RESUMO
UVBR-induced photolesions in genomic DNA of keratinocytes impair cellular functions and potentially determine the cell fate post-irradiation. The ability of insulin-like growth factor-I (IGF-I) to rescue epidermal keratinocytes after photodamage via apoptosis prevention and photolesion removal was recently demonstrated using in vitro two-dimensional and three-dimensional skin models. Given the limited knowledge of specific signalling cascades contributing to post-UVBR IGF-I effects, we used inhibitors to investigate the impact of blockade of various signalling mediators on IGF-I photoprotection. IGF-I treatment, in the presence of signalling inhibitors, particularly TDRL-505, which targets replication protein A (RPA), impaired activation of IGF-1R downstream signalling, diminished cyclobutane pyrimidine dimer removal, arrested growth, reduced cell survival and increased apoptosis. Further, the transient partial knockdown of RPA was found to abrogate IGF-I-mediated responses in keratinocytes, ultimately affecting photoprotection and, thereby, establishing that RPA is required for IGF-I function. Our findings thus elucidate the importance of RPA in linking the damage response activation, cell cycle regulation, repair and survival pathways, separately initiated by IGF-I upon UVBR-induced damage. This information is potentially imperative for the development of effective sunburn and photodamage repair strategies. This article has an associated First Person interview with the first author of the paper.
Assuntos
Fator de Crescimento Insulin-Like I , Proteína de Replicação A , Apoptose , Dano ao DNA , Queratinócitos , Transdução de Sinais , Raios Ultravioleta/efeitos adversosRESUMO
Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane-type-1 matrix metalloproteinase (MT1-MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1-MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1-MMP. Upon lipopolysaccharide (LPS) activation, MT1-MMP synthesis dramatically increases 10-fold at the surface by 15 hours. MT1-MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R-SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q-SNARE complex Stx4/SNAP23 to regulate MT1-MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1-MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1-MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.
Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Ativação de Macrófagos , Metaloproteinase 14 da Matriz/metabolismo , Animais , Movimento Celular , Complexo de Golgi/metabolismo , Camundongos , Transporte Proteico , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Células RAW 264.7RESUMO
Ultraviolet radiation (UVR), in particular the UVB spectrum, is a risk factor for skin cancer development. The generation and accumulation of UVB-induced genetic mutations are fundamental premalignant events. Keratinocyte interactions between other cutaneous cell populations and the surrounding microenvironment determine cell fate and acute photoresponses. In this study, the importance of the insulin-like growth factor (IGF) system, in particular the insulin-like growth factor-I (IGF-I), on influencing key processes in the keratinocyte acute photoresponse was investigated. Exogenous IGF-I and other growth factors present in dermal fibroblast-conditioned media (CM) were found to significantly enhance keratinocyte survival following UVB irradiation in vitro. This pretreatment was also shown to cause a shift in the expression levels of various DNA damage response proteins. Consequently, this was associated with accelerated rates of UVB-induced cyclobutane pyrimidine dimer removal in these samples. Finally, activation of the IGF system influenced cell cycle progression in UVB-irradiated keratinocytes. Taken together, these results highlight the importance of the IGF signalling network in initiating the repair of potentially mutagenic DNA damage in human keratinocytes. The dysregulation of these processes may therefore have significant implications in the aetiology of skin cancers and other cutaneous diseases.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Queratinócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Quinase 1 do Ponto de Checagem , Meios de Cultivo Condicionados/farmacologia , Fibroblastos , Histonas/metabolismo , Humanos , Queratinócitos/efeitos da radiação , Proteínas Quinases/metabolismo , Dímeros de Pirimidina/metabolismo , Transdução de Sinais , Raios UltravioletaRESUMO
The majority of the population experience successful wound-healing outcomes; however, 1-3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard-to-heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24-hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound-healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound-healing purposes.
Assuntos
Alicerces Teciduais , Vitronectina/uso terapêutico , Ferimentos Penetrantes/terapia , Animais , Técnicas de Cultura de Células , Derme/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Suínos , Vitronectina/farmacocinética , CicatrizaçãoRESUMO
Numerous studies have reported associations between IGF-I and other extra cellular matrix (ECM) proteins, including fibronectin (FN), integrins, IGF-binding proteins (IGFBPs) and through IGFBPs, with vitronectin (VN). Nevertheless, the precise nature and mechanisms of these interactions are still being characterised. In this paper, we discuss transglutaminases (TGases) as a constituent of the ECM and provide evidence for the first time that IGF-I is a lysine (K)-donor substrate to TGases. When IGF-I was incubated with an alpha-2 plasmin inhibitor-derived Q peptide in the presence of tissue transglutaminase (TG2), an IGF-I:Q peptide cross-linked species was detected using Western immunoblotting and confirmed by mass spectrometry. Similar findings were observed in the presence of Factor XIIIa (FXIIIa) TGase. To identify the precise location of this K-donor TGase site/s on IGF-I, all the three IGF-I K-sites, individually and collectively (K27, K65 and K68), were substituted to arginine (R) using site-directed mutagenesis. Incubation of these KâR IGF-I analogues with Q peptide in the presence of TG2 or FXIIIa resulted in the absence of cross-linking in IGF-I analogues bearing arginine substitution at site 68. This established that K68 within the IGF-I D-domain was the principal K-donor site to TGases. We further annotated the functional significance of these KâR IGF-I analogues on IGF-I mediated actions. IGF-I analogues with KâR substitution within the D-domain at K65 and K68 hindered migration of MCF-7 breast carcinoma cells and correspondingly reduced PI3-K/AKT activation. Therefore, this study also provides first insights into a possible functional role of the previously uncharacterised IGF-I D-domain.
Assuntos
Fator de Crescimento Insulin-Like I/química , Fator de Crescimento Insulin-Like I/metabolismo , Lisina/metabolismo , Transglutaminases/metabolismo , Sequência de Aminoácidos , Movimento Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator XIIIa/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Cinética , Células MCF-7 , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Vitronectina/farmacologiaRESUMO
Presently, global rates of skin cancers induced by ultraviolet radiation (UVR) exposure are on the rise. In view of this, current knowledge gaps in the biology of photocarcinogenesis and skin cancer progression urgently need to be addressed. One factor that has limited skin cancer research has been the need for a reproducible and physiologically-relevant model able to represent the complexity of human skin. This review outlines the main currently-used in vitro models of UVR-induced skin damage. This includes the use of conventional two-dimensional cell culture techniques and the major animal models that have been employed in photobiology and photocarcinogenesis research. Additionally, the progression towards the use of cultured skin explants and tissue-engineered skin constructs, and their utility as models of native skin's responses to UVR are described. The inherent advantages and disadvantages of these in vitro systems are also discussed.
Assuntos
Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Técnicas de Cultura de Células , Humanos , Modelos Animais , Técnicas de Cultura de Órgãos , Engenharia TecidualRESUMO
Several different advanced treatments have been used to improve healing in chronic wounds, but none have shown sustained success. The application of topical growth factors (GFs) has displayed some potential, but the varying results, high doses and high costs have limited their widespread adoption. Many treatments have ignored the evidence that wound healing is driven by interactions between extracellular matrix proteins and GFs, not just GFs alone. We report herein that a clinical Good Manufacturing Practice-grade vitronectin:growth factor (cVN:GF) complex is able to stimulate functions relevant to wound repair in vitro, such as enhanced cellular proliferation and migration. Furthermore, we assessed this complex as a topical wound healing agent in a single-arm pilot study using venous leg ulcers, as well as several 'difficult to heal' case studies. The cVN:GF complex was safe and re-epithelialisation was observed in all but 1 of the 30 patients in the pilot study. In addition, the case studies show that this complex may be applied to several ulcer aetiologies, such as venous leg ulcers, diabetic foot ulcers and pressure ulcers. These findings suggest that further evaluation is warranted to determine whether the cVN:GF complex may be an effective topical treatment for chronic wounds.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Úlcera por Pressão/tratamento farmacológico , Úlcera Varicosa/tratamento farmacológico , Vitronectina/administração & dosagem , Cicatrização/efeitos dos fármacos , Administração Tópica , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Pé Diabético/tratamento farmacológico , Quimioterapia Combinada , Feminino , Humanos , Projetos Piloto , Úlcera por Pressão/patologia , Resultado do Tratamento , Úlcera Varicosa/patologiaRESUMO
Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.
Assuntos
Movimento Celular/efeitos dos fármacos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Vitronectina/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Domínios e Motivos de Interação entre Proteínas , Vitronectina/metabolismoRESUMO
Chronic exposure of human skin to solar ultraviolet radiation (UVR) induces a range of biological reactions which may directly or indirectly lead to the development of skin cancer. In order to overcome these damaging effects of UVR and to reduce photodamage, the skin's endogenous defence system functions in concert with the various exogenous photoprotectors. Growth factors, particularly insulin-like growth factor-I (IGF-I), produced within the body as a result of cellular interaction in response to UVR demonstrates photoprotective properties in human skin. This review summarises the impact of UVR-induced photolesions on human skin, discusses various endogenous as well as exogenous approaches of photoprotection described to date and explains how IGF-I mediates UVR photoprotective responses at the cellular and mitochondrial level. Further, we describe the current interventions using growth factors and propose how the knowledge of the IGF-I photoprotection signalling cascades may direct the development of improved UVR protection and remedial strategies.
Assuntos
Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like I/uso terapêutico , Neoplasias Cutâneas/prevenção & controle , Neoplasias Cutâneas/terapia , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Humanos , Camundongos , Transdução de Sinais , Pele/efeitos dos fármacos , Pele/patologiaRESUMO
Ultraviolet B radiation (UVBR) induces the formation of photolesions in epidermal keratinocytes, potentially affecting cellular function and contributing towards malignant transformation. Insulin-like growth factor-I (IGF-I) contributes to protection of keratinocytes against UVBR-induced damage. Studies have shown that exogenous IGF-I or dermal fibroblast conditioned media pre-UVBR contributes to protection in primary keratinocytes by preventing apoptosis, modulating cell cycle progression and affecting photolesion removal through its damage preventative effects, however, the efficacy of IGF-I post-UVBR has not been sufficiently addressed. Using 2D and 3D photobiology skin models, the ability of IGF-I post-UVBR to rescue primary keratinocytes from photodamage was investigated. The photoprotective effect of IGF-I, both pre- and post-UVBR on cellular functions of irradiated keratinocytes was examined. IGF-I application, either pre- or post-UVBR, was found to alter keratinocyte survival, apoptosis, cell cycle progression and damage removal responses to UVBR. In particular, IGF-I application post-UVBR was found to promote increased keratinocyte survival, prevent apoptosis, shift cell cycle progression and reduce photodamage in all the skin models. Furthermore, marked differences were observed in activation of signalling cascades upon IGF-I treatment post-UVBR. Taken together, these findings indicate that in addition to a previously known photodamage preventative effect, IGF-I treatment post-UVBR has a photoreparative role suggesting it may hold potential in the development of effective remedial strategies against sunburns and photodamage.
Assuntos
Fator de Crescimento Insulin-Like I/metabolismo , Queratinócitos/efeitos da radiação , Raios Ultravioleta , Sobrevivência Celular/efeitos da radiação , Técnicas de Cocultura , Humanos , Queratinócitos/metabolismo , Pele/metabolismo , Pele/efeitos da radiaçãoRESUMO
IGF-I can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4, and -5. Because IGF-I and VN have established roles in tumor cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGF binding proteins (IGFBPs), and VN in tumor cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and normal nontumorgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I-IGFBP-VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogs determined this stimulation to be dependent on both heterotrimeric IGF-I-IGFBP-VN complex formation and the involvement of the IGF-I receptor (IGF-IR). Furthermore, the enhanced cellular migration was abolished on incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signaling pathway while simultaneously producing a sustained activation of the phosphatidylinositide 3-kinase/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for phosphatidylinositide 3-kinase/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I-IGFBP-VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I-IGFBP-VN complexes and adds further evidence to support the involvement of VN-binding integrins and their cooperativity with the IGF-IR in the promotion of tumor cell migration.
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Movimento Celular/fisiologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Vitronectina/fisiologia , Mama/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Integrina alfaV/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismoRESUMO
The insulin receptor (IR) plays critical roles in metabolism and growth, directed by the binding of insulin. Decades of research to understand the mechanism of insulin binding and activation of the IR have identified a region of the receptor, the C-terminal (CT) peptide, to be crucial for insulin binding. In particular, a truncated IR consisting of the first three domains fused to the CT peptide was found to bind insulin with nanomolar affinity, with undetectable binding in the absence of fused or soluble CT peptide. Problematically, all current crystal structures of the IR indicate the fusion point of the CT peptide to the three domains is located far from the position of the CT peptide as resolved in such structures. We have attempted to address this problem using molecular modelling and dynamics simulations. The results led to the identification of a potential inter-domain interaction between the L2 domain and the CT peptide that is not observed in any of the crystal structures of the IR. Investigations into this new interaction found a conformational change that could potentially be in response to insulin binding. Additionally, further simulation work with the new conformation demonstrated its compatibility with the position and orientation of insulin from the latest insulin-bound IR crystal structure.
Assuntos
Antígenos CD/química , Insulina/química , Fragmentos de Peptídeos/química , Receptor de Insulina/química , Antígenos CD/metabolismo , Sítios de Ligação , Humanos , Insulina/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Fosforilação , Conformação Proteica , Receptor de Insulina/metabolismoRESUMO
The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline in both Australia and globally. Hence, the cellular and molecular pathways that are associated with UVR-induced photocarcinogenesis need to be urgently elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular, the highly mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which, to some extent, have limited their relevance to the in vivo situation. To address this, we characterized a three-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53, and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration after photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.
Assuntos
Transformação Celular Neoplásica/metabolismo , Queratinócitos/metabolismo , Modelos Biológicos , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Cutâneas/metabolismo , Engenharia Tecidual , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos da radiação , Transformação Celular Neoplásica/patologia , Células Cultivadas , Citocinas/metabolismo , Derme/metabolismo , Derme/patologia , Feminino , Humanos , Queratinócitos/patologia , Masculino , Camundongos , Neoplasias Induzidas por Radiação/patologia , Neoplasias Cutâneas/patologia , Alicerces Teciduais/química , Proteína Supressora de Tumor p53/metabolismoRESUMO
Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.
Assuntos
Fibroblastos/efeitos da radiação , Queratinócitos/efeitos da radiação , Pele/efeitos da radiação , Raios Ultravioleta , Apoptose , Sequência de Bases , Células Cultivadas , Técnicas de Cocultura , Primers do DNA , Reparo do DNA , Humanos , Técnicas In Vitro , Pele/citologiaRESUMO
Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (guanine-nucleotide exchange factor-H1)-RhoA signaling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signaling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Interleucina-8/biossíntese , NF-kappa B/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Western Blotting , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-8/genética , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação/efeitos dos fármacos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho , Fatores de Tempo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
The therapeutic use of human embryonic stem (hES) cells is severely limited by safety concerns regarding their culture in media containing animal-derived or nondefined factors and on animal-derived feeder cells. Thus, there is a pressing need to develop culture techniques that are xeno-free, fully defined, and synthetic. Our laboratory has discovered that insulin-like growth factor (IGF) and vitronectin (VN) bind to each other resulting in synergistic short-term functional effects in several cell types, including keratinocytes and breast epithelial cells. We have further refined this complex into a single chimeric VN:IGF-I protein that functionally mimics the effects obtained upon binding of IGF-I to VN. The aim of the current study was to determine whether hES cells can be serially propagated in feeder-cell-free and serum-free conditions using medium containing our novel chimeric VN:IGF-I protein. Here we demonstrate that hES cells can be serially propagated and retain their undifferentiated state in vitro for up to 35 passages in our feeder-cell-free, serum-free, chemically defined media. We have utilized real-time polymerase chain reaction (PCR), immunofluorescence, and fluorescence-activated cell sorter (FACS) analysis to show that the hES cells have maintained an undifferentiated phenotype. In vitro differentiation assays demonstrated that the hES cells retain their pluripotent potential and the karyotype of the hES cells remains unchanged. This study demonstrates that the novel, fully defined, synthetic VN:IGF-I chimera-containing medium described herein is a viable alternative to media containing serum, and that in conjunction with laminin-coated plates facilitates feeder-cell-free and serum-free growth of hES.
Assuntos
Proliferação de Células/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Vitronectina/farmacologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Humanos , Fator de Crescimento Insulin-Like I/química , Cariotipagem , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Vitronectina/químicaRESUMO
Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies.