RESUMEN
Fibroblasts are central to wound healing and fibrosis through TGFß1-triggered differentiation into contractile, α-smooth muscle actin (α-SMA)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localization of CD44 with the epidermal growth factor receptor (EGFR). Interactions of HA with hyaladherins, such as inter-α-inhibitor (IαI) and tumor necrosis factor-stimulated gene-6 (TSG-6), are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and α-SMA had different kinetics of induction by TGFß1, with TSG-6 peaking before α-SMA Si CD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing IαI heavy chain (HC) transfer), HA-oligosaccharides, cobalt, or Si bikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG-6/IαI HC interaction was necessary for the effect of TSG-6 and that HC stabilization of HA initiated the CD44/EGFR association. The newly described HC5 was shown to be the principal HC expressed, and its cell surface expression was prevented by siRNA inhibition of TSG-6 or bikunin. HC5 was released by hyaluronidase treatment, confirming its association with cell surface HA. Finally, HC5 knockdown by siRNA confirmed its role in myofibroblast differentiation. The current study describes a novel mechanism linking the TSG-6 transfer of the newly described HC5 to the HA-dependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGFß1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/fisiología , Miofibroblastos/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/genética , Actinas/metabolismo , alfa-Globulinas/farmacología , Anticuerpos Monoclonales de Origen Murino/farmacología , Moléculas de Adhesión Celular/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/farmacología , Miofibroblastos/citología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
INTRODUCTION: CD44 is a cell-surface glycoprotein involved in various cellular functions. Recent studies have suggested that CD44 is involved in early mineralization of odontoblasts. Hyaluronic acid (HA) is the principal ligand for receptor CD44. Whether and how HA regulated the mineralization process of dental pulp cells were investigated. METHODS: The effects of high-molecular-weight HA on differentiation and mineral deposition of dental pulp cells were tested by using alkaline phosphatase (ALP) activity assay and alizarin red S staining. Osteogenesis real-time polymerase chain reaction array, quantitative polymerase chain reaction, and Western blotting were performed to identify downstream molecules involved in the mineralization induction of HA. CD44 was knocked down and examined to confirm whether the mineralization effect of HA was mediated by receptor CD44. Immunohistochemistry was used to understand the localization patterns of CD44 and the identified downstream proteins in vivo. RESULTS: Pulse treatment of HA enhanced ALP activity and mineral deposition in dental pulp cells. Tissue-nonspecific ALP, bone morphogenetic protein 7 (BMP7), and type XV collagen (Col15A1) were upregulated via the HA-CD44 pathway in vitro. Immunohistochemistry of tooth sections showed that the staining pattern of BMP7 was very similar to that of CD44. CONCLUSIONS: Results of this study indicated that high-molecular-weight HA enhanced early mineralization of dental pulp cells mediated via CD44. The process involved important mineralization-associated molecules including tissue-nonspecific ALP, BMP7, and Col15A1. The findings may help develop new strategies in regenerative endodontics.
Asunto(s)
Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Pulpa Dental/metabolismo , Receptores de Hialuranos/farmacología , Ácido Hialurónico/farmacología , Calcificación de Dientes/efectos de los fármacos , Adulto , Fosfatasa Alcalina/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Antraquinonas , Western Blotting , Proteína Morfogenética Ósea 7/efectos de los fármacos , Proteína Morfogenética Ósea 7/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/administración & dosificación , Sialoproteína de Unión a Integrina/efectos de los fármacos , Sialoproteína de Unión a Integrina/metabolismo , Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Metaloproteinasa 13 de la Matriz/metabolismo , Tercer Molar/citología , Odontoblastos/efectos de los fármacos , Osteogénesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Epidermolytic ichthyosis (EI) is an autosomal dominant epidermal skin fragility disorder caused by mutations in keratin 1 and 10 (K1 and K10) genes. Mutated keratins form characteristic aggregates in vivo and in vitro. Some patients benefit from retinoid therapy, although the mechanism is not fully understood. Our aim was to demonstrate whether retinoids affect the formation of keratin aggregates in immortalized EI cells in vitro. EI keratinocytes were seeded on cover slips, pre-treated or not with retinoids, heat-stressed, and keratin aggregate formation monitored. K10 aggregates were detected in 5% of cells in the resting state, whereas heat stress increased this proportion to 25%. When cells were pre-incubated with all-trans-retinoic acid (ATRA) or retinoic acid receptor (RAR)-α agonists the aggregates decreased in a dose-dependent manner. Furthermore, ATRA decreased the KRT10 transcripts 200-fold as well as diminished the ratio of mutant to wild-type transcripts from 0.41 to 0.35, thus providing a plausible rational for retinoid therapy of EI due to K10 mutations.
Asunto(s)
Hiperqueratosis Epidermolítica/patología , Queratinocitos/efectos de los fármacos , Queratolíticos/farmacología , Retinoides/farmacología , Tretinoina/farmacología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas del Choque Térmico HSC70/metabolismo , Calor , Humanos , Receptores de Hialuranos/farmacología , Hiperqueratosis Epidermolítica/genética , Queratina-10/genética , Queratinocitos/metabolismo , Leupeptinas/farmacología , Masculino , Mutación , Receptor 2 Gatillante de la Citotoxidad Natural , ARN Mensajero/metabolismo , Receptores de Ácido Retinoico/agonistas , Ubiquitina/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND/AIMS: The aim of our study was to reveal the role of CD44-Hyaluronic acid (HA) in the homing and improving renal function of systemically transplanted MSCs in chronic renal failure. METHODS: First, a remnant kidney model was established in rats and the expression of HA was determined using immunohistochemistry (IHC) and western blotting. Next, chemotaxis assay using flow cytometry, and cell migration assay of MSCs were performed in vitro. Then, MSCs were transplanted into rats, thus, sprague-Dawley (SD) rats were randomly divided into sham group, 5/6 nephrectomy (5/6 Nx) group, MSC group and MSC/Anti-CD44 group (n = 8 for all groups). Migration of MSCs to the kidney in these rats was assessed by using cell tracking experiments, and tissue damage was evaluated by morphological analysis using Masson's trichrome staining and periodic acid Schiff staining. RESULTS: HA was significantly observed in 5/6 Nx group, but not in sham group. Meanwhile, HA was discovered induced MSCs migration remarkably (p < 0.05) and anti-CD44 antibody inhibited the migration significantly (p < 0.05) in vitro. In vivo, the GFP-MSCs were observed in MSC group and the cells reduced in MSC/Anti-CD44 groups, especially, in the tubulointerstitium. CONCLUSION: Our findings reveal that CD44-HA has the potential to induce MSCs homing to injured tissue, while its effect on the ability of MSCs, improving tissue function, is not significant.
Asunto(s)
Receptores de Hialuranos/farmacología , Ácido Hialurónico/farmacología , Enfermedades Renales/terapia , Riñón/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Animales , Movimiento Celular/efectos de los fármacos , Creatinina/sangre , Riñón/efectos de los fármacos , Corteza Renal/citología , Enfermedades Renales/sangre , Enfermedades Renales/fisiopatología , Masculino , Nefrectomía , Ratas , Ratas Sprague-Dawley , Urea/sangreRESUMEN
Novel approaches targeting the host's immune response to treat Staphylococcus aureus infections have significant potential to improve clinical outcomes, in particular during infection with antibiotic-resistant strains. The hyaluronic acid-binding peptide (HABP) PEP35 was assessed for its ability to treat S. aureus infections using a clinically relevant murine model of surgical wound infection. PEP35 demonstrated no direct antimicrobial activity against a range of antibiotic-susceptible and antibiotic-resistant clinical isolates of Staphylococcus aureus. However, when this peptide was administered at the onset of infection and up to 4 h postchallenge with a methicillin-susceptible (MSSA) or a methicillin-resistant (MRSA) strain of S. aureus, it significantly reduced the bacterial burden at the wound infection site. PEP35 reduced the tissue bacterial burden by exclusively modulating the local neutrophil response. PEP35 administration resulted in a significant early increase in local CXCL1 and CXCL2 production, which resulted in a more rapid influx of neutrophils to the infection site. Importantly, neutrophil influx was not sustained after treatment with PEP35, and administration of PEP35 alone did not induce a local inflammatory response. The immunomodulatory effects of PEP35 on CXC chemokine production were TLR2 and NF-κB dependent. We propose a novel role for a HABP as an innate immunomodulator in the treatment of MSSA and MRSA surgical wound infection through enhancement of the local CXC chemokine-driven neutrophil response.
Asunto(s)
Receptores de Hialuranos/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infección de Heridas/tratamiento farmacológico , Animales , Línea Celular , Quimiocinas CXC/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Factores Inmunológicos/farmacología , Staphylococcus aureus Resistente a Meticilina/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Infecciones Estafilocócicas/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Infección de Heridas/microbiologíaRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive brain tumor that, by virtue of its resistance to chemotherapy and radiotherapy, is currently incurable. Identification of molecules whose targeting may eliminate GBM cells and/or sensitize glioblastoma cells to cytotoxic drugs is therefore urgently needed. CD44 is a major cell surface hyaluronan receptor and cancer stem cell marker that has been implicated in the progression of a variety of cancer types. However, the major downstream signaling pathways that mediate its protumor effects and the role of CD44 in the progression and chemoresponse of GBM have not been established. Here we show that CD44 is upregulated in GBM and that its depletion blocks GBM growth and sensitizes GBM cells to cytotoxic drugs in vivo. Consistent with this observation, CD44 antagonists potently inhibit glioma growth in preclinical mouse models. We provide the first evidence that CD44 functions upstream of the mammalian Hippo signaling pathway and that CD44 promotes tumor cell resistance to reactive oxygen species-induced and cytotoxic agent-induced stress by attenuating activation of the Hippo signaling pathway. Together, our results identify CD44 as a prime therapeutic target for GBM, establish potent antiglioma efficacy of CD44 antagonists, uncover a novel CD44 signaling pathway, and provide a first mechanistic explanation as to how upregulation of CD44 may constitute a key event in leading to cancer cell resistance to stresses of different origins. Finally, our results provide a rational explanation for the observation that functional inhibition of CD44 augments the efficacy of chemotherapy and radiation therapy.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Receptores de Hialuranos/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Células COS , Procesos de Crecimiento Celular/fisiología , Chlorocebus aethiops , Glioblastoma/genética , Glioblastoma/patología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/farmacología , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Ratones , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Transducción de Señal , Regulación hacia ArribaRESUMEN
Hyaluronan is a glycosaminoglycan polymer prominent in embryogenesis and in tissues undergoing repair. It is responsible for the water content of skin, where half the hyaluronan of the body is present. As in other tissues, it undergoes rapid turnover. Its biology is vastly different between dermis and epidermis. Levels do not diminish with age but instead become increasingly associated with tissues and resistant to extraction in vitro. Hyaluronan-binding proteins are involved, most of which remain unidentified. Hyaluronan size is critical for its various functions. High molecular size reflects intact tissues and antiangiogenic and immunosuppressive state, whereas smaller polymers are distress signals and potent inducers of inflammation and angiogenesis.
Asunto(s)
Envejecimiento/fisiología , Ácido Hialurónico/farmacología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/fisiología , Adyuvantes Inmunológicos/uso terapéutico , Matriz Extracelular/metabolismo , Humanos , Receptores de Hialuranos/farmacología , Ácido Hialurónico/química , Ácido Hialurónico/fisiología , Ácido Hialurónico/uso terapéutico , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/fisiologíaRESUMEN
High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031-1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27(kip1) in vascular SMCs. p27(kip1) messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27(kip1), was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury.
Asunto(s)
Arterias/metabolismo , Ciclinas/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Mesodermo/metabolismo , Músculo Liso Vascular/metabolismo , Animales , Arterias/citología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina D , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/efectos de los fármacos , Ciclinas/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Fibroblastos/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/farmacología , Ácido Hialurónico/farmacología , Mesodermo/citología , Mesodermo/efectos de los fármacos , Ratones , Ratones Noqueados , Mitógenos/metabolismo , Mitógenos/farmacología , Músculo Liso Vascular/citología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is associated with increased apoptosis and defective phagocytosis in the airway. As uncleared cells can undergo secondary necrosis and perpetuate inflammation, strategies to improve clearance would have therapeutic significance. There is evidence that the 15-member macrolide antibiotic azithromycin has anti-inflammatory properties. Its effects may be increased in the lung due to its ability to reach high concentrations in alveolar macrophages (AMs). The present study investigated the effects of low-dose (500 ng x mL(-1)) azithromycin on the phagocytosis of apoptotic bronchial epithelial cells and neutrophils by AMs. Flow cytometry was applied to measure phagocytosis and receptors involved in AM recognition of apoptotic cells. Cytokines were investigated using cytometric bead array. Baseline phagocytosis was reduced in COPD subjects compared with controls. Azithromycin significantly improved the phagocytosis of epithelial cells or neutrophils by AMs from COPD subjects by 68 and 38%, respectively, often up to levels comparable with controls. The increase in phagocytosis was partially inhibited by phosphatidylserine, implicating the phosphatidylserine pathway in the pro-phagocytic effects of azithromycin. Azithromycin had no effect on other recognition molecules (granulocyte-macrophage colony-stimulating factor, CD44, CD31, CD36, CD91, alphavbeta3 integrin). At higher doses, azithromycin decreased levels of pro-inflammatory cytokines. Thus, low-dose azithromycin therapy could provide an adjunct therapeutic option in chronic obstructive pulmonary disease.
Asunto(s)
Antibacterianos/farmacología , Azitromicina/farmacología , Bronquios/inmunología , Macrófagos Alveolares/efectos de los fármacos , Fagocitosis , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Antibacterianos/uso terapéutico , Antígenos CD/metabolismo , Apoptosis , Azitromicina/uso terapéutico , Bronquios/citología , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Técnicas de Cocultivo , Citocinas/metabolismo , Células Epiteliales/inmunología , Humanos , Receptores de Hialuranos/farmacología , Lisosomas , Macrófagos Alveolares/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Fagocitosis/efectos de los fármacos , Fosfatidilserinas/farmacología , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismoRESUMEN
The prostanoid prostacyclin (PGI2) inhibits proliferation of cultured vascular SMCs by inhibiting cell cycle progression from G1 to S phase. Progression through G1 phase is regulated by the sequential activation of the G1 phase cyclin-dependent kinases (cdks). Recent studies have shown that PGI2-dependent activation of its receptor, IP, inhibits G1 phase progression by blocking the degradation of p27 and the activation of cyclin E-cdk2. High Density Lipoproteins (HDL) and its associated apolipoprotein, ApoE, also inhibit S phase entry of vascular SMCs, and the effects of HDL and ApoE are, at least in part, also mediated by the production of PGI2. The antimitogenic effects of hyaluronan may also be controlled by PGI2. This review summarizes the effects of PGI2 on the G1 phase cyclin-cdks and discusses the potential role of PGI2 as a common component of multiple extracellular signals that attenuate the proliferation of vascular SMCs.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Fase G1 , Mitógenos/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Ciclina A/genética , Estrógenos/farmacología , Humanos , Receptores de Hialuranos/farmacología , Ácido Hialurónico/farmacología , Fosforilación , Proteínas Quinasas Asociadas a Fase-S/genéticaRESUMEN
PURPOSE: The ectodomain of CD44, the principal receptor for hyaluronic acid (HA), is shed as a 32-kDa fragment-soluble CD44 (sCD44)-which is cytotoxic to trabecular meshwork (TM) cells and retinal ganglion cells (RGCs) in culture. The purpose of this study was to characterize sCD44 further by determining the phosphorylation of aqueous humor sCD44 in normal and primary open-angle glaucoma (POAG). METHODS: Aqueous humor samples of patients were subjected to CD44 enzyme-linked immunosorbent assay (ELISA) and two-dimensional (2-D) polyacrylamide gel electrophoresis, followed by Western blot analysis with anti-CD44, anti-serine/threonine, and anti-tyrosine phosphospecific antibodies, to determine sCD44 concentration, isoelectric point (pI), and phosphorylation, respectively. The bioactivity of hypophosphorylated sCD44 was tested in cell culture and HA affinity columns. RESULTS: Two-dimensional Western blot analysis revealed that the representative pI of the 32-kDa sCD44 was 6.96 +/- 0.07 in POAG versus 6.38 +/- 0.08 in normal (P < 0.0004). Enzymatic dephosphorylation of sCD44 resulted in a basic shift in the pI. The normal aqueous humor sCD44 was positive for serine-threonine phosphorylation; however, POAG sCD44 was hypophosphorylated. Hypophosphorylated sCD44 was more toxic to TM and RGC cells than standard sCD44, and hypophosphorylated sCD44 had decreased affinity to HA, particularly with increased pressure. CONCLUSIONS: POAG aqueous is characterized by posttranslational change in the pI of sCD44 and hypophosphorylation, which clearly distinguished POAG from normal aqueous humor. The high toxicity and low HA-binding affinity of hypophosphorylated sCD44 may represent specific pathophysiologic features of the POAG disease process.
Asunto(s)
Humor Acuoso/metabolismo , Glaucoma de Ángulo Abierto/metabolismo , Receptores de Hialuranos/metabolismo , Western Blotting , Técnicas de Cultivo de Célula , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Humanos , Receptores de Hialuranos/farmacología , Ácido Hialurónico/metabolismo , Inmunoprecipitación , Punto Isoeléctrico , Fosforilación , Células Ganglionares de la Retina/efectos de los fármacos , Malla Trabecular/efectos de los fármacosRESUMEN
Multidrug resistance is a potent barrier to effective, long term therapy in cancer patients. It is frequently attributed to enhanced expression of multidrug transporters or to the action of receptor kinases, such as ErbB2, and downstream anti-apoptotic signaling pathways, such as the phosphoinositide 3-kinase/Akt pathway. However, very few connections have been made between receptor kinases or anti-apoptotic pathways and multidrug transporter expression or function. Data presented herein show that constitutive interaction of the pericellular polysaccharide, hyaluronan, with its receptor, CD44, regulates assembly and activation of an ErbB2-containing signaling complex, which in turn stimulates phosphoinositide 3-kinase activity in multidrug-resistant MCF-7/Adr human breast carcinoma cells. Phosphoinositide 3-kinase activates Akt and downstream anti-apoptotic events, which contribute to drug resistance. However, hyaluronan and phosphoinositide 3-kinase stimulate expression of the multidrug transporter, MDR1 (P-glycoprotein), in an interdependent, but Akt-independent, manner. Furthermore, constitutively active phosphoinositide 3-kinase, but not Akt, stimulates hyaluronan production. These Akt-independent effects are dominant over the effects of Akt on doxorubicin resistance in MCF-7/Adr cells. Thus hyaluronan, phosphoinositide 3-kinase, and ErbB2 form a positive feedback loop that strongly amplifies MDR1 expression and regulates drug resistance in these cells. This pathway may also be important in progression of other malignant characteristics. These results illustrate the potential importance of hyaluronan as a therapeutic target in multidrug-resistant carcinomas.
Asunto(s)
Resistencia a Antineoplásicos/genética , Regulación de la Expresión Génica , Genes MDR/genética , Ácido Hialurónico/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Receptor ErbB-2/fisiología , Neoplasias de la Mama , Brevicano , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Doxorrubicina , Retroalimentación Fisiológica , Glucuronosiltransferasa/genética , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/farmacología , Receptores de Hialuranos/fisiología , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/farmacología , Lectinas Tipo C , Proteínas del Tejido Nervioso/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , ARN Interferente Pequeño/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
Evidence is accumulating to suggest that TFPI-2 is involved in regulating pericellular proteases implicated in a variety of physiologic and pathologic processes including cancer cell invasion, vascular inflammation, and atherosclerosis. Recent immunohistochemical studies of advanced atherosclerotic lesions, demonstrated a similar tissue distribution for TFPI-2, High Molecular Weight Kininogen (HK), and gC1qR/p33 (gC1qR), a ubiquitously expressed, multicompartmental cellular protein involved in modulating complement, coagulation, and kinin cascades. Further studies to evaluate TFPI-2 interactions with gC1qR demonstrated direct interactions between gC1qR and TFPI-2 using immunoprecipitation and solid phase binding studies. Specific and saturable binding between TFPI-2 and gC1qR (estimated Kd: approximately 70 nM) was observed by ELISA and surface plasmon resonance (Biacore) binding assays. Binding was inhibited by antibodies to gC1qR, and was strongly dependent on the Kunitz-2 domain of TFPI-2, as deletion of this domain reduced gC1qR-TFPI-2 interactions by approximately 75%. Deletion of gC1qR amino acids 74-95, involved in C1q binding, had no effect on gC1qR binding to TFPI-2, although antibodies to this region and purified C1q both inhibited binding, most likely via allosteric effects. In contrast, HK did not affect TFPI-2 binding to gC1qR. Binding of TFPI-2 to gC1qR produced statistically significant but modest reductions in TFPI-2 inhibition of plasmin, but had no effect on kallikrein inhibition in fluid phase chromogenic assays. Taken together, these data suggest that gC1qR may participate in tissue remodeling and inflammation by localizing TFPI-2 to the pericellular environment to modulate local protease activity and regulate HK activation.
Asunto(s)
Endotelio Vascular/patología , Glicoproteínas/metabolismo , Receptores de Hialuranos/metabolismo , Inflamación/etiología , Proteínas Portadoras , Complemento C1q/farmacología , Fibrinolisina/antagonistas & inhibidores , Glicoproteínas/farmacología , Humanos , Receptores de Hialuranos/farmacología , Calicreínas/antagonistas & inhibidores , Quininógeno de Alto Peso Molecular/farmacología , Proteínas Mitocondriales , Unión Proteica/efectos de los fármacosRESUMEN
CD44 is the main cellular receptor for hyaluronic acid (HA). We previously found that overexpression of CD44 inhibited tumor growth of mouse fibrosarcoma cells in mice. Here, we show that soluble recombinant CD44 HA-binding domain (CD44-HABD) acts directly onto endothelial cells by inhibiting endothelial cell proliferation in a cell-specific manner. Consequently, soluble recombinant CD44-HABD also blocked angiogenesis in vivo in chick and mouse, and thereby inhibited tumor growth of various origins at very low doses (0.25 mg/kg x day). The antiangiogenic effect of CD44 is independent of its HA-binding capacity, since mutants deficient in HA binding still maintain their antiangiogenic and antiproliferative properties. Recombinant CD44-HABD represents a novel class of angiogenesis inhibitors based on a cell-surface receptor.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Receptores de Hialuranos/genética , Ácido Hialurónico/metabolismo , Animales , Sitios de Unión , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Pollos , Fibrosarcoma , Glioblastoma , Humanos , Receptores de Hialuranos/farmacología , Ácido Hialurónico/farmacología , Melanoma , Ratones , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Chorioallantoic membranes (CAMs) were used to determine which extracellular matrix molecules play essential roles in growth and vascular development in vivo and whether expression of critical molecules is affected by cigarette smoke exposure. Treatment of CAMs on day 5 of development with antibodies to CD44 or tenascin, but not to other matrix molecules, inhibited CAM growth and affected various aspects of blood vessel development including normal growth and branching of vessels, migration of vessels, and formation and differentiation of the capillary plexus. DNA synthesis was inhibited by antibodies to both C44 and tenascin which probably accounted for many of the phenotypic changes observed in treated CAMs. CD44 was located on all cells in day 5 CAMs, and tenascin, while present throughout the CAM, was especially abundant around large, non-migratory mesodermal blood vessels and endodermal cells that were positioned away from the direction of blood vessel migration. These data suggest that while tenascin is required for normal blood vessel migration, high levels of tenascin inhibit migration. The different distributions of CD44 and tenascin in CAMs and the observation that antibodies to either CD44 or tenascin produced similar phenotypes indicate that CD44 and tenascin were not functionally redundant. Mainstream smoke solutions, which produce a phenotype similar to that seen with anti-tenascin and anti-CD44, inhibited expression of CD44 mRNA and increased tenascin mRNA expression. 3-Ethylpyridine, a chemical in cigarette smoke that produced changes in CAM development similar to anti-CD44 and anti-tenascin treatment, also increased tenascin mRNA expression, but did not affect CD44. Together these data show that tenascin and CD44 play critical roles in early growth and vascular development of the CAM and support the idea that 3-ethylpyridine in mainstream smoke impairs CAM growth and vascular development by targeting expression of tenascin. 3-Ethylpyridine is generally regarded as safe and is used in many consumer products including food and tobacco.
Asunto(s)
Membrana Corioalantoides/crecimiento & desarrollo , Receptores de Hialuranos/farmacología , Neovascularización Fisiológica , Tenascina/farmacología , Contaminación por Humo de Tabaco/efectos adversos , Animales , Embrión de Pollo , ADN/biosíntesis , Modelos Animales de Enfermedad , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/inmunología , Fenotipo , Piridinas/efectos adversos , ARN Mensajero/biosíntesis , Tenascina/inmunologíaRESUMEN
A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis of tumor cells. Taken together, these results suggest that the HA binding motif that is present in HA binding proteins may be responsible for the antitumor effect exerted by the members of this family.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Receptores de Hialuranos/farmacología , Melanoma/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Sitios de Unión , División Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Melanoma/genética , Melanoma/patología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Relación Estructura-Actividad , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hyaluronan binding protein 1 (HABP1) is a ubiquitously expressed multifunctional phospho-protein that interacts with a wide range of ligands and is implicated in cell signalling. Recently, we have reported that HABP1 is an endogenous substrate for MAP kinase and upon mitogenic stimulation it is translocated to the nucleus in a MAP kinase-dependent manner (Biochem. Biophys. Res. Commun. 291(4) (2002) 829-837). This prompted us to investigate the role of HABP1 in cell growth or otherwise in low MAP kinase background. We demonstrate that HABP1, when overexpressed in normal rat skin fibroblasts, remained in the cytosol, primarily concentrated around the nuclear periphery. However, HABP1 overexpressing cells showed extensive vacuolation and reduced growth rate, which was corrected by frequent medium replenishment. Further investigation revealed that HABP1 overexpressing cells undergo apoptosis, as detected by TUNEL assay, induction of Bax expression, and FACS analysis, and they failed to enter into the S-phase. Periodic medium supplementation prevented these cells from undergoing apoptotic death. We also demonstrate that upon induction of apoptosis in HeLa cells by cisplatin, HABP1 level is upregulated, indicating a correlation between HABP1 and cell death in a normal cellular environment.
Asunto(s)
Fibroblastos/metabolismo , Receptores de Hialuranos/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras , División Celular/efectos de los fármacos , Línea Celular , Medios de Cultivo/farmacología , Citoplasma/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HeLa , Humanos , Receptores de Hialuranos/farmacología , Proteínas Mitocondriales , Ratas , Estrés Fisiológico/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: In an earlier study, the authors demonstrated that tumor budding was useful for predicting lymph node metastasis in patients with early invasive (T1) colorectal carcinoma. This study was undertaken to clarify the associations between tumor budding, E-cadherin-catenin complex, and CD44 variant 6 abnormalities. METHODS: In 51 T1 colorectal carcinomas, tumor budding (the number of dedifferentiation units at the invasive margin) on hematoxylin and eosin-stained slides was counted under light microscopy. Immunostaining for E-cadherin, alpha-catenin, beta-catenin, and CD44 variant 6 was performed on formalin fixed, paraffin embedded sections. The associations between locoregional failure (lymph node metastasis or local recurrence) and tumor budding and clinicopathologic parameters and immunoreactivity were examined statistically. RESULTS: In univariate analysis, tumor budding and nuclear beta-catenin expression were associated significantly with locoregional failure (P = 0.004, 0.01). Multivariate analysis showed that tumor budding alone was associated significantly with locoregional failure (P = 0.02), and the association between nuclear beta-catenin expression and locoregional failure was marginally significant (P = 0.07). Analysis of variance showed that lymphatic invasion alone was associated significantly with tumor budding (P = 0.02), and there was a significant interaction effect for tumor budding between CD44 variant 6 expression and nuclear beta-catenin expression (P = 0.01). There was a significant correlation between expression patterns of these two molecules and locoregional failure (P = 0.01). CONCLUSIONS: The current results suggest that the up-regulation of CD44 variant 6 through nuclear beta-catenin activation may contribute to the formation of tumor budding, and immunostaining of these two adhesion molecules may be useful in identifying those at high-risk for locoregional failure among patients with T1 colorectal carcinoma.
Asunto(s)
Carcinoma/patología , Diferenciación Celular , Neoplasias Colorrectales/patología , Proteínas del Citoesqueleto/biosíntesis , Glicoproteínas/biosíntesis , Receptores de Hialuranos/biosíntesis , Invasividad Neoplásica/fisiopatología , Transactivadores , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/genética , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/farmacología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/análisis , Glicoproteínas/farmacología , Humanos , Receptores de Hialuranos/análisis , Receptores de Hialuranos/farmacología , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Factores de Riesgo , Regulación hacia Arriba , beta CateninaRESUMEN
The inhibitor for the serine protease activity of plasma hyaluronan binding protein (PHBP) was purified from human plasma by polyethylene glycol (PEG) fractionation, diethylaminoethyl (DEAE)-Sephacel ion-exchange chromatography, Phenyl Toyopearl 650M hydrophobic chromatography, Bio Gel A-0.5 m gel-filtration and hydroxyapatite chromatography. The serine protease activity of PHBP was measured with Boc-Phe-Ser-Arg-methylcoumarine amide (MCA) as the synthetic substrate of PHBP. The results of the amino acid sequence analyses of the purified PHBP inhibitor indicated that it was C1 inhibitor of the serpin family. C1 inhibitor formed a complex with PHBP, suggesting that it is the actual inhibitor of PHBP in human plasma. On the other hand, dextran sulfate and phosphatidylethanolamine enhanced the auto-fragmentation and the serine protease activity of pro-PHBP, but kaolin did not. These results suggested that the serine protease activity of PHBP was regulated in a similar manner to that of factor XII of the coagulation system.
Asunto(s)
Receptores de Hialuranos/aislamiento & purificación , Receptores de Hialuranos/farmacología , Serina Endopeptidasas/sangre , Inhibidores de Serina Proteinasa/aislamiento & purificación , Inhibidores de Serina Proteinasa/farmacología , Aminoácidos/análisis , Western Blotting , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Factor XII/química , Humanos , Serina Endopeptidasas/metabolismoRESUMEN
Proliferation of vascular smooth muscle cells (VSMC) is crucial to the progression of arteriosclerosis. In this study, we examined the role that interactions among endothelin-1 (ET-1), CD44, and hyaluronic acid (HA) play in VSMC proliferation. Co-localization of ET-1, CD44 and HA positive areas, as well as proliferating cell nuclear antigen positive nuclei, were observed in mouse neointima induced by endothelial injury. As found in intimal VSMC, cultured mouse VSMC secreted ET-1. The endothelin-converting enzyme (ECE) inhibitor, phosphoramidon, and endothelin type-A (ETA) receptor antagonist BQ-123 reduced expression of CD44 in VSMC. ET-1 reversed the reduction of CD44 expression by phosphoramidon. Because CD44 is a receptor for HA, we investigated the effects of phosphoramidon, BQ-123 or ET-1 on the mitogenic activity of HA in VSMC. Among the different molecular weights of this polysaccharide, oligosaccharides of HA (oHA) stimulated VSMC proliferation most effectively. Phosphoramidon and BQ-123 inhibited this oHA-induced DNA synthesis in VSMC. ET-1 reversed the suppression of oHA-induced proliferation by phosphoramidon. In conclusion, endogenously secreted ET-1 enhances oHA-stimulated VSMC growth via the ETA receptor in an autocrine manner. Thus it is suggested that the CD44-inducing activity of ET-1 is responsible for its stimulating effect on oHA-dependent growth of VSMC. These findings support the hypothesis that the interactions among ET-1, CD44 and HA promote the progression of arteriosclerosis.