Lack of integrin alpha-chain endoproteolytic cleavage in furin-deficient human colon adenocarcinoma cells LoVo.
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ABSTRACT:
SUBMITTER: Lehmann M
PROVIDER: S-EPMC1217556 | biostudies-other | 1996 Aug
REPOSITORIES: biostudies-other
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Project description:The integrin α (ITGA) subfamily genes play a fundamental role in various cancers. However, the potential mechanism and application values of ITGA genes in colon adenocarcinoma (COAD) remain elusive. The present study investigated the significance of the expression of ITGA genes in COAD from the perspective of diagnosis and prognosis. A COAD RNA‑sequencing dataset was obtained from The Cancer Genome Atlas. The present study investigated the biological function of the ITGA subfamily genes through bioinformatics analysis. Reverse transcription‑quantitative polymerase chain reaction was applied to investigate the distribution of integrin α8 (ITGA8) expression in COAD tumors and adjacent normal tissues. Bioinformatics analysis indicated that ITGA genes were noticeably enriched in cell adhesion and the integrin‑mediated signaling pathway, and co‑expressed with each other. It was also revealed through observation that the majority of gene expression was significantly low in tumor tissues (P<0.05), and diagnostic receiver operating characteristic curves revealed that most of the genes could serve as significant diagnostic markers in COAD (P<0.05), especially ITGA8 which had a high diagnostic value with an area under curve (AUC) of 0.989 [95% confidence interval (CI) 0.980‑0.997] in COAD (P<0.0001). In addition, ITGA8 expression was verified in clinical samples and it was revealed that it was higher in adjacent normal tissues (P=0.041) compared to COAD tissues, and the AUC was 0.704 (95% CI, 0.577‑0.831; P<0.0085). Multivariate survival analysis indicated that integrin α (ITGA5) may be an independent prognostic indicator for COAD overall survival. Gene set enrichment analysis indicated that ITGA5 may participate in multiple biological processes and pathways. The present study revealed that ITGA genes were associated with the diagnosis and prognosis of COAD. The mRNA expression of ITGA8 may be a potential diagnosis biomarker and ITGA5 may serve as an independent prognosis indicator for COAD.
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Project description:Cleavage of the viral surface protein prM by the proprotein convertase furin is a key step in the maturation process of flavivirus particles. A mutant of tick-borne encephalitis virus (TBEV) carrying a deletion mutation within the furin recognition motif of protein prM (changing R-T-R-R to R-T-R) was previously shown to be noninfectious in BHK-21 cells. We now demonstrate how natural selection can overcome this lethal defect in two different growth systems by distinct resuscitating mutations. In BHK-21 cells, a spontaneous codon duplication created a minimal furin cleavage motif (R-R-T-R). This mutation restored infectivity by enabling intracellular prM cleavage. A completely different mutation pattern was observed when the mutant virus was passaged in mouse brains. The "pr" part of protein prM, which is removed by cleavage, contains six conserved Cys residues. The mutations selected in mice changed the number of Cys residues to five or seven by substitution mutations near the original cleavage site, probably causing a major perturbation of the structural integrity of protein prM. Although viable in mice, such Cys mutants could not be passaged in BHK-21 cells under normal growth conditions (37 degrees C), but one of the mutants exhibited a low level of infectivity at a reduced incubation temperature (28 degrees C). No evidence for the cleavage of protein prM in BHK-21 cells was obtained. This suggests that under certain growth conditions, the structural perturbation of protein prM can restore the infectivity of TBEV by circumventing the need for intracellular furin-mediated cleavage. This is the first example of a flavivirus using such a molecular mechanism.
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Project description:TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.
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Project description:Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.
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Project description:The spike protein is a focused target of COVID-19, a pandemic caused by SARS-CoV-2. A 12-nt insertion at S1/S2 in the spike coding sequence yields a furin cleavage site, which raised controversy views on origin of the virus. Here we analyzed the phylogenetic relationships of coronavirus spike proteins and mapped furin recognition motif on the tree. Furin cleavage sites occurred independently for multiple times in the evolution of the coronavirus family, supporting the natural occurring hypothesis of SARS-CoV-2.
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Project description:Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.
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Project description:Cancer cell invasion is a key element in metastasis that requires integrins for adhesion/de-adhesion, as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Herein we show that MMP-2 is up-regulated in resected colorectal tumors and degrades ?1 integrins with the release of fragments containing the ?1 I-domain. The ?1 cleavage pattern is similar to that produced by digestion of ?5?1 and ?2?1 with MMP-2. Two such fragments, at 25 and 75 kDa, were identified after immunoprecipitation, with monoclonal antibody BD610468 reacting with the NH(2)-terminal I-like ectodomain followed by SDS-PAGE and microsequencing using electrospray (ISI-Q-TOF-Micromass) spectrometry. Cleavage of the ?1 integrin can be abolished by inhibition of MMP-2 activity; it can be induced by up-regulation of MMP-2 expression, as exemplified by HT29 colon cancer cells transfected with pCMV6-XL5-MMP-2. Co-immunoprecipitation studies of colon cancer cells showed that the ?1 integrin subunit is associated with MMP-2. The MMP-2-mediated shedding of the I-like domain from ?1 integrins resulted in decreased adhesion of colon cancer cells to collagen and fibronectin, thus abolishing their receptivity. Furthermore, such cells showed enhanced motility as evaluated by a "wound healing-like" assay and time-lapse microscopy, indicating their increased invasiveness. Altogether, our data demonstrate that MMP-2 amplifies the motility of colon cancer cells, not only by digesting the extracellular matrix components in the vicinity of cancer cells but also by inactivating their major ?1 integrin receptors.
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Project description:Intact fibroblast growth factor 23 (iFGF23) is a phosphaturic hormone that is cleaved by furin into N-terminal and C-terminal fragments. Several studies have implicated vitamin D in regulating furin in infections. Thus, we investigated the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D] and the vitamin D receptor (VDR) on furin-mediated iFGF23 cleavage. Mice lacking VDR (Vdr-/-) had a 25-fold increase in iFGF23 cleavage, with increased furin levels and activity compared with wild-type (WT) littermates. Inhibition of furin activity blocked the increase in iFGF23 cleavage in Vdr-/- animals and in a Vdr-knockdown osteocyte OCY454 cell line. Chromatin immunoprecipitation revealed VDR binding to DNA upstream of the Furin gene, with more transcription in the absence of VDR. In WT mice, furin inhibition reduced iFGF23 cleavage, increased iFGF23, and reduced serum phosphate levels. Similarly, 1,25(OH)2D reduced furin activity, decreased iFGF23 cleavage, and increased total FGF23. In a post hoc analysis of a randomized clinical trial, we found that ergocalciferol treatment, which increased serum 1,25(OH)2D, significantly decreased serum furin activity and iFGF23 cleavage, compared with placebo. Thus, 1,25(OH)2D inhibits iFGF23 cleavage via VDR-mediated suppression of Furin expression, thereby providing a mechanism by which vitamin D can augment phosphaturic iFGF23 levels.
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Project description:BackgroundMesothelial E- and P-selectins substantially mediate the intraperitoneal spread of Pancreatic ductal adenocarcinoma (PDA) cells in xenograft models. In the absence of selectins in the host, the integrin subunit alpha-V (ITGAV, CD51) was upregulated in the remaining metastatic deposits. Here we present the first experimental study to investigate if ITGAV plays a functional role in PDA tumor growth and progression with a particular focus on intraperitoneal carcinomatosis.MethodsKnockdown of ITGAV was generated using an RNA interference-mediated approach in two PDA cell lines. Tumor growth, intraperitoneal and distant metastasis were analyzed in a xenograft model. Cell lines were characterized in vitro. Gene expression of the xenograft tumors was analyzed. Patient samples were histologically classified and associations to survival were evaluated.ResultsThe knockdown of ITGAV in PDA cells strongly reduces primary tumor growth, peritoneal carcinomatosis and spontaneous pulmonary metastasis. ITGAV activates latent TGF-β and thereby drives epithelial-mesenchymal transition. Combined depletion of ITGAV on the tumor cells and E- and P-selectins in the tumor-host synergistically almost abolishes intraperitoneal spread. Accordingly, high expression of ITGAV in PDA cells was associated with reduced survival in patients.ConclusionCombined depletion of ITGAV in PDA cells and E- and P-selectins in host mice massively suppresses intraperitoneal carcinomatosis of PDA cells xenografted into immunodeficient mice, confirming the hypothesis of a partly redundant adhesion cascade of metastasizing cancer cells. Our data strongly encourage developing novel therapeutic approaches for the combined targeting of E- and P-selectins and ITGAV in PDA.
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