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.
Asunto(s)
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
Thrombospondin-1 (TSP1) binding to cell surface calreticulin (CRT) stimulates the association of CRT with low-density lipoprotein (LDL) receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. A recent study demonstrated that membrane rafts are necessary for TSP1-mediated focal adhesion disassembly, but the molecular role of membrane rafts in mediating TSP1-CRT-LRP1 signaling is unknown. In this study, we investigated the effect of lipid bilayer environments on TSP1 and CRT interactions via atomically detailed molecular dynamics simulations. Results showed that the microscopic structural properties of lipid molecules and mesoscopic mechanical properties and electrostatic potential of the bilayer were significantly different between a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and a raftlike lipid bilayer [a POPC/cholesterol (CHOL) raftlike lipid bilayer or a POPC/CHOL/sphingomyelin (SM) raftlike lipid bilayer], and the difference was enhanced by SM lipids in a raftlike lipid bilayer. These bilayer property differences affect the interactions of CRT with the bilayer, further influencing CRT conformation and TSP1-CRT interactions. A raftlike lipid bilayer stabilized CRT conformation as compared to a POPC bilayer environment. TSP1 binding to CRT resulted in a conformation for the CRT N-domain more "open" than that of the CRT P-domain in a raftlike lipid bilayer environment, which could facilitate binding of CRT to LRP1 to engage downstream signaling. The open conformational changes of CRT by binding to TSP1 in a raftlike lipid bilayer were enhanced by SM lipids in a lipid bilayer. The direct interactions of both the N- and P-domains of CRT with the bilayer contribute to the more open conformation of CRT in the TSP1-CRT complex on a raftlike lipid bilayer as compared to that on a POPC bilayer. The interactions of CRT or the TSP1-CRT complex with the lipid bilayer also caused CHOL molecules and/or lipids to be more coordinated and to aggregate into patchlike regions in the raftlike lipid bilayers. The lipid and CHOL molecule coordination and aggregation could in turn affect the interactions of CRT with the membrane raft, thereby altering TSP1-CRT interactions and CRT conformational changes that potentially regulate its interactions with LRP1. This study provides molecular insights into the role of lipid bilayer environments in TSP1-CRT interactions and in the CRT conformational changes that are predicted to facilitate binding of CRT to LRP1 to engage downstream signaling events.
Asunto(s)
Calreticulina/química , Membrana Dobles de Lípidos/química , Trombospondina 1/química , Sitios de Unión , Humanos , Enlace de Hidrógeno , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de ProteínaRESUMEN
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
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.
Asunto(s)
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
Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation that could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptor to bleomycin-induced lung injury and measured efferocytosis, inflammation, and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days postinjury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days postinjury, 51% increase in lung macrophages 14 days postinjury), and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days postinjury, respectively). Reduced fibrosis in CD36(-/-) mice was associated with lower levels of profibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1, and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 h following zymosan administration in G2A(-/-) mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.
Asunto(s)
Bleomicina/toxicidad , Antígenos CD36/metabolismo , Proteínas de Ciclo Celular/metabolismo , Inflamación/metabolismo , Lesión Pulmonar/inducido químicamente , Receptores Acoplados a Proteínas G/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Antígenos CD36/genética , Proteínas de Ciclo Celular/genética , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inflamación/genética , Lesión Pulmonar/inmunología , Lesión Pulmonar/metabolismo , Ratones , Ratones Noqueados , Receptores Acoplados a Proteínas G/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Transforming growth factor-ß (TGF-ß) is a critical regulator of bone development and remodeling. TGF-ß must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-ß activation and TSP1 control of TGF-ß activation is critical for regulation of TGF-ß activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-ß inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-ß activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-ß. Cultured MSCs express TSP1 and both TSP1 expression and TGF-ß activity decrease during osteoblast differentiation. TSP1 and active TGF-ß block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-ß, since a peptide which blocks TSP1 TGF-ß activation reduced TGF-ß activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-ß neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-ß activity is a critical determinant of osteoblast differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Osteogénesis/fisiología , Trombospondina 1/fisiología , Factor de Crecimiento Transformador beta/agonistas , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/efectos de los fármacos , Péptidos/farmacología , Trombospondina 1/análisis , Trombospondina 1/farmacología , Factor de Crecimiento Transformador beta/análisis , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Human cytomegalovirus (HCMV) infection is associated epidemiologically with poor outcome of renal allografts due to mechanisms which remain largely undefined. Transforming growth factor-ß1 (TGF-ß1), a potent fibrogenic cytokine, is more abundant in rejecting renal allografts that are infected with either HCMV or rat CMV as compared to uninfected, rejecting grafts. TGF-ß1 induces renal fibrosis via epithelial-to-mesenchymal transition (EMT) of renal epithelial cells, a process by which epithelial cells acquire mesenchymal characteristics and a migratory phenotype, and secrete molecules associated with extracellular matrix deposition and remodeling. We report that human renal tubular epithelial cells infected in vitro with HCMV and exposed to TGF-ß1 underwent morphologic and transcriptional changes of EMT, similar to uninfected cells. HCMV infected cells after EMT also activated extracellular latent TGF-ß1 via induction of MMP-2. Renal epithelial cells transiently transfected with only the HCMV IE1 or IE2 open reading frames and stimulated to undergo EMT also induced TGF-ß1 activation associated with MMP-2 production, suggesting a role for these viral gene products in MMP-2 production. Consistent with the function of these immediate early gene products, the antiviral agents ganciclovir and foscarnet did not inhibit TGF-ß1 production after EMT by HCMV infected cells. These results indicate that HCMV infected renal tubular epithelial cells can undergo EMT after exposure to TGF-ß1, similar to uninfected renal epithelial cells, but that HCMV infection by inducing active TGF-ß1 may potentiate renal fibrosis. Our findings provide in vitro evidence for a pathogenic mechanism that could explain the clinical association between HCMV infection, TGF-ß1, and adverse renal allograft outcome.
Asunto(s)
Infecciones por Citomegalovirus/metabolismo , Citomegalovirus/patogenicidad , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/virología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Infecciones por Citomegalovirus/patología , Infecciones por Citomegalovirus/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Transición Epitelial-Mesenquimal , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Túbulos Renales Proximales/citología , ARN Mensajero/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Transforming growth factor-ß (TGF-ß) is key in the pathogenesis of diabetic nephropathy. Thrombospondin 1 (TSP1) expression is increased in diabetes, and TSP1 regulates latent TGF-ß activation in vitro and in diabetic animal models. Herein, we investigate the effect of blockade of TSP1-dependent TGF-ß activation on progression of renal disease in a mouse model of type 1 diabetes (C57BL/6J-Ins2(Akita)) as a targeted treatment for diabetic nephropathy. Akita and control C57BL/6 mice who underwent uninephrectomy received 15 weeks of thrice-weekly i.p. treatment with 3 or 30 mg/kg LSKL peptide, control SLLK peptide, or saline. The effects of systemic LSKL peptide on dermal wound healing was assessed in type 2 diabetic mice (db/db). Proteinuria (urinary albumin level and albumin/creatinine ratio) was significantly improved in Akita mice treated with 30 mg/kg LSKL peptide. LSKL treatment reduced urinary TGF-ß activity and renal phospho-Smad2/3 levels and improved markers of tubulointerstitial injury (fibronectin) and podocytes (nephrin). However, LSKL did not alter glomerulosclerosis or glomerular structure. LSKL did not increase tumor incidence or inflammation or impair diabetic wound healing. These data suggest that selective targeting of excessive TGF-ß activity through blockade of TSP1-dependent TGF-ß activation represents a therapeutic strategy for treating diabetic nephropathy that preserves the homeostatic functions of TGF-ß.
Asunto(s)
Nefropatías Diabéticas/complicaciones , Riñón/patología , Proteinuria/complicaciones , Proteinuria/prevención & control , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Albuminuria/complicaciones , Albuminuria/patología , Albuminuria/orina , Secuencia de Aminoácidos , Animales , Creatinina/orina , Dermis/efectos de los fármacos , Dermis/patología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/patología , Nefropatías Diabéticas/orina , Modelos Animales de Enfermedad , Fibronectinas/metabolismo , Inflamación/complicaciones , Inflamación/patología , Inyecciones Intraperitoneales , Riñón/efectos de los fármacos , Riñón/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/química , Péptidos/farmacología , Péptidos/uso terapéutico , Fosforilación/efectos de los fármacos , Proteinuria/orina , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/orina , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Thrombospondin-1 (TSP-1) is a matricellular extracellular matrix protein. Matricellular proteins are components of the extracellular matrix (ECM) that regulate key cellular functions and impact ECM organization, but which lack direct primary structural roles in the ECM. TSP-1 expression is upregulated in response to injury, hypoxia, growth factor stimulation, inflammation, glucose, and by reactive oxygen species. Relevant to glaucoma, TSP-1 is also a mechanosensitive molecule upregulated by mechanical stretch. TSP-1 expression is increased in ocular remodeling in glaucoma in both the trabecular meshwork and in the optic nerve head. The exact roles of TSP-1 in glaucoma remain to be defined, however. It plays important roles in cell behavior and in ECM remodeling during wound healing, fibrosis, angiogenesis, and in tumorigenesis and metastasis. At the cellular level, TSP-1 can modulate cell adhesion and migration, protease activity, growth factor activity, anoikis resistance, apoptosis, and collagen secretion and matrix assembly and cross-linking. These multiple functions and macromolecular and receptor interactions have been ascribed to specific domains of the TSP-1 molecule. In this review, we will focus on the cell regulatory activities of the TSP-1 N-terminal domain (NTD) sequence that binds to cell surface calreticulin (Calr) and which regulates cell functions via signaling through Calr complexed with LDL receptor related protein 1 (LRP1). We will describe TSP-1 actions mediated through the Calr/LRP1 complex in regulating focal adhesion disassembly and cytoskeletal reorganization, cell motility, anoikis resistance, and induction of collagen secretion and matrix deposition. Finally, we will consider the relevance of these TSP-1 functions to the pathologic remodeling of the ECM in glaucoma.
RESUMEN
Multiple myeloma is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib is one of the most effective first-line chemotherapeutic drugs for multiple myeloma; however, 15% to 20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that multiple myeloma cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OB), and that this OB-Runx2 deficiency induces a cytokine-rich and immunosuppressive microenvironment in the BM. In the current study, we assessed the impact of OB-Runx2 deficiency on the outcome of bortezomib treatment using OB-Runx2+/+ and OB-Runx2-/- mouse models of multiple myeloma. In vitro and in vivo experiments revealed that OB-Runx2 deficiency induces multiple myeloma cell resistance to bortezomib via the upregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs), downregulation of cytotoxic T cells, and activation of TGFß1 in the BM. In multiple myeloma tumor-bearing OB-Runx2-/- mice, treatment with SRI31277, an antagonist of thrombospondin-1 (TSP-1)-mediated TGFß1 activation, reversed the BM immunosuppression and significantly reduced tumor burden. Furthermore, treatment with SRI31277 combined with bortezomib alleviated multiple myeloma cell resistance to bortezomib-induced apoptosis caused by OB-Runx2 deficiency in cocultured cells and produced a synergistic effect on tumor burden in OB-Runx2-/- mice. Depletion of MDSCs by 5-fluorouracil or gemcitabine similarly reversed the immunosuppressive effects and bortezomib resistance induced by OB-Runx2 deficiency in tumor-bearing mice, indicating the importance of the immune environment for drug resistance and suggesting new strategies to overcome bortezomib resistance in the treatment of multiple myeloma.
Asunto(s)
Médula Ósea/metabolismo , Bortezomib/uso terapéutico , Subunidad alfa 1 del Factor de Unión al Sitio Principal/deficiencia , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Osteoblastos/metabolismo , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Bortezomib/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Mieloma Múltiple/patologíaRESUMEN
Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface signals focal adhesion disassembly, leading to the intermediate adhesive phenotype, cell migration, anoikis resistance, and collagen stimulation. Residues Lys 24 and 32 in TSP1 and amino acids 24-26 and 32-34 in CRT have been shown through biochemical and cell-based approaches to be critical for TSP1-CRT binding and signaling. This study investigated the molecular and structural basis for these key TSP1 and CRT residues in TSP1-CRT binding. On the basis of a validated TSP1-CRT complex structure, we adopted steered molecular dynamics simulations to determine the effect of mutation of these key residues on TSP1-CRT binding and validated the simulation results with experimental observations. We further performed 30 ns molecular dynamics simulations for wild-type TSP1, CRT, K24A/K32A mutant TSP1, and mutant CRT (residues 24-26 and 32-34 mutated to Ala) and studied the conformational and structural changes in TSP1 and CRT as the result of mutation of these critical residues. Results showed that mutation of residues 24 and 32 to Ala in TSP1 and of amino acids 24-26 and 32-34 to Ala in CRT results in a shortened ß-strand in the binding site, decreased hydrogen bond occupancy for ß-strand pairs that are located within or near the binding site, increased conformational flexibility of the binding site, a changed degree of dynamically correlated motion between the residues in the binding site and the other residues in protein, and a changed degree of overall correlated motion between the residues in the protein. These changes could directly contribute to the loss or weakened binding between TSP1 and CRT and the resultant effects on TSP1-CRT binding-induced cellular activities. Results from this study provide a molecular and structural insight into the role of these critical residues of TSP1 and CRT in TSP1-CRT binding.
Asunto(s)
Calreticulina/química , Calreticulina/metabolismo , Simulación de Dinámica Molecular , Trombospondina 1/química , Trombospondina 1/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Calreticulina/genética , Metabolismo Energético/genética , Entropía , Adhesiones Focales/genética , Humanos , Lisina/genética , Datos de Secuencia Molecular , Mutación , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Transducción de Señal/genética , Electricidad Estática , Relación Estructura-Actividad , Trombospondina 1/genéticaRESUMEN
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
Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.
Asunto(s)
Diferenciación Celular , Fibroblastos/metabolismo , Pulmón/citología , Mioblastos/citología , Receptores de Vitronectina/metabolismo , Antígenos Thy-1/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Secuencias de Aminoácidos , Animales , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Sistema Libre de Células , Endotelina-1/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Proteínas Inmovilizadas/metabolismo , Ratones , Contracción Muscular/efectos de los fármacos , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Oxazoles/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Solubilidad/efectos de los fármacos , Antígenos Thy-1/química , Antígenos Thy-1/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
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.
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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
Calreticulin (CRT), when localized to the endoplasmic reticulum (ER), has important functions in directing proper conformation of proteins and glycoproteins, as well as in homeostatic control of cytosolic and ER calcium levels. There is also steadily accumulating evidence for diverse roles for CRT localized outside the ER, including data suggesting important roles for CRT localized to the outer cell surface of a variety of cell types, in the cytosol, and in the extracellular matrix (ECM). Furthermore, the addition of exogenous CRT rescues numerous CRT-driven functions, such as adhesion, migration, phagocytosis, and immunoregulatory functions of CRT-null cells. Recent studies show that topically applied CRT has diverse and profound biological effects that enhance cutaneous wound healing in animal models. This evidence for extracellular bioactivities of CRT has provided new insights into this classically ER-resident protein, despite a lack of knowledge of how CRT exits from the ER to the cell surface or how it is released into the extracellular milieu. Nonetheless, it has become clear that CRT is a multicompartmental protein that regulates a wide array of cellular responses important in physiological and pathological processes, such as wound healing, the immune response, fibrosis, and cancer.-Gold, L. I., Eggleton, P., Sweetwyne, M. T., Van Duyn, L. B., Greives, M. R., Naylor, S.-M., Michalak, M., Murphy-Ullrich, J. E. Calreticulin: non-endoplamic reticulum functions in physiology and disease.
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Calreticulina/fisiología , Retículo Endoplásmico/metabolismo , Animales , Calreticulina/metabolismo , Calreticulina/farmacología , Movimiento Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Fagocitosis/efectos de los fármacosRESUMEN
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
Diabetes is a major predictor of in-stent restenosis, which is associated with fibroproliferative remodeling of the vascular wall due to increased transforming growth factor-beta (TGF-beta) action. It is well established that thrombospondin1 (TSP1) is a major regulator of TGF-beta activation in renal and cardiac complications of diabetes. However, the role of the TSP1-TGF-beta pathway in macrovascular diabetic complications, including restenosis, has not been addressed. In mesangial cells, high glucose concentrations depress protein kinase G (PKG) activity, but not PKG-I protein, thereby downregulating transcriptional repression of TSP1. Previously, we showed that high glucose downregulates PKG-I protein expression by vascular smooth muscle cells (VSMCs) through altered NADPH oxidase signaling. In the present study, we investigated whether high glucose regulation of PKG protein and activity in VSMCs similarly regulates TSP1 expression and downstream TGF-beta activity. These studies showed that high glucose stimulates both TSP1 expression and TGF-beta bioactivity in primary murine aortic smooth muscle cells (VSMCs). TSP1 is responsible for the increased TGF-beta bioactivity under high glucose conditions, because treatment with anti-TSP1 antibody, small interfering RNA-TSP1, or an inhibitory peptide blocked glucose-mediated increases in TGF-beta activity and extracellular matrix protein (fibronectin) expression. Overexpression of constitutively active PKG, but not the PKG-I protein, inhibited glucose-induced TSP1 expression and TGF-beta bioactivity, suggesting that PKG protein expression is insufficient to regulate TSP1 expression. Together, these data establish that glucose-mediated downregulation of PKG levels stimulates TSP1 expression and enhances TGF-beta activity and matrix protein expression, which can contribute to vascular remodeling in diabetes.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Glucosa/farmacología , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Diabetes Mellitus , Regulación hacia Abajo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/genética , Regulación hacia ArribaRESUMEN
Thrombospondin-1 (TSP1) binding to calreticulin (CRT) on the cell surface stimulates association of CRT with LDL receptor-related protein (LRP1) to signal focal adhesion disassembly and engagement of cellular activities. The structural basis for this phenomenon is unknown. We studied the binding thermodynamics of the TSP1-CRT complex and the conformational changes in CRT induced by binding to TSP1 with combined binding free energy analysis, molecular dynamics simulation, and anisotropic network model restrained molecular dynamics simulation. Results showed that mutations of Lys 24 and Lys 32 in TSP1 to Ala and of amino acids 24-26 and 32-34 in CRT to Ala significantly weakened the binding of TSP1 and CRT, which is consistent with experimental results. Upon validation of the calculated binding affinity changes of the TSP1-CRT complex by mutations in key residues in TSP1 and CRT with the experimental results, we performed conformational analyses to understand the role of TSP1 binding to CRT in the induction of conformational changes in CRT. Conformational analyses showed that TSP1 binding to CRT resulted in a more "open" conformation and a significant rotational change for the CRT N-domain with respect to the CRT P-domain, which could expose the potential binding site(s) in CRT for binding to LRP1 to signal focal adhesion disassembly. Results offer structural insight into the role of TSP1 binding to CRT in CRT-induced focal adhesion disassembly.