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α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8+/+), Fut8 heterozygous knockout (Fut8+/-), and Fut8 knockout (Fut8-/-) mice. The IgG levels in serum were lower in Fut8+/- and Fut8-/- mice compared with Fut8+/+ mice. Exogenous L-fucose increased IgG levels in Fut8+/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor â £ (FcγRâ £), mainly expressed on macrophages and neutrophils, were increased in Fut8+/- mice compared to Fut8+/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRâ £ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRâ £-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8+/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation.
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α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8-/-) or heterozygous KO (Fut8+/-) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/- mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/- mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/- mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/- mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation.
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Fucosa , Inflamación , Lipopolisacáridos , Animales , Humanos , Ratones , Receptor gp130 de Citocinas , Fucosa/farmacología , Fucosa/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Interleucina-6/genética , Lipopolisacáridos/toxicidad , Enfermedades Neuroinflamatorias , ARN MensajeroRESUMEN
Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.
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Colitis Ulcerosa , Fucosiltransferasas , Animales , Ratones , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Fucosiltransferasas/genética , Inflamación , Mucina 2/genética , Mucina 2/metabolismo , Células HT29RESUMEN
The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , FN-kappa B , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Transducción de Señal , Doxorrubicina/farmacología , Polisacáridos/metabolismo , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
Sialylation is a terminal glycosylated modification of glycoproteins that regulates critical biological events such as cell adhesion and immune response. Our previous study showed that integrin α3ß1 plays a crucial role in regulating the sialylation of N-glycans. However, the underlying mechanism for the regulation remains unclear. This study investigated how sialylation is affected by focal adhesion kinase (FAK), which is a critical downstream signal molecule of integrin ß1. We established a stable FAK knockout (KO) cell line using the CRISPR/Cas9 system in HeLa cells. The results obtained from lectin blot, flow cytometric analysis, and MS showed that the sialylation levels were significantly decreased in the KO cells compared with that in wild-type (WT) cells. Moreover, phosphatidylinositol 4-phosphate (PI4P) expression levels were also reduced in the KO cells due to a decrease in the stability of phosphatidylinositol 4-kinase-IIα (PI4KIIα). Notably, the decreased levels of sialylation, PI4P, and the complex formation between GOLPH3 and ST3GAL4 or ST6GAL1, which are the main sialyltransferases for modification of N-glycans, were significantly restored by the re-expression of FAK. Furthermore, the decreased sialylation and phosphorylation of Akt and cell migration caused by FAK deficiency all were restored by overexpressing PI4KIIα, which suggests that PI4KIIα is one of the downstream molecules of FAK. These findings indicate that FAK regulates sialylation via the PI4P synthesis pathway and a novel mechanism is suggested for the integrin-FAK-PI4KIIα-GOLPH3-ST axis modulation of sialylation in N-glycans.
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Quinasa 1 de Adhesión Focal , Polisacáridos , Transducción de Señal , Humanos , Quinasa 1 de Adhesión Focal/metabolismo , Células HeLa , Proteínas de la Membrana/metabolismo , Fosforilación , Polisacáridos/metabolismoRESUMEN
Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.
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Anticuerpos Monoclonales , Fucosa , Inmunoglobulina G , Enfermedades Pulmonares , Polisacáridos , Humanos , Células A549 , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Linfocitos B/inmunología , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Fucosa/sangre , Fucosa/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Técnicas de Inactivación de Genes , Inmunoensayo/normas , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Enfermedades Pulmonares/diagnóstico , Enfermedades Pulmonares/inmunología , Polisacáridos/metabolismo , Animales , Ratones , Células CHO , Células HEK293 , CricetulusRESUMEN
Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.
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Leucemia Mieloide Aguda , Tirosina Quinasa 3 Similar a fms , Humanos , Tirosina Quinasa 3 Similar a fms/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Transducción de Señal/fisiología , Mutación , Estabilidad ProteicaRESUMEN
For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1ß, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.
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Leucemia Promielocítica Aguda , Humanos , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Glicosilación , Tretinoina/farmacología , Tretinoina/metabolismo , Diferenciación Celular , Células HL-60 , Línea Celular TumoralRESUMEN
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily. BMPs play crucial roles in embryogenesis and bone remodeling. Recently, BMP signaling has been found to have diverse effects on different types of tumors. In this review, we summarized the effects of BMP signaling on gynecologic cancer. BMP signaling has tumor-promoting effects on ovarian cancer (OC) and endometrial cancer (EC), whereas it has tumor-suppressing effects on uterine cervical cancer (UCC). Interestingly, EC has frequent gain-of-function mutations in ACVR1, encoding one of the type I BMP receptors, which are also observed in fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. Little is known about the relationship between BMP signaling and other gynecologic cancers. Tumor-promoting effects of BMP signaling in OC and EC are dependent on the promotion of cancer stemness and epithelial-mesenchymal transition (EMT). In accordance, BMP receptor kinase inhibitors suppress the cell growth and migration of OC and EC. Since both cancer stemness and EMT are associated with chemoresistance, BMP signaling activation might also be an important mechanism by which OC and EC patients acquire chemoresistance. Therefore, BMP inhibitors are promising for OC and EC patients even if they become resistant to standard chemotherapy. In contrast, BMP signaling inhibits UCC growth in vitro. However, the in vivo effects of BMP signaling have not been elucidated in UCC. In conclusion, BMP signaling has a variety of functions, depending on the types of gynecologic cancer. Therefore, targeting BMP signaling should improve the treatment of patients with gynecologic cancer.
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Miositis Osificante , Neoplasias , Humanos , Femenino , Proteínas Morfogenéticas Óseas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Miositis Osificante/genética , Miositis Osificante/metabolismo , Miositis Osificante/patología , Transición Epitelial-MesenquimalRESUMEN
N-Linked glycosylation and O-linked N-acetylglucosamine (O-GlcNAc) are important protein post-translational modifications that are orchestrated by a diverse set of gene products. Thus far, the relationship between these two types of glycosylation has remained elusive, and it is unclear whether one influences the other via UDP-GlcNAc, which is a common donor substrate. Theoretically, a decrease in O-GlcNAcylation may increase the products of GlcNAc-branched N-glycans. In this study, via examination by lectin blotting, HPLC, and mass spectrometry analysis, however, we found that the amounts of GlcNAc-branched tri-antennary N-glycans catalyzed by N-acetylglucosaminyltransferase IV (GnT-IV) and tetra-antennary N-glycans were significantly decreased in O-GlcNAc transferase knockdown cells (OGT-KD) compared with those in wild type cells. We examined this specific alteration by focusing on SLC35A3, which is the main UDP-GlcNAc transporter in mammals that is believed to modulate GnT-IV activation. It is interesting that a deficiency of SLC35A3 specifically leads to a decrease in the amounts of GlcNAc-branched tri- and tetra-antennary N-glycans. Furthermore, co-immunoprecipitation experiments have shown that SLC35A3 interacts with GnT-IV, but not with N-acetylglucosaminyltransferase V. Western blot and chemoenzymatic labeling assay have confirmed that OGT modifies SLC35A3 and that O-GlcNAcylation contributes to its stability. Furthermore, we found that SLC35A3-KO enhances cell spreading and suppresses both cell migration and cell proliferation, which is similar to the phenomena observed in the OGT-KD cells. Taken together, these data are the first to demonstrate that O-GlcNAcylation specifically governs the biosynthesis of tri- and tetra-antennary N-glycans via the OGT-SLC35A3-GnT-IV axis.
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Acetilglucosamina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Polisacáridos/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Glicosilación , Células HEK293 , Células HeLa , HumanosRESUMEN
Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression. TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.
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Proteínas Morfogenéticas Óseas , Neoplasias , Animales , Ratones , Humanos , Proteínas Morfogenéticas Óseas/metabolismo , Transducción de Señal , Ratones Noqueados , Desarrollo Embrionario/genética , Neoplasias/genéticaRESUMEN
As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8+ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8+/+ OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8-/- OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8-/- CTL, resulted in enhanced Fut8-/- CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.
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Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/terapia , Antineoplásicos/inmunología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T Citotóxicos/inmunología , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , Fucosiltransferasas/inmunología , Glicosilación , Células HEK293 , Humanos , Activación de Linfocitos/inmunología , Ratones , Transducción de Señal/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML.
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Fucosa/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Glicosilación , Células HEK293 , Humanos , Interleucina-3/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Dominios Proteicos , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT5/metabolismo , Tirosina Quinasa 3 Similar a fms/química , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
The α2,3-sialylation of N-glycans is considered important but complicated because the functions of the three ß-galactoside α2,3-sialyltransferases, ST3GAL3, ST3GAL4, and ST3GAL6, could be compensating for one another. To distinguish their specific functions, we established each individual knockout (KO) cell line. Loss of either the ST3GAL3 or ST3GAL6 genes decreased cell proliferation and colony formation, as opposed to the effect in the ST3GAL4 KO cells. The phosphorylation levels of ERK and AKT were significantly suppressed in the ST3GAL6 KO and ST3GAL3 KO cells, respectively. The cell aggregations were clearly observed in the KO cells, particularly the ST3GAL3 KO and ST3GAL6 KO cells, and the expression levels of E-cadherin and claudin-1 were enhanced in both those cell lines, but were suppressed in the ST3GAL4 KO cells. Those alterations were reversed with an overexpression of each corresponding gene in rescued cells. Of particular interest, the α2,3-sialylation levels of ß1 integrin were clearly suppressed in the ST3GAL4 KO cells, but these were increased in the ST3GAL3 KO and ST3GAL6 KO cells, whereas the α2,3-sialylation levels of EGFR were significantly decreased in the ST3GAL6 KO cells. The decrease in α2,3-sialylation increased the α2,6-sialylation on ß1, but not EGFR. Furthermore, a cross-restoration of each of the three genes in ST3GAL6 KO cells showed that overexpression of ST3GAL6 sufficiently rescued the total α2,3-sialylation levels, cell morphology, and α2,3-sialylation of EGFR, whereas the α2,3-sialylation levels of ß1 were greatly enhanced by an overexpression of ST3GAL4. These results clearly demonstrate that the three α2,3-sialyltransferases modify characteristic target proteins and regulate cell biological functions in different ways.
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Sialiltransferasas/metabolismo , Cadherinas/metabolismo , Línea Celular , Proliferación Celular/fisiología , Glicosilación , Humanos , Fosforilación/fisiología , Transporte de Proteínas , Sialiltransferasas/genética , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
The effects of bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, in endometrial cancer (EC) have yet to be determined. In this study, we analyzed the TCGA and MSK-IMPACT datasets and investigated the effects of BMP2 and of TWSG1, a BMP antagonist, on Ishikawa EC cells. Frequent ACVR1 mutations and high mRNA expressions of BMP ligands and receptors were observed in EC patients of the TCGA and MSK-IMPACT datasets. Ishikawa cells secreted higher amounts of BMP2 compared with ovarian cancer cell lines. Exogenous BMP2 stimulation enhanced EC cell sphere formation via c-KIT induction. BMP2 also induced EMT of EC cells, and promoted migration by induction of SLUG. The BMP receptor kinase inhibitor LDN193189 augmented the growth inhibitory effects of carboplatin. Analyses of mRNAs of several BMP antagonists revealed that TWSG1 mRNA was abundantly expressed in Ishikawa cells. TWSG1 suppressed BMP7-induced, but not BMP2-induced, EC cell sphere formation and migration. Our results suggest that BMP signaling promotes EC tumorigenesis, and that TWSG1 antagonizes BMP7 in EC. BMP signaling inhibitors, in combination with chemotherapy, might be useful in the treatment of EC patients.
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Biomarcadores de Tumor/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 7/metabolismo , Carcinógenos/metabolismo , Neoplasias Endometriales/patología , Regulación Neoplásica de la Expresión Génica , Apoptosis , Biomarcadores de Tumor/genética , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 7/genética , Proliferación Celular , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Femenino , Humanos , Pronóstico , Transducción de Señal , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Aberrant N-glycan sialylation of glycoproteins is closely associated with malignant phenotypes of cancer cells and metastatic potential, which includes cell adhesion, migration, and growth. Recently, phosphatidylinositol 4-kinase IIα (PI4KIIα), which is localized to the trans-Golgi network, was identified as a regulator of Golgi phosphoprotein 3 (GOLPH3) and of vesicle transport in the Golgi apparatus. GOLPH3 is a target of PI4KIIα and helps anchor sialyltransferases and thereby regulates sialylation of cell surface receptors. However, how PI4KIIα-mediated sialyation of cell surface proteins is regulated remains unclear. In this study, using several cell lines, CRISPR/Cas9-based gene knockout and short hairpin RNA-mediated silencing, RT-PCR, lentivirus-mediated overexpression, and immunoblotting methods, we confirmed that PI4KIIα knockdown suppresses the sialylation of N-glycans on the cell surface, in Akt phosphorylation and activation, and integrin α3-mediated cell migration of MDA-MB-231 breast cancer cells. Interestingly, both integrin α3ß1 and PI4KIIα co-localized to the trans-Golgi network, where they physically interacted with each other, and PI4KIIα specifically associated with integrin α3 but not α5. Furthermore, overexpression of both integrin α3ß1 and PI4KIIα induced hypersialylation. Conversely, integrin α3 knockout significantly inhibited the sialylation of membrane proteins, such as the epidermal growth factor receptor, as well as in total cell lysates. These findings suggest that the malignant phenotype of cancer cells is affected by a sialylation mechanism that is regulated by a complex between PI4KIIα and integrin α3ß1.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Integrina alfa3beta1/metabolismo , Ácido N-Acetilneuramínico/metabolismo , 1-Fosfatidilinositol 4-Quinasa/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Movimiento Celular , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfa3beta1/genética , Proteínas de la Membrana/metabolismo , Fosforilación , Polisacáridos/metabolismo , Unión Proteica , Transducción de Señal , Red trans-Golgi/metabolismoRESUMEN
O-GlcNAcylation is a post-translational modification of a protein serine or threonine residue catalyzed by O-GlcNAc transferase (OGT) in the nucleus and cytoplasm. O-GlcNAcylation plays important roles in the cellular signaling that affect the different biological functions of cells, depending upon cell type. However, whether or not O-GlcNAcylation regulates cell adhesion and migration remains unclear. Here, we used the doxycycline-inducible short hairpin RNA (shRNA) system to establish an OGT knockdown (KD) HeLa cell line and found that O-GlcNAcylation is a key regulator for cell adhesion, migration, and focal adhesion (FA) complex formation. The expression levels of OGT and O-GlcNAcylation were remarkably suppressed 24 h after induction of doxycycline. Knockdown of OGT significantly promoted cell adhesion, but it suppressed the cell migration on fibronectin. The immunostaining with paxillin, a marker for FA plaque, clearly showed that the number of FAs was increased in the KD cells compared with that in the control cells. The O-GlcNAcylation levels of paxillin, talin, and focal adhesion kinase were down-regulated in KD cells. Interestingly, the complex formation between integrin ß1, focal adhesion kinase, paxillin, and talin was greatly increased in KD cells. Consistently, levels of active integrin ß1 were significantly enhanced in KD cells, whereas they were decreased in cells overexpressing OGT. The data suggest a novel regulatory mechanism for O-GlcNAcylation during FA complex formation, which thereby affects integrin activation and integrin-mediated functions such as cell adhesion and migration.
Asunto(s)
Acetilglucosamina/metabolismo , Adhesión Celular , Movimiento Celular , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Quinasa 1 de Adhesión Focal/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , N-Acetilglucosaminiltransferasas/deficiencia , N-Acetilglucosaminiltransferasas/genética , Paxillin/metabolismo , Talina/metabolismoRESUMEN
The N-glycosylation of integrin α5ß1 is involved in multiple cell biological functions. Our group previously reported that the N-glycosylation of the Calf-1,2 domain on α5 subunit (S3-5,10-14) was important for its inhibitory effect on EGFR signaling through regulating α5-EGFR complex formation. In this follow-up study, we provide evidence that the N-glycosylation on integrin ß1 subunit suppress cell growth by promoting its association with EGFR under fibronectin (FN)-coated conditions. Expression of wild-type (WT) ß1, but not the N-glycosylation mutant S4-6 ß1, which contains fewer N-glycans, inhibited EGFR signaling and cell proliferation after cell adhesion to FN. Furthermore, consistent restoration of the N-glycans on sites 1-3 of ß1 reinstated the inhibitory effects. Mechanistically, the N-glycosylation mutant of ß1 (S4-6+1-3) inhibited the EGFR response upon EGF stimulation via facilitating the α5ß1-EGFR complex formation. Moreover, we identified the N-glycosylation of sites 10-14 on α5 and 1-3 on ß1 were most important for EGFR signaling. Taken together, these data indicate that α5S3-5+10-14ß1S4-6+1-3 mutant represents the minimal N-glycosylation required for its regulation on EGFR signaling and cell proliferation, providing a plausible mechanism for the crosstalk between with α5ß1 and EGFR.
Asunto(s)
Integrina alfa5beta1/metabolismo , Polisacáridos/farmacología , Transducción de Señal/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Glicosilación , Humanos , Integrina alfa5beta1/genética , MutaciónRESUMEN
The epithelial cell adhesion molecule (EpCAM) is one of the most frequently and intensely expressed of tumor-associated antigens, but the role that EpCAM plays in the proliferation, adhesion and migration properties of cancer cells remains unclear. In the present study, we screened several tumor cell lines and found that colorectal cancer CW-2 and epidermoid carcinoma A431 cells expressed relatively higher levels of EpCAM. In order to assess the biological functions of EpCAM expression in cell adhesion and migration, we established a knock out (KO) of EpCAM genes in both of these types of cancer cells via a CRISPR/Cas9 system. The elongated cell morphology was converted to a rounded morphology in the EpCAM-KO cells. These cells showed decreases in cell proliferation and migration into extracellular matrix proteins, as well as decreases in cellular signaling elements such as phosphorylated focal adhesion kinase (FAK), AKT and ERK. Moreover, the cell growth and the colony formation abilities were significantly decreased in EpCAM-KO cells. Importantly, co-immunoprecipitation analysis revealed that EpCAM associated with integrin ß1. Also, the expression levels of integrin α5 were decreased in EpCAM-KO cells, compared with that in the wild-type cells. Taken together, these data clearly demonstrate that EpCAM associates with integrin ß1 to regulate FAK/ERK signaling pathways in controlling cell adhesion, migration and proliferation via extracellular matrix adhesion, which provides novel mechanisms for EpCAM-mediated biological functions and cancer phenotypes.
Asunto(s)
Molécula de Adhesión Celular Epitelial/metabolismo , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Neoplasias/patología , Adhesión Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Integrina alfa5/genética , Integrina beta1/genética , Neoplasias/genética , Transducción de SeñalRESUMEN
N-Glycans are involved in numerous biologic processes, such as cell adhesion, migration, and invasion. To distinguish the functions of complex high-mannose types of N-glycans, we used the clustered, regularly interspaced, short palindromic repeats/Cas9 system to establish N-acetylglucosaminyltransferase (GnT)-I-knockout (KO) cells. Loss of GnT-I greatly induced cell-cell adhesion and decreased cell migration. In addition, the expression levels of epithelial-mesenchymal transition (EMT) markers such as α-SMA, vimentin, and N-cadherin were suppressed, whereas the expression of claudin-1 was promoted, suggesting a mesenchymal-epithelial transition-like phenotype, an opposite process to the EMT, was occurred in the KO cells. The phosphorylation levels of Smad-2, epidermal growth factor receptor, and integrin-mediated focal adhesion kinase (FAK) were consistently suppressed. Furthermore, the restoration of GnT-I in the KO cells suppressed the cell-cell adhesion and augmented the expression of EMT markers as well as that of FAK activation. The expression levels of integrins were upregulated in the KO cells, although their functions were decreased, whereas their expression levels were downregulated in the rescued cells, which suggests a negative feedback loop between function and expression. Finally, we also found that the expression of GnT-I was important for cell survival, resistance to cancer drugs, and increased colony formation. The results of the present study demonstrate that GnT-I works as a switch to turn on/off EMT, which further supports the notion that on most surface receptors, the N-glycans differentially play essential roles in biologic functions.-Zhang, G., Isaji, T., Xu, Z., Lu, X., Fukuda, T., Gu, J. N-acetylglucosaminyltransferase-I as a novel regulator of epithelial-mesenchymal transition.