RESUMEN
The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.
Asunto(s)
Colágeno Tipo I/metabolismo , Decorina/metabolismo , Fibromodulina/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Lumican/metabolismo , Mapas de Interacción de Proteínas , Animales , Línea Celular , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Células 3T3 NIHRESUMEN
BACKGROUND: Chemotherapeutic efficacy can be improved by targeting the structure and function of the extracellular matrix (ECM) in the carcinomal stroma. This can be accomplished by e.g. inhibiting TGF-ß1 and -ß3 or treating with Imatinib, which results in scarcer collagen fibril structure in xenografted human KAT-4/HT29 (KAT-4) colon adenocarcinoma. METHODS: The potential role of αVß6 integrin-mediated activation of latent TGF-ß was studied in cultured KAT-4 and Capan-2 human ductal pancreatic carcinoma cells as well as in xenograft carcinoma generated by these cells. The monoclonal αVß6 integrin-specific monoclonal antibody 3G9 was used to inhibit the αVß6 integrin activity. RESULTS: Both KAT-4 and Capan-2 cells expressed the αVß6 integrin but only KAT-4 cells could utilize this integrin to activate latent TGF-ß in vitro. Only when Capan-2 cells were co-cultured with human F99 fibroblasts was the integrin activation mechanism triggered, suggesting a more complex, fibroblast-dependent, activation pathway. In nude mice, a 10-day treatment with 3G9 reduced collagen fibril thickness and interstitial fluid pressure in KAT-4 but not in the more desmoplastic Capan-2 tumors that, to achieve a similar effect, required a prolonged 3G9 treatment. In contrast, a 10-day direct inhibition of TGF-ß1 and -ß3 reduced collagen fibril thickness in both tumor models. CONCLUSION: Our data demonstrate that the αVß6-directed activation of latent TGF-ß plays a pivotal role in modulating the stromal collagen network in carcinoma, but that the sensitivity to αVß6 inhibition depends on the simultaneous presence of alternative paths for latent TGF-ß activation and the extent of desmoplasia.
Asunto(s)
Antígenos de Neoplasias/inmunología , Colágeno/química , Integrinas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Colágeno/metabolismo , Líquido Extracelular/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Integrinas/metabolismo , Ratones , Presión , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
NEW FINDINGS: What is the central question of this study? Collagen-binding ß1 -integrins function physiologically in cellular control of dermal interstitial fluid pressure (PIF ) in vivo and thereby participate in control of extravascular fluid volume. During anaphylaxis, simulated by injection of compound 48/80, integrin αV ß3 takes over this physiological function. Here we addressed the question whether integrin αV ß3 can replace collagen-binding ß1 -integrin to maintain a long-term homeostatic PIF . What is the main finding and its importance? Mice lacking the collagen-binding integrin α11 ß1 show a complex dermal phenotype with regard to the interstitial physiology apparent in the control of PIF . Notably dermal PIF is not lowered with compound 48/80 in these animals. Our present data imply that integrin αV ß3 is the likely candidate that has taken over the role of collagen-binding ß1 -integrins for maintaining a steady-state homeostatic PIF . A better understanding of molecular processes involved in control of PIF is instrumental for establishing novel treatment regimens for control of oedema formation in anaphylaxis and septic shock. ABSTRACT: Accumulated data indicate that cell-mediated contraction of reconstituted collagenous gels in vitro can serve as a model for cell-mediated control of interstitial fluid pressure (PIF ) in vivo. A central role for collagen-binding ß1 -integrins in both processes has been established. Furthermore, integrin αV ß3 takes over the role of collagen-binding ß1 -integrins in mediating contraction after perturbations of collagen-binding ß1 -integrins in vitro. Integrin αV ß3 is also instrumental for normalization of dermal PIF that has been lowered due to mast cell degranulation with compound 48/80 (C48/80) in vivo. Here we demonstrate a role of integrin αV ß3 in maintaining a long term homeostatic dermal PIF in mice lacking the collagen-binding integrin α11 ß1 (α11-/- mice). Measurements of PIF were performed after circulatory arrest. Furthermore, cell-mediated integrin αV ß3 -directed contraction of collagenous gels in vitro depends on free access to a collagen site known to bind several extracellular matrix (ECM) proteins that form substrates for αV ß3 -directed cell attachment, such as fibronectin and fibrin. A streptococcal collagen-binding protein, CNE, specifically binds to and blocks this site on the collagen triple helix. Here we show that whereas CNE perturbed αV ß3 -directed and platelet-derived growth factor BB-induced normalization of dermal PIF after C48/80, it did not affect αV ß3 -dependent maintenance of a homeostatic dermal PIF . These data imply that dynamic modification of the ECM structure is needed during acute patho-physiological modulations of PIF but not for long-term maintenance of a homeostatic PIF . Our data thus show that collagen-binding ß1 -integrins, integrin αV ß3 and ECM structure are potential targets for novel therapy aimed at modulating oedema formation and hypovolemic shock during anaphylaxis.
Asunto(s)
Colágeno/metabolismo , Líquido Extracelular/metabolismo , Integrina alfaVbeta3/metabolismo , Integrina beta1/metabolismo , Animales , Edema/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Mastocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Derivado de Plaquetas/metabolismo , PresiónRESUMEN
Cell-mediated contraction of collagenous matrices is modulated by various growth factors and cytokines, such as platelet-derived growth factor-BB (PDGF-BB). Here we used a genetic cell model to delineate defined signaling pathways that enhance collagen gel contraction downstream of ligand-stimulated platelet-derived growth factor receptor-ß (PDGF-Rß). Our data show that PDGF BB-enhanced activations of phosphatidylinositol 3'-kinase (PI3K) and phospholipase Cγ (PLCγ) were necessary for PDGF-enhanced collagen gel contraction. Importantly, other defined signaling pathways down-stream of PDGF-Rß were, however, dispensable. The decisive roles for PI3K and PLCγ were corroborated by experiments using selective inhibitors. Furthermore, we show that de-phosphorylation and thereby activation of cofilin that is important for the turnover of actin filaments, is depended on PI3K and PLCγ down-stream of PDGF-Rß. Moreover, inhibition of protein kinase C (PKC) by GÖ6976 and bisindolylmaleimide-II abolished cofilin de-phosphorylation, as well as PDGF-enhanced contraction. In contrast, activation of the PKC protein family by 4ß-phorbol 12-myristate 13-acetate (PMA) did not accelerate collagen gel contraction although it induced long-term cofilin de-phosphorylation, showing the need of a dynamic control of cofilin de-phosphorylation for PDGF-enhanced collagen gel contraction. Taken together, our data point to the involvement of a PI3K/PLCγ-PKC-cofilin pathway in both PDGF-enhanced cofilin de-phosphorylation and PDGF-enhanced collagen gel contraction.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Becaplermina/metabolismo , Colágeno/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Factores Despolimerizantes de la Actina/genética , Fibroblastos , Geles , Técnicas de Silenciamiento del Gen , Humanos , Modelos Biológicos , FosforilaciónRESUMEN
Tumor barrier function in carcinoma represents a major challenge to treatment and is therefore an attractive target for increasing drug delivery. Variables related to tumor barrier include aberrant blood vessels, high interstitial fluid pressure, and the composition and structure of the extracellular matrix. One of the proteins associated with dense extracellular matrices is fibromodulin, a collagen fibrillogenesis modulator expressed in tumor stroma but scarce in normal loose connective tissues. Here, we investigated the effects of fibromodulin on stroma ECM in a syngeneic murine colon carcinoma model. We show that fibromodulin deficiency decreased collagen fibril thickness but glycosaminoglycan content and composition were unchanged. Furthermore, vascular density, pericyte coverage and macrophage amount were unaffected. Fibromodulin can therefore be a unique effector of dense collagen matrix assembly in tumor stroma and, without affecting other major matrix components or the cellular composition, can function as a main agent in tumor barrier function.
Asunto(s)
Colágeno/metabolismo , Neoplasias del Colon/metabolismo , Modelos Animales de Enfermedad , Fibromodulina/deficiencia , Glicosaminoglicanos/metabolismo , Animales , Línea Celular Tumoral , Neoplasias del Colon/patología , Fibromodulina/genética , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Imatinib causes increased turnover of stromal collagen, reduces collagen fibril diameter, enhances extracellular fluid turnover and lowers interstitial fluid pressure (IFP) in the human colonic carcinoma KAT-4/HT-29 (KAT-4) xenograft model. METHODS: We compared the effects of imatinib on oxygen levels, vascular morphology and IFP in three experimental tumor models differing in their content of a collagenous extracellular matrix. RESULTS: Neither the KAT4 and CT-26 colonic carcinoma models, nor B16BB melanoma expressed PDGF ß-receptors in the malignant cells. KAT-4 tumors exhibited a well-developed ECM in contrast to the other two model systems. The collagen content was substantially higher in KAT-4 than in CT-26, while collagen was not detectable in B16BB tumors. The pO2 was on average 5.4, 13.9 and 19.3 mmHg in KAT-4, CT-26 and B16BB tumors, respectively. Treatment with imatinib resulted in similar pO2-levels in all three tumor models but only in KAT-4 tumors did the increase reach statistical significance. It is likely that after imatinib treatment the increase in pO2 in KAT-4 tumors is caused by increased blood flow due to reduced vascular resistance. This notion is supported by the significant reduction observed in IFP in KAT-4 tumors after imatinib treatment. Vessel area varied between 4.5 and 7% in the three tumor models and was not affected by imatinib treatment. Imatinib had no effect on the fraction of proliferating cells, whereas the fraction of apoptotic cells increased to a similar degree in all three tumor models. CONCLUSION: Our data suggest that the effects of imatinib on pO2-levels depend on a well-developed ECM and provide further support to the suggestion that imatinib acts by causing interstitial stroma cells to produce a less dense ECM, which would in turn allow for an increased blood flow. The potential of imatinib treatment to render solid tumors more accessible to conventional treatments would therefore depend on the degree of tumor desmoplasia.
Asunto(s)
Neoplasias del Colon/metabolismo , Matriz Extracelular/metabolismo , Mesilato de Imatinib/farmacología , Neoplasias Experimentales/metabolismo , Oxígeno/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Colágeno/metabolismo , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , Ratones SCID , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/patología , Presión , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Células del Estroma/metabolismo , Carga Tumoral/efectos de los fármacos , AguaRESUMEN
A typical obstacle to cancer therapy is the limited distribution of low molecular weight anticancer drugs within the carcinoma tissue. In experimental carcinoma, imatinib (STI571) increases efficacy of synchronized chemotherapy, reduces tumor interstitial fluid pressure, and increases interstitial fluid volume. STI571 also increases the water-perfusable fraction in metastases from human colorectal adenocarcinomas. Because the mechanism(s) behind these effects have not been fully elucidated, we investigated the hypothesis that STI571 alters specific properties of the stromal extracellular matrix. We analyzed STI571-treated human colorectal KAT-4/HT-29 experimental carcinomas, known to have a well-developed stromal compartment, for solute exchange and glycosaminoglycan content, as well as collagen content, structure, and synthesis. MRI of STI571-treated KAT-4/HT-29 experimental carcinomas showed a significantly increased efficacy in dynamic exchanges of solutes between tumor interstitium and blood. This effect was paralleled by a distinct change of the stromal collagen network architecture, manifested by a decreased average collagen fibril diameter, and increased collagen turnover. The glycosaminoglycan content was unchanged. Furthermore, the apparent effects on the stromal cellular composition were limited to a reduction in an NG2-positive stromal cell population. The current data support the hypothesis that the collagen network architecture influences the dynamic exchanges of solutes between blood and carcinoma tissue. It is conceivable that STI571 reprograms distinct nonvascular stromal cells to produce a looser extracellular matrix, ultimately improving transport characteristics for traditional chemotherapeutic agents. Mol Cancer Ther; 15(10); 2455-64. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma/metabolismo , Colágeno/metabolismo , Líquido Extracelular/metabolismo , Mesilato de Imatinib/farmacología , Agregado de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Animales , Carcinoma/tratamiento farmacológico , Carcinoma/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Ratones , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Células del Estroma/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Tumor stroma is similar to the connective tissue of chronic inflammation. The extracellular matrix of tumors is formed by cancer-associated fibroblasts that also modulate the inflammatory response. MATERIALS AND METHODS: We studied the ability of oral keratinocytes (NOK) and oral squamous cell carcinoma cells (SCC) to induce an innate immune response in fibroblasts. Co-cultures with fibroblasts in collagen gels and keratinocytes in inserts were used. Pentraxin 3 (PTX3) was used as an indicator of an innate immune response. RESULTS: SCC and NOK up-regulated fibroblast mRNA expression and protein release of PTX3. mRNA levels were more pronounced in cultures with malignant cells. The induction of PTX3 was abrogated by an interleukin-1 receptor antagonist CONCLUSION: Keratinocytes have the capacity to induce an interleukin-1-dependent innate immune response by fibroblasts in vitro. This could be important for subsequent fibroblast modulation of the inflammatory reaction in non-malignant and malignant disease processes.
Asunto(s)
Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Queratinocitos/patología , Boca/citología , Proteína C-Reactiva/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Fibroblastos/metabolismo , Fibroblastos/patología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Interleucina-1alfa/biosíntesis , Queratinocitos/metabolismo , Boca/metabolismo , Receptores de Interleucina-1/biosíntesis , Componente Amiloide P Sérico/metabolismo , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.
Asunto(s)
Colágeno/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Colágeno/análisis , Activación Enzimática , Proteínas de la Matriz Extracelular/genética , Fibromodulina , Eliminación de Gen , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mapas de Interacción de Proteínas , Proteína-Lisina 6-Oxidasa/análisis , Proteoglicanos/genéticaRESUMEN
UNLABELLED: High interstitial fluid pressure (IFP) in colorectal cancer metastases may decrease the uptake and, thus, the effects of antitumor drugs. Imatinib, a selective inhibitor of platelet-derived growth factor receptors, and anakinra, an interleukin-1 receptor antagonist, respectively, increase drug uptake or decrease IFP in preclinical models of carcinoma. Drug-induced decrease in IFP in human metastases has not been objectively shown but should be reflected by an increase in water-perfusable tissue fraction (PTF) or tumor blood flow (TBF) using (15)O-water PET/CT and kinetic modeling. Hence, the aim of this study was to assess the effects of imatinib and anakinra on PTF and TBF in colorectal cancer metastases in patients. METHODS: Nine patients with documented progressive disease despite all established therapy underwent (15)O-water PET/CT at baseline and at 2 d and 6-7 d after the start of oral administration of imatinib (400 mg/d). After a washout period of 1 wk, the protocol was repeated with anakinra (100 mg/d) subcutaneously. Six patients underwent a second baseline scan on the same day to assess reproducibility of PTF and TBF measurements. Volumes of interest were drawn over liver metastases and aorta. PTF and TBF were calculated using the standard single-tissue-compartment model. RESULTS: Imatinib administration during 6-7 d increased PTF from 0.62 ± 0.12 to 0.69 ± 0.13, compared with baseline and day 2 (P = 0.02, Wilcoxon test). No significant changes were found in TBF. PTF values were no longer significantly different from baseline 1 wk after the last imatinib dosage. Anakinra induced no significant change in PTF or TBF. The repeatability coefficients of PTF and TBF in liver lesions were 22% and 28%, respectively. CONCLUSION: Imatinib increases PTF of colorectal cancer metastases in patients and hence may increase the delivery of antitumor drugs. (15)O-water PET/CT and kinetic modeling provide insights into the microenvironment of human cancers.
Asunto(s)
Antineoplásicos/administración & dosificación , Benzamidas/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Piperazinas/administración & dosificación , Tomografía de Emisión de Positrones/métodos , Pirimidinas/administración & dosificación , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Administración Oral , Adulto , Anciano , Neoplasias Colorrectales/patología , Femenino , Humanos , Mesilato de Imatinib , Cinética , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Radioisótopos de Oxígeno , Perfusión , Reproducibilidad de los Resultados , Factores de Tiempo , Agua/químicaRESUMEN
BACKGROUND: While carcinoma-associated fibroblasts (CAFs) support tumorigenesis, normal tissue fibroblasts suppress tumor progression. Mechanisms behind conversion of fibroblasts into a CAF phenotype are largely unrevealed. MATERIALS AND METHODS: Transwell co-cultures with fibroblasts in collagen gels and squamous-cell carcinoma (SCC) cells or normal oral keratinocytes (NOKs) in inserts. Differences in fibroblast global gene expression were analyzed using Affymetrix arrays and subsequent functional annotation and cluster analysis, as well as gene set enrichment analysis were performed. RESULTS: There were 52 up-regulated and 30 down-regulated transcript IDs (>2-fold, p<0.05) in fibroblasts co-cultured with SCC compared to NOKs. Functional analysis demonstrated an enrichment of collagen-related genes. There were similarities with gene sets reflecting a non-specific, innate-type response with activation of both interferon pathways and connective tissue turnover. CONCLUSION: There were distinct differences in fibroblast gene expression between the co-culture types. Many were in genes related to an innate-type of response and to connective tissue turnover.
Asunto(s)
Carcinoma de Células Escamosas/genética , Transformación Celular Neoplásica/genética , Fibroblastos/patología , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , Queratinocitos/patología , Proteínas de Neoplasias/biosíntesisRESUMEN
The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVß3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVß3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVß3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.
Asunto(s)
Colágeno Tipo I/metabolismo , Fibrina/metabolismo , Integrina alfaVbeta3/metabolismo , Animales , Línea Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Geles , Ratones , Trombina/farmacologíaRESUMEN
The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.
Asunto(s)
Colágeno Tipo I/química , Proteínas de la Matriz Extracelular/deficiencia , Péptidos/química , Proteoglicanos/deficiencia , Tendones/metabolismo , Aldehídos/metabolismo , Secuencia de Aminoácidos , Animales , Colágeno Tipo I/metabolismo , Fibromodulina , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 (m1J) mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 (m1J) mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 (m1J) mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.
Asunto(s)
Carcinoma/genética , Carcinoma/patología , Melanoma/genética , Melanoma/patología , NADPH Oxidasas/genética , Neoplasias/genética , Neoplasias/patología , Animales , Carcinoma/metabolismo , Carcinoma/mortalidad , Carcinoma Pulmonar de Lewis , Modelos Animales de Enfermedad , Inflamación/genética , Inflamación/metabolismo , Melanoma/metabolismo , Melanoma/mortalidad , Melanoma Experimental , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , NADPH Oxidasas/deficiencia , NADPH Oxidasas/metabolismo , Neoplasias/metabolismo , Neoplasias/mortalidad , Especies Reactivas de Oxígeno/inmunología , Carga Tumoral/genéticaRESUMEN
BACKGROUND: To investigate possible differences in the effects of soluble factors from oral squamous cell carcinoma (SCC) cells (UT-SCC-87) and normal oral keratinocytes (NOK) on fibroblast expression of genes involved in tumor stroma turnover. MATERIALS AND METHODS: Transwell co-cultures with fibroblasts in collagen gels, and SCC cells or NOK in inserts were carried out. Fibroblast gene expression was measured with real-time polymerase chain reaction (PCR). RESULTS: The expression of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) was up-regulated in co-cultures with SCC cells but not with NOK. In contrast, both SCC cells and NOK regulated matrix metalloproteinase-1 (MMP1) and -3, and tissue inhibitor of metalloproteinases-2 (TIMP2) and -3 to a similar extent, while MMP2 and TIMP1 were largely unaffected. Interleukin 1 alpha (IL1α) up-regulated both MMP1 and MMP3 and down-regulated PAI-1, TIMP2 and -3. CONCLUSION: SCC and NOK regulate fibroblast expression of genes involved in tumor stroma turnover differentially in vitro. These observations may contribute to a better understanding of the mechanisms behind extracellular matrix turnover in tumors.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Queratinocitos/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Queratinocitos/patología , Masculino , Inhibidor 1 de Activador Plasminogénico/genética , Carcinoma de Células Escamosas de Cabeza y Cuello , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
OBJECTIVES: The composition of tumor stroma and the activity of tumor associated fibroblasts are important for tumor growth. Interactions between carcinoma cells and fibroblasts regulate the turnover of extracellular matrix (ECM). Here, the in vitro effects of oral squamous cell carcinoma (SCC) cells (UT-SCC-30 and UT-SCC-87) on fibroblast expression of genes for ECM components and connective tissue growth factor (CTGF/CCN2), were compared to those of normal oral keratinocytes (NOK). MATERIALS AND METHODS: Cocultures with fibroblasts in collagen gels and keratinocytes with the two cell types separated by a semi permeable membrane were used, and relative gene expression was measured with real-time PCR. RESULTS: All investigated genes were regulated by NOK and the SCCs. The downregulation of pro-collagens α1(I) and α1(III) was more pronounced in cocultures with NOK, while the expression of CCN2 and fibronectin was downregulated by both NOK and the SCCs to a similar extent. UT-SCC-87, but not UT-SCC-30, secreted significantly more IL-1α than NOK. A recombinant interleukin-1 receptor antagonist reversed many of the observed effects on fibroblast gene expression suggesting involvement of IL-1 in cocultures with NOK as well as with SCCs. CONCLUSION: The observed differential effects on fibroblast gene expression suggest that NOK are more antifibrotic compared to UT-SCC-30 and UT-SCC-87. These findings may contribute to a better understanding of the mechanisms behind ECM turnover in tumors.
Asunto(s)
Carcinoma de Células Escamosas/patología , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Interleucina-1/fisiología , Queratinocitos/efectos de los fármacos , Boca/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Stroma properties affect carcinoma physiology and direct malignant cell development. Here we present data showing that α(V)ß(3) expressed by stromal cells is involved in the control of interstitial fluid pressure (IFP), extracellular volume (ECV) and collagen scaffold architecture in experimental murine carcinoma. IFP was elevated and ECV lowered in syngeneic CT26 colon and LM3 mammary carcinomas grown in integrin ß(3)-deficient compared to wild-type BALB/c mice. Integrin ß(3)-deficiency had no effect on carcinoma growth rate or on vascular morphology and function. Analyses by electron microscopy of carcinomas from integrin ß(3)-deficient mice revealed a coarser and denser collagen network compared to carcinomas in wild-type littermates. Collagen fibers were built from heterogeneous and thicker collagen fibrils in carcinomas from integrin ß(3)-deficient mice. The fibrotic extracellular matrix (ECM) did not correlate with increased macrophage infiltration in integrin ß(3)-deficient mice bearing CT26 tumors, indicating that the fibrotic phenotype was not mediated by increased inflammation. In conclusion, we report that integrin ß(3)-deficiency in tumor stroma led to an elevated IFP and lowered ECV that correlated with a more fibrotic ECM, underlining the role of the collagen network for carcinoma physiology.
Asunto(s)
Carcinoma/metabolismo , Fibrosis/patología , Regulación Neoplásica de la Expresión Génica , Integrina beta3/genética , Animales , Línea Celular Tumoral , Colágeno/metabolismo , Líquido Extracelular , Femenino , Hidroxiprolina/metabolismo , Integrina alfaVbeta3/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Fenotipo , PresiónRESUMEN
Whereas the roles of proangiogenic factors in carcinogenesis are well established, those of endogenous angiogenesis inhibitors (EAIs) remain to be fully elaborated. We investigated the roles of three EAIs during de novo tumorigenesis to further test the angiogenic balance hypothesis, which suggests that blood vessel development in the tumor microenvironment can be governed by a net loss of negative regulators of angiogenesis in addition to the well-established principle of up-regulated angiogenesis inducers. In a mouse model of pancreatic neuroendocrine cancer, administration of endostatin, thrombospondin-1, and tumstatin peptides, as well as deletion of their genes, reveal neoplastic stage-specific effects on angiogenesis, tumor progression, and survival, correlating with endothelial expression of their receptors. Deletion of tumstatin and thrombospondin-1 in mice lacking the p53 tumor suppressor gene leads to increased incidence and reduced latency of angiogenic lymphomas associated with diminished overall survival. The results demonstrate that EAIs are part of a balance mechanism regulating tumor angiogenesis, serving as intrinsic microenvironmental barriers to tumorigenesis.
Asunto(s)
Autoantígenos/metabolismo , Colágeno Tipo IV/metabolismo , Endostatinas/metabolismo , Neoplasias/metabolismo , Trombospondina 1/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Autoantígenos/genética , Línea Celular , Colágeno Tipo IV/química , Colágeno Tipo IV/genética , Progresión de la Enfermedad , Endostatinas/química , Endostatinas/genética , Femenino , Humanos , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Estadificación de Neoplasias , Neoplasias/genética , Neoplasias/prevención & control , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevención & control , Péptidos/farmacología , Propionatos/farmacología , Análisis de Supervivencia , Trombospondina 1/química , Trombospondina 1/genética , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin α(V)ß(3)-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited α(V)ß(3)-dependent cell-mediated collagen gel contraction, PDGF BB-induced and α(V)ß(3)-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding ß(1) integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited α(V)ß(3)-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced α(V)ß(3)-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colágeno/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Colágeno/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Becaplermina , Unión Competitiva , Rastreo Diferencial de Calorimetría , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Colágeno/química , Colágeno/ultraestructura , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Integrina alfaVbeta3/genética , Microscopía Electrónica , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-sis , Interferencia de ARN , Receptores de Colágeno/genética , Streptococcus/genética , Streptococcus/metabolismo , TransfecciónRESUMEN
This review will summarize current knowledge on the role of the extracellular matrix (ECM) in general and on the interstitial fluid pressure (P(if)) in particular with regard to their importance in transcapillary exchange. The fluid volume in the interstitial space is normally regulated within narrow limits by automatic re-adjustment of the interstitial hydrostatic and colloid osmotic pressures in response to perturbations in capillary filtration and by the lymphatics. Contrary to this commonly accepted view, P(if) can become an active force and create a fluid flux across the capillaries in several inflammatory reactions and trauma situations rather than limit the changes occurring. The molecular mechanisms involved in the lowering of P(if) include the release of cellular tension exerted on the collagen and microfibril networks in the connective tissue via the collagen-binding beta(1)-integrins, thereby allowing the glycosaminoglycan ground substance, which is normally underhydrated, to expand and take up fluid. Several growth factors and cytokines, including the platelet-derived growth factor BB, are able to reverse a lowering of P(if) and restore the normal compaction of the ECM. The magnitude of the lowering of P(if) varies with the inflammatory response. In several inflammatory reactions, a lowering of P(if) to -5 to -10 mmHg is seen, which will increase capillary filtration by 10-20 times since the normal capillary filtration pressure is usually 0.5-1 mmHg (skin and skeletal muscle). Unless this lowering of P(if) is taken into account, the enhanced solute flux resulting from an inflammatory response will be ascribed to an increased capillary permeability.