on the normal age-dependent decline in brain CSP levels in neonates maintained normothermic post-injury. HI robustly disrupts the expected correlation in RNA-binding motif 3 (RBM3) and reticulon-3 (RTN3). The potent neuroprotectant RBM3 is not increased 1-4 days after HI in a mouse model of neonatal encephalopathy (NE) in the term newborn and in which rigorous temperature control prevents the manifestation of endogenous post-insult hypothermia.

Abstract WP24: Pharmacological Hypothermia (PH) Promotes Plasticity Through RNA Binding Motif 3 (RBM3)-Mediated Cold Shock Pathway Following Ischemic Stroke

Background: Phenothiazine, including chlorpromazine and promethazine (C+P), induce hypothermia and neuroprotection after stroke. Immediately after stroke onset, there is a neuroplasticity window within which rehabilitation is especially effective. Recent studies have indicated that hypothermia-induced neuroplasticity involves the RBM3-mediated cold shock pathway, but it is unknown whether this pathway participates in neuroprotection and neuroplasticity in the context of pharmacological hypothermia (PH) induced by C+P after stroke. Methods: Adult Sprague-Dawley rats were divided in 3 groups: (1) sham; (2) stroke; (3) stroke treated with C+P before reperfusion. Long-term functional outcomes were determined by grid walk tests, rota-rod tests, beam balance tests, forelimb placing tests, adhesive tape touch and adhesive tape removal at 7 through 28 days. mRNA and protein quantities of cold shock pathway [RBM3, eukaryotic initiation factor 2 alpha (eIF2α), and reticulon 3 (RTN3)] and neuroplasticity [synaptophysin (SYN), post-synaptic density protein-95 (PSD-95), Tau, and recombinant growth associated protein 43 (GAP43)] were assessed by real-time PCR and western blot at day 1, 7 and 28 after reperfusion. Additionally, expression of RTN3 and eIF2α, and the co-localization of SYN and PSD95 was measured in primary neurons which subjected to oxygen/glucose deprivation (OGD) with RBM3 knockdown. Results: PH significantly improved functional outcomes at day 7 and 28. At day 1, mRNA and protein expression of RBM3 and RTN3 were significantly increased, and eIF2α was dephosphorylated by PH after stroke. mRNA and protein levels of SYN, Tau and GAP43 were all increased by PH at day 7 and 28. Furthermore, in OGD with shRBM3 group, levels of RTN3 and eIF2α dephosphorylation were significantly decreased, and the co-localization of SYN and PSD95 were significantly reduced compared with OGD group at 24 h without shRBM3. Conclusion: Our study has provided evidence, for the first time, for a novel mechanism underlying hypothermia in which the neuroplasticity and rehabilitation were achieved after ischemic stroke. Our findings suggest that the RBM3-induce cold shock pathway is a potential target of hypothermia for neuroprotection and neurorehabilitation.

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia.

Objective Cold-inducible RNA-binding protein (CIRBP) has been shown to be involved not only in cooling-induced cellular protection but also as a mediator of sterile inflammation, a critical mechanism of the innate immune response in ischemia/reperfusion (I/R) injury. The role of microglia and its activation in cerebral I/R injury warrants further investigation as both detrimental and regenerative properties have been described. Therefore, we investigated the effects of cooling, specifically viability, activation, and release of damage associated molecular patterns (DAMPs) on oxygen glucose deprivation/reperfusion- (OGD/R-) induced injury in murine BV-2 microglial cells. Methods Murine BV-2 microglial cells were exposed to 2 to 6 h OGD (0.2% O2 in glucose- and serum-free medium) followed by up to 19 h of reperfusion, simulated by restoration of oxygen (21% O2) and nutrients. Cells were maintained at either normothermia (37°C) or cooled to 33.5°C, 1 h after experimental start. Cultured supernatants were harvested after exposure to OGD for analysis of DAMP secretions, including high-mobility group box 1 (HMGB1), heat shock protein 70 (HSP70), and CIRBP, and cytotoxicity was assessed by lactate dehydrogenase releases after exposure to OGD and reperfusion. Intracellular cold-shock proteins CIRBP and RNA-binding motif 3 (RBM3) as well as caspases 9, 8, and 3 were also analyzed via Western blot analysis. Furthermore, inducible nitric oxide synthase (iNOS), ionized calcium-binding adaptor molecule 1 (Iba1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-1α (IL-1α), monocyte chemotactic protein 1 (MCP-1), transforming growth factor β (TGFβ), CIRBP, and RBM3 gene expressions were assessed via reverse transcription polymerase chain reaction, and TNF-α, IL-6, and IL-1β releases into the cultured supernatants were assessed via enzyme-linked immunosorbent assays (ELISA). Results Prolonged exposure to OGD resulted in increased BV-2 necrotic cell death, which was attenuated by cooling. Cooling also significantly induced cold-shock proteins CIRBP and RBM3 gene expressions, with CIRBP expression more rapidly regulated than RBM3 and translatable to significantly increased protein expression. DAMPs including HMGB-1, HSP70, and CIRBP could be detected in cultured supernatants after 6 h of OGD with CIRBP release being significantly attenuated by cooling. Exposure to OGD suppressed cytokine gene expressions of IL-1β, TNF-α, MCP-1, and TGFβ independently of temperature management, whereas cooling led to a significant increase in IL-1α gene expression after 6 h of OGD. In the reperfusion phase, TNF-α and MCP-1 gene expressions were increased, and cooling was associated with significantly lower TGFβ gene expression. Interestingly, cooled Normoxia groups had significant upregulations of microglial activation marker, Iba1, IL-1β, and TNF-α gene expressions. Conclusion BV-2 microglial cells undergo necrotic cell death resulting in DAMP release due to OGD/R-induced injury. Cooling conveyed neuroprotection in OGD/R-injury as observable in increased cell viability as well as induced gene expressions of cold shock proteins. As cooling alone resulted in both upregulation of microglial activation, expression of proinflammatory cytokines, and cold shock protein transcript and protein expression, temperature management might have ambiguous effects in sterile inflammation. However, cooling resulted in a significant decrease of extracellular CIRBP, which has recently been characterized as a novel DAMP and a potent initiator and mediator of inflammation.

TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection.

Increasing levels of the cold-shock protein, RNA-binding motif 3 (RBM3), either through cooling or by ectopic over-expression, prevents synapse and neuronal loss in mouse models of neurodegeneration. To exploit this process therapeutically requires an understanding of mechanisms controlling cold-induced RBM3 expression. Here, we show that cooling increases RBM3 through activation of TrkB via PLCγ1 and pCREB signaling. RBM3, in turn, has a hitherto unrecognized negative feedback on TrkB-induced ERK activation through induction of its specific phosphatase, DUSP6. Thus, RBM3 mediates structural plasticity through a distinct, non-canonical activation of TrkB signaling, which is abolished in RBM3-null neurons. Both genetic reduction and pharmacological antagonism of TrkB and its downstream mediators abrogate cooling-induced RBM3 induction and prevent structural plasticity, whereas TrkB inhibition similarly prevents RBM3 induction and the neuroprotective effects of cooling in prion-diseased mice. Conversely, TrkB agonism induces RBM3 without cooling, preventing synapse loss and neurodegeneration. TrkB signaling is, therefore, necessary for the induction of RBM3 and related neuroprotective effects and provides a target by which RBM3-mediated synapse-regenerative therapies in neurodegenerative disorders can be used therapeutically without the need for inducing hypothermia.

Characterization of RNA-Binding Motif 3 (RBM3) Protein Levels and Nuclear Architecture Changes in Aggressive and Recurrent Prostate Cancer.

The RNA-binding motif protein 3 (RBM3) has been shown to be up-regulated in several types of cancer, including prostate cancer (PCa), compared to normal tissues. Increased RBM3 nuclear expression has been linked to improved clinical outcomes. Given that RBM3 has been hypothesized to play a role in critical nuclear functions such as chromatin remodeling, DNA damage response, and other post-transcriptional processes, we sought to: (1) quantify RBM3 protein levels in archival PCa samples; (2) develop a nuclear morphometric model to determine if measures of RBM3 protein levels and nuclear features could be used to predict disease aggressiveness and biochemical recurrence. This study utilized two tissue microarrays (TMAs) stained for RBM3 that included 80 total cases of PCa stratified by Gleason score. A software-mediated image processing algorithm identified RBM3-positive cancerous nuclei in the TMA samples and calculated twenty-two features quantifying RBM3 expression and nuclear architecture. Multivariate logistic regression (MLR) modeling was performed to determine if RBM3 levels and nuclear structural changes could predict PCa aggressiveness and biochemical recurrence (BCR). Leave-one-out cross validation (LOOCV) was used to provide insight on how the predictive capabilities of the feature set might behave with respect to an independent patient cohort to address issues such as model overfitting. RBM3 expression was found to be significantly downregulated in highly aggressive GS ≥ 8 PCa samples compared to other Gleason scores (P < 0.0001) and significantly down-regulated in recurrent PCa samples compared to non-recurrent samples (P = 0.0377). An eleven-feature nuclear morphometric MLR model accurately identified aggressive PCa, yielding a receiver operating characteristic area under the curve (ROC-AUC) of 0.90 (P < 0.0001) in the raw data set and 0.77 (95% CI: 0.83-0.97) for LOOCV testing. The same eleven-feature model was then used to predict recurrence, yielding a ROC-AUC of 0.92 (P = 0.0004) in the raw data set and 0.76 (95% CI: 0.64-0.87) for LOOCV testing. The RBM3 biomarker alone is a strong prognostic marker for the prediction of aggressive PCa and biochemical recurrence. Further, RBM3 appears to be down-regulated in aggressive and recurrent tumors.

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.

Age-related responses to a bout of mechanotherapy in skeletal muscle of rats.

Cyclic compressive loading (CCL) is a massage mimetic that improves muscle regrowth from atrophy in adult rats. Therefore, we tested if a single bout of CCL increases anabolic signaling and protein synthesis in muscle during normal, weight-bearing conditions in gastrocnemius muscle from adult and aged rats. Male Brown Norway/F344 rats at 10 (adult) and 30 (aged) months of age were assigned control or CCL (receiving a single bout of CCL). Twenty-four hours following a single bout of CCL there was no change in protein synthesis, Akt, or GSK3β signaling at either age, despite adult rats having higher abundance and activation of mechanosensitive pathways (integrins and integrin-linked kinase). Murf1 was elevated in response to CCL in both age groups, potentially indicating muscle remodeling. Muscle from aged rats exhibited an increase in heat shock protein (HSP) 25 and HSP70 and in the cold shock protein RNA-binding motif 3 (RBM3), demonstrating a unique stress response to CCL in aged muscle only. Finally, muscle from aged rats exhibited higher basal protein synthesis that was corroborated by elevated eIF2Bε and rpS6 signaling, without an additional effect of CCL. In summary, a single bout of CCL does not have anabolic effects on skeletal muscle during normal, weight-bearing conditions, even though it has previously been shown to improve regrowth from atrophy. These data demonstrate that interventions that may help recover from atrophy do not necessarily induce muscle hypertrophy in unperturbed conditions.NEW & NOTEWORTHY Massage has been demonstrated to be an effective mechanotherapy to improve recovery from atrophy in adult skeletal muscle; however, this study shows that a single bout of massage fails to increase protein synthesis or anabolic signaling in adult or aged skeletal muscle during normal, weight-bearing conditions. Altogether, our data suggest massage is a useful mechanotherapy for preserving skeletal muscle when combined with other interventions but is not an anabolic stimulus on its own.

Robust RBM3 and β-klotho expression in developing neurons in the human brain

RNA binding motif 3 (RBM3) is a powerful neuroprotectant that inhibits neurodegenerative cell death in vivo and is a promising therapeutic target in brain ischemia. RBM3 is increased by the hormone fibroblast growth factor 21 (FGF21) in an age- and temperature-dependent manner in rat cortical neurons. FGF21 receptor binding is controlled by the transmembrane protein β-klotho, which is mostly absent in the adult brain. We discovered that RBM3/β-klotho is unexpectedly high in the human infant vs. adult brain (hippocampus/prefrontal cortex). The use of tissue homogenates in that study precluded a comparison of RBM3/β-klotho expression among different CNS cell-types, thus, omitted key evidence (i.e. confirmation of neuronal expression) that would otherwise provide a critical link to support their possible direct neuroprotective effects in humans. This report addresses that knowledge gap. High-quality fixed human hippocampus, cortex, and hypothalamic tissues were acquired from the NIH Neurobiobank (<1 yr (premature born) infants, 1 yr, 4 yr, and 34 yr). Dual labeling of cell-type markers vs. RBM3/β-klotho revealed enriched staining of targets in neurons in the developing brain. Identifying that RBM3/β-klotho is abundant in neurons in the immature brain is fundamentally important to guide protocol design and conceptual frameworks germane to future testing of these neuroprotective pathways in humans.

Infants Uniquely Express High Levels of RBM3 and Other Cold-Adaptive Neuroprotectant Proteins in the Human Brain

Neuroprotective cold-shock proteins (CSPs) are abundant in the normothermic neonatal rodent brain but decrease with advancing neurodevelopmental age and are low or absent in the adult brain. It has not been established if neurodevelopmental age alters the baseline expression of CSPs in the human brain. Here, we tested the hypothesis that protein levels of RNA-binding motif 3 (RBM3), reticulon-3 (RTN3), and cold-induced RNA-binding protein (CIRBP) are abundant in the normothermic developing human brain but low-to-absent in adults. We also tested if β-klotho (KLB) is expressed in the developing brain; KLB functions as a coreceptor that controls tissue-specific binding and activity of the systemically circulating thermogenic hormone fibroblast growth factor 21 (FGF21), and is predominantly expressed in the liver, pancreas, and in adipose cells. Methods: Hippocampi and anterior prefrontal cortices (aPFCs/BA10) from a total of 20 male and 20 female subjects were obtained from the NIH NeuroBioBank. CSP and KLB levels were measured in: infants < 1 year old (n = 8), toddlers aged 1–2 years (n = 8), children aged 3–5 years (n = 7), 18-year-old adolescents (n = 8), and adults aged 31–34 years (n = 8). An equal number of male and female (n = 4 each) samples were pooled into each age group, except in the 3- to 5-year-olds which comprised 3 male and 4 female specimens due to sample availability. In total, 78 whole-brain tissues were dissociated using a bead-based Precellys homogenizer to generate equivalent homogenates, and levels of protein targets subsequently analyzed by Western blotting. Results: Infants had the highest levels of RBM3 and other CSPs in the brain compared to all other ages. In the hippocampus, CSPs were detected predominantly in infants. In the aPFC, CSP levels were highest in infants, moderate-to-low in toddlers/children, and below assay detection limits in adolescents/adults. Germane to the thermogenic FGF21/KLB signaling axis, our results confirm that KLB is absent in the adult hippocampus/aPFC as reported by others. In contrast, we report for the first time that KLB is abundant in the early developing human brain; KLB levels were highest in the infant hippocampus/aPFC and moderately expressed in toddlers. RBM3 is a potent neuroprotective CSP. Thus, the impact of these findings on the observed efficacy of therapeutic hypothermia in neonatal brain injury merits further investigation.

Impact of RNA‑binding motif 3 expression on the whole transcriptome of prostate cancer cells: An RNA sequencing study.

RNA‑binding motif 3 (RBM3) is a cold‑shock protein that has been previously shown to attenuate cancer stem cell‑like features in prostate cancer (PCa) cells. However, the mechanism underlying RBM3 regulation in PCa cells is largely unknown. The present study investigated the impact of RBM3 expression on the whole transcriptome of PCa cells using high‑throughput RNA sequencing (RNA‑seq). Differentially expressed genes (DEGs) that were identified through RNA‑seq were applied to Gene Ontology (GO), pathway analysis, pathway‑action networks and protein‑protein interaction network analysis. GO and pathway ananlyses showed that RBM3 expression was associated with several metabolism pathways. Combining GO analysis and pathway analysis, certain DEGs, including phospholipase A2 group IIA (PLA2G2A), PLA2G2F, PLA2G4C, endothelin1, cytochrome P450 family2 subfamilyB member6, Gprotein subunitγ5, nitric oxide synthase3 and CD38 molecule, were shown to be closely associated with RBM3 regulation in PCa cells. Furthermore, the changes in expression of selected genes upon RBM3‑knockdown in RNA‑seq were confirmed by separate reverse transcription‑quantitative‑polymerase chain reaction, validating the results of RNA‑seq. Thus, the present study provides a series of valuable reference genes and pathways for the future study of the pathogenic role of RBM3 in the development of PCa.

Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons.

ObjectiveTherapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression.MethodsExperiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis.ResultsExposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels.ConclusionModerate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.

Podocalyxin-like and RNA-binding motif protein 3 are prognostic biomarkers in urothelial bladder cancer: a validatory study

BackgroundUrothelial bladder cancer (UBC) is a disease that often is discovered when the tumour is non-muscle invasive, i.e. in Ta or T1 stage. Some patients will progress into muscle-invasive disease, a potentially deadly condition. Although there are some prognostic models, the need for prognostic and predictive biomarkers is considerate and urgent. Membranous expression of podocalyxin-like protein 1 (PODXL) and low expression of the RNA-binding motif 3 (RBM3) has previously been shown to be associated with an aggressive tumour phenotype and poor prognosis in several forms of cancer, including UBC. In this study, we sought to validate the prognostic impact of PODXL and RBM3 in an independent cohort of UBC.MethodsUsing tissue microarrays and immunohistochemistry, PODXL and RBM3 expression was evaluated in 272 incident UBC cases from the prospective, population-based cohort study Malmö Diet and Cancer. Kaplan-Meier analysis and Cox proportional hazards modelling were used to evaluate the prognostic impact of these markers on 5-year overall survival (OS).ResultsIn line with previous studies, both membranous PODXL expression and low RBM3 expression was significantly associated with disadvantageous clinicopathological features. Membranous PODXL expression was significantly associated with a reduced 5-year overall survival in the entire cohort (univariable HR 3.28; 95% CI 1.89–5.69), but this association did not remain significant in multivariable analysis. In T1 tumours, PODXL was significantly associated with reduced survival in univariable analysis (HR = 2.83; 95% CI 1.04–7.72) and borderline significant in multivariable analysis (HR = 2.60; 95% CI 0.91–7.39). Low RBM3 expression was an independent predictor of a reduced survival in the entire cohort (univariable HR 3.19; 95% CI 2.02–5.04, and multivariable HR 1.85; 95% CI 1.11–3.09), and in T1 tumours (univariable HR 2.64; 95% CI 1.11–6.27, and multivariable HR 2.63; 95% CI 1.01–6.84).ConclusionsA link between membranous PODXL expression and clinically more aggressive tumours was further confirmed, but PODXL expression was not an independent prognostic biomarker in this study. Low RBM3 expression was validated as an independent factor of poor prognosis in UBC, including T1 disease. These findings suggest that these biomarkers could be useful in stratifying patients with non-muscle invasive disease for more aggressive first line treatment.