RESUMO
Integrins link the extracellular environment to the actin cytoskeleton in cell migration and adhesiveness. Rapid coordination between events outside and inside the cell is essential. Single-molecule fluorescence dynamics show that ligand binding to the bent-closed integrin conformation, which predominates on cell surfaces, is followed within milliseconds by two concerted changes, leg extension and headpiece opening, to give the high-affinity integrin conformation. The extended-closed integrin conformation is not an intermediate but can be directly accessed from the extended-open conformation and provides a pathway for ligand dissociation. In contrast to ligand, talin, which links the integrin ß-subunit cytoplasmic domain to the actin cytoskeleton, modestly stabilizes but does not induce extension or opening. Integrin activation is thus initiated by outside-in signaling and followed by inside-out signaling. Our results further imply that talin binding is insufficient for inside-out integrin activation and that tensile force transmission through the ligand-integrin-talin-actin cytoskeleton complex is required.
Assuntos
Integrinas , Talina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Adesão Celular , Células CHO , Cricetulus , Integrinas/metabolismo , Integrinas/química , Ligantes , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Imagem Individual de Molécula , Talina/metabolismo , Talina/químicaRESUMO
Understanding cellular force transmission dynamics is crucial in mechanobiology. We developed the DNA-based ForceChrono probe to measure force magnitude, duration, and loading rates at the single-molecule level within living cells. The ForceChrono probe circumvents the limitations of in vitro single-molecule force spectroscopy by enabling direct measurements within the dynamic cellular environment. Our findings reveal integrin force loading rates of 0.5-2 pN/s and durations ranging from tens of seconds in nascent adhesions to approximately 100 s in mature focal adhesions. The probe's robust and reversible design allows for continuous monitoring of these dynamic changes as cells undergo morphological transformations. Additionally, by analyzing how mutations, deletions, or pharmacological interventions affect these parameters, we can deduce the functional roles of specific proteins or domains in cellular mechanotransduction. The ForceChrono probe provides detailed insights into the dynamics of mechanical forces, advancing our understanding of cellular mechanics and the molecular mechanisms of mechanotransduction.
Assuntos
Mecanotransdução Celular , Imagem Individual de Molécula , Animais , Humanos , Camundongos , Fenômenos Biomecânicos , Adesão Celular , DNA/química , DNA/metabolismo , Adesões Focais/metabolismo , Integrinas/metabolismo , Microscopia de Força Atômica/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Sobrevivência Celular , Pareamento de Bases , CalibragemRESUMO
When the filtrate of the glomerulus flows through the renal tubular system, various microscopic sediment particles, including mineral crystals, are generated. Dislodging these particles is critical to ensuring the free flow of filtrate, whereas failure to remove them will result in kidney stone formation and obstruction. However, the underlying mechanism for the clearance is unclear. Here, using high-resolution microscopy, we found that the juxtatubular macrophages in the renal medulla constitutively formed transepithelial protrusions and "sampled" urine contents. They efficiently sequestered and phagocytosed intraluminal sediment particles and occasionally transmigrated to the tubule lumen to escort the excretion of urine particles. Mice with decreased renal macrophage numbers were prone to developing various intratubular sediments, including kidney stones. Mechanistically, the transepithelial behaviors of medulla macrophages required integrin ß1-mediated ligation to the tubular epithelium. These findings indicate that medulla macrophages sample urine content and remove intratubular particles to keep the tubular system unobstructed.
Assuntos
Cálculos Renais , Rim , Camundongos , Animais , MacrófagosRESUMO
Basal stem cells of the epidermis continuously differentiate into keratinocytes and replenish themselves via self-renewal to maintain skin homeostasis. Numerous studies have attempted to reveal how basal cells undergo differentiation or self-renewal; however, this has been hampered by a lack of robust basal cell markers and analytical platforms that allow single-cell tracking. Here, we report that zebrafish integrin beta 4 is a useful marker for basal cell labelling, irrespective of the body region, stage and regenerative status. We employed Cre-loxP recombination in combination with live cell tracking of single basal clones in the caudal fin and investigated the embryonic origin and behaviour of basal cells during fish growth and homeostasis. Although most basal cells, including those in fins, became quiescent in the adult stage, genetic cell ablation showed that basal cells were reactivated to either self-renew or differentiate, depending on the injured cell type. Our study provides a simple and easy-to-use platform for quantitative in vivo imaging of basal stem cells at wider stages and under various conditions.
Assuntos
Epiderme , Peixe-Zebra , Animais , Células Epidérmicas , Queratinócitos , HomeostaseRESUMO
Integrin activation resulting in enhanced adhesion to the extracellular matrix plays a key role in fundamental cellular processes. Although integrin activation has been extensively studied in circulating cells such as leukocytes and platelets, much less is known about the regulation and functional impact of integrin activation in adherent cells such as smooth muscle. Here, we show that two different asthmagenic cytokines, IL-13 and IL-17A, activate type I and IL-17 cytokine receptor families, respectively, to enhance adhesion of airway smooth muscle. These cytokines also induce activation of ß1 integrins detected by the conformation-specific antibody HUTS-4. Moreover, HUTS-4 binding is increased in the smooth muscle of patients with asthma compared to nonsmokers without lung disease, suggesting a disease-relevant role for integrin activation in smooth muscle. Indeed, integrin activation induced by the ß1-activating antibody TS2/16, the divalent cation manganese, or the synthetic peptide ß1-CHAMP that forces an extended-open integrin conformation dramatically enhances force transmission in smooth muscle cells and airway rings even in the absence of cytokines. We demonstrate that cytokine-induced activation of ß1 integrins is regulated by a common pathway of NF-κB-mediated induction of RhoA and its effector Rho kinase, which in turn stimulates PIP5K1γ-mediated synthesis of PIP2 at focal adhesions, resulting in ß1 integrin activation. Taken together, these data identify a pathway by which type I and IL-17 cytokine receptor family stimulation induces functionally relevant ß1 integrin activation in adherent smooth muscle and help to explain the exaggerated force transmission that characterizes chronic airway diseases such as asthma.
Assuntos
Asma , Integrina beta1 , Interleucina-13 , Interleucina-17 , Músculo Liso , NF-kappa B , Quinases Associadas a rho , Humanos , Integrina beta1/metabolismo , Interleucina-17/metabolismo , Músculo Liso/metabolismo , NF-kappa B/metabolismo , Quinases Associadas a rho/metabolismo , Interleucina-13/metabolismo , Asma/metabolismo , Transdução de Sinais , Adesão Celular , Miócitos de Músculo Liso/metabolismo , AnimaisRESUMO
Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.
Assuntos
Adesões Focais , Proteínas Ativadoras de GTPase , Mecanotransdução Celular , Proteínas Supressoras de Tumor , Proteínas de Sinalização YAP , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Movimento Celular , Retroalimentação Fisiológica , Adesões Focais/metabolismo , Adesões Focais/genética , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mecanotransdução Celular/genética , Neovascularização Fisiológica , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.
Assuntos
Antígeno CD11b , Proteínas de Membrana , Camundongos Knockout , Sepse , Transdução de Sinais , Animais , Humanos , Camundongos , Antígeno CD11b/metabolismo , Antígeno CD11b/genética , Adesão Celular/genética , Movimento Celular/genética , Regulação para Baixo , Endossomos/metabolismo , Deleção de Genes , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Fagócitos/metabolismo , Fagócitos/imunologia , Fagocitose , Sepse/genética , Sepse/imunologia , Sepse/metabolismoRESUMO
Isthmin-1 (Ism1) was first described to be syn-expressed with Fgf8 in Xenopus. However, its biological role has not been elucidated until recent years. Despite of accumulated evidence that Ism1 participates in angiogenesis, tumor invasion, macrophage apoptosis, and glucose metabolism, the cognate receptors for Ism1 remain largely unknown. Ism1 deficiency in mice results in renal agenesis (RA) with a transient loss of Gdnf transcription and impaired mesenchyme condensation at E11.5. Ism1 binds to and activates Integrin α8ß1 to positively regulate Gdnf/Ret signaling, thus promoting mesenchyme condensation and ureteric epithelium branching morphogenesis. Here, we propose the hypothesis underlying the mechanism by which Ism1 regulates branching morphogenesis during early kidney development.
Assuntos
Estruturas Embrionárias , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Néfrons/embriologia , Ureter , Camundongos , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Rim/anormalidades , Rim/metabolismo , Rim/patologia , Ureter/metabolismo , MorfogêneseRESUMO
The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell-dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.
Assuntos
Caspases , Linfoma Difuso de Grandes Células B , Camundongos , Humanos , Animais , Adulto , Tensinas/genética , Caspases/metabolismo , NF-kappa B/metabolismo , Adesão Celular/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Linfoma Difuso de Grandes Células B/genética , IntegrinasRESUMO
We are interested in the contribution of integrins and the extracellular matrix to epithelial differentiation in carcinomas. This study was motivated by our finding that the Hippo effectors YAP and TAZ can sustain the expression of laminin 332 (LM332), the predominant ECM ligand for the integrin ß4, in breast carcinoma cells with epithelial differentiation. More specifically, we observed that YAP and TAZ regulate the transcription of the LAMC2 subunit of LM332. Given that the ß4-LM332 axis is associated with epithelial differentiation and YAP/TAZ have been implicated in carcinoma de-differentiation, we sought to resolve this paradox. Here, we observed that the ß4 integrin sustains the expression of miR-200s that target the transcription factor ZEB1 and that ZEB1 has a pivotal role in determining the nature of YAP/TAZ-mediated transcription. In the presence of ß4, ZEB1 expression is repressed enabling YAP/TAZ/TEAD-mediated transcription of LAMC2. The absence of ß4, however, induces ZEB1, and ZEB1 binds to the LAMC2 promoter to inhibit LAMC2 transcription. YAP/TAZ-mediated regulation of LAMC2 has important functional consequences because we provide evidence that LM332 enables carcinoma cells to resist ferroptosis in concert with the ß4 integrin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ferroptose , Integrina beta4 , Calinina , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Feminino , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Integrina beta4/metabolismo , Integrina beta4/genética , Calinina/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Transativadores/metabolismo , Transativadores/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
Integrin-mediated activation of the profibrotic mediator transforming growth factor-ß1 (TGF-ß1), plays a critical role in idiopathic pulmonary fibrosis (IPF) pathogenesis. Galectin-3 is believed to contribute to the pathological wound healing seen in IPF, although its mechanism of action is not precisely defined. We hypothesized that galectin-3 potentiates TGF-ß1 activation and/or signaling in the lung to promote fibrogenesis. We show that galectin-3 induces TGF-ß1 activation in human lung fibroblasts (HLFs) and specifically that extracellular galectin-3 promotes oleoyl-L-α-lysophosphatidic acid sodium salt-induced integrin-mediated TGF-ß1 activation. Surface plasmon resonance analysis confirmed that galectin-3 binds to αv integrins, αvß1, αvß5, and αvß6, and to the TGFßRII subunit in a glycosylation-dependent manner. This binding is heterogeneous and not a 1:1 binding stoichiometry. Binding interactions were blocked by small molecule inhibitors of galectin-3, which target the carbohydrate recognition domain. Galectin-3 binding to ß1 integrin was validated in vitro by coimmunoprecipitation in HLFs. Proximity ligation assays indicated that galectin-3 and ß1 integrin colocalize closely (≤40 nm) on the cell surface and that colocalization is increased by TGF-ß1 treatment and blocked by galectin-3 inhibitors. In the absence of TGF-ß1 stimulation, colocalization was detectable only in HLFs from IPF patients, suggesting the proteins are inherently more closely associated in the disease state. Galectin-3 inhibitor treatment of precision cut lung slices from IPF patients' reduced Col1a1, TIMP1, and hyaluronan secretion to a similar degree as TGF-ß type I receptor inhibitor. These data suggest that galectin-3 promotes TGF-ß1 signaling and may induce fibrogenesis by interacting directly with components of the TGF-ß1 signaling cascade.
Assuntos
Fibroblastos , Galectina 3 , Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Humanos , Fator de Crescimento Transformador beta1/metabolismo , Galectina 3/metabolismo , Galectina 3/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Transdução de Sinais , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Galectinas/metabolismo , Colágeno Tipo I/metabolismo , Células Cultivadas , Proteínas SanguíneasRESUMO
Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.
Assuntos
Membrana Basal , Laminina , Humanos , Laminina/metabolismo , Laminina/química , Laminina/genética , Animais , Membrana Basal/metabolismo , Distrofias Musculares/metabolismo , Distrofias Musculares/genética , Deformidades Congênitas dos Membros/metabolismo , Deformidades Congênitas dos Membros/genética , Mutação , Síndrome Nefrótica , Distúrbios Pupilares , Síndromes Miastênicas CongênitasRESUMO
SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor-binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent but integrin- and clathrin-dependent manner and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9, and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to the entry of SARS-CoV-2.
Assuntos
Ligação Proteica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Domínios Proteicos , Células HEK293 , COVID-19/metabolismo , COVID-19/virologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Interações Hospedeiro-PatógenoRESUMO
Heterotrimeric G proteins (Gαßγ) are molecular switches that relay signals from 7-transmembrane receptors located at the cell surface to the cytoplasm. The function of these receptors is so intimately linked to heterotrimeric G proteins that they are named G protein-coupled receptors (GPCRs), showcasing the interdependent nature of this archetypical receptor-transducer axis of transmembrane signaling in eukaryotes. It is generally assumed that activation of heterotrimeric G protein signaling occurs exclusively by the action of GPCRs, but this idea has been challenged by the discovery of alternative mechanisms by which G proteins can propagate signals in the cell. This review will focus on a general principle of G protein signaling that operates without the direct involvement of GPCRs. The mechanism of G protein signaling reviewed here is mediated by a class of G protein regulators defined by containing an evolutionarily conserved sequence named the Gα-binding-and-activating (GBA) motif. Using the best characterized proteins with a GBA motif as examples, Gα-interacting vesicle-associated protein (GIV)/Girdin and dishevelled-associating protein with a high frequency of leucine residues (DAPLE), this review will cover (i) the mechanisms by which extracellular cues not relayed by GPCRs promote the coupling of GBA motif-containing regulators with G proteins, (ii) the structural and molecular basis for how GBA motifs interact with Gα subunits to facilitate signaling, (iii) the relevance of this mechanism in different cellular and pathological processes, including cancer and birth defects, and (iv) strategies to manipulate GBA-G protein coupling for experimental therapeutics purposes, including the development of rationally engineered proteins and chemical probes.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP , Receptores Acoplados a Proteínas G , Motivos de Aminoácidos , Membrana Celular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Humanos , Animais , Engenharia de ProteínasRESUMO
BACKGROUND & AIMS: Dysbiosis of the gut microbiota is considered a key contributor to inflammatory bowel disease (IBD) etiology. Here, we investigated potential associations between microbiota composition and the outcomes to biological therapies. METHODS: The study prospectively recruited 296 patients with active IBD (203 with Crohn's disease, 93 with ulcerative colitis) initiating biological therapy. Quantitative microbiome profiles of pretreatment and posttreatment fecal samples were obtained combining flow cytometry with 16S amplicon sequencing. Therapeutic response was assessed by endoscopy, patient-reported outcomes, and changes in fecal calprotectin. The effect of therapy on microbiome variation was evaluated using constrained ordination methods. Prediction of therapy outcome was performed using logistic regression with 5-fold cross-validation. RESULTS: At baseline, 65.9% of patients carried the dysbiotic Bacteroides2 (Bact2) enterotype, with a significantly higher prevalence among patients with ileal involvement (76.8%). Microbiome variation was associated with the choice of biological therapy rather than with therapeutic outcome. Only anti-tumor necrosis factor-α treatment resulted in a microbiome shift away from Bact2, concomitant with an increase in microbial load and butyrogen abundances and a decrease in potentially opportunistic Veillonella. Remission rates for patients hosting Bact2 at baseline were significantly higher with anti-tumor necrosis factor-α than with vedolizumab (65.1% vs 35.2%). A prediction model, based on anthropometrics and clinical data, stool features (microbial load, moisture, and calprotectin), and Bact2 detection predicted treatment outcome with 73.9% accuracy for specific biological therapies. CONCLUSION: Fecal characterization based on microbial load, moisture content, calprotectin concentration, and enterotyping may aid in the therapeutic choice of biological therapy in IBD.
Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Humanos , Disbiose , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/tratamento farmacológico , Fezes , Terapia Biológica , Fator de Necrose Tumoral alfa , Complexo Antígeno L1 Leucocitário , NecroseRESUMO
The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Nefrite Hereditária , Podócitos , Humanos , Camundongos , Animais , Integrina alfa1/genética , Integrina alfa1/metabolismo , Ramipril/farmacologia , Ramipril/metabolismo , Longevidade , Membrana Basal Glomerular/metabolismo , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/genética , Nefrite Hereditária/metabolismo , Podócitos/metabolismo , Laminina/genética , Laminina/metabolismo , Camundongos Knockout , Proteinúria/tratamento farmacológico , Proteinúria/genética , Proteinúria/metabolismo , Análise de Sequência de RNARESUMO
BACKGROUND: Hypospadias is a common congenital abnormality of the penile. Abnormal regulation of critical genes involved in urethral development leads to hypospadias. We used the Rab25-/- mice and foreskin fibroblasts transfected with lentivirus in vitro and in vivo to investigate the role of Rab25 in hypospadias. METHODS: The expression levels of various molecules in tissue samples and foreskin fibroblasts were confirmed using molecular biology methods (western blotting, PCR, immunohistochemistry, etc.). A scanning electron microscope (SEM) was used to visualize the external morphology of genital tubercles (GTs) of gestation day (GD) 18.5 male wild-type (WT) and Rab25-/- mice. RESULTS: An expanded distal cleft and V-shaped urethral opening were observed in GD 18.5 Rab25-/- mice. We demonstrated that Rab25 mediated hypospadias through the ß1 integrin/EGFR pathway. In addition, silencing Rab25 inhibited cell proliferation and migration and promoted apoptosis in the foreskin fibroblasts; Ki-67- and TUNEL-positive cells were mainly concentrated near the urethral seam. CONCLUSION: These findings suggest that Rab25 plays an essential role in hypospadias by activation of ß1 integrin/EGFR pathway, and Rab25 is a critical mediator of urethral seam formation in GD18.5 male fetal mice.
Assuntos
Hipospadia , Humanos , Masculino , Camundongos , Animais , Hipospadia/genética , Hipospadia/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Uretra/metabolismo , Pênis/metabolismo , Receptores ErbB/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
A microRNA miR-200c-3p is a regulator of epithelial-mesenchymal transition to control adhesion and migration of epithelial and mesenchymal cells. However, little is known about whether miR-200c-3p affects lymphocyte adhesion and migration mediated by integrins. Using TK-1 (a T lymphoblast cell) as a model of T cell, here we show that repressed expression of miR-200c-3p upregulated α4 integrin-mediated adhesion to and migration across mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Conversely, overexpression of miR-200c-3p downregulated α4 integrin-mediated adhesion and migration. Unlike in epithelial cells, miR-200c-3p did not target talin, a conformation activator of integrin, but, targeted E26-transformation-specific sequence 1 (ETS1), a transcriptional activator of α4 integrin, in T cells. Treatment of the miR-200c-3p-low-expressing TK-1 cells that possessed elevated α4 integrin with ETS1 small interfering RNA (siRNA) resulted in the reversion of the α4 integrin expression, supporting that ETS1 is a target of miR-200c-3p. A potential proinflammatory immune-modulator retinoic acid (RA) treatment of TK-1 cells elicited a significant reduction of miR-200c-3p and simultaneously a marked increase in ETS1 and α4 integrin expression. An anti-inflammatory cytokine TGF-ß1 treatment elevated miR-200c-3p, thereby downregulating ETS1 and α4 integrin expression. These results suggest that miR-200c-3p is an important regulator of α4 integrin expression and functions and may be controlled by RA and TGF-ß1 in an opposite way. Overexpression of miR-200c-3p could be a novel therapeutic option for treatment of gut inflammation through suppressing α4 integrin-mediated T cell migration.
Assuntos
Adesão Celular , Movimento Celular , Integrina alfa4 , MicroRNAs , Linfócitos T , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , Integrina alfa4/metabolismo , Integrina alfa4/genética , Movimento Celular/genética , Adesão Celular/genética , Linfócitos T/metabolismo , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Mucoproteínas/genética , Mucoproteínas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/genética , Linhagem CelularRESUMO
PRELP is thought to be an inhibitor of the development and progression of a variety of malignancies. Metastasis is a major cause of death in patients with colorectal cancer, but the mechanism underlying the role of PRELP in colorectal cancer metastasis remains poorly understood. In this study, we found that PRELP was significantly higher in metastatic tissues than in non-metastatic tissues of colorectal cancer and was closely associated with poor prognosis of colorectal cancer patients. PRELP promotes growth and metastasis of colorectal cancer cells. PRELP reduces cell stiffness and adhesion. PRELP promoted EMT in colorectal cancer cells and that PRELP bind to integrin α5 to activate the integrin α5/FAK/AKT signaling pathway. In conclusion, we demonstrate that PRELP is upregulated in metastatic colorectal cancer, providing a potential prognostic marker and therapeutic target for the clinical management of metastatic colorectal cancer from a biomechanical perspective.
Assuntos
Adesão Celular , Proliferação de Células , Neoplasias Colorretais , Proteínas da Matriz Extracelular , Glicoproteínas , Integrina alfa5 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Integrina alfa5/metabolismo , Integrina alfa5/genética , Integrinas , Camundongos Nus , Metástase Neoplásica , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismoRESUMO
The acute respiratory virus infection can induce uncontrolled inflammatory responses, such as cytokine storm and viral pneumonia, which are the major causes of death in clinical cases. Cyclophilin A (CypA) is mainly distributed in the cytoplasm of resting cells and released into the extracellular space in response to inflammatory stimuli. Extracellular CypA (eCypA) is upregulated and promotes inflammatory response in severe COVID-19 patients. However, how eCypA promotes virus-induced inflammatory response remains elusive. Here, we observe that eCypA is induced by influenza A and B viruses and SARS-CoV-2 in cells, mice, or patients. Anti-CypA mAb reduces pro-inflammatory cytokines production, leukocytes infiltration, and lung injury in virus-infected mice. Mechanistically, eCypA binding to integrin ß2 triggers integrin activation, thereby facilitating leukocyte trafficking and cytokines production via the focal adhesion kinase (FAK)/GTPase and FAK/ERK/P65 pathways, respectively. These functions are suppressed by the anti-CypA mAb that specifically blocks eCypA-integrin ß2 interaction. Overall, our findings reveal that eCypA-integrin ß2 signaling mediates virus-induced inflammatory response, indicating that eCypA is a potential target for antibody therapy against viral pneumonia.