RESUMEN
CD8 tissue-resident memory T (TRM) cells provide frontline protection at barrier tissues; however, mechanisms regulating TRM cell development are not completely understood. Priming dictates the migration of effector T cells to the tissue, while factors in the tissue induce in situ TRM cell differentiation. Whether priming also regulates in situ TRM cell differentiation uncoupled from migration is unclear. Here, we demonstrate that T cell priming in the mesenteric lymph nodes (MLN) regulates CD103+ TRM cell differentiation in the intestine. In contrast, T cells primed in the spleen were impaired in the ability to differentiate into CD103+ TRM cells after entry into the intestine. MLN priming initiated a CD103+ TRM cell gene signature and licensed rapid CD103+ TRM cell differentiation in response to factors in the intestine. Licensing was regulated by retinoic acid signaling and primarily driven by factors other than CCR9 expression and CCR9-mediated gut homing. Thus, the MLN is specialized to promote intestinal CD103+ CD8 TRM cell development by licensing in situ differentiation.
Asunto(s)
Linfocitos T CD8-positivos , Tretinoina , Linfocitos T CD8-positivos/metabolismo , Intestinos , Diferenciación Celular , Transducción de Señal , Memoria InmunológicaRESUMEN
Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFß) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFß signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFß signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.
RESUMEN
Fibrotic posterior capsular opacification (PCO), a major complication of cataract surgery, is driven by transforming growth factor-ß (TGF-ß). Previously, αV integrins were found to be critical for the onset of TGF-ß-mediated PCO in vivo; however, the functional heterodimer was unknown. Here, ß8 integrin-conditional knockout (ß8ITG-cKO) lens epithelial cells (LCs) attenuated their fibrotic responses, while both ß5 and ß6 integrin-null LCs underwent fibrotic changes similar to WT at 5 days post cataract surgery (PCS). RNA-Seq revealed that ß8ITG-cKO LCs attenuated their upregulation of integrins and their ligands, as well as known targets of TGF-ß-induced signaling, at 24 hours PCS. Treatment of ß8ITG-cKO eyes with active TGF-ß1 at the time of surgery rescued the fibrotic response. Treatment of WT mice with an anti-αVß8 integrin function blocking antibody at the time of surgery ameliorated both canonical TGF-ß signaling and LC fibrotic response PCS, and treatment at 5 days PCS, after surgically induced fibrotic responses were established, largely reversed this fibrotic response. These data suggest that αVß8 integrin is a major regulator of TGF-ß activation by LCs PCS and that therapeutics targeting αVß8 integrin could be effective for fibrotic PCO prevention and treatment.
Asunto(s)
Opacificación Capsular/prevención & control , Catarata/prevención & control , Integrinas/uso terapéutico , Animales , Humanos , RatonesRESUMEN
αvß8 integrin, a key activator of transforming growth factor ß (TGF-ß), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvß8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvß8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of ß8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-ß and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvß8 integrin as a promising target for cancer immunotherapy.
Asunto(s)
Inmunidad , Inmunoterapia , Integrinas/metabolismo , Modelos Biológicos , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T/inmunología , Animales , Anticuerpos Antineoplásicos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/inmunología , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Granzimas/metabolismo , Interferón gamma/metabolismo , Depleción Linfocítica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Transducción de Señal , Proteína smad3/metabolismo , Análisis de Supervivencia , Linfocitos T Citotóxicos/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Recent fate-mapping studies in mice have provided substantial evidence that mature adult hepatocytes are a major source of new hepatocytes after liver injury. In other systems, integrin αvß8 has a major role in activating transforming growth factor (TGF)-ß, a potent inhibitor of hepatocyte proliferation. We hypothesized that depletion of hepatocyte integrin αvß8 would increase hepatocyte proliferation and accelerate liver regeneration after injury. Using Itgb8flox/flox;Alb-Cre mice to deplete hepatocyte αvß8, after partial hepatectomy, hepatocyte proliferation and liver-to-body weight ratio were significantly increased in Itgb8flox/flox;Alb-Cre mice compared with control mice. Antibody-mediated blockade of hepatocyte αvß8 in vitro, with assessment of TGF-ß signaling pathways by real-time quantitative PCR array, supported the hypothesis that integrin αvß8 inhibition alters hepatocyte TGF-ß signaling toward a pro-regenerative phenotype. A diethylnitrosamine-induced model of hepatocellular carcinoma, used to examine the possibility that this pro-proliferative phenotype might be oncogenic, revealed no difference in either tumor number or size between Itgb8flox/flox;Alb-Cre and control mice. Immunohistochemistry for integrin αvß8 in healthy and injured human liver demonstrated that human hepatocytes express integrin αvß8. Depletion of hepatocyte integrin αvß8 results in increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in mice. These data demonstrate that targeting integrin αvß8 may represent a promising therapeutic strategy to drive liver regeneration in patients with a broad range of liver diseases.
Asunto(s)
Proliferación Celular , Hepatocitos/metabolismo , Integrinas/deficiencia , Regeneración Hepática , Hígado/metabolismo , Transducción de Señal , Animales , Hepatocitos/patología , Hígado/patología , Ratones , Ratones Transgénicos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Activated fibroblasts are deemed the main executors of organ fibrosis. However, regulation of the pathologic functions of these cells in vivo is poorly understood. PDGF receptor ß (PDGFRß) is highly expressed in activated pericytes, a main source of fibroblasts. Studies using a PDGFRß promoter-driven Cre system to delete αv integrins in activated fibroblasts identified these integrins as core regulators of fibroblast activity across solid organs, including the kidneys. Here, we used the same PDGFRß-Cre line to isolate and study renal fibroblasts ex vivo We found that renal fibroblasts express three αv integrins, namely αvß1, αvß3, and αvß5. Blockade of αvß1 prevented direct binding of fibroblasts to the latency-associated peptide of TGF-ß1 and prevented activation of the latent TGF-ß complex. Continuous administration of a recently described potent small molecule inhibitor of αvß1, compound 8, starting the day of unilateral ureteral obstruction operation, inhibited collagen deposition in the kidneys of mice 14 days later. Compound 8 also effectively attenuated renal failure, as measured by BUN levels in mice fed an adenine diet known to cause renal injury followed by fibrosis. Inhibition of αvß1 integrin could thus hold promise as a therapeutic intervention in CKD characterized by renal fibrosis.
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Guanidinas/farmacología , Guanidinas/uso terapéutico , Riñón/patología , Receptores de Vitronectina/antagonistas & inhibidores , Insuficiencia Renal/prevención & control , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Animales , Fibrosis/etiología , Fibrosis/prevención & control , Masculino , Ratones , Receptores de Vitronectina/fisiología , Insuficiencia Renal/etiologíaRESUMEN
The efficacy and feasibility of targeting transforming growth factor-ß (TGFß) in pulmonary fibrosis and lung vascular remodeling in systemic sclerosis (SSc) have not been well elucidated. In this study we analyzed how blocking TGFß signaling affects pulmonary abnormalities in Fos-related antigen 2 (Fra-2) transgenic (Tg) mice, a murine model that manifests three important lung pathological features of SSc: fibrosis, inflammation, and vascular remodeling. To interrupt TGFß signaling in the Fra-2 Tg mice, we used a pan-TGFß-blocking antibody, 1D11, and Tg mice in which TGFß receptor type 2 (Tgfbr2) is deleted from smooth muscle cells and myofibroblasts (α-SMA-CreER;Tgfbr2flox/flox). Global inhibition of TGFß by 1D11 did not ameliorate lung fibrosis histologically or biochemically, whereas it resulted in a significant increase in the number of immune cells infiltrating the lungs. In contrast, 1D11 treatment ameliorated the severity of pulmonary vascular remodeling in Fra-2 Tg mice. Similarly, genetic deletion of Tgfbr2 from smooth muscle cells resulted in improvement of pulmonary vascular remodeling in the Fra-2 Tg mice, as well as a decrease in the number of Ki67-positive vascular smooth muscle cells, suggesting that TGFß signaling contributes to development of pulmonary vascular remodeling by promoting the proliferation of vascular smooth muscle cells. Deletion of Tgfbr2 from α-smooth muscle actin-expressing cells had no effect on fibrosis or inflammation in this model. These results suggest that efforts to target TGFß in SSc will likely require more precision than simply global inhibition of TGFß function.
Asunto(s)
Neumonía/patología , Fibrosis Pulmonar/patología , Esclerodermia Sistémica/patología , Factor de Crecimiento Transformador beta/farmacología , Remodelación Vascular/efectos de los fármacos , Actinas , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Modelos Animales de Enfermedad , Antígeno 2 Relacionado con Fos/metabolismo , Eliminación de Gen , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Granulocitos/patología , Ratones Transgénicos , Miocitos del Músculo Liso/metabolismo , Neumonía/complicaciones , Proteínas Serina-Treonina Quinasas/genética , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/complicaciones , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Esclerodermia Sistémica/complicaciones , Transducción de Señal/efectos de los fármacos , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
Treatment options are limited for severe asthma, and the need for additional therapies remains great. Previously, we demonstrated that integrin αvß6-deficient mice are protected from airway hyperresponsiveness, due in part to increased expression of the murine ortholog of human chymase. Here, we determined that chymase protects against cytokine-enhanced bronchoconstriction by cleaving fibronectin to impair tension transmission in airway smooth muscle (ASM). Additionally, we identified a pathway that can be therapeutically targeted to mitigate the effects of airway hyperresponsiveness. Administration of chymase to human bronchial rings abrogated IL-13-enhanced contraction, and this effect was not due to alterations in calcium homeostasis or myosin light chain phosphorylation. Rather, chymase cleaved fibronectin, inhibited ASM adhesion, and attenuated focal adhesion phosphorylation. Disruption of integrin ligation with an RGD-containing peptide abrogated IL-13-enhanced contraction, with no further effect from chymase. We identified α5ß1 as the primary fibronectin-binding integrin in ASM, and α5ß1-specific blockade inhibited focal adhesion phosphorylation and IL-13-enhanced contraction, with no additional effect from chymase. Delivery of an α5ß1 inhibitor into murine airways abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. Finally, α5ß1 blockade enhanced the effect of the bronchodilator isoproterenol on airway relaxation. Our data identify the α5ß1 integrin as a potential therapeutic target to mitigate the severity of airway contraction in asthma.
Asunto(s)
Asma/metabolismo , Asma/fisiopatología , Integrina alfa5beta1/metabolismo , Isoproterenol/farmacología , Relajación Muscular/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Animales , Asma/tratamiento farmacológico , Asma/genética , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular , Quimasas/toxicidad , Modelos Animales de Enfermedad , Humanos , Integrina alfa5beta1/genética , Interleucina-13/genética , Interleucina-13/metabolismo , Ratones , Miocitos del Músculo Liso/patología , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , ConejosRESUMEN
Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-ß receptor (LTßR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvß8-mediated activation of transforming growth factor-ß (TGFß). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFß activation and induction of mucosal IgA responses.
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Linfocitos B/inmunología , Células Dendríticas/inmunología , Inmunoglobulina A Secretora/biosíntesis , Cambio de Clase de Inmunoglobulina , Ganglios Linfáticos Agregados/inmunología , Receptores CCR6/inmunología , Animales , Comunicación Celular/inmunología , Movimiento Celular/inmunología , Inmunoglobulina A Secretora/genética , Integrinas/inmunología , Mucosa Intestinal/inmunología , Activación de Linfocitos , Receptor beta de Linfotoxina/genética , Receptor beta de Linfotoxina/inmunología , Ratones , Ratones Mutantes , Receptores CCR6/genéticaRESUMEN
Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9ß6KDFyn cell line, which expresses full-length ß6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9ß6D1 cells which lack the ß6 cytoplasmic tail and, do not activate Fyn. SCC9ß6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.
Asunto(s)
Carcinoma de Células Escamosas/patología , Cadenas beta de Integrinas/biosíntesis , Neoplasias de la Boca/patología , Proteínas Proto-Oncogénicas c-fyn/biosíntesis , Esferoides Celulares/patología , Cadherinas/biosíntesis , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Humanos , Proteínas Proto-Oncogénicas c-raf/biosíntesis , Transducción de Señal , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
OBJECTIVE: Integrin αvß5 has been identified as a regulator of vascular leak and endothelial permeability. We hypothesized that targeting αvß5 could represent a viable treatment strategy for sepsis. DESIGN: Integrin ß5 subunit knockout and wild-type 129/svJae mice and wild-type mice treated with αvß5 blocking or control antibodies were tested in models of intraperitoneal lipopolysaccharide and cecal ligation and puncture. Human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers were treated with αvß5 antibodies to assess for effects on lipopolysaccharide-induced changes in transendothelial resistance and on patterns of cytoskeletal reorganization. SETTING: Laboratory-based research. SUBJECTS: Mice and endothelial cell monolayers. INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: Measurements taken after intraperitoneal lipopolysaccharide and/or cecal ligation and puncture included mortality, vascular leak, hematocrit, quantification of a panel of serum cytokines/chemokines, and assessment of thioglyccolate-induced leukocyte migration. ß5 knockout mice had decreased mortality after intraperitoneal lipopolysaccharide and cecal ligation and puncture and decreased vascular leak, as measured by extravasation of an I-labeled intravascular tracer. Treating clinically ill mice with αvß5 antibodies, up to 20 hrs after intraperitoneal lipopolysaccharide and cecal ligation and puncture, also resulted in decreased mortality. αvß5 antibodies attenuated lipopolysaccharide-induced transendothelial resistance changes and cytoskeletal stress fiber formation in both human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers. αvß5 antibodies had no effect on cytokine/chemokine serum levels after cecal ligation and puncture. ß5 knockout mice and wild-type controls did not exhibit differences in thioglyccolate-induced leukocyte migration. CONCLUSIONS: Our studies suggest that αvß5 is an important regulator of the vascular endothelial leak response in sepsis and that αvß5 blockade may provide a novel approach to treating this devastating disease syndrome.
Asunto(s)
Anticuerpos/farmacología , Receptores de Vitronectina/antagonistas & inhibidores , Sepsis/terapia , Animales , Ciego/cirugía , Movimiento Celular , Quimiocinas/sangre , Citocinas/sangre , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Humanos , Inyecciones Intraperitoneales , Leucocitos/fisiología , Ligadura , Lipopolisacáridos/administración & dosificación , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Punciones , Receptores de Vitronectina/genéticaRESUMEN
Exaggerated contraction of airway smooth muscle is the major cause of symptoms in asthma, but the mechanisms that prevent exaggerated contraction are incompletely understood. Here, we showed that integrin α9ß1 on airway smooth muscle localizes the polyamine catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) in close proximity to the lipid kinase PIP5K1γ. As PIP5K1γ is the major source of PIP2 in airway smooth muscle and its activity is regulated by higher-order polyamines, this interaction inhibited IP3-dependent airway smooth muscle contraction. Mice lacking integrin α9ß1 in smooth muscle had increased airway responsiveness in vivo, and loss or inhibition of integrin α9ß1 increased in vitro airway narrowing and airway smooth muscle contraction in murine and human airways. Contraction was enhanced in control airways by the higher-order polyamine spermine or by cell-permeable PIP2, but these interventions had no effect on airways lacking integrin α9ß1 or treated with integrin α9ß1-blocking antibodies. Enhancement of SSAT activity or knockdown of PIP5K1γ inhibited airway contraction, but only in the presence of functional integrin α9ß1. Therefore, integrin α9ß1 appears to serve as a brake on airway smooth muscle contraction by recruiting SSAT, which facilitates local catabolism of polyamines and thereby inhibits PIP5K1γ. Targeting key components of this pathway could thus lead to new treatment strategies for asthma.
Asunto(s)
Integrinas/fisiología , Músculo Liso/fisiología , Músculos Respiratorios/fisiología , Acetiltransferasas/fisiología , Animales , Asma/etiología , Asma/fisiopatología , Señalización del Calcio , Expresión Génica , Humanos , Integrinas/deficiencia , Integrinas/genética , Pulmón/fisiopatología , Ratones , Ratones Transgénicos , Modelos Biológicos , Contracción Muscular/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Poliaminas/metabolismoRESUMEN
RATIONALE: Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms. OBJECTIVES: We hypothesized that integrin αvß3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI. METHODS: ß3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of ß3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvß3 antibodies were evaluated for sphingosine-1 phosphate (S1P)mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization. MEASUREMENTS AND MAIN RESULTS: ß3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvß3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvß3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvß3 localization after S1P was decreased by αvß3 antibodies, suggesting that ligation of the αvß3 with its extracellular matrix ligands is required to stabilize cortical αvß3 focal adhesions. CONCLUSIONS: Our studies identify a novel mechanism by which αvß3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvß3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.
Asunto(s)
Actinas/metabolismo , Lesión Pulmonar Aguda/metabolismo , Endotelio Vascular/metabolismo , Integrina alfaVbeta3/antagonistas & inhibidores , Sepsis/metabolismo , Enfermedades Vasculares/metabolismo , Lesión Pulmonar Aguda/complicaciones , Animales , Modelos Animales de Enfermedad , Femenino , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Edema Pulmonar/complicaciones , Edema Pulmonar/metabolismo , Sepsis/complicaciones , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
The α9ß1 integrin is a multifunctional receptor that interacts with a variety of ligands including vascular cell adhesion molecule 1, tenascin-C, and osteopontin. A 2.3-kb truncated form of α9 integrin subunit cDNA was identified by searching the Medline database. This splice variant, which we called the short form of α9 integrin (SFα9), encodes a 632-aa isoform lacking transmembrane and cytoplasmic domains, and its authentic expression was verified by PCR and Western blotting. SFα9 is expressed on the cell surface but cannot bind ligand in the absence of the full-length α9 subunit. Over-expression of SFα9 in cells expressing full-length α9 promotes α9-dependent cell adhesion. This promoting effect of SFα9 requires the authentic cytoplasmic domain of the co-expressed full-length α9 subunit. Thus, SFα9 is a novel functional modulator of α9ß1 integrin by inside-out signaling.
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Empalme Alternativo , Adhesión Celular , Movimiento Celular , Cadenas alfa de Integrinas/genética , Integrinas/genética , Tenascina/genética , Animales , Western Blotting , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Citometría de Flujo , Humanos , Cadenas alfa de Integrinas/metabolismo , Integrinas/metabolismo , Unión Proteica , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tenascina/metabolismoRESUMEN
Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.
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Quitinasas/genética , Haplotipos , Polimorfismo de Nucleótido Simple , Negro o Afroamericano/genética , Animales , Asma/etnología , Asma/genética , Sitios de Unión/genética , Catálisis , Línea Celular , Quitinasas/química , Quitinasas/metabolismo , Disacáridos/química , Disacáridos/metabolismo , Frecuencia de los Genes , Genotipo , Hispánicos o Latinos/genética , Humanos , Concentración de Iones de Hidrógeno , Insectos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Desequilibrio de Ligamiento , México , Estructura Molecular , Estructura Terciaria de Proteína , Puerto Rico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The extracellular matrix metalloproteinase inducer (EMMPRIN), found on the surface of many tumor cells, stimulates the production of matrix metalloproteinases (MMPs) by both fibroblasts and the tumor cells themselves. To evaluate its possible role as a tumor promoter, we first overexpressed EMMPRIN, by retroviral transduction, into poorly invasive squamous cell carcinoma (SCC) cells. Secondly, we knocked down its expression using small interfering RNA (siRNA) in invasive SCC cells. The cell lines were then re-evaluated for migration on fibronectin (FN). Overexpression of EMMPRIN, promoted motility, whereas the siRNA decreased migration. The MMP expression by these variant SCC cell lines was also manipulated by EMMPRIN. The expression of MMP-2, -3, and -9 coincided with the expression of EMMPRIN. Cocultures of SCC/peritumor fibroblasts (PTF) were used to investigate tenascin-C (TN-C) matrix deposition. The cocultures overexpressing EMMPRIN, deposited several fold greater levels of TN-C compared to the control cocultures. In addition, the siRNA cocultures deposited minimal amounts of TN-C. In the presence of the broad spectrum MMP inhibitor, GM6001, TN-C deposition by the EMMPRIN overexpressing cocultures was suppressed. Thus EMMPRIN regulates migration, MMP production by SCC cells and deposition of the TN-C matrix.
Asunto(s)
Basigina/biosíntesis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Movimiento Celular/fisiología , Tenascina/metabolismo , Neoplasias de la Lengua/metabolismo , Neoplasias de la Lengua/patología , Basigina/genética , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Humanos , Isoenzimas , Metaloproteinasas de la Matriz/biosíntesis , ARN Interferente Pequeño/genética , Neoplasias de la Lengua/enzimologíaRESUMEN
Vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis. We now show that VEGF-A-induced adhesion and migration of human endothelial cells are dependent on the integrin alpha9beta1 and that VEGF-A is a direct ligand for this integrin. Adhesion and migration of these cells on the 165 and 121 isoforms of VEGF-A depend on cooperative input from alpha9beta1 and the cognate receptor for VEGF-A, VEGF receptor 2 (VEGF-R2). Unlike alpha3beta1or alphavbeta3 integrins, alpha9beta1 was also found to bind the 121 isoform of VEGF-A. This interaction appears to be biologically significant, because alpha9beta1-blocking antibody dramatically and specifically inhibited angiogenesis induced by VEGF-A165 or -121. Together with our previous findings that alpha9beta1 directly binds to VEGF-C and -D and contributes to lymphangiogenesis, these results identify the integrin alpha9beta1 as a potential pharmacotherapeutic target for inhibition of pathogenic angiogenesis and lymphangiogenesis.
Asunto(s)
Movimiento Celular/fisiología , Células Endoteliales/metabolismo , Integrinas/metabolismo , Neovascularización Fisiológica , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Integrinas/agonistas , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Factor A de Crecimiento Endotelial Vascular/farmacología , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/farmacología , Factor D de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Increased lung vascular permeability is an important contributor to respiratory failure in acute lung injury (ALI). We found that a function-blocking antibody against the integrin alphavbeta5 prevented development of lung vascular permeability in two different models of ALI: ischemia-reperfusion in rats (mediated by vascular endothelial growth factor [VEGF]) and ventilation-induced lung injury (VILI) in mice (mediated, at least in part, by transforming growth factor-beta [TGF-beta]). Knockout mice homozygous for a null mutation of the integrin beta5 subunit were also protected from lung vascular permeability in VILI. In pulmonary endothelial cells, both the genetic absence and blocking of alphavbeta5 prevented increases in monolayer permeability induced by VEGF, TGF-beta, and thrombin. Furthermore, actin stress fiber formation induced by each of these agonists was attenuated by blocking alphavbeta5, suggesting that alphavbeta5 regulates induced pulmonary endothelial permeability by facilitating interactions with the actin cytoskeleton. These results identify integrin alphavbeta5 as a central regulator of increased pulmonary vascular permeability and a potentially attractive therapeutic target in ALI.
Asunto(s)
Barrera Alveolocapilar/metabolismo , Permeabilidad Capilar/fisiología , Endotelio Vascular/metabolismo , Integrinas/metabolismo , Receptores de Vitronectina/metabolismo , Amidas/farmacología , Animales , Anticuerpos/inmunología , Barrera Alveolocapilar/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Bovinos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Integrinas/antagonistas & inhibidores , Integrinas/inmunología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Enfermedades Pulmonares/inducido químicamente , Enfermedades Pulmonares/patología , Ratones , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Vitronectina/antagonistas & inhibidores , Receptores de Vitronectina/inmunología , Daño por Reperfusión , Fibras de Estrés/efectos de los fármacos , Ventiladores Mecánicos , Quinasas Asociadas a rhoRESUMEN
The integrin alpha9beta1 is widely expressed on neutrophils, smooth muscle, hepatocytes, endothelia, and some epithelia. We now show that mice lacking this integrin have a dramatic defect in neutrophil development, with decreased numbers of granulocyte precursors in bone marrow and impaired differentiation of bone marrow cells into granulocytes. In response to granulocyte colony-stimulating factor (G-CSF), alpha9-deficient bone marrow cells or human bone marrow cells incubated with alpha9beta1-blocking antibody demonstrated decreased phosphorylation of signal transducer and activator of transcription 3 and extracellular signal-regulated protein kinase. These effects depended on the alpha9 subunit cytoplasmic domain, which was required for formation of a physical complex between alpha9beta1 and ligated G-CSF receptor. Integrin alpha9beta1 was required for granulopoiesis and played a permissive role in the G-CSF-signaling pathway, suggesting that this integrin could play an important role in disorders of granulocyte development and other conditions characterized by defective G-CSF signaling.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/metabolismo , Granulocitos/citología , Hematopoyesis/inmunología , Integrinas/metabolismo , Transducción de Señal/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células CHO , Diferenciación Celular/inmunología , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/inmunología , Granulocitos/inmunología , Granulocitos/metabolismo , Immunoblotting , Integrinas/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Factor Estimulante de Colonias de Granulocito/inmunología , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , TransfecciónRESUMEN
The expression of the integrin alpha(v)beta6 has been correlated with oral SCC invasion. We evaluated its expression in three 4NQO transformed murine oral keratinocyte cell lines (B7E3, B7E11 and B4B8). The B7E3 cells were negative for beta6, whereas the B7E11 and the B4B8 cells were both positive. The beta6 negative B7E3 cells were fibroblast-like in appearance, whereas the B7E11 cells were more epithelial-like. The B4B8 cells were a mixture of the two cell types. Using immunofluorescent microscopy, we found that vimentin was highly expressed in the B7E3 cells, whereas the B7E11 cells keratin positive. The B4B8 cells expressed both filaments. The B7E3 cells formed large tumors when injected into nude mice, whereas the B4B8 cells formed small tumors and the B7E11 cells formed none. These results suggest that the expression of the alpha(v)beta6 integrin suppresses tumor formation and may promote the epithelial phenotype in 4NQO-transformed murine oral keratinocytes.