RESUMEN
TGF-ß has been a target of interest for the treatment of fibrotic diseases and certain cancers. Approaches to target TGF-ß include antagonists of the active ligand or TGF-ß receptor kinase activity. These approaches have failed in clinical trials due to a lack of effectiveness and a limited therapeutic window. In this context, newer and more selective approaches to target TGF-ß are needed. We previously reported that the matricellular protein, thrombospondin 1, activates the latent TGF-ß complex and that antagonism of this pathway using tri/tetrapeptides in various animal models reduces fibrosis. The tripeptide, SRI-31277 (1), is effective in vivo but has a short plasma half life (0.2 h). Herein we describe the design and synthesis SRI-31277 analogs, specifically smaller peptides that retain potency and have improved bioavailability. We identified SRI-35241 (36) with a single chiral center, which blocks TGF-ß activation (pIC50 = 8.12 nM) and has a plasma half life of 1.8 h (iv).
RESUMEN
Previously, our lab showed that the endoplasmic reticulum (ER) and calcium regulatory protein, calreticulin (CRT), is important for collagen transcription, secretion, and assembly into the extracellular matrix (ECM) and that ER CRT is critical for TGF-ß stimulation of type I collagen transcription through stimulation of ER calcium release and NFAT activation. Diabetes is the leading cause of end stage renal disease. TGF-ß is a key factor in the pathogenesis of diabetic nephropathy. However, the role of calreticulin (Calr) in fibrosis of diabetic nephropathy has not been investigated. In current work, we used both in vitro and in vivo approaches to assess the role of ER CRT in TGF-ß and glucose stimulated ECM production by renal tubule cells and in diabetic mice. Knockdown of CALR by siRNA in a human proximal tubular cell line (HK-2) showed reduced induction of soluble collagen when stimulated by TGF-ß or high glucose as compared to control cells, as well as a reduction in fibronectin and collagen IV transcript levels. CRT protein is increased in kidneys of mice made diabetic with streptozotocin and subjected to uninephrectomy to accelerate renal tubular injury as compared to controls. We used renal-targeted ultrasound delivery of Cre-recombinase plasmid to knockdown specifically CRT expression in the remaining kidney of uninephrectomized Calr fl/fl mice with streptozotocin-induced diabetes. This approach reduced CRT expression in the kidney, primarily in the tubular epithelium, by 30-55%, which persisted over the course of the studies. Renal function as measured by the urinary albumin/creatinine ratio was improved in the mice with knockdown of CRT as compared to diabetic mice injected with saline or subjected to ultrasound and injected with control GFP plasmid. PAS staining of kidneys and immunohistochemical analyses of collagen types I and IV show reduced glomerular and tubulointerstitial fibrosis. Renal sections from diabetic mice with CRT knockdown showed reduced nuclear NFAT in renal tubules and treatment of diabetic mice with 11R-VIVIT, an NFAT inhibitor, reduced proteinuria and renal fibrosis. These studies identify ER CRT as an important regulator of TGF-ß stimulated ECM production in the diabetic kidney, potentially through regulation of NFAT-dependent ECM transcription.
RESUMEN
Transforming growth factor (TGF)-ß supports multiple myeloma progression and associated osteolytic bone disease. Conversion of latent TGF-ß to its biologically active form is a major regulatory node controlling its activity. Thrombospondin1 (TSP1) binds and activates TGF-ß. TSP1 is increased in myeloma, and TSP1-TGF-ß activation inhibits osteoblast differentiation. We hypothesized that TSP1 regulates TGF-ß activity in myeloma and that antagonism of the TSP1-TGF-ß axis inhibits myeloma progression. Antagonists (LSKL peptide, SRI31277) derived from the LSKL sequence of latent TGF-ß that block TSP1-TGF-ß activation were used to determine the role of the TSP1-TGF-ß pathway in mouse models of myeloma. TSP1 binds to human myeloma cells and activates TGF-ß produced by cultured human and mouse myeloma cell lines. Antagonists delivered via osmotic pump in an intratibial severe combined immunodeficiency CAG myeloma model or in a systemic severe combined immunodeficiency CAG-heparanase model of aggressive myeloma reduced TGF-ß signaling (phospho-Smad 2) in bone sections, tumor burden, mouse IL-6, and osteoclasts, increased osteoblast number, and inhibited bone destruction as measured by microcomputed tomography. SRI31277 reduced tumor burden in the immune competent 5TGM1 myeloma model. SRI31277 was as effective as dexamethasone or bortezomib, and SRI31277 combined with bortezomib showed greater tumor reduction than either agent alone. These studies validate TSP1-regulated TGF-ß activation as a therapeutic strategy for targeted inhibition of TGF-ß in myeloma.
Asunto(s)
Mieloma Múltiple/tratamiento farmacológico , Osteólisis/tratamiento farmacológico , Péptidos/farmacología , Trombospondina 1/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Bortezomib/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mieloma Múltiple/patología , Osteogénesis/efectos de los fármacos , Osteólisis/patología , Péptidos/uso terapéutico , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND/AIMS: The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-ß-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-ß regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition. METHODS: Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery. RESULTS: In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-ß. TGF-ß stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. CONCLUSIONS: This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation.
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Calbindina 2/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Colágeno Tipo I/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima , Animales , Calbindina 2/deficiencia , Calbindina 2/genética , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Modelos Animales de Enfermedad , Ligadura , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Músculo Liso Vascular/cirugía , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Transducción de Señal , Factores de Tiempo , Transfección , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
Type 2 diabetes significantly elevates the risk of cardiovascular disease. This can be largely attributed to the adverse effects of hyperglycemic conditions on normal endothelial cell (EC) function. ECs in both large and small vessels are influenced by hyperglycemic conditions, which increase susceptibility to EC dysfunction and atherosclerotic lesion formation. Fluid shear stress and flow patterns play an essential role in atherogenesis: lesions form only at locations where fluid flow behavior can be classified as "disturbed flow" (i.e., low shear stress recirculation and/or retrograde flow). Since regions of disturbed flow are the focal points of atherosclerotic cardiovascular disease, we hypothesized that the combinatorial effects of high glucose and disturbed flow conditions elicit significantly different responses from ECs than high glucose alone. To validate our hypothesis, we used our endothelial cell culture model (ECCM) to establish vascular niches associated with "normal" and "disturbed" flow conditions typically seen in vivo along with physiological pressure and stretch. We subjected human aortic endothelial cells (HAECs) to hyperglycemic conditions under both "normal" and "disturbed" flow. Our results confirm significant and quantifiable differences in phenotypic and functional markers between cells cultured under conditions of "normal" and "disturbed flow" under hyperglycemic conditions suggesting that elevated glucose in conjunction with "disturbed" flow conditions results in significantly higher level of EC dysfunction. The ECCM can therefore be used as a physiologically relevant model to study early stage hyperglycemia induced atherosclerosis for basic research, drug discovery, and screening and toxicity studies.
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Arterias/fisiopatología , Aterosclerosis/fisiopatología , Hiperglucemia/fisiopatología , Modelos Biológicos , Western Blotting , Células Cultivadas , Glucosa/administración & dosificación , Humanos , Técnicas In Vitro , Microscopía Fluorescente , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca(2+) signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-ß. These studies show that CRT-/- mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-ß stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-ß. The lack of CRT does not impact canonical TGF-ß signaling as TGF-ß was able to stimulate Smad reporter activity in CRT-/- MEFs. CRT regulation of TGF-ß-stimulated Ca(2+) signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca(2+) levels in response to TGF-ß. NFAT activity is required for ECM stimulation by TGF-ß. In CRT-/- MEFs, TGF-ß stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-ß stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-ß stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca(2+)-dependent pathways as a critical molecular link between ER stress and TGF-ß fibrotic signaling.
Asunto(s)
Calreticulina/metabolismo , Retículo Endoplásmico/metabolismo , Matriz Extracelular/metabolismo , Factores de Transcripción NFATC/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Calcio/metabolismo , Calreticulina/genética , Línea Celular , Colágeno/metabolismo , Fibroblastos/metabolismo , Humanos , Pulmón/metabolismo , Ratones , Ratas , Transducción de Señal , Transcripción GenéticaRESUMEN
Thrombospondin-1 (TSP1) is a multidomain protein that contains epidermal growth factor (EGF)-like repeats that indirectly activate the EGF receptor (EGFR) and selected downstream signaling pathways. In these studies, we show that TSP1 opens the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls) in a dose-, time-, and protein tyrosine kinase (PTK)-dependent manner. TSP1 increased tyrosine phosphorylation of proteins enriched to intercellular boundaries including the zonula adherens (ZA) proteins, vascular endothelial-cadherin, γ-catenin, and p120 catenin. In HMVEC-Ls, EGFR and ErbB2 are expressed at low levels, and both heterodimerize and tyrosine autophosphorylate in response to TSP1. Prior EGFR-selective PTK inhibition with AG1478 or ErbB2-selective PTK inhibition with AG825 protected against TSP1-induced tyrosine phosphorylation of ZA proteins and barrier disruption. Preincubation of HMVEC-Ls with an EGFR ectodomain-blocking antibody also prevented TSP1-induced opening of the paracellular pathway. Therefore, in HMVEC-Ls, TSP1 increases tyrosine phosphorylation of ZA proteins and opens the paracellular pathway, in part, through EGFR/ErbB2 activation. Surprisingly, recombinant TSP1 EGF-like repeats 1-3 and the high-affinity EGFR ligands, EGF, TGF-α, and amphiregulin, each failed to increase paracellular permeability. However, HMVEC-Ls in which EGFR was overexpressed became responsive to the EGF-like repeats of TSP1 as well as to EGF. These studies indicate that TSP1 disrupts the endothelial barrier through EGFR/ErbB2 activation although additional signals are necessary in cells with low receptor expression.
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Células Endoteliales/enzimología , Receptores ErbB/metabolismo , Pulmón/irrigación sanguínea , Microvasos/citología , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Trombospondina 1/farmacología , Uniones Adherentes/efectos de los fármacos , Uniones Adherentes/metabolismo , Radioisótopos de Carbono , Células Endoteliales/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Receptores ErbB/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Multimerización de Proteína/efectos de los fármacos , Receptor ErbB-2/genética , Secuencias Repetitivas de Aminoácido , Albúmina Sérica Bovina/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Trombospondina 1/química , Factores de TiempoRESUMEN
Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling. Surgical sponges impregnated with a plasmid encoding the secreted calreticulin-binding sequence [NTD (1-35)-EGFP] or a control sequence [mod NTD (1-35)-EGFP] tagged with enhanced green fluorescent protein were implanted subcutaneously in mice. Sponges expressing NTD (1-35)-EFGP formed a highly organized capsule despite no differences in cellular composition, suggesting stimulation of collagen deposition by the calreticulin-binding sequence of TSP1. TSP1, recombinant NTD, or a peptide of the TSP1 calreticulin-binding sequence (hep I) increased both collagen expression and matrix deposition by fibroblasts in vitro. TSP1 stimulation of collagen was inhibited by a peptide that blocks TSP1 binding to calreticulin, demonstrating the requirement for cell surface calreticulin. Collagen stimulation was independent of TGF-ß activity and Smad phosphorylation but was blocked by an Akt inhibitor, suggesting that signaling through the Akt pathway is important for regulation of collagen through TSP1 binding to calreticulin. These studies identify a novel function for the NTD of TSP1 as a mediator of collagen expression and deposition during tissue remodeling.
Asunto(s)
Calreticulina/metabolismo , Colágeno/metabolismo , Embrión de Mamíferos/citología , Endotelio Vascular/citología , Fibroblastos/citología , Trombospondina 1/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Apoptosis , Western Blotting , Bovinos , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos/metabolismo , Endotelio Vascular/metabolismo , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Proteína Smad2/genética , Proteína Smad2/metabolismo , Trombospondina 1/genética , Andamios del Tejido , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signalling. Electrophilic lipids affect redox signalling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In the present study, we investigate the effect of the electrophilic prostaglandin 15d-PGJ2 (15-deoxy-Delta12,14-prostaglandin J2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal-adhesion disassembly and cause extensive F-actin (filamentous actin) reorganization at low concentrations (0.03-0.3 microM). Importantly, these effects seem to be independent of PPARgamma (peroxisome-proliferator-activated receptor gamma) and modification of actin or Keap1 (Kelch-like ECH-associated protein 1), which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal-adhesion disassembly. Taken together, the results of the present study suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity.
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Antineoplásicos/farmacología , Prostaglandina D2/análogos & derivados , Actinas/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas del Citoesqueleto/fisiología , Quinasa 1 de Adhesión Focal/fisiología , Adhesiones Focales/efectos de los fármacos , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Metástasis de la Neoplasia/tratamiento farmacológico , Prostaglandina D2/farmacología , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Calreticulin (CRT), a chaperone and Ca(2+) regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts deficient in CRT (CRT(-/-) MEFs) have reduced transcript levels of fibrillar collagen I and III and less soluble collagen as compared with wild type MEFs. Correspondingly, fibroblasts engineered to overexpress CRT have increased collagen type I transcript and protein. Collagen expression appears to be regulated by endoplasmic reticulum (ER) calcium levels and intracellular CRT, because thapsigargin treatment reduced collagen expression, whereas addition of exogenous recombinant CRT had no effect. CRT(-/-) MEFs exhibited increased ER retention of collagen, and collagen and CRT were co-immunoprecipitated from isolated cell lysates, suggesting that CRT is important for trafficking of collagen through the ER. CRT(-/-) MEFs also have reduced type I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis.
Asunto(s)
Calreticulina/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Colágenos Fibrilares/metabolismo , Animales , Ácido Ascórbico/farmacología , Calcio/metabolismo , Calreticulina/genética , Fraccionamiento Celular , Células Cultivadas , Colágeno Tipo I/genética , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/metabolismo , Colágenos Fibrilares/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Humanos , Ratones , Ratones Noqueados , Transducción de Señal/fisiología , Fracciones Subcelulares/metabolismo , Tapsigargina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND/AIMS: Despite advances in stent design, in-stent restenosis (ISR) remains a significant clinical problem. All implant metals exhibit corrosion, which results in release of metal ions. Stainless steel (SS), a metal alloy widely used in stents, releases ions to the vessel wall and induces reactive oxygen species, inflammation and fibroproliferative responses. The molecular mechanisms are unknown. TGF-beta is known to be involved in the fibroproliferative responses of vascular smooth muscle cells (VSMCs) in restenosis, and TGF-beta antagonists attenuate ISR. We hypothesized that SS ions induce the latent TGF-beta activator, thrombospondin-1 (TSP1), through altered oxidative signaling to stimulate increased TGF-beta activation and VSMC phenotype change. METHODS: VSMCs were treated with SS metal ion cocktails, and morphology, TSP1, extracellular matrix production, desmin and TGF-beta activity were assessed by immunoblotting. RESULTS: SS ions stimulate the synthetic phenotype, increased TGF-beta activity, TSP1, increased extracellular matrix and downregulation of desmin in VSMCs. Furthermore, SS ions increase hydrogen peroxide and decrease cGMP-dependent protein kinase (PKG) signaling, a known repressor of TSP1 transcription. Catalase blocks SS ion attenuation of PKG signaling and increased TSP1 expression. CONCLUSIONS: These data suggest that ions from stent alloy corrosion contribute to ISR through stimulation of TSP1-dependent TGF-beta activation.
Asunto(s)
Angioplastia Coronaria con Balón/instrumentación , Reestenosis Coronaria/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Acero Inoxidable , Stents , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Angioplastia Coronaria con Balón/efectos adversos , Animales , Aorta/metabolismo , Aorta/patología , Catalasa/metabolismo , Forma de la Célula , Células Cultivadas , Reestenosis Coronaria/etiología , Reestenosis Coronaria/patología , Vasos Coronarios/metabolismo , Vasos Coronarios/patología , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Desmina/metabolismo , Matriz Extracelular/metabolismo , Femenino , Humanos , Peróxido de Hidrógeno/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Estrés Oxidativo , Fenotipo , Diseño de Prótesis , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Thrombospondin (TSP) 1 is a trimeric multidomain protein that contains motifs that recognize distinct host cell receptors coupled to multiple signaling pathways. Selected TSP1-induced cellular responses are tyrosine kinase-dependent, and TSP1 contains epidermal growth factor (EGF)-like repeats. Specific receptor interactions or functions for the EGF-like repeats have not been identified. We asked whether one or more biological responses to TSP1 might be explained through EGF receptor (EGFR) activation. In A431 cells, TSP1 increased autophosphorylation of Tyr-1068 of EGFR in a dose- and time-dependent manner. The ability of TSP1 to activate EGFR was replicated by the tandem EGF-like repeats as a recombinant protein. The three EGF-like repeats alone produced a high level of Tyr-1068 phosphorylation. EGF-like repeats from TSP2 and TSP4 also activated EGFR. Tyr-1068 phosphorylation was less when individual EGF-like repeats were tested or flanking sequences were added to the three EGF-like repeats. TSP1 and its EGF-like repeats also increased phosphorylation of EGFR Tyr-845, Tyr-992, Tyr-1045, Tyr-1086, and Tyr-1173, activated phospholipase Cgamma, and increased cell migration. No evidence was found for binding of the EGF-like repeats to EGFR. Instead, EGFR activation in response to TSP1 or its EGF-like repeats required matrix metalloprotease activity, including activity of matrix metalloprotease 9. Access to the ligand-binding portion of the EGFR ectodomain was also required. These findings suggest release of an endogenous EGFR ligand in response to ligation of a second unknown receptor by the TSPs.
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Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Fosfolipasa C gamma/metabolismo , Trombospondinas/farmacología , Secuencias de Aminoácidos/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Células Epiteliales/citología , Receptores ErbB/genética , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Trombospondinas/metabolismo , Factores de TiempoRESUMEN
Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N-terminal domain of TSP1 binds to the calreticulin-LRP1 receptor co-complex to signal down-regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin-LRP1 activates cell survival signals such as PI3-kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion-independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N-terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin-LRP1 binding site [amino acids 17-35 (hep I)] in the N-terminal domain promotes fibroblast survival under anchorage-independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1-mediated anchorage-independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage-independent survival. These data define a novel role for TSP1 signaling through the calreticulin/LRP1 co-complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.-Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy-Ullrich, J. E. Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.
Asunto(s)
Anoicis/fisiología , Calreticulina/agonistas , Fibroblastos/metabolismo , Péptidos/farmacología , Receptores de LDL/agonistas , Transducción de Señal/efectos de los fármacos , Trombospondina 1/farmacología , Proteínas Supresoras de Tumor/agonistas , Animales , Sitios de Unión/fisiología , Calreticulina/metabolismo , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fibroblastos/citología , Humanos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Ratones , Ratones Noqueados , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Estructura Terciaria de Proteína/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de LDL/metabolismo , Transducción de Señal/fisiología , Trombospondina 1/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Choque Térmico HSP20/metabolismo , Relajación Muscular/fisiología , Músculo Liso/fisiología , Fenómenos Fisiológicos Respiratorios , Animales , Bovinos , Colforsina/farmacología , Isoproterenol/farmacología , Cinética , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/enzimología , Fosfopéptidos/metabolismo , Serotonina/farmacologíaRESUMEN
In diabetes and hypertension, the induction of increased transforming growth factor-beta (TGF-beta) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-beta activator thrombospondin-1 (TSP1). Because activation of latent TGF-beta is a major means of regulating TGF-beta, we addressed the role of TSP1-mediated TGF-beta activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-beta activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-beta activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-beta activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-beta activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-beta activation as a potential antifibrotic therapeutic strategy are warranted.
Asunto(s)
Angiotensina II/sangre , Cardiomiopatías , Miocardio/patología , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta , Animales , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/prevención & control , Diabetes Mellitus Experimental , Fibrosis/patología , Fibrosis/prevención & control , Hemodinámica , Humanos , Masculino , Miocardio/citología , Miocardio/metabolismo , Péptidos/metabolismo , Ratas , Ratas Endogámicas WKY , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.
Asunto(s)
Citoesqueleto/metabolismo , Proteínas de Choque Térmico/metabolismo , Fosfopéptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas 14-3-3/metabolismo , Células 3T3/química , Células 3T3/metabolismo , Factores Despolimerizantes de la Actina , Actinina/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión , Línea Celular , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Proteínas del Choque Térmico HSP20 , Proteínas de Choque Térmico/química , Ratones , Proteínas de Microfilamentos/metabolismo , Paxillin , Péptidos/química , Péptidos/metabolismo , Fosfopéptidos/síntesis química , Fosfoproteínas/química , Fosforilación , Fosfoserina/metabolismo , Estructura Terciaria de Proteína , Vinculina/metabolismoRESUMEN
The hep I peptide of thrombospondin-1 is known to induce the disassembly of focal adhesions, a critical step in regulating cellular adhesive changes needed for cell motility. Fibroblasts that are heterogeneous with respect to the surface expression of Thy-1 differ markedly in morphology, cytoskeletal organization, and migration, suggesting differential regulation of focal adhesion dynamics. Here we demonstrate that disassembly of focal adhesions mediated by both full-length thrombospondin-1 and the hep I peptide in fibroblasts requires the expression of Thy-1, although it does not appear to function as a stable member of the hep I receptor complex. Consistent with a known function of Thy-1 in regulating lipid raft-associated signaling, intact lipid rafts are necessary for hep I-mediated focal adhesion disassembly. Furthermore, we establish Src family kinase (SFK) activation as a novel component required for hep I-induced signaling leading to focal adhesion disassembly. hep I induces transient phosphorylation of SFKs in Thy-1-expressing fibroblasts only. Therefore, we conclude that Thy-1 surface expression is required for thrombospondin-1-induced focal adhesion disassembly in fibroblasts through an SFK-dependent mechanism. This represents a novel role for Thy-1 in the regulation of fibroblast-matrix interactions critical to tissue homeostasis and remodeling.