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Smoking and tetramer tryptase accelerate intervertebral disc degeneration by inducing METTL14-mediated DIXDC1 m6 modification
Abstract
Although cigarette smoke (CS) and low back pain (LBP) are common worldwide, their correlations and the mechanisms of action remain unclear. We had shown that excessive activation of Mast Cells (MCs) and their proteases play key roles in CS associated diseases, like asthma, chronic obstructive pulmonary disease (COPD), blood coagulation and lung cancer. Previous studies also show that MCs and their proteases induce degenerative musculoskeletal disease. By using mice custom-designed smoke-exposure system, we demonstrated that CS results in intervertebral disc (IVDs) degeneration and the release of MC-restricted tetramer tryptases (TT) inside the IVDs. TT was found to regulate the expression of methyltransferase 14 (METTL14) at the epigenetic level by inducing N6-methyladenosine (m6A) deposition in the 3'-untranslated region (3'-UTR) of the transcript that encodes DIX Domain Containing 1 (DIXDC1). That reaction increases the mRNA stability and expression of Dixdc1. DIXDC1 functionally interacts with 'Disrupted in Schizophrenia-1' (DISC1) to accelerate the degeneration and senescence of nucleus pulposus (NP) cells by activating a canonical Wnt pathway. Our study demonstrates the association between CS, MC-derived TTs, and LBP. These findings raise the possibility that METTL14-medicated DIXDC1 m6A modification could serve as a potential therapeutic target to block the development of degeneration of the NP in LBP patients.
... Smoking and LBP are global public health concerns. Numerous studies [56][57][58] indicate that smoking increases the risk of LBP. Using a passive smoking model in rats, Nemoto et al. [56] found cracks, tears, and dislocations in the intervertebral discs. ...
... Using a passive smoking model in rats, Nemoto et al. [56] found cracks, tears, and dislocations in the intervertebral discs. A recent study [58] has also shown that smoking and tetramer tryptase accelerate intervertebral disc degeneration by inducing methyltransferase 14-mediated dishevelled-axin domaincontaining 1 m6 modifications. Smokers experience significantly greater levels of lumbar pain and dysfunction than non-smokers [57]. ...
Background
In China, the world’s largest developing country, low back pain (LBP) is a common public health issue affecting workability. This meta-analysis aimed to systematically assess the risk factors of LBP in the Chinese population.
Methods
Four English language and four Chinese databases were searched, and cross-sectional studies on the risk factors for LBP in Chinese populations were identified and collected. The search timeframe covered the period from the establishment of the database to November 2023. Two researchers independently reviewed the literature, extracted the data, and evaluated the risk of bias. Begg’s and Egger’s tests were used to evaluate publication bias.
Results
Fifteen cross-sectional studies involving 86,575 people were included. Seven risk factors for LBP were identified. Six risk factors were statistically significant: Cigarette smoking (odds ratio [OR] = 1.55; 95% confidence interval [CI]: 1.15, 2.08, P = 0.004, I² = 72%), body mass index (BMI) ≥ 28 kg/m² (OR = 4.51; 95% CI: 3.36, 6.07, P < 0.00001, I² = 8%), female sex (OR = 1.54; 95% CI: 1.25, 1.90, P < 0.0001, I² = 63%), vibration exposure at work (OR = 1.65; 95% CI: 1.16, 2.34, P = 0.006, I² = 84%), working overtime (OR = 2.57; 95% CI: 1.12, 5.91, P = 0.03, I² = 85%), and lack of exercise (OR = 2.48; 95% CI: 1.62, 3.78, P < 0.0001, I² = 0%). One risk factor that was not statistically significant was standing for long periods (OR = 1.02; 95% CI: 0.82, 1.26, P = 0.88, I² = 73%).
Conclusions
This study found that smoking, a BMI ≥ 28 kg/m², female sex, vibration exposure at work, working overtime, and lack of exercise may be risk factors for LBP in the Chinese population. Because the included studies were cross-sectional and the certainty of the evidence was very low, the results need to be interpreted cautiously. Multicentre, high-quality studies should be conducted in the future. To reduce the prevalence of LBP, the Chinese government and hospitals must develop early screening programs and implement effective preventive and interventional measures.
Trial registration
This study is registered in the PROSPERO database (No. CRD42023447857).
... Intervertebral disc degeneration (IDD) has been recognised as the main pathology contributing to Low Back Pain (LBP) which has resulted in an enormous socio-economic burden worldwide. Multiple factors have been reported to be involved in the aetiology, including excessive mechanical loading, nutritional deficiency, aging, smoking, trauma et al. [1][2][3][4]. Though the complicated mechanisms underlying the aetiology remain to be illustrated, there is a growing consensus that severe IDD is generally characterised by decreased disc height and torn annulus fibrosus, accompanied by impaired segmental stability, with an enhanced risk of radiculopathy, in which the disruption of extracellular matrix (ECM) homeostasis plays a pivotal role in promoting the whole IDD. ...
Intervertebral Disc Degeneration (IDD) is a multifactorial result contributing to Low Back Pain (LBP) while Cartilage Intermediate Layer Protein‐1 (CILP‐1) is gradually up‐regulated along with IDD. Whether CILP‐1 acts in a direct role promoting IDD via regulating matrix metabolism remains to be elucidated. Herein, we firstly detected the expression level of matrix‐related phenotypes in nucleus pulposus (NP) cells treated with CILP‐1, including ADAMTS, MMPs, IL‐6, Collagens, Aggrecan (ACAN) and SOX9. Meanwhile, the phosphorylation levels of MAPKs and NF‐κB were detected to explore the involved signalling pathways, which were further validated by inhibition experiments. Furthermore, molecular docking analysis was employed to evaluate the possibility of CD47 acting as the direct receptor mediating CILP's regulation above, which was further validated by immunoprecipitation and inhibition experiment. Our findings have made a comprehensive investigation into the regulatory effect of CILP‐1 on the matrix metabolism of NP cells and explored the underlying mechanism.
... METTL14 is highly present in NP cells from IVD patients, which stabilize NLRP3 mRNA in an IGFBP2-dependent manner and trigger pyroptotic NP cell death [29]. METTL14-medicated m6A modification of DIXDC1 has been reported as a potential therapeutic target to restrain the degeneration of NP in patients with LBP [35]. Our results revealed that METTL14 expression was increased with the increase of TNF-α concentration, decreased with the decrease of ZFAS1 expression, and increased with the decrease of miR-155-3p expression. ...
Background
This study investigates the mechanism of lncRNA ZFAS1 in pyroptosis of TNF-α-induced nucleus pulposus cells (NPCs) in intervertebral disc degeneration (IDD).
Methods
Mouse NPCs were isolated and induced by TNF-α to establish a cell model of IDD. The cell viability was evaluated by MTT assay. NLRP3, GSDMD-N, and cleaved-Caspase1 expressions were detected by Western blot. IL-1β and IL-18 contents were detected by ELISA. RT-qPCR was performed to determine ZFAS1, miR-155-3p, and METTL14 expressions. After intervening in ZFAS1 expression, the effect of ZFAS1 on pyroptosis was verified by Western blot and ELISA assays. RNA pull down or dual luciferase assay verified the binding between ZFAS1, miR-155-3p, and METTL14.
Results
TNF-α induced pyroptosis of NPCs and promoted ZFAS1 expression. Silence of ZFAS1 repressed pyroptosis of TNF-α-induced NPCs. Mechanistically, ZFAS1 upregulated the transcription of METTL14 by competitively binding to miR-155-3p, thus enhancing NLRP3/Caspase-1-mediated NPC pyroptosis. Inhibition of miR-155-3p or overexpression of METTL14 alleviated the inhibitory effect of ZFAS1 silencing on TNF-α-treated NPC pyroptosis.
Conclusion
ZFAS1 facilitates NLRP3/Caspase-1-mediated pyroptosis of NPCs in IDD via miR-155-3p/METTL14 axis.
... This response increases mRNA stability and hence DIXDC1 expression. DIXDC1 interacts functionally with schizophrenia 1 interrupted cells (DISC1), thereby accelerating the degeneration and senescence of NP cells through activation of the classical Wingless/Integrated (Wnt) pathway [33]. In view of the limited number of studies on IDD and m6A-related pathways, further research is required to gain a better understanding of the mechanisms involved. ...
Background
The mechanism for RNA methylation during disc degeneration is unclear. The aim of this study was to identify N6-methyladenosine (m6A) markers and therapeutic targets for the prevention and treatment of intervertebral disc degeneration (IDD).
Methods
Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and quantitative reverse transcription PCR (RT-qPCR) were employed to analyze m6A modifications of IDD-related gene expression. Bioinformatics was used to identify enriched gene pathways in IDD. m6A-RIP-qPCR was used to validate potential targets and markers.
Results and Conclusion
Human IDD samples exhibited a distinct m6A modification pattern that allowed associated genes and pathways to be identified. These genes had functions such as “nuclear factor kappa-B (NF-κB) binding” and “extracellular matrix components”, which are crucial for IDD pathogenesis. ANXA2 showed increased m6A modification in IDD, while SLC3A2 and PBX3 showed decreased m6A methylation. The results of this study offer novel insights for the prevention and treatment of IDD.
... Unhealthy lifestyles, notably characterized by insufficient physical activity, prolonged periods of sitting, and poor posture, impose an increased burden and pressure on intervertebral discs, thereby exacerbating the degenerative process [46]. Furthermore, smoking has been identified as a risk factor, as it diminishes the blood supply to intervertebral discs, influencing their overall health [47]. Occupational exposures, such as heavy lifting, prolonged periods of poor posture, and exposure to vibrations, may also contribute to the development of IVDD [48,49]. ...
Background
Intervertebral disc degeneration (IVDD) is widely recognized as one of the leading causes of low back pain. Intervertebral disc cells are the main components of the intervertebral disc (IVD), and their functions include synthesizing and secreting collagen and proteoglycans to maintain the structural and functional stability of the IVD. In addition, IVD cells are involved in several physiological processes. They help maintain nutrient metabolism balance in the IVD. They also have antioxidant and anti-inflammatory effects. Because of these roles, IVD cells are crucial in IVDD. When IVD cells are subjected to oxidative stress, mitochondria may become damaged, affecting normal cell function and accelerating degenerative changes. Mitochondria are the energy source of the cell and regulate important intracellular processes. As a key site for redox reactions, excessive oxidative stress and reactive oxygen species can damage mitochondria, leading to inflammation, DNA damage, and apoptosis, thus accelerating disc degeneration.
Aim of review
Describes the core knowledge of IVDD and oxidative stress. Comprehensively examines the complex relationship and potential mechanistic pathways between oxidative stress, mitochondrial dysfunction and IVDD. Highlights potential therapeutic targets and frontier therapeutic concepts. Draws researchers' attention and discussion on the future research of all three.
Key scientific concepts of review
Origin, development and consequences of IVDD, molecular mechanisms of oxidative stress acting on mitochondria, mechanisms of oxidative stress damage to IVD cells, therapeutic potential of targeting mitochondria to alleviate oxidative stress in IVDD.
The translational potential of this article
Targeted therapeutic strategies for oxidative stress and mitochondrial dysfunction are particularly critical in the treatment of IVDD. Using antioxidants and specific mitochondrial therapeutic agents can help reduce symptoms and pain. This approach is expected to significantly improve the quality of life for patients. Individualized therapeutic approaches, on the other hand, are based on an in-depth assessment of the patient's degree of oxidative stress and mitochondrial functional status to develop a targeted treatment plan for more precise and effective IVDD management. Additionally, we suggest preventive measures like customized lifestyle changes and medications. These are based on understanding how IVDD develops. The aim is to slow down the disease and reduce the chances of it coming back. Actively promoting clinical trials and evaluating the safety and efficacy of new therapies helps translate cutting-edge treatment concepts into clinical practice. These measures not only improve patient outcomes and quality of life but also reduce the consumption of healthcare resources and the socio-economic burden, thus having a positive impact on the advancement of the IVDD treatment field.
... 36 In addition, smoking promotes the exacerbation of IDD by a mechanism that may be related to the promotion of MCs hyperactivation and the release of proteases through the induction of mettl14-mediated modification of DIXDC1 m6. 37 The cellular and humoral immune responses are the two main subtypes of immune responses that occur in IDD. The development of vascular endothelial cells around the damaged disc can lead to lymphocyte aggregation to the injured area, recognition of antigens and activation of T lymphocytes. ...
Intervertebral disc degeneration is an important pathological basis for spinal degenerative diseases. The imbalance of the immune microenvironment and the involvement of immune cells has been shown to lead to nucleus pulposus cells death. This article presents a bibliometric analysis of studies on immune cells in IDD in order to clarify the current status and hotspots. We searched the WOSCC, Scopus and PubMed databases from 01/01/2001 to 08/03/2023. We analyzed and visualized the content using software such as Citespace, Vosviewer and the bibliometrix. This study found that the number of annual publications is increasing year on year. The journal study found that Spine had the highest number of articles and citations. The country/regions analysis showed that China had the highest number of publications, the USA had the highest number of citations and total link strength. The institutional analysis found that Shanghai Jiao Tong University and Huazhong University of Science Technology had the highest number of publications, Tokai University had the highest citations, and the University of Bern had the highest total link strength. Sakai D and Risbud MV had the highest number of publications. Sakai D had the highest total link strength, and Risbud MV had the highest number of citations. The results of the keyword analysis suggested that the current research hotspots and future directions continue to be the study of the mechanisms of immune cells in IDD, the therapeutic role of immune cells in IDD and the role of immune cells in tissue engineering for IDD.
Low back pain is a widespread condition that significantly impacts quality of life, with intervertebral disc degeneration (IDD) being a major contributing factor. However, the underlying mechanisms of IDD remain poorly understood, necessitating further investigation. Environmental risk factors, such as mechanical stress and cigarette smoke, elevate reactive oxygen species levels from both endogenous and exogenous sources, leading to redox imbalance and oxidative stress. The endoplasmic reticulum (ER) and mitochondria, two key organelles responsible for protein folding and energy production, respectively, are particularly vulnerable to oxidative stress. Under oxidative stress conditions, ER stress and mitochondrial dysfunction occur, resulting in unfolded protein response activation, impaired biosynthetic processes, and disruptions in the tricarboxylic acid cycle and electron transport chain, ultimately compromising energy metabolism. Prolonged and excessive ER stress can further trigger apoptosis through ER–mitochondrial crosstalk. Given the unique microenvironment of the intervertebral disc (IVD)—characterized by hypoxia, glucose starvation, and region-specific cellular heterogeneity—the differential effects of environmental stressors on distinct IVD cell populations require further investigation. This review explores the potential mechanisms through which environmental risk factors alter IVD cell activities, contributing to IDD progression, and discusses future therapeutic strategies aimed at mitigating disc degeneration.
There is still a lack of high-level evidence regarding the causal relationship between smoking and intervertebral disc degenerative diseases. This study utilized data from genome wide analysis studies and conducted two-sample Mendelian randomization analyses across multiple heterogeneous datasets. We evaluated the causal relationships between smoking behavior, serum inflammatory factors, serum chemokines, and intervertebral disc degeneration. Sensitivity analysis was performed to examine data heterogeneity and the pleiotropy of causal effects. The results indicated that smokers were liable to develop intervertebral disc degeneration (OR 1.770; 95 % CI, 1.519–2.064; p = 2.992 × 10–13), and long-term smoking behavior increased the risk of intervertebral disc degeneration (OR 1.715; 95 % CI 1.475–1.994; P = 2.220 × 10–12). Additionally, a causal relationship was confirmed between serum IL-1β level and intervertebral disc degeneration (OR 1.087; 95 % CI, 1.023–1.154; p = 0.007). The “smoking index” representing lifelong smoking habit was also found to be causally related to serum MCP-3 level(β = 0.292; SE = 0.093; p = 0.002). All of the causality mentioned above remained stable in sensitivity tests. Based on the analysis results and fundamental medicine theories around macrophage-induced inflammation in degenerative intervertebral discs, we have constructed a new mechanism that long-term smoking could induce an increase in serum MCP-3 level, promoting the gathering and activation of monocyte macrophages. Furthermore, the recruited macrophages led to an increase in local IL-1β within the intervertebral disc, ultimately exacerbating the process of intervertebral disc degeneration. What we have found is expected to accelerate the development of prevention and treatment of intervertebral disc degeneration.
Chronic obstructive pulmonary disease (COPD) ranks as the third leading cause of global illness and mortality. It is commonly triggered by exposure to respiratory irritants like cigarette smoke or biofuel pollutants. This multifaceted condition manifests through an array of symptoms and lung irregularities, characterized by chronic inflammation and reduced lung function. Present therapies primarily rely on maintenance medications to alleviate symptoms, but fall short in impeding disease advancement. COPD’s diverse nature, influenced by various phenotypes, complicates diagnosis, necessitating precise molecular characterization. Omics-driven methodologies, including biomarker identification and therapeutic target exploration, offer a promising avenue for addressing COPD’s complexity. This analysis underscores the critical necessity of improving molecular profiling to deepen our comprehension of COPD and identify potential therapeutic targets. Moreover, it advocates for tailoring treatment strategies to individual phenotypes. Through comprehensive exploration-based molecular characterization and the adoption of personalized methodologies, innovative treatments may emerge that are capable of altering the trajectory of COPD, instilling optimism for efficacious disease-modifying interventions.
Background:
Currently, many emerging polycyclic aromatic hydrocarbons (PAHs) have been found to be widely present in the environment. However, little has been reported about their toxicity, particularly in relation to CYP1A1.
Objectives:
This study aimed to explore the toxicity of naphtho[2,1-a]pyrene (N21aP) and elucidate the mechanism underlying N21aP-induced expression of CYP1A1.
Methods:
The concentration and sources of N21aP were detected and analyzed by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) and diagnostic ratio analysis. Then the effects of CYP1A1 on the toxicity of N21aP were conducted in male wild-type (WT) and Cyp1a1 knockout mice exposed to N21aP (0.02, 0.2, and 2mg/kg) through intratracheal instillation. Further, the aryl hydrocarbon receptor (AhR) pathway was examined through luciferase and chromatin immunoprecipitation (ChIP) assays. N6-methyladenosine (m6A) modification levels were measured on global RNA and specifically on CYP1A1 mRNA using dot blotting and methylated RNA immunoprecipitation-quantitative real-time polymerase chain reaction (MeRIP qRT-PCR), with validation by m6A inhibitors, DAA and SAH. m6A sites on CYP1A1 were identified by bioinformatics and luciferase assays, and CYP1A1 mRNA's interaction with IGF2BP3 was confirmed by RNA pull-down, luciferase, and RNA binding protein immunoprecipitation (RIP) assays.
Results:
N21aP was of the same environmental origin as benzo[a]pyrene (BaP) but was more stably present in the environment. N21aP could be metabolically activated by CYP1A1 to produce epoxides, causing DNA damage and further leading to lung inflammation. Importantly, in addition to the classical AhR pathway (i.e., BaP), N21aP also induced CYP1A1 expression with a posttranscriptional modification of m6A in CYP1A1 mRNA via the METTL14-IGF2BP3-CYP1A1 axis. Specifically, in the two recognition sites of METTL14 on the CYP1A1 mRNA transcript (position at 2700 and 5218), a methylation site (position at 5218) in the 3'-untranslated region (UTR) was recognized by IGF2BP3, enhanced the stability of CYP1A1 mRNA, and finally resulted in an increase in CYP1A1 expression.
Discussion:
This study systematically demonstrated that in addition to AhR-mediated transcriptional regulation, N21aP, had a new additional mechanism of m6A-mediated posttranscriptional modification, jointly contributing to CYP1A1 expression. Given that PAHs are the metabolic substrates of CYP1A1, this study not only helps to understand the significance of environment-genetic interactions for the toxicity of PAHs but also helps to better understand the health risks of the emerging PAHs at environmental exposure levels. https://doi.org/10.1289/EHP14055.
Background:
Low back pain (LBP) is a debilitating phenomenon that significantly impacts quality-of-life (QoL). The PainDETECT questionnaire (PD-Q) is a screening tool aimed at distinguishing nociceptive pain (NoP) and neuropathic pain (NeP) classifications. Associations between these classifications and patient-reported outcome measures (PROMs) and sociodemographic parameters are yet to be established.
Objective:
The study aimed to determine the relationship between NeP as assessed by the PD-Q and pain, disability, QoL, and sociodemographic factors.
Methods:
A retrospective analysis of an ongoing prospectively collected database was conducted involving 512 patients aged >18 years who presented to a tertiary spine clinic for LBP having completed the PainDETECT questionnaire, Oswestry Disability Index (ODI), EuroQol Five-Dimensional (EQ-5D) questionnaire, or answered questions regarding sociodemographic status.
Results:
The NeP group had a higher mean numerical rating scale (NRS) score (7.96±1.54 vs. 5.76±2.27, p <.001) and lower age (55±15.6 vs. 59±17.8, p <.05) compared to the NoP group. When confounded for NRS, analysis of covariance demonstrated an 89.5% higher total ODI score (p <.001) and 50.5% lower EQ-5D utility score (p <.001) in the NeP compared to NoP group. Smokers and individuals with a no partner marital status were 2.373 (OR = 2.373, 95% CI = 1.319-4.266, p <.01) and 2.384 times (OR = 2.384, 95% CI = 1.390-4.092, p <.01) more likely to have NeP compared to NoP, respectively. Patients with NeP were also of lower income class compared to patients with NoP (Z = -2.45, p <.05).
Conclusion:
NeP was associated with higher levels of disability and lower QoL. Smokers, individuals with a no partner marital status, and individuals with a lower income class were more likely to suffer NeP rather than NoP. These findings have illuminated a crucial notion: in patients with elevated NRS, the detrimental impact of NeP on patient wellbeing underscores the fundamental need to represent pain on a nociceptive-neuropathic continuum, permitting more accurate differentiation of pain components.
Cigarette smoking was known to accelerate the occurrence and development of bladder cancer by regulating RNA modification. However, the association between the combination of cigarette smoking and RNA modification‐related single nucleotide polymorphisms (RNAm‐SNPs) and bladder cancer risk remains unclear. In this study, 1681 participants, including 580 cases and 1101 controls, were recruited for genetic association analysis. In total, 1 287 990 RNAm‐SNPs involving nine RNA modifications (m ⁶ A, m ¹ A, m ⁶ Am, 2'‐O‐Me, m ⁵ C, m ⁷ G, A‐to‐I, m ⁵ U, and pseudouridine modification) were obtained from the RMVar database. The interactive effect of cigarette smoking and RNAm‐SNPs on bladder cancer risk was assessed through joint analysis. The susceptibility analysis revealed that 89 RNAm‐SNPs involving m ⁶ A, m ¹ A, and A‐to‐I modifications were associated with bladder cancer risk. Among them, m ⁶ A‐related rs2273058 in CRNKL1 was associated with bladder cancer risk (odds ratios (OR) = 1.35, p adj = 1.78 × 10 ⁻⁴ ), and CRNKL1 expression was increased in bladder cancer patients ( p = 0.035). Cigarette smoking combined with the A allele of rs2273058 increased bladder cancer risk compared with nonsmokers with the G allele of rs2273058 (OR = 2.40, p adj = 3.11 × 10 ⁻⁹ ). Mechanistically, the A allele of rs2273058 endowed CRNKL1 with an additional m ⁶ A motif, facilitating recognition by m ⁶ A reader IGF2BP1, thereby promoting CRNKL1 expression under cigarette smoking ( r = 0.142, p = 0.017). Moreover, elevated CRNKL1 expression may accelerate cell cycle and proliferation, thereby increasing bladder cancer risk. In summary, our study demonstrated that cigarette smoking combined with RNAm‐SNPs contributes to bladder cancer risk, which provides a potential target for bladder cancer prevention.
N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during TGF-β-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and non-redundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by METTL3 m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3’UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during TGF-β-induced EMT of lung cancer cells.
Background
COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase-1 (hCMA1) and it's ortholog mCMA1/mouse mast cell (MC) protease-5 (mMCP5) are exocytosed from activated MCs and have adverse roles in numerous disorders, but their role in COPD is unknown.
Methods
We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5 -deficient ( −/− ) mice to evaluate this proteases’ role and potential for therapeutic targeting in CS-induced experimental COPD. We also used ex vivo/in vitro studies to define mechanisms.
Results
The levels of hCMA1 mRNA and CMA1 ⁺ MCs were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated MC numbers and mMCP5 protein were increased in lung tissues of wild-type (WT) mice with experimental COPD. mmcp5 −/− mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of MCs from WT but not mmcp5 −/− mice with WT lung macrophages increased in TNF-α release. It also caused the release of CMA1 from human MCs, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD.
Conclusion
CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.
Objective:
Increased inflammasome responses are strongly implicated in inflammatory diseases; however, their specific roles are incompletely understood. Therefore, we sought to examine the roles of NLRP3 and AIM2 inflammasomes in cigarette smoke-induced inflammation in a model of experimental chronic obstructive pulmonary disease (COPD).
Methods:
We targeted NLRP3 with the inhibitor MCC950 given prophylactically or therapeutically and examined Aim2-/- mice in cigarette smoke-induced experimental COPD.
Results:
MCC950 treatment had minimal effects on disease development and/or progression. Aim2-/- mice had increased airway neutrophils with decreased caspase-1 levels, independent of changes in lung neutrophil chemokines. Suppressing neutrophils with anti-Ly6G in experimental COPD in wild-type mice reduced neutrophils in bone marrow, blood, and lung. In contrast, anti-Ly6G treatment in Aim2-/- mice with experimental COPD had no effect on neutrophils in bone marrow, partially reduced neutrophils in the blood, and had no effect on neutrophils or neutrophil caspase-1 levels in the lungs.
Conclusion:
These findings identify that following cigarette smoke exposure, Aim2 is important for anti-Ly6G-mediated depletion of neutrophils, suppression of neutrophil recruitment and mediates activation of caspase-1 in neutrophils.
Objective
To study the N6-methyladenosine (m⁶A) modification pattern of nucleus pulposus (NP) tissue during intervertebral disc degeneration (IDD).
Methods
A standing mouse model was generated, and staining and imaging methods were used to evaluate the IDD model. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) was used to analyze m⁶A methylation-associated transcripts in the NP, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of methylation-related enzymes and conduct bio-informatics analysis.
Results
The standing mouse model caused IDD. Continuous axial pressure changed the expression of related methylases in degenerated NP tissue. Relative to the control group, the expression levels of KIAA1429, METTL14, METTL3, METTL4, WTAP, DGCR8, EIF3A and YTHDC1 in the experimental group were higher, while those of FTO, ELAVL1, HNRNPC1 and SRSF2 were lower. We identified 985 differentially expressed genes through MeRIP-Seq, among which 363 genes were significantly up-regulated, and 622 genes were significantly down-regulated. In addition, among the 9648 genes counted, 1319 m⁶A peaks with significant differences in methylation were identified, among which 933 were significantly up-regulated, and 386 were significantly down-regulated. Genes and pathways that were enriched in IDD have been identified.
Conclusion
The results of this study elucidated the m⁶A methylation pattern of NP tissue in degenerated lumbar intervertebral disc of mice and provided new perspectives and clues for research on and the treatment of lumbar disc degeneration.
The Translational potential of this article
As one of the important causes of low back and leg pain, intervertebral disc degeneration brings a huge economic burden to the society, family and medical system. Therefore, understanding the molecular and cellular mechanisms of intervertebral disc degeneration is of great significance for guiding clinical treatment. In this study, methylated RNA immunoprecipitation with next-generation sequencing on mice lumbar nucleus pulposus tissues found that differentially expressed genes and changes in the expression of related methylases, confirming that RNA methylation is involved in intervertebral disc degeneration. The process provides new vision and clues for future research on intervertebral disc degeneration.
Although degeneration of the nucleus pulposus (NP) is a major contributor to intervertebral disc degeneration (IVDD) and low back pain, the underlying molecular complexity and cellular heterogeneity remain poorly understood. Here, a comprehensive single-cell resolution transcript landscape of human NP is reported. Six novel human NP cells (NPCs) populations are identified by their distinct molecular signatures. The potential functional differences among NPC subpopulations are analyzed. Predictive transcripts, transcriptional factors, and signal pathways with respect to degeneration grades are explored. It is reported that fibroNPCs is the subpopulation for end-stage degeneration. CD90+NPCs are observed to be progenitor cells in degenerative NP tissues. NP-infiltrating immune cells comprise a previously unrecognized diversity of cell types, including granulocytic myeloid-derived suppressor cells (G-MDSCs). Integrin M (CD11b) and oxidized low density lipoprotein receptor 1 (OLR1) as surface markers of NP-derived G-MDSCs are uncovered. The G-MDSCs are found to be enriched in mildly degenerated (grade II and III) NP tissues compared to severely degenerated (grade IV and V) NP tissues. Their immunosuppressive function and alleviation effects on NPCs' matrix degradation are revealed in vitro. Collectively, this study reveals the NPC-type complexity and phenotypic characteristics in NP, thereby providing new insights and clues for IVDD treatment.
Protein structures can provide invaluable information, both for reasoning about biological processes and for enabling interventions such as structure-based drug development or targeted mutagenesis. After decades of effort, 17% of the total residues in human protein sequences are covered by an experimentally-determined structure1. Here we dramatically expand structural coverage by applying the state-of-the-art machine learning method, AlphaFold2, at scale to almost the entire human proteome (98.5% of human proteins). The resulting dataset covers 58% of residues with a confident prediction, of which a subset (36% of all residues) have very high confidence. We introduce several metrics developed by building on the AlphaFold model, and use them to interpret the dataset, identifying strong multi-domain predictions as well as regions likely to be disordered. Finally, we provide some case studies illustrating how high-quality predictions may be used to generate biological hypotheses. Importantly, we are making our predictions freely available to the community via a public database (hosted by the European Bioinformatics Institute at https://alphafold.ebi.ac.uk/). We anticipate that routine large-scale and high-accuracy structure prediction will become an important tool, allowing new questions to be addressed from a structural perspective.
Background
Rats are a widely accepted preclinical model for evaluating intervertebral disc (IVD) degeneration and regeneration. IVD morphology is commonly assessed using histology, which forms the foundation for quantifying the state of IVD degeneration. IVD degeneration severity is evaluated using different grading systems that focus on distinct degenerative features. A standard grading system would facilitate more accurate comparison across laboratories and more robust comparisons of different models and interventions.
Aims
This study aimed to develop a histology grading system to quantify IVD degeneration for different rat models.
Materials & Methods
This study involved a literature review, a survey of experts in the field, and a validation study using 25 slides that were scored by 15 graders from different international institutes to determine inter- and intra-rater reliability.
Results
A new IVD degeneration grading system was established and it consists of eight significant degenerative features, including nucleus pulposus (NP) shape, NP area, NP cell number, NP cell morphology, annulus fibrosus (AF) lamellar organization, AF tears/fissures/disruptions, NP-AF border appearance, as well as endplate disruptions/microfractures and osteophyte/ossification. The validation study indicated this system was easily adopted, and able to discern different severities of degenerative changes from different rat IVD degeneration models with high reproducibility for both experienced and inexperienced graders. In addition, a widely-accepted protocol for histological preparation of rat IVD samples based on the survey findings include paraffin embedding, sagittal orientation, section thickness < 10 μm, and staining using H&E and/or SO/FG to facilitate comparison across laboratories.
Conclusion
The proposed histological preparation protocol and grading system provide a platform for more precise comparisons and more robust evaluation of rat IVD degeneration models and interventions across laboratories.
Study design:
Translational review encompassing basic science and clinical evidence.
Objectives:
Multiple components of the lumbar spine interact during its normal and pathological function. Bony stress in the lumbar spine is recognized as a factor in the development of pars interarticularis defect and stress fractures, but its relationship with intervertebral disc (IVD) degeneration is not well understood. Therefore, we conducted a systematic review to examine the relationship between bony stress and IVD degeneration.
Methods:
Online databases Scopus, PubMed and MEDLINE via OVID were searched for relevant studies published between January 1980-February 2020, using PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. Two authors independently analyzed the data, noting characteristics and biases in various studies.
Results:
Thirty-two articles were included in the review: 8 clinical studies, 9 finite element modeling studies, 3 in-vivo biomechanical testing studies, and 12 in-vitro biomechanical testing studies. Of the 32 articles, 19 supported, 4 rejected and 9 made no conclusion on the hypothesis that there is a positive associative relationship between IVD degeneration and bony stress. However, sufficient evidence was not available to confirm or reject a causal relationship.
Conclusions:
Most studies suggest that the prevalence of IVD degeneration increases in the presence of bony stress; whether a causal relationship exists is unclear. The literature recommends early diagnosis and clinical suspicion of IVD degeneration and bony stress. Longitudinal studies are required to explore causal relationships between IVD degeneration and bony stress.
In inflamed tissue sites characterized by on-going matrix degradation, the matrix metalloproteinases are secreted as latent precursors which are capable of proteolysis only after extracellular activation. Such areas often contain locally increased numbers of mast cells capable of releasing complexes between heparin proteoglycans and fully active endopeptidases with either tryptic (tryptase) or both tryptic and chymotryptic (chymase) activity. We have examined the ability of purified human skin chymase to activate human interstitial procollagenase (matrix metalloproteinase-1) in the absence and presence of heparin, the physiologic associate of chymase. Our studies indicate that chymase activates procollagenase in a time- and concentration-dependent manner. Heparin was found to increase markedly the rate at which chymase activates procollagenase both by accelerating the cleavage of procollagenase and also by preventing its further degradation. Moreover, we found that chymase activates procollagenase directly by cleaving the Leu83-Thr84 bond, without formation of any intermediate species. This is a novel mechanism for procollagenase activation, which contrasts sharply with the activation mechanisms of other activators studied so far. The ability of chymase to activate procollagenase suggests that chymase plays an active role in matrix degradation at tissue sites where mast cells coexist with extracellular procollagenase.
N6-Methyladenosine (m⁶A) is an epigenetic modification associated with various tumors, but its role in tumorigenesis remains unexplored. Here, as confirmed by MeRIP-Seq and RNA-Seq analyses, exposure of human bronchial epithelial (HBE) cells to cigarette smoke extract (CSE) caused an m⁶A modification in the 3’UTR of ZBTB4, a transcriptional repressor. For these cells, CSE also elevated METTL3 levels, which increased the m⁶A modification of ZBTB4. RIP-qPCR illustrated that ZBTB4 is the intent gene of YTHDF2 and that levels of ZBTB4 were decreased in an YTHDF2-dependent mechanism. The lower levels of ZBTB4 were associated with up-regulation of EZH2, which enhanced H3K27me3 combining with E-cadherin promoter, causing lower E-cadherin levels and induction of the epithelial-mesenchymal transition (EMT). Further, in the lungs of mice, downregulation of METTL3 alleviated the cigarette smoke (CS)-induced EMT. Further, the expression of METTL3 was high in the lung tissues of smokers and inversely correlated with ZBTB4. Overall, our results show that the METTL3-mediated m⁶A modification of ZBTB4 via EZH2 is involved in the CS-induced EMT and in lung cancer. These results indicate that m⁶A modifications are a potential therapeutic target of lung damage induced by CS.
PANTHER (Protein Analysis Through Evolutionary Relationships, http://www.pantherdb.org) is a resource for the evolutionary and functional classification of protein-coding genes from all domains of life. The evolutionary classification is based on a library of over 15,000 phylogenetic trees, and the functional classifications include Gene Ontology terms and pathways. Here, we analyze the current coverage of genes from genomes in different taxonomic groups, so that users can better understand what to expect when analyzing a gene list using PANTHER tools. We also describe extensive improvements to PANTHER made in the past two years. The PANTHER Protein Class ontology has been completely refactored, and 6101 PANTHER families have been manually assigned to a Protein Class, providing a high level classification of protein families and their genes. Users can access the TreeGrafter tool to add their own protein sequences to the reference phylogenetic trees in PANTHER, to infer evolutionary context as well as fine-grained annotations. We have added human enhancer-gene links that associate non-coding regions with the annotated human genes in PANTHER. We have also expanded the available services for programmatic access to PANTHER tools and data via application programming interfaces (APIs). Other improvements include additional plant genomes and an updated PANTHER GO-slim.
In addition to their well characterized role in mediating IgE-dependent allergic diseases, aberrant accumulation and activation of mast cells (MCs) is associated with many non-allergic inflammatory diseases, whereby their activation is likely triggered by non-IgE stimuli (e.g., IL-33). Siglec-8 is an inhibitory receptor expressed on MCs and eosinophils that has been shown to inhibit IgE-mediated MC responses and reduce allergic inflammation upon ligation with a monoclonal antibody (mAb). Herein, we evaluated the effects of an anti-Siglec-8 mAb (anti-S8) in non-allergic disease models of experimental cigarette-smoke-induced chronic obstructive pulmonary disease and bleomycin-induced lung injury in Siglec-8 transgenic mice. Therapeutic treatment with anti-S8 inhibited MC activation and reduced recruitment of immune cells, airway inflammation, and lung fibrosis. Similarly, using a model of MC-dependent, IL-33-induced inflammation, anti-S8 treatment suppressed neutrophil influx, and cytokine production through MC inhibition. Transcriptomic profiling of MCs further demonstrated anti-S8-mediated downregulation of MC signaling pathways induced by IL-33, including TNF signaling via NF-κB. Collectively, these findings demonstrate that ligating Siglec-8 with an antibody reduces non-allergic inflammation and inhibits IgE-independent MC activation, supporting the evaluation of an anti-Siglec-8 mAb as a therapeutic approach in both allergic and non-allergic inflammatory diseases in which MCs play a role.
Background
Post-transcriptional modifications of RNA constitute fundamental mechanisms of gene regulation. N⁶-methyladenosine (m⁶A) is critical for health and disease and is modulated by cellular stressors. However, associations between environmental exposures and m⁶A have not been studied in humans. We aimed to examine associations between tobacco smoking and particulate air pollution with m⁶A and mRNA expression levels of its reader, writer and eraser (RWE) genes in blood.
Methods
Using the Beijing Truck Driver Air Pollution Study, we investigated global m⁶A in RNA from peripheral blood collected from 106 human subjects in Beijing, China, in 2008. We measured m⁶A with nano-flow liquid chromatography-tandem mass spectrometry and investigated gene expression of six m⁶A RWEs with real-time-quantitative PCR. Using linear models, we examined associations with smoking status, pack-years, and smoking on day of visit in men, and with environmental tobacco smoke in nonsmokers. We also examined associations with ambient PM10 (particulate matter ≤ 10 µm in diameter), and personal black carbon (BC) and PM2.5 measured with a portable monitor.
Results
Smoking in men was significantly associated with a relative 10.7% decrease in global m⁶A levels in comparison to nonsmokers (p = 0.02). In men, smoking greater than 3.8 pack-years was associated with a 14.9% lower m⁶A than in nonsmokers. BC exposure trended towards positive associations with m⁶A (5.95% per 10 μg/m³ increase in BC; 95% CI: −0.96, 13.3). Global m⁶A levels were not correlated with RWE gene expression levels. No associations were detected between smoking or air pollutants and m⁶A RWE gene expression.
Discussion
m⁶A was negatively associated with long-term smoking, yet positively associated with short-term BC exposure. These results indicate variable m⁶A responses to environmental stressors, providing early evidence into the impacts of toxicants on RNA modifications and suggesting potential for m⁶A as a biomarker or mechanism in environmental health research.
Background Context
Abnormal Wnt signaling in intervertebral discs (IVDs) progresses degenerative disc disease (DDD) pathogenesis by impairing nucleus pulposus (NP) cell function, decreasing matrix deposition, and accelerating fibrosis.
Purpose
This study was conducted to evaluate the effects of lorecivivint (LOR; SM04690), a small-molecule Wnt pathway inhibitor, on IVD cells and in an animal model of DDD.
Study Design
We used in vitro assays and a rat model of DDD to test the effects of LOR on NP cell senescence and viability, annulus fibrosus (AF) cell fibrosis, and cartilage regeneration and protection.
Methods
Wnt pathway gene expression was measured in human NP and AF cell cultures treated with LOR or DMSO (vehicle). Chondrocyte-like differentiation of rat and human NP cells, NP cell senescence and protection, and AF cell fibrosis were assessed using gene expression and immunocytochemistry. Disc and plasma pharmacokinetics were analyzed following intradiscal LOR injection in rats. In vivo effects of LOR and vehicle on AF integrity, AF/NP junction, NP cellularity and matrix, and disc height (DH) were compared using histopathology and radiography in a rat coccygeal IVD needle-puncture model of DDD.
Results
In NP and AF cell cultures, LOR inhibited Wnt pathway gene expression compared with vehicle. In NP cells, LOR inhibited senescence, decreased catabolism, and induced differentiation into chondrocyte-like cells; in AF cells, LOR decreased catabolism and inhibited fibrosis. A single intradiscal LOR injection in rats resulted in therapeutic disc concentrations (∼30 nM) for >180 days and minimal systemic exposure. DDD-model rats receiving LOR qualitatively demonstrated increased cartilage matrix and reduced AF lamellar disorganization and fragmentation with significantly (P<0.05) improved histology scores and increased DH compared with vehicle.
Conclusions
LOR showed beneficial effects on IVD cells in vitro and reduced disease progression in a rat model of DDD compared with vehicle, suggesting that LOR may have disease-modifying therapeutic potential.
Clinical Significance
The current therapeutic options for DDD are pain management and surgical intervention; there are no approved therapies that alter the progression of DDD. Our data support advancing LOR into clinical development as an injectable, small-molecule, potential disease-modifying treatment for DDD in humans.
Abstract N6-methyladenosine (m6A) is a well-known post-transcriptional modification that is the most common type of methylation in eukaryotic mRNAs. The regulation of m6A is dynamic and reversible, which is erected by m6A methyltransferases (“writers”) and removed by m6A demethylases (“erasers”). Notably, the effects on targeted mRNAs resulted by m6A predominantly depend on the functions of different m6A-binding proteins (“readers”) including YT521-B homology (YTH) domain family, heterogeneous nuclear ribonucleoproteins (HNRNPs), and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs). Indeed, m6A readers not only participate in multiple procedures of RNA metabolism, but also are involved in a variety of biological processes. In this review, we summarized the specific functions and underlying mechanisms of m6A-binding proteins in tumorigenesis, hematopoiesis, virus replication, immune response, and adipogenesis.
Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte‐like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young‐adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40‐75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre‐osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young‐adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5‐deficiency in osterix‐expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45‐70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60‐80%. Overall, these data indicate that age‐related inactivation of Wnt signaling in osterix‐expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte‐like cells.
RNA methylation to form N6-methyladenosine (m6A) in mRNA accounts for the most abundant mRNA internal modification and has emerged as a widespread regulatory mechanism that controls gene expression in diverse physiological processes. Transcriptome-wide m6A mapping has revealed the distribution and pattern of m6A in cellular RNAs, referred to as the epitranscriptome. These maps have revealed the specific mRNAs that are regulated by m6A, providing mechanistic links connecting m6A to cellular differentiation, cancer progression and other processes. The effects of m6A on mRNA are mediated by an expanding list of m6A readers and m6A writer-complex components, as well as potential erasers that currently have unclear relevance to m6A prevalence in the transcriptome. Here we review new and emerging methods to characterize and quantify the epitranscriptome, and we discuss new concepts — in some cases, controversies — regarding our understanding of the mechanisms and functions of m6A readers, writers and erasers. N6-methyladenosine (m6A) is the most abundant mRNA internal modification. The recent mapping of m6A has provided insights into which and how mRNAs are modified, how m6A affects gene expression and how it is linked to cellular differentiation, cancer progression and other biological processes.
Objectives:
Wnt pathway upregulation contributes to knee osteoarthritis (OA) through osteoblast differentiation, increased catabolic enzymes, and inflammation. The small-molecule Wnt pathway inhibitor, lorecivivint (SM04690), which previously demonstrated chondrogenesis and cartilage protection in an animal OA model, was evaluated to elucidate its mechanism of action.
Design:
Biochemical assays measured kinase activity. Western blots measured protein phosphorylation in human mesenchymal stem cells (hMSCs), chondrocytes, and synovial fibroblasts. siRNA knockdown effects in hMSCs and BEAS-2B cells on Wnt pathway, chondrogenic genes, and LPS-induced inflammatory cytokines was measured by qPCR. In vivo anti-inflammation, pain, and function were evaluated following single intra-articular (IA) lorecivivint or vehicle injection in the monosodium iodoacetate (MIA)-induced rat OA model.
Results:
Lorecivivint inhibited intranuclear kinases CDC-like kinase 2 (CLK2) and dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Lorecivivint inhibited CLK2-mediated phosphorylation of serine/arginine-rich (SR) splicing factors and DYRK1A-mediated phosphorylation of SIRT1 and FOXO1. siRNA knockdowns identified a role for CLK2 and DYRK1A in Wnt pathway modulation without affecting β-catenin with CLK2 inhibition inducing early chondrogenesis and DYRK1A inhibition enhancing mature chondrocyte function. NF-κB and STAT3 inhibition by lorecivivint reduced inflammation. DYRK1A knockdown was sufficient for anti-inflammatory effects, while combined DYRK1A/CLK2 knockdown enhanced this effect. In the MIA model, lorecivivint inhibited production of inflammatory cytokines and cartilage degradative enzymes, resulting in increased joint cartilage, decreased pain, and improved weight-bearing function.
Conclusions:
Lorecivivint inhibition of CLK2 and DYRK1A suggested a novel mechanism for Wnt pathway inhibition, enhancing chondrogenesis, chondrocyte function, and anti-inflammation. Lorecivivint shows potential to modify structure and improve symptoms of knee OA.
Osteoarthritis is characterized by articular cartilage breakdown, and emerging evidence suggests that dysregulated innate immunity is likely involved. Here, we performed proteomic, transcriptomic, and electron microscopic analyses to demonstrate that mast cells are aberrantly activated in human and murine osteoarthritic joint tissues. Using genetic models of mast cell deficiency, we demonstrate that lack of mast cells attenuates osteoarthritis in mice. Using genetic and pharmacologic approaches, we show that the IgE/FcεRI/Syk signaling axis is critical for the development of osteoarthritis. We find that mast cell-derived tryptase induces inflammation, chondrocyte apoptosis, and cartilage breakdown. Our findings demonstrate a central role for IgE-dependent mast cell activation in the pathogenesis of osteoarthritis, suggesting that targeting mast cells could provide therapeutic benefit in human osteoarthritis.
Editorial note:
This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
DNA and histone modifications have notable effects on gene expression¹. Being the most prevalent internal modification in mRNA, the N⁶-methyladenosine (m⁶A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2–4 and has crucial roles in various normal and pathological processes5–12. However, it is unclear how m⁶A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m⁶A deposition globally. We show that m⁶A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m⁶A methyltransferase complex (MTC), which in turn facilitates the binding of the m⁶A MTC to adjacent RNA polymerase II, thereby delivering the m⁶A MTC to actively transcribed nascent RNAs to deposit m⁶A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m⁶A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m⁶A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.
The Cistrome Data Browser (DB) is a resource of human and mouse cis-regulatory information derived from ChIP-seq, DNase-seq and ATAC-seq chromatin profiling assays, which map the genome-wide locations of transcription factor binding sites, histone post-translational modifications and regions of chromatin accessible to endonuclease activity. Currently, the Cistrome DB contains approximately 47,000 human and mouse samples with about 24,000 newly collected datasets compared to the previous release two years ago. Furthermore, the Cistrome DB has a new Toolkit module with several features that allow users to better utilize the large-scale ChIP-seq, DNase-seq, and ATAC-seq data. First, users can query the factors which are likely to regulate a specific gene of interest. Second, the Cistrome DB Toolkit facilitates searches for factor binding, histone modifications, and chromatin accessibility in any given genomic interval shorter than 2Mb. Third, the Toolkit can determine the most similar ChIP-seq, DNase-seq, and ATAC-seq samples in terms of genomic interval overlaps with user-provided genomic interval sets. The Cistrome DB is a user-friendly, up-to-date, and well maintained resource, and the new tools will greatly benefit the biomedical research community. The database is freely available at http://cistrome.org/db, and the Toolkit is at http://dbtoolkit.cistrome.org.
The idea that epigenetic determinants such as DNA methylation, histone modifications or RNA can be passed to the next generation through meiotic products (gametes) is long standing. Such meiotic epigenetic inheritance (MEI) is fairly common in yeast, plants and nematodes, but its extent in mammals has been much debated. Advances in genomics techniques are now driving the profiling of germline and zygotic epigenomes, thereby improving our understanding of MEI in diverse species. Whereas the role of DNA methylation in MEI remains unclear, insights from genome-wide studies suggest that a previously underappreciated fraction of mammalian genomes bypass epigenetic reprogramming during development. Notably, intergenerational inheritance of histone modifications, tRNA fragments and microRNAs can affect gene regulation in the offspring. It is important to note that MEI in mammals rarely constitutes transgenerational epigenetic inheritance (TEI), which spans multiple generations. In this Review, we discuss the examples of MEI in mammals, including mammalian epigenome reprogramming, and the molecular mechanisms of MEI in vertebrates in general. We also discuss the implications of the inheritance of histone modifications and small RNA for embryogenesis in metazoans, with a particular focus on insights gained from genome-wide studies.
Background
There is a close relationship between the vertebral trabecular morphology and the condition of the associated disc.
Objective
The relationship between disc degeneration and vertebral trabecular inhomogeneity is unclear. This study aimed to analyse the regional changes of vertebral trabecular morphology after disc degeneration.
Methods
Thirty male Sprague–Dawley rats were randomly assigned to five groups. Group 1 served as an experimental group for the assessment of disc degeneration induced by needle puncture. Group 2 served as a sham group for trabecular morphology analysis. In Group 3, rats had their tail bent between the eighth and tenth coccygeal vertebrae. In Group 4, the tail of rats was bent with a compression load of 4.5 N. In Group 5, rats first underwent disc degeneration induced by a needle puncture before their tail was bent with a compressive load of 4.5 N. Magnetic resonance imaging was performed on all groups, and histological examination was performed on rodents from Group 1. The ninth coccygeal vertebrae of rats from Groups 2–5 were scanned by Micro-computed tomography. Trabecular morphologic changes were assessed in the concave and convex regions by bone volume fraction, trabecular number, trabecular thickness and trabecular separation.
Results
Vertebral trabecular morphology in the concave region improved significantly, whereas the convex region was of significantly lower trabecular morphologic parameters with disc degeneration. The difference in trabecular morphologic parameters between the convex and concave regions increased significantly after disc degeneration.
Conclusion
Disc degeneration promotes regional inhomogeneity in the vertebral trabecular morphology, with the convex region of the vertebrae having the worse trabecular bone morphology than the concave region.
The translational potential of this article
Our study indicates that disc degeneration promotes regional inhomogeneity in the vertebral trabecular morphology. Regional variations in trabecular microarchitecture are helpful to predict vertebral fragility. This may help to elucidate the mechanisms by which disc degeneration contributes to vertebral fracture.
B-Catenin, transcription factor of Wnt signaling, is promoted in patients with intervertebral disc (IVD) degeneration, but Wnt signaling decreases with aging. We hypothesize that IVD degeneration is associated with decreased Wnt signaling despite more b-Catenin. Chronic compression of tail IVDs of young-adult and aged Wnt-reporter (TOPGAL) animals initiated an age-related cascade of degenerative-like changes, which included reduced Wnt ligand expression and Wnt signaling in nucleus pulposus cells, despite elevation of b-Catenin protein and gene expression. To determine the effect of upregulated and downregulated Wnt signaling in adult discs, b-Catenin in the nucleus pulposus was stabilized (Shh-CreErT2/b-Cateninfl(Ex3)/fl(Ex3), cACT) or knocked out (Shh-CreErT2/b-Cateninfl/fl, cKO). cACT discs had promoted expression of Wnt-targets and -ligands, brachyury, extracellular matrix production and 34% greater compressive stiffness than WT (b-Cateninfl(Ex3)/fl(Ex3)) discs, but 50% less tensile stiffness. By contrast, knockout reversed the cACT phenotype: less protein expression of b-catenin in the nucleus pulposus, less expression of brachyury, heightened expression of extracellular matrix breakdown and 46% less compressive stiffness than wild-type (b-Cateninfl/fl,WT) discs. These data suggest that intervertebral disc degeneration is associated with loss of Wnt signaling and that the concomitant increase in b-catenin is a regenerative response, potentially offering a therapeutic approach to degeneration.
Degenerative changes in the spine have high medical and socioeconomic significance. Imaging of the degenerative spine is a frequent challenge in radiology. The pathogenesis of this degenerative process represents a biomechanically related continuum of alterations, which can be identified with different imaging modalities. The aim of this article is to review radiological findings involving the intervertebral discs, end plates, bone marrow changes, facet joints and the spinal canal in relation to the pathogenesis of degenerative changes in the spine. Findings are described in association with the clinical symptoms they may cause, with a brief review of the possible treatment options. The article provides an illustrated review on the topic for radiology residents.
Teaching Points
• The adjacent vertebrae, intervertebral disc, ligaments and facet joints constitute a spinal unit.
• Degenerative change is a response to insults, such as mechanical or metabolic injury.
• Spine degeneration is a biomechanically related continuum of alterations evolving over time.
N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.
Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract (GIT). Cigarette smoke (CS) exposure and chronic obstructive pulmonary disease (COPD) are risk factors for CD, although the mechanisms involved are poorly understood. We employed a mouse model of CS-induced experimental COPD and clinical studies to examine these mechanisms. Concurrent with the development of pulmonary pathology and impaired gas exchange, CS-exposed mice developed CD-associated pathology in the colon and ileum, including gut mucosal tissue hypoxia, HIF-2 stabilization, inflammation, increased microvasculature, epithelial cell turnover, and decreased intestinal barrier function. Subsequent smoking cessation reduced GIT pathology, particularly in the ileum. Dimethyloxaloylglycine, a pan-prolyl hydroxylase inhibitor, ameliorated CS-induced GIT pathology independently of pulmonary pathology. Prior smoke exposure exacerbated intestinal pathology in 2,4,6-trinitrobenzenesulfonic acid-induced (TNBS-induced) colitis. Circulating vascular endothelial growth factor, a marker of systemic hypoxia, correlated with CS exposure and CD in mice and humans. Increased mucosal vascularisation was evident in ileum biopsies from CD patients who smoke compared with nonsmokers, supporting our preclinical data. We provide strong evidence that chronic CS exposure and, for the first time to our knowledge, associated impaired gas exchange cause systemic and intestinal ischemia, driving angiogenesis and GIT epithelial barrier dysfunction, resulting in increased risk and severity of CD.
Smoking is a common risk factor for both chronic obstructive pulmonary disease (COPD) and osteoporosis. In patients with COPD, severe emphysema is a risk factor for vertebral fracture; however, the effects of smoking or emphysema on bone health remain largely unknown. We report bone deterioration in a mouse model of emphysema induced by nose-only cigarette smoke (CS) exposure. Unexpectedly, short-term exposure for 4-weeks decreased bone turnover and increased bone volume in mice. However, prolonged exposure for 20- and 40-weeks reversed the effects from suppression to promotion of bone resorption. This long-term CS exposure increased osteoclast number and impaired bone growth, while it increased bone volume. Strikingly, long-term CS exposure deteriorated bone quality of the lumbar vertebrae as illustrated by disorientation of collagen fibers and the biological apatite c-axis. This animal model may provide a better understanding of the mechanisms underlying the deterioration of bone quality in pulmonary emphysema caused by smoking.
Objectives:
Osteoarthritis (OA) is a degenerative disease characterized by loss of cartilage and increased subchondral bone within synovial joints. Wnt signaling affects the pathogenesis of OA as this pathway modulates both the differentiation of osteoblasts and chondrocytes, and production of catabolic proteases. A novel small molecule Wnt pathway inhibitor, SM04690, was evaluated in a series of in vitro and in vivo animal studies to determine its effects on chondrogenesis, cartilage protection and synovial-lined joint pathology.
Design:
A high-throughput screen was performed using a cell-based reporter assay for Wnt pathway activity to develop a small molecule designated SM04690. Its properties were evaluated in bone marrow derived human mesenchymal stem cells (hMSCs) to assess chondrocyte differentiation and effects on cartilage catabolism by immunocytochemistry and gene expression, and glycosaminoglycan breakdown. In vivo effects of SM04690 on Wnt signaling, cartilage regeneration and protection were measured using biochemical and histopathological techniques in a rodent acute cruciate ligament tear and partial medial meniscectomy (ACLT+pMMx) OA model.
Results:
SM04690 induced hMSC differentiation into mature, functional chondrocytes and decreased cartilage catabolic marker levels compared to vehicle. A single SM04690 intra-articular (IA) injection was efficacious in a rodent OA model, with increased cartilage thickness, evidence for cartilage regeneration, and protection from cartilage catabolism observed, resulting in significantly improved Osteoarthritis Research Society International (OARSI) histology scores and biomarkers, compared to vehicle.
Conclusions:
SM04690 induced chondrogenesis and appeared to inhibit joint destruction in a rat OA model, and is a candidate for a potential disease modifying therapy for OA.
Orthology assignment is ideally suited for functional inference. However, because predicting orthology is computationally intensive at large scale, and most pipelines are relatively inaccessible (e.g. new assignments only available through database updates), less precise homology-based functional transfer is still the default for (meta-)genome annotation. We therefore developed eggNOG-mapper, a tool for functional annotation of large sets of sequences based on fast orthology assignments using precomputed clusters and phylogenies from the eggNOG database. To validate our method, we benchmarked Gene Ontology predictions against two widely used homology-based approaches: BLAST and InterProScan. Orthology filters applied to BLAST results reduced the rate of false positive assignments by 11%, and increased the ratio of experimentally validated terms recovered over all terms assigned per protein by 15%. Compared to InterProScan, eggNOG-mapper achieved similar proteome coverage and precision while predicting, on average, 41 more terms per protein and increasing the rate of experimentally validated terms recovered over total term assignments per protein by 35%. EggNOG-mapper predictions scored within the top-5 methods in the three Gene Ontology categories using the CAFA2 NK-partial benchmark. Finally, we evaluated eggNOG-mapper for functional annotation of metagenomics data, yielding better performance than interProScan. eggNOG-mapper runs ∼15x faster than BLAST and at least 2.5x faster than InterProScan. The tool is available standalone and as an online service at http://eggnog-mapper.embl.de.
The constitutive heparin+ (HP) mast cells (MCs) in mice express mouse MC protease (mMCP)-5 and carboxypeptidase A (mMC CPA). The amino acid sequence of mMCP-5 is most similar to that of human chymase-1, as is the nucleotide sequences of their genes and transcripts. Using a homologous recombination approach, a C57BL/6 mouse line was created that possessed a disrupted mMCP-5 gene. The resulting mice were fertile and had no obvious developmental abnormality. Lack of mMCP-5 protein did not alter the granulation of the IL-3/IL-9-dependent mMCP 2+ MCs in the jejunal mucosa of Trichinella spiralis-infected mice. In contrast, the constitutive HP+ MCs in the tongues of mMCP-5-null mice were poorly granulated and lacked mMC-CPA protein. Bone marrow-derived MCs were readily developed from the transgenic mice using IL-3. Although these MCs contained high levels of mMC-CPA mRNA, they also lacked the latter exopeptidase. mMCP 5 protein is therefore needed to target translated mMC CPA to the secretory granule along with HP-containing serglycin proteoglycans. Alternately, mMCP-5 is needed to protect mMC-CPA from autolysis in the cell granules. Fibronectin was identified as a target of mMCP 5, and the exocytosis of mMCP 5 from the MCs in the mouse peritoneal cavity resulted in the expression of metalloproteinase protease 9 which has been implicated in arthritis. In support of the latter finding, experimental arthritis was markedly reduced in mMCP-5-null mice relative to wild type mice in two disease models.
The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N(6)-methyladenosine (m(6)A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N(6)-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N(1)-methyladenosine (m(1)A), 5-methylcytosine (m(5)C) and pseudouridine, together with m(6)A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.
Mice lacking DIX domain containing-1 (DIXDC1), an intracellular Wnt/β-catenin signal pathway protein, have abnormal measures of anxiety, depression and social behavior. Pyramidal neurons in these animals' brains have reduced dendritic spines and glutamatergic synapses. Treatment with lithium or a glycogen synthase kinase-3 (GSK3) inhibitor corrects behavioral and neurodevelopmental phenotypes in these animals. Analysis of DIXDC1 in over 9000 cases of autism, bipolar disorder and schizophrenia reveals higher rates of rare inherited sequence-disrupting single-nucleotide variants (SNVs) in these individuals compared with psychiatrically unaffected controls. Many of these SNVs alter Wnt/β-catenin signaling activity of the neurally predominant DIXDC1 isoform; a subset that hyperactivate this pathway cause dominant neurodevelopmental effects. We propose that rare missense SNVs in DIXDC1 contribute to psychiatric pathogenesis by reducing spine and glutamatergic synapse density downstream of GSK3 in the Wnt/β-catenin pathway.Molecular Psychiatry advance online publication, 18 October 2016; doi:10.1038/mp.2016.184.
Intervertebral discs (IVDs) are complex structures that consist of three parts, namely, nucleus pulposus, annulus fibrosus and cartilage endplates. With aging, IVDs gradually degenerate as a consequence of many factors, such as microenvironment changes and cell death. Human clinical trial and animal model studies have documented that cell death, particularly apoptosis and autophagy, significantly contribute to IVD degeneration. The mechanisms underlying this phenomenon include the activation of apoptotic pathways and the regulation of autophagy in response to nutrient deprivation and multiple stresses. In this review, we briefly summarize recent progress in understanding the function and regulation of apoptosis and autophagy signaling pathways. In particular, we focus on studies that reveal the functional mechanisms of these pathways in IVD degeneration.
N6-methyladenosine (m6A) is a prevalent RNA methylation modification involved in the regulation of degradation, subcellular localization, splicing
and local conformation changes of RNA transcripts. High-throughput experiments have demonstrated that only a small fraction
of the m6A consensus motifs in mammalian transcriptomes are modified. Therefore, accurate identification of RNA m6A sites becomes emergently important. For the above purpose, here a computational predictor of mammalian m6A site named SRAMP is established. To depict the sequence context around m6A sites, SRAMP combines three random forest classifiers that exploit the positional nucleotide sequence pattern, the K-nearest
neighbor information and the position-independent nucleotide pair spectrum features, respectively. SRAMP uses either genomic
sequences or cDNA sequences as its input. With either kind of input sequence, SRAMP achieves competitive performance in both
cross-validation tests and rigorous independent benchmarking tests. Analyses of the informative features and overrepresented
rules extracted from the random forest classifiers demonstrate that nucleotide usage preferences at the distal positions,
in addition to those at the proximal positions, contribute to the classification. As a public prediction server, SRAMP is
freely available at http://www.cuilab.cn/sramp/.
Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labeled nascent RNA molecules derived from nuclear isolates. Widespread use of this technique has been limited because of its technical difficulty relative to steady-state total mRNA analyses. Here we describe a detailed protocol for the quantification of transcriptional activity in human cell cultures. Nuclei are first isolated and NRO transcription is performed in the presence of bromouridine. Labeled nascent transcripts are purified by immunoprecipitation, and transcript levels are determined by reverse-transcription quantitative PCR (RT-qPCR). Data are then analyzed using standard techniques described elsewhere. This method is rapid (the protocol can be completed in 2 d) and cost-effective, exhibits negligible detection of background noise from unlabeled transcripts, requires no radioactive materials and can be performed from as few as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR.
Objective
Tension stimulation is an important inducer of endplate cartilage degeneration, but the specific regulatory mechanism remains unclear. This study was the first to reveal the mechanism by which methyltransferase-like 3 (METTL3)-mediated N(6)-methyladenosine (m6A) modification affected the extracellular matrix anabolism by tension-induced endplate chondrocytes.
Method
We examined the differences in METTL3 expression and m6A methylation levels in human endplate chondrocytes and human cartilage endplate tissues under in vitro tension. The effect on endplate cartilage degeneration was evaluated by manipulating m6A methylation mediated by METTL3 in vivo and in vitro. The effect of METTL3-mediated m6A methylation on the stability of sex-determining region Y-box transcription factor 9 (SOX9) gene expression was determined experimentally.
Results
METTL3 expression and m6A methylation levels were significantly increased in degenerative human endplate cartilage tissue. Similarly, tension stimulation inhibited the ability of human endplate chondrocytes to synthesize extracellular matrix, which was accompanied by an increase in METTL3-mediated m6A methylation. The ability of endplate chondrocytes to resist tension was significantly enhanced by inhibiting METTL3 expression and subsequently downregulating m6A methylation in vitro and in vivo, thereby reducing intervertebral disc degeneration. Furthermore, METTL3 mediated SOX9 RNA methylation and disrupted SOX9 mRNA stability, thereby inhibiting the gene expression of the downstream collagen type II alpha 1 chain.
Conclusion
Tension stimulation downregulated SOX9 expression through METTL3-mediated m6A methylation, thereby inhibiting the synthesis of extracellular matrix in endplate chondrocytes.
Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death worldwide. Inhalation of cigarette smoke (CS) is the major cause in developed countries. Current therapies have limited efficacy in controlling disease or halting its progression. Aberrant expression of microRNAs (miRNAs) is associated with lung disease, including COPD. We performed miRNA microarray analyses of the lungs of mice with CS-induced experimental COPD. miR-21 was the second highest up-regulated miRNA, particularly in airway epithelium and lung macrophages. Its expression in human lung tissue correlated with reduced lung function in COPD. Prophylactic and therapeutic treatment with a specific miR-21 inhibitor (Ant-21) inhibited CS-induced lung miR-21 expression in mice; suppressed airway macrophages, neutrophils, and lymphocytes; and improved lung function, as evidenced by decreased lung hysteresis, transpulmonary resistance, and tissue damping in mouse models of COPD. In silico analyses identified a potential miR-21/special AT-rich sequence–binding protein 1 (SATB1)/S100 calcium binding protein A9 (S100A9)/nuclear factor κB (NF-κB) axis, which was further investigated. CS exposure reduced lung SATB1 in a mouse model of COPD, whereas Ant-21 treatment restored SATB1 and reduced S100A9 expression and NF-κB activity. The beneficial effects of Ant-21 in mice were reversed by treatment with SATB1-targeting small interfering RNA. We have identified a pathogenic role for a miR-21/SATB1/S100A9/NF-κB axis in COPD and defined miR-21 as a therapeutic target for this disease.
Background:
Mast cells (MC) play a role in inflammation and both innate and adaptive immunity but their involvement in severe asthma (SA) remains undefined.
Objective:
We investigated the phenotypic characteristics of the U-BIOPRED asthma cohort by applying published MC activation signatures to the sputum cell transcriptome.
Methods:
84 SA, 20 mild/moderate (MMA) asthma, and 16 non-asthmatic healthy participants were studied. We calculated enrichment scores (ES) for nine MC activation signatures by asthma severity, sputum granulocyte status and three previously-defined sputum molecular phenotypes or transcriptome-associated clusters (TAC1, 2, 3) using gene-set variation analysis.
Results:
MC signatures except unstimulated, repeated FcεR1-stimulated and IFNγ-stimulated were enriched in SA. A FcεR1-IgE-stimulated and a single cell signature from asthmatic bronchial biopsies were highly enriched in eosinophilic asthma and in the TAC1 molecular phenotype. Subjects with a high ES for these signatures had elevated sputum levels of similar genes and pathways. IL-33- and LPS-stimulated MC signatures had greater ES in neutrophilic and mixed granulocytic asthma and in the TAC2 molecular phenotype. These subjects exhibited neutrophil, NF-κB, and IL-1β/TNFα pathway activation. The IFNγ-stimulated signature had the greatest ES in TAC2 and TAC3 that was associated with responses to viral infection. Similar results were obtained in an independent ADEPT asthma cohort.
Conclusions:
Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MC can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33-stimulated MCs signature was associated with severe neutrophilic asthma while IgE-activated MC with an eosinophilic phenotype.
Rationale:
Necroptosis, mediated by RIPK3 and MLKL, is a form of regulated necrosis that can drive tissue inflammation and destruction, however its contribution to COPD pathogenesis is poorly understood.
Objectives:
To determine the role of necroptosis in COPD.
Methods:
Levels of RIPK3, MLKL and activated phospho-MLKL were measured in lung tissues of COPD patients and non-COPD controls. Necroptosis-related mRNA and proteins and cell death were examined in the lungs and pulmonary macrophages of mice with cigarette smoke (CS)-induced experimental COPD. The responses of Ripk3- and Mlkl-deficient (-/-) mice to CS exposure were compared to wild-type mice. Combined inhibition of apoptosis (pan-caspase inhibitor qVD-OPh) and necroptosis (Mlkl-/- mice) was assessed.
Measurements and main results:
Protein levels of MLKL and pMLKL but not RIPK3 were increased in lung tissues of COPD patients compared to never smokers or smoker non-COPD controls. Necroptosis-related mRNA and protein levels were increased in lung tissue and macrophages in CS-exposed mice/experimental COPD. Ripk3 or Mlkl deletion prevented airway inflammation in response to acute CS-exposure. Ripk3 deficiency reduced airway inflammation and remodelling and development of emphysematous pathology following chronic CS-exposure. Mlkl deletion and qVD-OPh treatment reduced chronic CS-induced airway inflammation, but only Mlkl deletion prevented airway remodelling and emphysema. Ripk3 or Mlkl deletion and qVD-OPh treatment reduced CS-induced lung cell death.
Conclusions:
Necroptosis is induced by CS exposure and increased in COPD patient lungs and experimental COPD. Inhibiting necroptosis attenuates CS-induced airway inflammation, airway remodelling and emphysema. Targeted inhibition of necroptosis is a potential therapeutic strategy in COPD.
Aim: To explore the potential functions and mechanism of N ⁶ .methyladenosine (m ⁶ A) abnormality of RNAs in nucleus pulposus from the intervertebral disc degeneration (IDD). Materials & methods: We performed rat model, m ⁶ A epitranscriptomic microarray, bioinformatics analysis and metabolomics. Results: In IDD, most of the differentially methylated RNAs showed a significant demethylation situation. The competing endogenous RNA network LOC102555094/ miR-431/ GSK-3β combining downstream Wnt pathway were identified in bioinformatics analysis. For metabolomics, activation of Wnt pathway led to reprogramming of glucose metabolism and enzyme activation of PKM2. Finally, quantitative real-time PCR and methylated RNA immunoprecipitation coupled with quantitative real-time PCR revealed the positive correlation between demethylation of LOC102555094 and expression of both FTO and ZFP217. Conclusion: LOC102555094 might be demethylated by ZFP217, activating FTO and LOC102555094/ miR-431/ GSK-3β/Wnt played a crucial role in IDD.
Tissue remodeling/fibrosis is a major feature of all fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). It is underpinned by accumulating extracellular matrix (ECM) proteins. Fibulin-1c (Fbln1c) is a matricellular ECM protein associated with lung fibrosis in both humans and mice, and stabilizes collagen formation. Here we discovered that Fbln1c was increased in the lung tissues of IPF patients and experimental bleomycin-induced pulmonary fibrosis. Fbln1c-deficient (-/-) mice had reduced pulmonary remodeling/fibrosis and improved lung function after bleomycin challenge. Fbln1c interacted with fibronectin, periostin and tenascin-c in collagen deposits following bleomycin challenge. In a novel mechanism of fibrosis Fbln1c bound to latent transforming growth factor (TGF)-β binding protein-1 (LTBP1) to induce TGF-β activation, and mediated downstream Smad3 phosphorylation/signaling. This process increased myofibroblast numbers and collagen deposition. Fbln1 and LTBP1 co-localized in lung tissues from IPF patients. Thus, Fbln1c may be a novel driver of TGF-β-induced fibrosis involving LTBP1 and may be an upstream therapeutic target.
Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of morbidity and death globally. The lack of effective treatments results from an incomplete understanding of the underlying mechanisms driving COPD pathogenesis.
Interleukin (IL)-22 has been implicated in airway inflammation and is increased in COPD patients. However, its roles in the pathogenesis of COPD is poorly understood. Here, we investigated the role of IL-22 in human COPD and in cigarette smoke (CS)-induced experimental COPD.
IL-22 and IL-22 receptor mRNA expression and protein levels were increased in COPD patients compared to healthy smoking or non-smoking controls. IL-22 and IL-22 receptor levels were increased in the lungs of mice with experimental COPD compared to controls and the cellular source of IL-22 included CD4 ⁺ T-helper cells, γδ T-cells, Natural Killer T-cells and group 3 innate lymphoid cells. CS-induced pulmonary neutrophils were reduced in IL-22-deficient ( Il22−/− ) mice. CS-induced airway remodelling and emphysema-like alveolar enlargement did not occur in Il22−/− mice. Il22−/− mice also had improved lung function in terms of airway resistance, total lung capacity, inspiratory capacity, forced vital capacity and compliance.
These data highlight important roles for IL-22 and its receptors in human COPD and CS-induced experimental COPD.
The spinal cord receives, relays and processes sensory information from the periphery and integrates this information with descending inputs from supraspinal centres to elicit precise and appropriate behavioural responses and orchestrate body movements. Understanding how the spinal cord circuits that achieve this integration are wired during development is the focus of much research interest. Several families of proteins have well-established roles in guiding developing spinal cord axons, and recent findings have identified new axon guidance molecules. Nevertheless, an integrated view of spinal cord network development is lacking, and many current models have neglected the cellular and functional diversity of spinal cord circuits. Recent advances challenge the existing spinal cord axon guidance dogmas and have provided a more complex, but more faithful, picture of the ontogenesis of vertebrate spinal cord circuits. Spinal neural circuits are established through the navigation of multiple types of neuronal axon to their appropriate synaptic targets. Chédotal reviews the cellular and molecular mechanisms that control this complex wiring, incorporating recent discoveries of new guidance factors.
Although preconditioning strategies are growing areas of interest for therapies targeting intervertebral discs (IVDs), it is unknown whether the Wnt signals previously implicated in chondrogenesis, Wnt3A, Wnt5A, and Wnt11, play key roles in the promotion of human nucleus pulposus (NP) cell redifferentiation. In this study, NP cells isolated from herniated disc patients were transduced with lentiviral vectors to overexpress the WNT3A, WNT5A, or WNT11 genes, or CRISPR associated protein 9 (Cas9)/single guide RNA (sgRNA) vectors to knock out these genes. Following expansion, transduced NP cells were induced for redifferentiation toward the NP phenotype. The overexpression of specific WNT factors led to increases in both glycosaminoglycan (GAG) deposition and expression of redifferentiation genes. These effects were attenuated by knockout of the same WNT genes. These results indicate that specific WNT signals can regulate the expression of redifferentiation genes, unequally impact GAG deposition, and contribute to the redifferentiation of human NP cells. This article is protected by copyright. All rights reserved
N(6)-Methyladenosine (m(6)A) is the most prevalent post-transcriptional modification of eukaryotic mRNA and long noncoding RNA. m(6)A mediates its effects primarily by recruiting proteins, including the multiprotein eukaryotic initiation factor 3 complex and a set of proteins that contain the YTH domain. Here we describe the mechanisms by which YTH domain-containing proteins bind m(6)A and influence the fate of m(6)A-containing RNA in mammalian cells. We discuss the diverse, and occasionally contradictory, functions ascribed to these proteins and the emerging concepts that are influencing our understanding of these proteins and their effects on the epitranscriptome.
COPD is a major cause of global mortality and morbidity but current treatments are poorly effective. This is because the underlying mechanisms that drive the development and progression of COPD are incompletely understood. Animal models of disease provide a valuable, ethically and economically viable experimental platform to examine these mechanisms and identify biomarkers that may be therapeutic targets that would facilitate the development of improved standard of care. Here, we review the different established animal models of COPD and the various aspects of disease pathophysiology that have been successfully recapitulated in these models including chronic lung inflammation, airway remodelling, emphysema and impaired lung function. Furthermore, some of the mechanistic features, and thus biomarkers and therapeutic targets of COPD identified in animal models are outlined. Some of the existing therapies that suppress some disease symptoms that were identified in animal models and are progressing towards therapeutic development have been outlined. Further studies of representative animal models of human COPD have the strong potential to identify new and effective therapeutic approaches for COPD.
The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.
Countries have agreed on reduction targets for tobacco smoking stipulated in the WHO global monitoring framework, for achievement by 2025. In an analysis of data for tobacco smoking prevalence from nationally representative survey data, we aimed to provide comprehensive estimates of recent trends in tobacco smoking, projections for future tobacco smoking, and country-level estimates of probabilities of achieving tobacco smoking targets.
We used a Bayesian hierarchical meta-regression modelling approach using data from the WHO Comprehensive Information Systems for Tobacco Control to assess trends from 1990 to 2010 and made projections up to 2025 for current tobacco smoking, daily tobacco smoking, current cigarette smoking, and daily cigarette smoking for 173 countries for men and 178 countries for women. Modelling was implemented in Python with DisMod-MR and PyMC. We estimated trends in country-specific prevalence of tobacco use, projections for future tobacco use, and probabilities for decreased tobacco use, increased tobacco use, and achievement of targets for tobacco control from posterior distributions.
During the most recent decade (2000-10), the prevalence of tobacco smoking in men fell in 125 (72%) countries, and in women fell in 156 (88%) countries. If these trends continue, only 37 (21%) countries are on track to achieve their targets for men and 88 (49%) are on track for women, and there would be an estimated 1·1 billion current tobacco smokers (95% credible interval 700 million to 1·6 billion) in 2025. Rapid increases are predicted in Africa for men and in the eastern Mediterranean for both men and women, suggesting the need for enhanced measures for tobacco control in these regions.
Our findings show that striking between-country disparities in tobacco use would persist in 2025, with many countries not on track to achieve tobacco control targets and several low-income and middle-income countries at risk of worsening tobacco epidemics if these trends remain unchanged. Immediate, effective, and sustained action is necessary to attain and maintain desirable trajectories for tobacco control and achieve global convergence towards elimination of tobacco use.
Ministry of Health, Labour and Welfare, Japan; Ministry of Education, Culture, Sports and Technology, Japan; Department of Health, Australia; Bloomberg Philanthropies.
Copyright © 2015 World Health Organization. Published by Elsevier Ltd/Inc/BV. All rights reserved. Published by Elsevier Ltd. All rights reserved.