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JCI Insight Jul 2023The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we...
The RNA-binding protein LIN28B is overexpressed in over 30% of patients with colorectal cancer (CRC) and is associated with poor prognosis. In the present study, we unraveled a potentially novel mechanism by which LIN28B regulates colonic epithelial cell-cell junctions and CRC metastasis. Using human CRC cells (DLD-1, Caco-2, and LoVo) with either knockdown or overexpression of LIN28B, we identified claudin 1 (CLDN1) tight junction protein as a direct downstream target and effector of LIN28B. RNA immunoprecipitation revealed that LIN28B directly binds to and posttranscriptionally regulates CLDN1 mRNA. Furthermore, using in vitro assays and a potentially novel murine model of metastatic CRC, we show that LIN28B-mediated CLDN1 expression enhances collective invasion, cell migration, and metastatic liver tumor formation. Bulk RNA sequencing of the metastatic liver tumors identified NOTCH3 as a downstream effector of the LIN28B/CLDN1 axis. Additionally, genetic and pharmacologic manipulation of NOTCH3 signaling revealed that NOTCH3 was necessary for invasion and metastatic liver tumor formation. In summary, our results suggest that LIN28B promotes invasion and liver metastasis of CRC by posttranscriptionally regulating CLDN1 and activating NOTCH3 signaling. This discovery offers a promising new therapeutic option for metastatic CRC to the liver, an area where therapeutic advancements have been relatively scarce.
Topics: Humans; Animals; Mice; Colorectal Neoplasms; Claudin-1; Caco-2 Cells; Colonic Neoplasms; Liver Neoplasms; Receptor, Notch3; RNA-Binding Proteins
PubMed: 37318881
DOI: 10.1172/jci.insight.167310 -
Molecular Medicine (Cambridge, Mass.) Oct 2023Ferroptosis plays an essential role in lipopolysaccharide (LPS)-induced acute lung injury (ALI). Meteorin-like/Meteorin-β (Metrnβ) is a protein secreted by skeletal...
BACKGROUND
Ferroptosis plays an essential role in lipopolysaccharide (LPS)-induced acute lung injury (ALI). Meteorin-like/Meteorin-β (Metrnβ) is a protein secreted by skeletal muscle and adipose tissue and plays a role in cardiovascular diseases. However, its role in acute lung injury has not been elucidated.
METHODS
In this study, we used an adenovirus (Ad) delivery system to overexpress or knockdown Metrnβ in lung tissue to examine the role of Metrnβ in LPS-induced acute lung injury.
RESULTS
We found that ferroptosis was increased during LPS-induced ALI. The expression of Metrnβ was reduced in ALI lung tissue. Overexpression of Metrnβ in lung tissue alleviated LPS-induced lung injury, inflammation, and ferroptosis. Moreover, Metrnβ knockout in lung tissue accelerated LPS-induced ALI, inflammation, and ferroptosis. We also cultured MLE-12 cells and transfected the cells with Ad-Metrnβ or Metrnβ siRNA. Metrnβ overexpression ameliorated LPS-induced MLE cell death, inflammation and ferroptosis, while Metrnβ knockdown aggregated cell survival and decreased inflammation and ferroptosis. Moreover, we found that Metrnβ enhanced ferroptosis-related Gpx4 expression and reduced ferroportin and ferritin levels. Furthermore, we found that Metrnβ positively regulated SIRT1 transcription thus inhibited P53, increased SLC7A11 expression. When we used the ferroptosis inhibitor ferrostatin-1, the deteriorating effects of Metrnβ knockout were abolished in ALI mice. Moreover, SIRT1 knockout also abolished the protective effects of Metrnβ overexpression in vivo.
CONCLUSIONS
Taken together, Metrnβ could protect LPS-induced ALI by activating SIRT1-P53- SLC7A11 mediated ferroptosis inhibition.
Topics: Animals; Mice; Acute Lung Injury; Ferroptosis; Inflammation; Lipopolysaccharides; Sirtuin 1; Tumor Suppressor Protein p53
PubMed: 37880599
DOI: 10.1186/s10020-023-00714-6 -
CNS Neuroscience & Therapeutics Dec 2023Chromosome 9 open reading frame 72 (C9orf72) is one of the most dazzling molecules in neurodegenerative diseases, albeit that its role in Parkinson's disease (PD)...
AIMS
Chromosome 9 open reading frame 72 (C9orf72) is one of the most dazzling molecules in neurodegenerative diseases, albeit that its role in Parkinson's disease (PD) remains unknown. This article aimed to explore the potential mechanism of C9orf72 involved in the pathogenesis of PD.
METHODS
The expression and phosphorylation levels of C9orf72 were examined by Western blotting, RT-PCR, and immunoprecipitation using PD models. Multiple bioinformatics software was used to predict the potential phosphorylation sites of C9orf72 by Cdk5, followed by verification of whether Cdk5-inhibitor ROSCOVITINE could reverse the degradation of C9orf72 in PD. By constructing the sh-C9orf72-knockdown adenovirus and overexpressing the FLAG-C9orf72 plasmid, the effects of C9orf72 knockdown and overexpression, respectively, were determined. A short peptide termed Myr-C9orf72 was used to verify whether interfering with Cdk5 phosphorylation at the Ser9 site of the C9orf72 protein could alleviate autophagy disorder, neuronal death, and movement disorder in PD models.
RESULTS
The expression level of the C9orf72 protein was significantly reduced, albeit the mRNA expression was not changed in the PD models. Moreover, the phosphorylation level was enhanced, and its reduction was mainly degraded by the ubiquitin-proteasome pathway. The key nervous system kinase Cdk5 directly phosphorylated the S9 site of the C9orf72 protein, which promoted the degradation of the C9orf72 protein. The knockdown of C9orf72 aggravated autophagy dysfunction and increased neuronal loss and motor dysfunction in substantia nigra neurons of PD mice. The overexpression of C9orf72 alleviated autophagy dysfunction in PD neurons. Specifically, interference with Cdk5 phosphorylation at the S9 site of C9orf72 alleviated autophagy dysfunction, neuronal death, and motor dysfunction mediated by C9orf72 protein degradation during PD.
CONCLUSIONS
Cumulatively, our findings illustrate the importance of the role of C9orf72 in the regulation of neuronal death during PD progression via the Cdk5-dependent degradation.
Topics: Animals; Mice; C9orf72 Protein; Cell Death; Cyclin-Dependent Kinase 5; Neurons; Parkinson Disease; Phosphorylation
PubMed: 37353944
DOI: 10.1111/cns.14319 -
Free Radical Biology & Medicine Oct 2023Cytochrome b reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme,...
Cytochrome b reductase 3 (CYB5R3) activates respiratory metabolism in cellular systems and exerts a prolongevity action in transgenic mice overexpressing this enzyme, mimicking some of the beneficial effects of calorie restriction. The aim of our study was to investigate the role of sex on metabolic adaptations elicited by CYB5R3 overexpression, and how key markers related with mitochondrial function are modulated in skeletal muscle, one of the major contributors to resting energy expenditure. Young CYB5R3 transgenic mice did not exhibit the striking adaptations in carbon metabolism previously detected in older animals. CYB5R3 was efficiently overexpressed and targeted to mitochondria in skeletal muscle from transgenic mice regardless sex. Overexpression significantly elevated NADH in both sexes, although differences were not statistically significant for NAD, and increased the abundance of cytochrome c and the fission protein DRP-1 in females but not in males. Moreover, while mitochondrial biogenesis and function markers (as TFAM, NRF-1 and cleaved SIRT3) were markedly upregulated by CYB5R3 overexpression in females, a downregulation was observed in males. Ultrastructural changes were also highlighted, with an increase in the number of mitochondria per surface unit, and in the size of intermyofibrillar mitochondria in transgenic females compared with their wild-type controls. Our results support that CYB5R3 overexpression upregulates markers consistent with enhanced mitochondrial biogenesis and function, and increases mitochondrial abundance in skeletal muscle, producing most of these potentially beneficial actions in females.
Topics: Animals; Female; Male; Mice; Carrier Proteins; Cytochrome-B(5) Reductase; Energy Metabolism; Mice, Transgenic; Mitochondria; Muscle, Skeletal; Sex Factors
PubMed: 37463636
DOI: 10.1016/j.freeradbiomed.2023.07.012 -
BMC Biology Dec 2023Alzheimer's disease (AD) is the most common neurodegenerative disorder with clinical presentations of progressive cognitive and memory deterioration. The pathologic...
BACKGROUND
Alzheimer's disease (AD) is the most common neurodegenerative disorder with clinical presentations of progressive cognitive and memory deterioration. The pathologic hallmarks of AD include tau neurofibrillary tangles and amyloid plaque depositions in the hippocampus and associated neocortex. The neuronal aggregated tau observed in AD cells suggests that the protein folding problem is a major cause of AD. J-domain-containing proteins (JDPs) are the largest family of cochaperones, which play a vital role in specifying and directing HSP70 chaperone functions. JDPs bind substrates and deliver them to HSP70. The association of JDP and HSP70 opens the substrate-binding domain of HSP70 to help the loading of the clients. However, in the initial HSP70 cycle, which JDP delivers tau to the HSP70 system in neuronal cells remains unclear.
RESULTS
We screened the requirement of a diverse panel of JDPs for preventing tau aggregation in the human neuroblastoma cell line SH-SY5Y by a filter retardation method. Interestingly, knockdown of DNAJB6, one of the JDPs, displayed tau aggregation and overexpression of DNAJB6b, one of the isoforms generated from the DNAJB6 gene by alternative splicing, reduced tau aggregation. Further, the tau bimolecular fluorescence complementation assay confirmed the DNAJB6b-dependent tau clearance. The co-immunoprecipitation and the proximity ligation assay demonstrated the protein-protein interaction between tau and the chaperone-cochaperone complex. The J-domain of DNAJB6b was critical for preventing tau aggregation. Moreover, reduced DNAJB6 expression and increased tau aggregation were detected in an age-dependent manner in immunohistochemical analysis of the hippocampus tissues of a mouse model of tau pathology.
CONCLUSIONS
In summary, downregulation of DNAJB6b increases the insoluble form of tau, while overexpression of DNAJB6b reduces tau aggregation. Moreover, DNAJB6b associates with tau. Therefore, this study reveals that DNAJB6b is a direct sensor for its client tau in the HSP70 folding system in neuronal cells, thus helping to prevent AD.
Topics: Animals; Humans; Mice; Alternative Splicing; Alzheimer Disease; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Molecular Chaperones; Nerve Tissue Proteins; Neuroblastoma; Protein Folding; Protein Isoforms
PubMed: 38110916
DOI: 10.1186/s12915-023-01798-6 -
Chinese Medical Journal Sep 2023Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to...
BACKGROUND
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC.
METHODS
We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/β-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25.
RESULTS
USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/β-catenin pathway (β-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo .
CONCLUSIONS
In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the β-catenin signaling pathway.
Topics: Animals; Mice; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Liver Neoplasms; Ubiquitin Thiolesterase; Wnt Signaling Pathway
PubMed: 37439386
DOI: 10.1097/CM9.0000000000002714 -
Cell Communication and Signaling : CCS Aug 2023Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable malignancies to overcome clinically due to its insidious onset as well as rapid progression. It is...
BACKGROUND
Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable malignancies to overcome clinically due to its insidious onset as well as rapid progression. It is urgent to seek new diagnostic markers and therapeutic targets in order to furthest ameliorate the prognosis of patients with PDAC. V-set and immunoglobulin domain containing 2 (VSIG2) belongs to immunoglobulin superfamily (IgSF), which function as coinhibitory molecule to mediate immune evasion of tumors. Nevertheless, the role of VSIG2 in PDAC and related mechanism still keep unclear.
METHODS
Different expression of VSIG2 in PDAC tissues and cells were detected by bioinformatic analysis, immunohistochemistry, real-time quantitative PCR as well as western blotting. CCK-8, colony formation, Transwell assay, and scratch experiment were utilized to assess proliferation, invasion and migration properties of PDAC cells. The relationship of VSIG2 with late endosomal/lysosomal adaptor, MAPK and MTOR activator 2 (LAMTOR2) and mechanistic target of rapamycin (mTOR) was identified using mass spectrometry, co-immunoprecipitation and immunofluorescence. GO and KEGG enrichment analysis were performed for further pathway verification using western blotting. Additionally, subcutaneous xenograft tumor model and clinical samples analysis were implemented to further elucidate the oncogenic effect of VSIG2 on PDAC in vivo and clinically.
RESULTS
VSIG2 was highly expressed in PDAC tissues and cells. Overexpression of VSIG2 facilitated the proliferation, invasion and migration abilities of PDAC cells, while VSIG2-inhibition exerted opposite effects. Mechanistically, VSIG2 could simultaneously bind to LAMTOR2 and mTOR, thereby enhancing interaction between two molecules, which resulted in elevated phosphorylation-modificatory activation of mTOR and downstream key molecules. Clinically, up-regulation of VSIG2 was positively associated with advanced stage, overall survival and disease-free survival of PDAC patients.
CONCLUSIONS
Our study disclosed that VSIG2 was overexpressed in PDAC, which promoted the proliferation, invasion and metastasis. Mechanically, VSIG2 acted as a scaffold to recruit LAMTOR2 and mTOR simultaneously, stabilize the interaction between them, thus enhancing LAMTOR2-mediated mTOR phosphorylated activation. Collectively, VSIG2 could be exploited as a biomarker for diagnosis and prognosis monitor of PDAC in the future, meanwhile, targeting VSIG2 in PDAC management is expected to be a novel strategy. Video Abstract. Video Abstract.
Topics: Animals; Humans; Biological Assay; Carcinoma, Pancreatic Ductal; Computational Biology; Disease Models, Animal; Pancreatic Neoplasms; Membrane Glycoproteins; Adaptor Proteins, Signal Transducing
PubMed: 37626304
DOI: 10.1186/s12964-023-01209-x -
Biomolecules & Biomedicine Nov 2023Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains...
Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains largely unexplored. This study aimed to investigate the role of apoE in CRC metastasis and to identify the transcription factor and receptor of apoE involved in regulation of CRC metastasis. Bioinformatic analyses were conducted to examine the expression pattern and prognosis of apolipoproteins. APOE-overexpressing cell lines were utilized to explore the effects of apoE on proliferation, migration and invasion of CRC cells. Additionally, the transcription factor and receptor of apoE were screened via bioinformatics, and further validated through knockdown experiments. We discovered that the mRNA levels of APOC1, APOC2, APOD and APOE were higher in lymphatic invasion group, and a higher apoE level indicated poorer overall survival and progression-free interval. In vitro studies demonstrated that APOE-overexpression did not affect proliferation but promoted the migration and invasion of CRC cells. We also reported that APOE-expression was modulated by the transcription factor Jun by activating the proximal promoter region of APOE, and APOE-overexpression reversed the metastasis suppression of JUN knockdown. Furthermore, bioinformatics analysis suggested an interaction between apoE and low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 was highly expressed in both the lymphatic invasion group and the APOEHigh group. Additionally, we found that APOE-overexpression upregulated LRP1 protein levels, and LRP1 knockdown attenuated the metastasis-promoting function of APOE. Overall, our study suggests that the Jun-APOE-LRP1 axis contributes to tumor metastasis in CRC.
Topics: Humans; Apolipoproteins E; Low Density Lipoprotein Receptor-Related Protein-1; Transcription Factors; Cell Movement; Carrier Proteins; Colorectal Neoplasms
PubMed: 37310025
DOI: 10.17305/bb.2023.9248 -
Advanced Science (Weinheim,... Feb 2024Stanniocalcin-1 (STC1) is upregulated by inflammation and modulates oxidative stress-induced cell death. Herein, the function of STC1 in colitis and stress-induced...
Stanniocalcin-1 (STC1) is upregulated by inflammation and modulates oxidative stress-induced cell death. Herein, the function of STC1 in colitis and stress-induced parthanatos, a newly identified type of programmed necrotic cell death dependent on the activation of poly-ADP ribose polymerase-1 (PARP1) is investigated. Results show that STC1 expression is markedly increased in the inflamed colonic mucosa of Crohn's disease (CD) patients and chemically-induced mice colitis models. Evaluation of parthanatos severity and pro-inflammatory cytokine expression shows that intestinal-specific Stc1 knockout (Stc1 ) mice are resistant to dextran sulfate sodium (DSS)-induced colitis and exhibit lower disease severity. STC1-overexpressing cells show an increased degree of parthanatos and proinflammatory cytokine expression, whereas STC1-knockout cells show a decreased degree of parthanatos. Co-immunoprecipitation, mass spectrometry, and proteomic analyses indicate that STC1 interacts with PARP1, which activates the JNK pathway via PARP1-JNK interactions. Moreover, inhibition of PARP1 and JNK alleviates parthanatos and inflammatory injuries triggered by STC1 overexpression. Finally, following restoration of Stc1 and Parp1 expression by adeno-associated viruses, and overexpression of Stc1 and Parp1 aggravated DSS-induced colitis in Stc1 mice. In conclusion, STC1 mediates oxidative stress-associated parthanatos and aggravates inflammation via the STC1-PARP1-JNK interactions and subsequent JNK pathway activation in CD pathogenesis.
Topics: Animals; Humans; Mice; Apoptosis; Colitis; Cytokines; Glycoproteins; Inflammation; Poly (ADP-Ribose) Polymerase-1; Proteomics
PubMed: 38088577
DOI: 10.1002/advs.202304123 -
Hepatology Communications Aug 2023The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly...
BACKGROUND
The role of thioredoxin-interacting protein (TXNIP) in lipopolysaccharide-induced liver injury in mice has been reported, but the underlying mechanisms are poorly understood.
METHODS
We overexpressed deubiquitinase in cells overexpressing TXNIP and then detected the level of TXNIP to screen out the deubiquitinase regulating TXNIP; the interaction between TXNIP and deubiquitinase was verified by coimmunoprecipitation. After knockdown of a deubiquitinase and overexpression of TXNIP in Huh7 and HepG2 cells, lipopolysaccharide was used to establish a cellular inflammatory model to explore the role of deubiquitinase and TXNIP in hepatocyte inflammation.
RESULTS
In this study, we discovered that ubiquitin-specific protease 5 (USP5) interacts with TXNIP and stabilizes it through deubiquitylation in Huh-7 and HepG2 cells after treatment with lipopolysaccharide. In lipopolysaccharide-treated Huh-7 and HepG2 cells, USP5 knockdown increased cell viability, reduced apoptosis, and decreased the expression of inflammatory factors, including NLRP3, IL-1β, IL-18, ASC, and procaspase-1. Overexpression of TXNIP reversed the phenotype induced by knockdown USP5.
CONCLUSIONS
In summary, USP5 promotes lipopolysaccharide-induced apoptosis and inflammatory response by stabilizing the TXNIP protein.
Topics: Apoptosis; Deubiquitinating Enzymes; Lipopolysaccharides; NLR Family, Pyrin Domain-Containing 3 Protein; Signal Transduction; Humans; Hep G2 Cells; Endopeptidases; Carrier Proteins
PubMed: 37534934
DOI: 10.1097/HC9.0000000000000193