Subunit Of Kinase Research Articles

A Live-Cell Imaging Assay for Nuclear Entry of cAMP-Dependent Protein Kinase Catalytic Subunits Stimulated by Endogenous GPCR Activation.

Nuclear entry of cAMP-dependent protein kinase catalytic subunits is typically inferred from changes in net protein amount or kinase activity in the nucleus. Previous methods to directly assess nuclear entry require kinase subunit overexpression and/or supraphysiological cAMP elevation. We describe a method to detect nuclear entry of catalytic subunits expressed at an endogenous level in living cells, stimulated by cAMP in a physiological range, and in real time.

Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function

Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca2+ sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins’ (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.

Increased Pathogenicity of the Nematophagous Fungus Drechmeria coniospora Following Long-Term Laboratory Culture.

Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.

Abstract P199: Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo

Abstract In an initial safety evaluation in domestic cats suffering from oral squamous cell carcinomas (FOSCC), nine cat subjects were treated IV with 5-6 doses given twice weekly of s50 RNAi feCK2 at either 2 or 20 ug/kg (Cannon et. al., Hu Gene Ther Clin Devel 28(2):80-6, 2017). Treatment was generally well tolerated. In one case, a clear drug-related Grade 3 side effect was rapid tumor death (tumor lysis) resulting in an open wound. This issue has been addressed successfully in separate mice studies with longer, lower-dose SQ regimens. Protein kinase CK2, as a promising target in oncology, plays vital roles in cell growth, proliferation, and suppression of apoptosis (Ahmed et al., Trends Cell Biol 12: 226-30, 2002). Anti-feCK2aa’ is a tumor-targeted 20 nm crystalline capsule bearing single-stranded RNAi oligos against both CK2 catalytic kinase subunits with a ligand-coated shell derived from Tenascin-C (tenfibgen). Tenascin-C is upregulated in multiple solid tumors and is present in tumors throughout their lifecycle. The capsule’s ultrasmall size enables efficient raft-mediated delivery of oligos to the perinuclear space of the target cell, and the ability to reach metastases without reliance on EPR (Unger et. al, Mol Cancer Ther 13:2018-29, 2014). Initial mechanistic work focused on changes in clinically scored IHC of the CK2a subunit in study tumors. In an effort to develop a future working hypothesis, remaining paraffin-embedded tissues were retrospectively examined for both kinase subunits of CK2 as a mixture (CK2aa’), cleaved PARP (CP, apoptosis marker) and Ago2 (critical enzyme required for RNAi drug activity). Previous work in murine models showed variation of Ago2 levels in primary human and xenograft tumors. Further, model xenograft and syngeneic tumors responded with increased Ago2 levels and drug activity upon 10 mg/ml IV priming followed by low-dose SQ treatment (Cancer Res 2016;76(14 Suppl):3746). Using confocal fluorescence microscopy, we observed that decreased post-treatment CK2aa’ and increased CP corresponded positively with clinical tumor responses while non-responders showed an inverse relationship. Examination of arrayed human HNC samples also showed a variation in Ago2 levels but premalignant oral lesions from a human chemoprevention trial did not. These observations suggest a possible future path for feline patient therapy of advanced lesions using Ago2 stratification, potential IV priming and more convenient, longer-term SQ dosing administered by feline caregivers. A path for human therapy may lie through chemoprevention with much to be learned from feline studies. Funding: University of Minnesota Clinical and Translational Sciences Award (1UL1 RR033183-01), NIH SBIR Contract HHSN26120 1300030C, N01-CN-15000, Mod#5, V.A. Merit Review research funds (BX00173 and BX003282) and NCI Award CA150182. Citation Format: Frank G. Ondrey, Gretchen M. Unger, Claire M. Cannon, Janeen H. Trembley, BT Kren, Jaime F. Modiano, Khalil Ahmed. Tenfibgen nanoencapsulated RNAi feCK2 inhibits protein kinase CK2 and induces apoptosis in feline oral squamous cell carcinomas in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P199.

A Pan-Cancer Analysis of Clinical Prognosis and Immune Infiltration of CKS1B in Human Tumors.

Although more and more evidence supports CDC28 protein kinase subunit 1B (CKS1B) is involved significantly in the development of human cancers, most of the researches have focused on a single disease, and pan-cancer studies conducted from a holistic perspective of different tumor sources have not been reported yet. Here, for the first time, we investigated the potential oncogenic and prognostic role of CKS1B across 33 tumors based on public databases and further verified it in a small scale by RNA sequencing or quantitative real-time PCR. CKS1B was generally highly expressed in a majority of tumors and had a notable correlation with the prognosis of patients, but its prognostic significance in different tumors was not exactly the same. In addition, CKS1B expression was also closely related to the infiltration of cancer-associated fibroblasts in tumors such as breast invasive carcinoma, kidney chromophobe, lung adenocarcinoma, and tumor-infiltrating lymphocytes in tumors such as glioblastoma multiforme, bladder urothelial carcinoma, and brain lower grade glioma. Moreover, reduced CKS1B methylation was observed in certain tumors, for example, adrenocortical carcinoma. Cell cycle and kinase activity regulation and PI3K-Akt signaling pathway were found to be involved in the functional mechanism of CKS1B. In conclusion, our first pan-cancer analysis of CKS1B contributes to a better overall understanding of CKS1B and may provide a new target for future cancer therapy.

Role of Inflammation and Redox Status on Doxorubicin-Induced Cardiotoxicity in Infant and Adult CD-1 Male Mice.

Doxorubicin (DOX) is a topoisomerase II inhibitor commonly used in the treatment of several types of cancer. Despite its efficacy, DOX can potentially cause fatal adverse effects, like cardiotoxicity. This work aimed to assess the role of inflammation in DOX-treated infant and adult mice and its possible link to underlying cardiotoxicity. Two groups of CD-1 male mice of different ages (infants or adults) were subjected to biweekly DOX administrations, to reach a cumulative dose of 18.0 mg/kg, which corresponds approximately in humans to 100.6 mg/m2 for infants and 108.9 mg/m2 for adults a clinically relevant dose in humans. The classic plasmatic markers of cardiotoxicity increased, and that damage was confirmed by histopathological findings in both groups, although it was higher in adults. Moreover, in DOX-treated adults, an increase of cardiac fibrosis was observed, which was accompanied by an increase in specific inflammatory parameters, namely, macrophage M1 and nuclear factor kappa B (NF-κB) p65 subunit, with a trend toward increased levels of the tumor necrosis factor receptor 2 (TNFR2). On the other hand, the levels of myeloperoxidase (MPO) and interleukin (IL)-6 significantly decreased in DOX-treated adult animals. In infants, a significant increase in cardiac protein carbonylation and in the levels of nuclear factor erythroid-2 related factor 2 (Nrf2) was observed. In both groups, no differences were found in the levels of tumor necrosis factor (TNF-α), IL-1β, p38 mitogen-activated protein kinase (p38 MAPK) or NF-κB p52 subunit. In conclusion, using a clinically relevant dose of DOX, our study demonstrated that cardiac effects are associated not only with the intensity of the inflammatory response but also with redox response. Adult mice seemed to be more prone to DOX-induced cardiotoxicity by mechanisms related to inflammation, while infant mice seem to be protected from the damage caused by DOX, possibly by activating such antioxidant defenses as Nrf2.

Cyclin E1 in Murine and Human Liver Cancer: A Promising Target for Therapeutic Intervention during Tumour Progression

Simple SummaryThe cell cycle regulator Cyclin E1 is a key mediator and biomarker of liver cancer progression in mice and man independent of its canonical interacting partner Cyclin-dependent kinase 2. Over-expression of Cyclin E1 during hepatocarcinogenesis modulates several distinct biological processes such as proliferation, DNA damage response, stemness, invasion and the tumour microenvironment. Interventional depletion of Cyclin E1 in the course of liver cancer progression significantly reduces tumour burden. In contrast, the expression of Cyclin-dependent kinase 2 is dispensable for the progression of liver cancer in mice and lacked diagnostic or prognostic value in patients. Thus, specific inhibition of Cyclin E1 expression represents a promising strategy for the treatment of liver cancer.Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.

Multimodality Imaging and Genetics of Primary Mucosal Melanomas and Response to Treatment.

Mucosal melanomas (MMs) are rare and aggressive tumors that arise from melanocytes in the mucosal tissues that line the respiratory, gastrointestinal, and urogenital tracts. Most MMs occur during the 6th and 7th decades of life. MMs may be asymptomatic but may also cause bleeding, pain, and itching, depending on the site of origin. Because of their asymptomatic or oligosymptomatic nature and the difficulty of visualizing them in some cases, they are often advanced tumors at patient presentation. MM staging varies depending on the site of the primary tumor. A simplified staging system allows classification of clinically localized disease as stage I, regional nodal involvement as stage II, and distant metastasis as stage III. MM differs genetically from its cutaneous counterparts. Common drivers in cutaneous melanoma such as B-raf proto-oncogene serine/threonine kinase (BRAF) have a lower mutation rate in MM, whereas mutations of other genes including the KIT proto-oncogene, receptor tyrosine kinase (KIT) and splicing factor 3b subunit 1 gene (SF3B1) are more common in MM. Complete resection is the best curative option. However, surgical intervention with wide local excision and negative margins may be difficult to attain because of the local anatomy and the extent of disease. In addition, despite aggressive surgical resection, most patients develop local recurrence and metastatic disease. Recent advances in the treatment of melanoma include immunotherapy and targeted therapy. Unfortunately, MMs have a relatively poor prognosis, with an overall 5-year survival rate of 25%. Online supplemental material is available for this article. ©RSNA, 2021.

RRM1 Expression as a Prognostic Biomarker for Unresectable or Recurrent Biliary Tract Cancer Treated with Gemcitabine plus Cisplatin.

The combination of gemcitabine plus cisplatin (GP) is regarded as a first-line treatment for patients with unresectable or recurrent biliary tract cancer (BTC). Several proteins including human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (DCK), cytidine deaminase (CDA), and ribonucleotide reductase subunit 1 (RRM1) are known to be involved in gemcitabine uptake and metabolism. This study was aimed to identify the predictive and prognostic values of these biomarkers in patients who treated with GP for advanced BTC. Tumor samples were obtained from 34 patients with unresectable or recurrent BTC who were treated with GP between August 2015 and February 2018. Intratumoral expression of hENT1, DCK, CDA and RRM1 was determined by immunohistochemistry and analyzed for association with chemotherapy response, progression-free survival (PFS) and overall survival (OS). Median OS was significantly longer in the RRM1-negative group than in the RRM1-positive (9.9 months vs. 5.9 months, p = 0.037). Multivariate adjustment analyses also demonstrated RRM1 expression as an independent prognostic factor for OS in patients treated with GP chemotherapy. Increased intratumoral expression of RRM1 on immunohistochemical staining may be a biomarker predicting poor survival in patients with GP chemotherapy for advanced BTC. Large-scale well-predefined prospective research is needed to validate the utility of biomarkers in clinical practice.

Endocrine resistance in breast cancer: from molecular mechanisms to therapeutic strategies.

Estrogen receptor-positive (ER +) breast cancer accounts for approximately 75% of all breast cancers. Endocrine therapies, including selective ER modulators (SERMs), aromatase inhibitors (AIs), and selective ER down-regulators (SERDs) provide substantial clinical benefit by reducing the risk of disease recurrence and mortality. However, resistance to endocrine therapies represents a major challenge, limiting the success of ER + breast cancer treatment. Mechanisms of endocrine resistance involve alterations in ER signaling via modulation of ER (e.g., ER downregulation, ESR1 mutations or fusions); alterations in ER coactivators/corepressors, transcription factors (TFs), nuclear receptors and epigenetic modulators; regulation of signaling pathways; modulation of cell cycle regulators; stress signaling; and alterations in tumor microenvironment, nutrient stress, and metabolic regulation. Current therapeutic strategies to improve outcome of endocrine-resistant patients in clinics include inhibitors against mechanistic target of rapamycin (mTOR), cyclin-dependent kinase (CDK) 4/6, and the phosphoinositide 3-kinase (PI3K) subunit, p110α. Preclinical studies reveal novel therapeutic targets, some of which are currently tested in clinical trials as single agents or in combination with endocrine therapies, such as ER partial agonists, ER proteolysis targeting chimeras (PROTACs), next-generation SERDs, AKT inhibitors, epidermal growth factor receptor 1 and 2 (EGFR/HER2) dual inhibitors, HER2 targeting antibody-drug conjugates (ADCs) and histone deacetylase (HDAC) inhibitors. In this review, we summarize the established and emerging mechanisms of endocrine resistance, alterations during metastatic recurrence, and discuss the approved therapies and ongoing clinical trials testing the combination of novel targeted therapies with endocrine therapy in endocrine-resistant ER + breast cancer patients.

Correction of a knock-in mouse model of acrodysostosis with gene therapy using a rAAV9-CAG-human PRKAR1A vector.

Acrodysostosis is a rare skeletal dysplasia caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). In a knock-in mouse model (PRKAR1Awt/mut) expressing one copy of the recurrent R368X mutation, we tested the effects of a rAAV9-CAG-human PRKR1A (hPRKAR1A) vector intravenously administered at 4 weeks of age. Caudal vertebrae and tibial diaphyses contained 0.52 ± 0.7 and 0.13 ± 0.3 vector genome per cell (VGC), respectively, at 10 weeks of age and 0.22 ± 0.04 and 0.020 ± 0.04 at 16 weeks while renal cortex contained 0.57 ± 0.14 and 0.26 ± 0.05 VGC. Vector-mediated hPRKAR1A expression was found in growth plate chondrocytes, osteoclasts, osteoblasts, and kidney tubular cells. Chondrocyte architecture was restored in the growth plates. Body length, tail length, and body weight were improved in vector treated PRKAR1Awt/mut mice, not the bone length of their limbs. These results provide one of the few proofs for gene therapy efficacy in a mouse model of chondrodysplasia. In addition, the increased urinary cAMP of PRKAR1Awt/mut mice was corrected almost to normal. In conclusion, gene therapy with hPRKAR1A improved skeletal growth and kidney dysfunction, the hallmarks of acrodysostosis in R368X mutated mice and humans.

P70.14 PRKDC Mutations Recurrently Found in Non-Small Cell Lung Cancer in East Asian Patients

Protein kinase, DNA-activated, catalytic polypeptide (PRKDC) encodes a 465 kDa catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) that plays a pivotal role in the maintenance of genomic stability and is a critical component of DNA double-strand break repair and recombination. Because of the rarity of those mutations, associated clinical features and prognostic significance have not been thoroughly described so far. The aim of this study is to investigate mutations and prognosis of NSCLC harboring PRKDC mutations. A total of 328 patients with non-small-cell lung cancer were recruited between July 2012 and December 2018. The status of PRKDC mutations and other genes were detected by next generation sequencing. PRKDC gene mutation rate was 1.22% (4/328) in non-small cell lung cancer, including D3411N(1 patient), D2724H(1 patient), M4002L(1 patient) and A1634T(1 patient), and median overall survival (OS) for these patients was 14.0 months. Among them, all patients were PRKDC gene with co-occurring mutations. Briefly, patients with (n=2) or without (n=2) co-occurring BCOR mutations had a median OS of 15.5 months and 22.0 months respectively (P=0.64); patients with (n=2) or without (n=2) co-occurring TP53 mutations had a median OS of 12.5 months and 19.0 months respectively (P=0.23); patients with (n=2) or without (n=2) co-occurring ARID1A mutations had a median OS of 17.5 months and 14.0 months respectively (P=0.81); patients with (n=2) or without (n=2) co-occurring KRAS mutations had a median OS of 12.5 months and 19.0 months respectively (P=0.23). BCOR, TP53, ARID1A, KRAS gene accompanied may have less correlation with PRKDC mutation in NSCLC patients. Next generation sequencing provides a simplified strategy and reasonably high detection rate for PRKDC mutation, which suggested application of the strategies into clinical molecular diagnostics.

Acetylation of ANXA1 reduces caspase-3 activation by enhancing the phosphorylation of caspase-9 under OGD/R conditions

SIRT2, a Class III HDACs, aggravates cell damage and activates caspase-3 under oxygen-glucose deprivation/reoxygenation and glucose (OGD/R) conditions. In this paper, we demonstrated the adverse effects of SIRT2 on cells after OGD/R attacks, which were mediated by increased interactions between SIRT2 and ANXA1, and explicated the mechanisms by which acetylated ANXA1 affects the activation and cleavage of caspase-3. We found that the acetylation level of ANXA1 was decreased through the its increased interactions with SIRT2 after the OGD/R insult. The lysine 312 residue (K312) was selected as the target site in ANXA1 because it is associated with SIRT2, and its mimic (K312Q) and silent (K312R) mutants were then established through site mutagenesis. Under OGD/R conditions, the acetylation mimic of K312Q ANXA1 accumulated in the cytoplasm, decreasing the activity levels of caspase-3 and the upstream initiator caspase-9, compared with the levels of WT and K312R ANXA1. Furthermore, K312Q ANXA1 intervened in the interactions of caspase-3 to caspase-9 by increasing the phosphorylation levels of caspase-9 and inhibited its cleavage by downregulating PRKAR2B, a regulatory subunit of protein kinase A (PKA). In this process, K312Q ANXA1 was found to be directly associated with PRKAR2B, diminishing its restriction on the catalytic subunit of PKA.In conclusion, acetylated ANXA1 can promote the phosphorylation of caspase-9 to decrease the activation of caspase-3 by enhancing the expression of a kinase upstream of caspase-9 after the OGD/R stimulation.

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.

Exosomal transfer of activated neutrophil-derived lncRNA CRNDE promotes proliferation and migration of airway smooth muscle cells in asthma.

Activated neutrophil-derived exosomes reportedly contribute to the proliferation of airway smooth muscle cells (ASMCs), thereby aggravating the airway wall remodeling during asthma; however, the specific mechanism remains unclear. Lipopolysaccharide (LPS)-EXO and si-CRNDE-EXO were extracted from the media of human neutrophils treated with LPS and LPS + si-CRNDE (a siRNA targets long non-coding RNA CRNDE), respectively. Human ASMCs were co-cultured with LPS-EXO or si-CRNDE-EXO, and cell viability, proliferation and migration were measured. The interplay of colorectal neoplasia differentially expressed (CRNDE), inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) and nuclear receptor subfamily 2 group C member 2 (TAK1) was explored using RNA immunoprecipitation (RIP) and Co-IP assays. A mouse model of asthma was induced using ovalbumin. CRNDE was upregulated in LPS-EXO and successfully transferred from LPS-treated neutrophils to ASMCs through exosome. Mechanically, CRNDE loaded in LPS-EXO reinforced TAK1-mediated IKKβ phosphorylation, thereby activating the nuclear factor kappa B (NF-κB) pathway. Functionally, silencing CRNDE in LPS-EXO, an IKKβ inhibitor, and an NF-κB inhibitor all removed the upregulation of cell viability, proliferation and migration induced by LPS-EXO in ASMCs. In the end, the in vivo experiment demonstrated that CRNDE knockdown in neutrophils effectively reduced the thickness of bronchial smooth muscle in a mouse model for asthma. Activated neutrophils-derived CRNDE was transferred to ASMCs through exosomes and activated the NF-κB pathway by enhancing IKKβ phosphorylation. The latter promoted the proliferation and migration of ASMCs and then contributed to airway remodeling in asthma.

Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKK\u03b2 promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice

BackgroundDuring inflammatory demyelination, TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF (solTNF), whereas TNFR2 mediates beneficial effects of transmembrane TNF (tmTNF) through oligodendroglia, microglia, and possibly other cell types. This model supports the use of selective inhibitors of solTNF/TNFR1 as anti-inflammatory drugs for central nervous system (CNS) diseases. A potential obstacle is the neuroprotective effect of solTNF pretreatment described in cultured neurons, but the relevance in vivo is unknown.MethodsTo address this question, we generated mice with neuron-specific depletion of TNFR1, TNFR2, or inhibitor of NF-κB kinase subunit β (IKKβ), a main downstream mediator of TNFR signaling, and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of solTNF neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wild-type (WT) and TNF and TNFR knockout cells and measuring N-methyl-d-aspartate (NMDA) excitotoxicity in vitro.ResultsNeither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone-induced demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid (KA) excitotoxicity model in mice and limited hippocampal neuron death. The protective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. As previously described, pretreatment of astrocyte-neuron co-cultures with solTNF (and therefore TNFR1) protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte tmTNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist.ConclusionsThese results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously described neuroprotective effects of solTNF against glutamate excitotoxicity in vitro are indirect and mediated via astrocyte tmTNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of solTNF/TNFR1 with maintenance of TNFR2 function would have combined anti-inflammatory and neuroprotective properties required for safe treatment of CNS diseases.

The Clp1 R140H mutation alters tRNA metabolism and mRNA 3′ processing in mouse models of pontocerebellar hypoplasia

Homozygous mutation of the RNA kinase CLP1 (cleavage factor polyribonucleotide kinase subunit 1) causes pontocerebellar hypoplasia type 10 (PCH10), a pediatric neurodegenerative disease. CLP1 is associated with the transfer RNA (tRNA) splicing endonuclease complex and the cleavage and polyadenylation machinery, but its function remains unclear. We generated two mouse models of PCH10: one homozygous for the disease-associated Clp1 mutation, R140H, and one heterozygous for this mutation and a null allele. Both models exhibit loss of lower motor neurons and neurons of the deep cerebellar nuclei. To explore whether Clp1 mutation impacts tRNA splicing, we profiled the products of intron-containing tRNA genes. While mature tRNAs were expressed at normal levels in mutant mice, numerous other products of intron-containing tRNA genes were dysregulated, with pre-tRNAs, introns, and certain tRNA fragments up-regulated, and other fragments down-regulated. However, the spatiotemporal patterns of dysregulation do not correlate with pathogenicity for most altered tRNA products. To elucidate the effect of Clp1 mutation on precursor messenger RNA (pre-mRNA) cleavage, we analyzed poly(A) site (PAS) usage and gene expression in Clp1 R140H/- spinal cord. PAS usage was shifted from proximal to distal sites in the mutant mouse, particularly in short and closely spaced genes. Many such genes were also expressed at lower levels in the Clp1 R140H/- mouse, possibly as a result of impaired transcript maturation. These findings are consistent with the hypothesis that select genes are particularly dependent upon CLP1 for proper pre-mRNA cleavage, suggesting that impaired mRNA 3' processing may contribute to pathogenesis in PCH10.

Elongin functions as a loading factor for Mediator at ATF6α-regulated ER stress response genes

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.

Geniposide exerts protective effects on spinal cord injury in rats by inhibiting the IKKs/NF-κB signaling pathway

BackgroundSpinal cord injury (SCI) is a traumatic condition of the central nervous system , which can cause nerve injury and affect nerve regeneration, thus leading to severe dysfunction of motor and sensory pathways, and unfortunately these effects are irreversible. Inflammatory response constitutes one of the important mechanisms of spinal cord secondary injury. Geniposide (Gen) is reported to possess anti-inflammation and neuronal repair capacities. ObjectivesTo investigate the effect and mechanism of Gen on motor function and inflammatory response in SCI rats. MethodsSprague-Dawley (SD) rats were randomly grouped, and the SCI model was established by Allen’s method. The motor function of rats was evaluated by the Basso, Beattie, and Bresnahan (BBB) scale. The protective effect of Gen on the injured spinal cord tissues was evaluated by measuring the water content, myeloperoxidase (MPO) activity, and levels of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6. Moreover, the protein level of the inflammation-related pathway was detected by spectrometry and Western blot assays. ResultsGen significantly promoted the recovery of SCI rats, decreased the edema of spinal cord tissues, reduced the area of cavity, increased the number of NF-200-positive neurons, as well as increased the number of horseradish peroxidase (HRP) retrograde tracing-positive neurons and regenerated axons with myelin sheath. Additionally, compared with the control group, the neutrophil infiltration, contents of TNF-α, IL-1β, and IL-6, the activity of inhibitor of nuclear factor κB kinase subunit β (IKKβ) kinase, and protein levels of (nuclear factor κB) NF-κB p65 and phosphorylated inhibitor of NF-κB (p-I-κB) in the Gen experimental group were significantly decreased. ConclusionGen effectively alleviated inflammatory response after SCI by inhibiting the IKKs/NF-κB signaling pathway and promoted the recovery of motor function and axon regeneration in rats. SignificanceThis study can provide novel insights for the early and effective intervention of SCI and confer basic data for the treatment of spinal cord secondary injury.

IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα

Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) is one of important kinases in inflammation to phosphorylate inhibitor of nuclear factor kappa-B (IκBα) and then activate nuclear factor kappa-B (NF-κB). Inhibition of IKKβ has been a therapeutic strategy for inflammatory and autoimmune diseases. Here we report that IKKβ is constitutively activated in healthy donors and healthy IkkβC46A (cysteine 46 mutated to alanine) knock-in mice although they possess intensive IKKβ–IκBα–NF-κB signaling activation. These indicate that IKKβ activation probably plays homeostatic role instead of causing inflammation. Compared to IkkβWT littermates, lipopolysaccharides (LPS) could induce high mortality rate in IkkβC46A mice which is correlated to breaking the homeostasis by intensively activating p-IκBα–NF-κB signaling and inhibiting phosphorylation of 5ʹ adenosine monophosphate-activated protein kinase (p-AMPK) expression. We then demonstrated that IKKβ kinase domain (KD) phosphorylates AMPKα1 via interacting with residues Thr183, Ser184, and Thr388, while IKKβ helix–loop–helix motifs is essential to phosphorylate IκBα according to the previous reports. Kinase assay further demonstrated that IKKβ simultaneously catalyzes phosphorylation of AMPK and IκBα to mediate homeostasis. Accordingly, activation of AMPK rather than inhibition of IKKβ could substantially rescue LPS-induced mortality in IkkβC46A mice by rebuilding the homeostasis. We conclude that IKKβ activates AMPK to restrict inflammation and IKKβ mediates homeostatic function in inflammation via competitively phosphorylating AMPK and IκBα.