Comprehensive analyses of m6A RNA methylation patterns and related immune microenvironment in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease with a poor prognosis and high heritability, characterized by elevated pulmonary vascular resistance (PVR) and pulmonary artery pressure. N6-methyladenosine (m6A) RNA modification influences many RNA metabolism pathways. However, the position of m6A methylation regulators in IPAH remains unknown. Therefore, the study aims to disclose the function m6A regulators exert in the pathological mechanisms of IPAH and the immune microenvironment involved. The GSE117261 dataset was downloaded from the Gene Expression Omnibus (GEO) to screen the differentially expressed genes (DEGs) between normal and IPAH samples. Functional and pathway enrichment analyses of DEGs were then conducted by Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). We also identified the differentially-expressed m6A (DEm6A) regulators between normal and IPAH samples. Key m6A regulators related to the prediction of IPAH were selected using the random forest model. The results showed that FMR1, RBM15, HNRNPA2B1 and IGFBP3 were upregulated in IPAH. In contrast, LRPPRC was downregulated. The single sample gene set enrichment analysis (ssGSEA) method was then adopted to estimate the immune microenvironment in distinct m6A clusters and m6A phenotype-related genes (PRGs) clusters, respectively. Furthermore, we calculated the m6A score via principal component analysis (PCA), and the Sankey diagram was selected to present the correlation among the m6A clusters, m6A PRGs clusters and m6A score. Finally, quantitative RT-PCR and Western blotting were used to validate the key genes in human pulmonary artery smooth muscle cells (HPASMCs) treated by human platelet-derived growth factor-BB (PDGF-BB). The relative mRNA and protein expression levels of FMR1 were significantly elevated, however, the relative mRNA and protein expression levels of LRPPRC were downregulated. Besides, the relative mRNA level of HNRNPA2B1 was increased. Generally, this bioinformatics analysis might provoke more insights into diagnosing and treating IPAH

Axin determines cell fate by controlling the p53 activation threshold after DNA damage

Cells can undergo either cell-cycle arrest or apoptosis after genotoxic stress, based on p53 activity(1-6). Here we show that cellular fate commitment depends on Axin forming distinct complexes with Pirh2, Tip60, HIPK2 and p53. In cells treated with sublethal doses of ultra-violet (UV) radiation or doxorubicin (Dox), Pirh2 abrogates Axin-induced p53 phosphorylation at Ser 46 catalysed by HIPK2, by competing with HIPK2 for binding to Axin. However, on lethal treatment, Tip60 interacts with Axin and abrogates Pirh2-Axin binding, forming an Axin-Tip60-HIPK2-p53 complex that allows maximal p53 activation to trigger apoptosis. We also provide evidence that the ATM/ATR pathway mediates the Axin-Tip60 complex assembly. An axin mutation promotes carcinogenesis in Axin(Fu)/+ (Axin-Fused) mice, consistent with a dominant-negative role for Axin(Fu) in p53 activation. Thus, Axin is a critical determinant in p53-dependent tumour suppression in which Pirh2 and Tip60 have different roles in triggering cell-cycle arrest or apoptosis depending on the severity of genotoxic stress.973 Program and 863 Program National Natural Science Foundation of China Ministry of Education of China National Basic Research Program of the Ministry of Science and Technology National Science Foundation of Fujian Provinc

AIDA通过内质网相关的蛋白质降解途径选择性下调脂肪合成途径的代谢酶从而减缓肠道脂肪吸收并防止肥胖发生

文章简介肠道对膳食脂肪吸收的效率是个人是否易患肥胖的主要决定因素之一。然而,目前人们还不清楚脂肪吸收是如何受调控并导致肥胖的。本研究表明,抑制内质网相关的蛋白质降解途径会提高甘油三酯合成途径的数个代谢酶的水平,并促进小肠对脂肪的吸收。包含C2结构域的蛋白AIDA作为一个重要国家重点基础研发计划;;\n国家自然科学基金;;\n厦门大学校长基金等支

Protein phosphorylation-acetylation cascade connects growth factor deprivation to autophagy

Different from unicellular organisms, metazoan cells require the presence of extracellular growth factors to utilize environmental nutrients. However, the underlying mechanism was unclear. We have delineated a pathway, in which glycogen synthase kinase 3 (GSK3) in cells deprived of growth factors phosphorylates and activates the acetyltransferase KAT5/TIP60, which in turn stimulates the protein kinase ULK1 to elicit autophagy. Cells with the Kat5/Tip60 gene replaced with Kat5(S86A) that cannot be phosphorylated by GSK3 are resistant to serum starvation-induced autophagy. Acetylation sites on ULK1 were mapped to K162 and K606, and the acetylation-defective mutant ULK1(K162,606R) displays reduced kinase activity and fails to rescue autophagy in Ulk1(-/-) mouse embryonic fibroblasts, indicating that acetylation is vital to the activation of ULK1. The GSK3-KAT5-ULK1 cascade seems to be specific for cells to sense growth factors, as KAT5 phosphorylation is not enhanced under glucose deprivation. Distinct from the glucose starvation-autophagy pathway that is conserved in all eukaryotic organisms, the growth factor deprivation response pathway is perhaps unique to metazoan organisms.973 Program [2011CB910800]; NSFC [31130016, 30921005, 31000621]; Fundamental Research Funds for the Central Universities [2010121094]; MOE of China [B06016

AMP as a Low-Energy Charge Signal Autonomously Initiates Assembly of AXIN-AMPK-LKB1 Complex for AMPK Activation

The AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis by sensing cellular energy status. AMPK is mainly activated via phosphorylation by LKB1 when cellular AMP/ADP levels are increased. However, how AMP/ADP brings about AMPK phosphorylation remains unclear. Here, we show that it is AMP, but not ADP, that drives AXIN to directly tether LKB1 to phosphorylate AMPK. The complex formation of AXIN-AMPK-LKB1 is greatly enhanced in glucose-starved or AICAR-treated cells and in cell-free systems supplemented with exogenous AMP. Depletion of AXIN abrogated starvation-induced AMPK-LKB1 colocalization. Importantly, adenovirus-based knockdown of AXIN in the mouse liver impaired AMPK activation and caused exacerbated fatty liver after starvation, underscoring an essential role of AXIN in AMPK activation. These findings demonstrate an initiating role of AMP and demonstrate that AXIN directly transmits AMP binding of AMPK to its activation by LKB1, uncovering the mechanistic route for AMP to elicit AMPK activation by LKB1.http://news.xmu.edu.cn/s/13/t/542/22/a9/info139945.ht

The Lysosomal v-ATPase-Ragulator Complex Is a Common Activator for AMPK and mTORC1, Acting as a Switch between Catabolism and Anabolism

林圣彩教授课题组长期致力于细胞信号转导的研究。近年来，该课题组潜心研究，不断攻关，取得了一系列重大成果，如揭示细胞如何应对生长因子缺乏的内在机理，发现了细胞自噬“路线图”、还发现了细胞如何感应“饥饿”信号AMP的信号传导通路等。其中，“发现细胞自噬‘路线图’”成果曾登上《科学》杂志，并入选2012年度“中国科学十大进展”。AMPK and mTOR play principal roles in governing metabolic programs; however, mechanisms underlying the coordination of the two inversely regulated kinases remain unclear. In this study we found, most surprisingly, that the late endosomal/lysosomal protein complex v-ATPase-Ragulator, essential for activation of mTORC1, is also required for AMPK activation. We also uncovered that AMPK is a residential protein of late endosome/lysosome. Under glucose starvation, the v-ATPase-Ragulator complex is accessible to AXIN/LKB1 for AMPK activation. Concurrently, the guanine nucleotide exchange factor (GEF) activity of Ragulator toward RAG is inhibited by AXIN, causing dissociation from endosome and inactivation of mTORC1. We have thus revealed that the v-ATPase-Ragulator complex is also an initiating sensor for energy stress and meanwhile serves as an endosomal docking site for LKB1-mediated AMPK activation by forming the v-ATPase-Ragulator-AXIN/LKB1-AMPK complex, thereby providing a switch between catabolism and anabolism. Our current study also emphasizes a general role of late endosome/lysosome in controlling metabolic programs

Bioinformatics analysis of the immune cell infiltration characteristics and correlation with crucial diagnostic markers in pulmonary arterial hypertension

Abstract Background Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome, characterized by pulmonary vascular remodeling. Immunity and inflammation are progressively recognized properties of PAH, which are crucial for the initiation and maintenance of pulmonary vascular remodeling. This study explored immune cell infiltration characteristics and potential biomarkers of PAH using comprehensive bioinformatics analysis. Methods Microarray data of GSE117261, GSE113439 and GSE53408 datasets were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified in GSE117261 dataset. The proportions of infiltrated immune cells were evaluated by CIBERSORT algorithm. Feature genes of PAH were selected by least absolute shrinkage and selection operator (LASSO) regression analysis and validated by fivefold cross-validation, random forest and logistic regression. The GSE113439 and GSE53408 datasets were used as validation sets and logistic regression and receiver operating characteristic (ROC) curve analysis were performed to evaluate the prediction value of PAH. The PAH-associated module was identified by weighted gene association network analysis (WGCNA). The intersection of genes in the modules screened and DEGs was used to construct protein–protein interaction (PPI) network and the core genes were selected. After the intersection of feature genes and core genes, the hub genes were identified. The correlation between hub genes and immune cell infiltration was analyzed by Pearson correlation analysis. The expression level of LTBP1 in the lungs of monocrotaline-induced PAH rats was determined by Western blotting. The localization of LTBP1 and CD4 in lungs of PAH was assayed by immunofluorescence. Results A total of 419 DEGs were identified, including 223 upregulated genes and 196 downregulated genes. Functional enrichment analysis revealed that a significant enrichment in inflammation, immune response, and transforming growth factor β (TGFβ) signaling pathway. CIBERSORT analysis showed that ten significantly different types of immune cells were identified between PAH and control. Resting memory CD4+ T cells, CD8+ T cells, γδ T cells, M1 macrophages, and resting mast cells in the lungs of PAH patients were significantly higher than control. Seventeen feature genes were identified by LASSO regression for PAH prediction. WGCNA identified 15 co-expression modules. PPI network was constructed and 100 core genes were obtained. Complement C3b/C4b receptor 1 (CR1), thioredoxin reductase 1 (TXNRD1), latent TGFβ binding protein 1 (LTBP1), and toll-like receptor 1 (TLR1) were identified as hub genes and LTBP1 has the highest diagnostic efficacy for PAH (AUC = 0.968). Pearson correlation analysis showed that LTBP1 was positively correlated with resting memory CD4+ T cells, but negatively correlated with monocytes and neutrophils. Western blotting showed that the protein level of LTBP1 was increased in the lungs of monocrotaline-induced PAH rats. Immunofluorescence of lung tissues from rats with PAH showed increased expression of LTBP1 in pulmonary arteries as compared to control and LTBP1 was partly colocalized with CD4+ cells in the lungs. Conclusion LTBP1 was correlated with immune cell infiltration and identified as the critical diagnostic maker for PAH

The identification and verification of hub genes associated with pulmonary arterial hypertension using weighted gene co-expression network analysis

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure, with complex etiology, difficult treatment and poor prognosis. The objective of this study was to investigate the potential biomarkers for PAH based on bioinformatics analysis. Methods The GSE117261 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified by screening PAH patients and controls. Then the DEGs were analyzed using a Weighted Gene Co-expression Network Analysis (WGCNA) and the key modules were determined, and to further explore their potential biological functions via Gene Ontology analysis (GO), Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG), and Gene Set Enrichment Analysis (GSEA). Moreover, Protein–protein interaction (PPI) networks were constructed to identify hub gene candidates in the key modules. Finally, real-time quantitative polymerase chain reaction was supplied to detect the expressions of hub genes in human pulmonary arterial smooth cells treated with cobalt chloride (COCl2) which was used to mimic hypoxia. Results There were 2299 DEGs identified. WGCNA indicated that yellow module was the key one correlated with PAH. GO and KEGG analysis demonstrated that genes in the yellow module were mainly enriched in ‘Pathways in cancer’. GSEA revealed that ‘HALLMARK_MYC_TARGETS_V1’ was remarkably enriched in PAH. Based on the PPI network, vascular endothelial growth factor A, proto-oncogene receptor tyrosine kinase (KIT), PNN interacting serine and arginine rich protein (PNISR) and heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) were identified as the hub genes. Additionally, the PCR indicated that the elevated expressions of PNISR and HNRNPH1 were in line with the bioinformatics analysis. ROC analysis determined that PNISR and HNRNPH1 may be potential biomarkers to provide better diagnosis of PAH. Conclusion PNISR and HNRNPH1 were potential biomarkers to diagnosis PAH. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of PAH

MDM2 Inhibits Axin-Induced p53 Activation Independently of its E3 Ligase Activity

<div><p>MDM2 plays a crucial role in negatively regulating the functions of tumor suppressor p53. Here we show that MDM2 can inhibit Axin-stimulated p53-dependent apoptosis by suppressing p53 phosphorylation at Ser 46 and apoptosis-related p53 transactivational activity. Interestingly, the ubiquitin E3 ligase activity of MDM2 is not required for this inhibitory effect. Mechanically, either wildtype MDM2 or its E3-dead mutant, disrupts the Axin-based HIPK2/p53 complex formation by blocking the binding of p53 and HIPK2 to Axin. MDM2Δp53, a deletion mutant that lacks p53 binding domain fails to exert the inhibitory effect, demonstrating that the interaction of MDM2 and p53, but not its E3 ligase activity toward p53 plays key role in suppressing Axin-stimulated p53 activation. Our results thus have revealed a novel aspect of the mechanism by which MDM2 regulates p53 activities.</p></div