RESUMEN
Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.
Asunto(s)
Basigina/fisiología , Diferenciación Celular/fisiología , Receptores de Hialuranos/fisiología , Miofibroblastos/citología , Factor de Crecimiento Transformador beta1/fisiología , Basigina/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Interleucina-1beta/fisiología , Miofibroblastos/metabolismoRESUMEN
Hyaluronidase (HYAL)-2 is a weak, acid-active, hyaluronan-degrading enzyme broadly expressed in somatic tissues. Aberrant HYAL2 expression is implicated in diverse pathology. However, a significant proportion of HYAL2 is enzymatically inactive; thus the mechanisms through which HYAL2 dysregulation influences pathobiology are unclear. Recently, nonenzymatic HYAL2 functions have been described, and nuclear HYAL2 has been shown to influence mRNA splicing to prevent myofibroblast differentiation. Myofibroblasts drive fibrosis, thereby promoting progressive tissue damage and leading to multimorbidity. This study identifies a novel HYAL2 cytoplasmic function in myofibroblasts that is unrelated to its enzymatic activity. In fibroblasts and myofibroblasts, HYAL2 interacts with the GTPase-signaling small molecule ras homolog family member A (RhoA). Transforming growth factor beta 1-driven fibroblast-to-myofibroblast differentiation promotes HYAL2 cytoplasmic relocalization to bind to the actin cytoskeleton. Cytoskeletal-bound HYAL2 functions as a key regulator of downstream RhoA signaling and influences profibrotic myofibroblast functions, including myosin light-chain kinase-mediated myofibroblast contractility, myofibroblast migration, myofibroblast collagen/fibronectin deposition, as well as connective tissue growth factor and matrix metalloproteinase-2 expression. These data demonstrate that, in certain biological contexts, the nonenzymatic effects of HYAL2 are crucial in orchestrating RhoA signaling and downstream pathways that are important for full profibrotic myofibroblast functionality. In conjunction with previous data demonstrating the influence of HYAL2 on RNA splicing, these findings begin to explain the broad biological effects of HYAL2.
Asunto(s)
Fibroblastos/metabolismo , Hialuronoglucosaminidasa/metabolismo , Miofibroblastos/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Fibrosis/metabolismo , Humanos , Masculino , Empalme del ARN , RatasRESUMEN
Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = -0.396 (p = 0.002), r = -0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.
Asunto(s)
Adipogénesis , Ácido Hialurónico/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Índice de Masa Corporal , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Hialuronano Sintasas/metabolismo , PPAR gamma/metabolismoRESUMEN
Fibroblasts are central to wound healing and fibrosis through TGFß1-triggered differentiation into contractile, α-smooth muscle actin (α-SMA)-positive myofibroblasts. This is mediated by accumulation of a pericellular matrix of hyaluronan (HA) and the HA-dependent co-localization of CD44 with the epidermal growth factor receptor (EGFR). Interactions of HA with hyaladherins, such as inter-α-inhibitor (IαI) and tumor necrosis factor-stimulated gene-6 (TSG-6), are also essential for differentiation. This study investigated the mechanisms involved. TSG-6 and α-SMA had different kinetics of induction by TGFß1, with TSG-6 peaking before α-SMA Si CD44 or EGFR inhibition prevented differentiation but had no effect on TSG-6 expression. TSG-6 was essential for differentiation, and mAb A38 (preventing IαI heavy chain (HC) transfer), HA-oligosaccharides, cobalt, or Si bikunin prevented TSG-6 activity, preventing differentiation. A38 also prevented the EGFR/CD44 association. This suggested that TSG-6/IαI HC interaction was necessary for the effect of TSG-6 and that HC stabilization of HA initiated the CD44/EGFR association. The newly described HC5 was shown to be the principal HC expressed, and its cell surface expression was prevented by siRNA inhibition of TSG-6 or bikunin. HC5 was released by hyaluronidase treatment, confirming its association with cell surface HA. Finally, HC5 knockdown by siRNA confirmed its role in myofibroblast differentiation. The current study describes a novel mechanism linking the TSG-6 transfer of the newly described HC5 to the HA-dependent control of cell phenotype. The interaction of HC5 with cell surface HA was essential for TGFß1-dependent differentiation of fibroblasts to myofibroblasts, highlighting its importance as a novel potential therapeutic target.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular/fisiología , Miofibroblastos/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Actinas/genética , Actinas/metabolismo , alfa-Globulinas/farmacología , Anticuerpos Monoclonales de Origen Murino/farmacología , Moléculas de Adhesión Celular/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/farmacología , Miofibroblastos/citología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Oral mucosal wounds are characterized by rapid healing with minimal scarring, partly attributable to the "enhanced" wound healing properties of oral mucosal fibroblasts (OMFs). Hepatocyte growth factor (HGF) is a pleiotropic growth factor, with potential key roles in accelerating healing and preventing fibrosis. HGF can exist as full-length or truncated (HGF-NK), NK1 and NK2 isoforms. As OMFs display elevated HGF expression compared to dermal fibroblasts (DFs), this study investigated the extent to which HGF mediates the preferential cellular functions of OMFs, and the influence of pro-fibrotic, transforming growth factor-ß1 (TGF-ß1) on these responses. Knockdown of HGF expression in OMFs by short-interfering RNA (siHGF) significantly inhibited OMF proliferative and migratory responses. Supplementation with exogenous TGF-ß1 also significantly inhibited proliferation and migration, concomitant with significantly down-regulated HGF expression. In addition, knockdown abrogated OMF resistance to TGF-ß1-driven myofibroblast differentiation, as evidenced by increased α-smooth muscle actin (α-SMA) expression, F-actin reorganisation, and stress fibre formation. Responses were unaffected in siHGF-transfected DFs. OMFs expressed significantly higher full-length HGF and NK1 levels compared to patient-matched DFs, whilst NK2 expression was similar in both OMFs and DFs. Furthermore, NK2 was preferentially expressed over NK1 in DFs. TGF-ß1 supplementation significantly down-regulated full-length HGF and NK1 expression by OMFs, while NK2 was less affected. This study demonstrates the importance of HGF in mediating "enhanced" OMF cellular function. We also propose that full-length HGF and HGF-NK1 convey desirable wound healing properties, whilst fibroblasts preferentially expressing more HGF-NK2 readily undergo TGF-ß1-driven differentiation into myofibroblasts.
Asunto(s)
Diferenciación Celular , Factor de Crecimiento de Hepatocito/metabolismo , Miofibroblastos/citología , Miofibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas , Biomarcadores , Diferenciación Celular/genética , Proliferación Celular , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Factor de Crecimiento de Hepatocito/genética , Humanos , Mucosa Bucal/citología , Mucosa Bucal/metabolismo , Isoformas de ProteínasRESUMEN
Hyaluronan (HA) promotes transforming growth factor (TGF)-ß1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-ß1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-ß1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-ß1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-ß1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na(+)/H(+) exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-ß1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology.
Asunto(s)
Proteína Morfogenética Ósea 7/metabolismo , Ácido Hialurónico/metabolismo , Miofibroblastos/citología , Fenotipo , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Endosomas/metabolismo , Fibroblastos/citología , Regulación Enzimológica de la Expresión Génica , Glucuronosiltransferasa/genética , Humanos , Receptores de Hialuranos/genética , Hialuronano Sintasas , Hialuronoglucosaminidasa/genética , Miofibroblastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-ß1 (TGF-ß1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-ß1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-ß1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca(2+)/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-ß1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-ß1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.
Asunto(s)
Diferenciación Celular/fisiología , Receptores ErbB/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Microdominios de Membrana/metabolismo , Miofibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular Transformada , Activación Enzimática/fisiología , Receptores ErbB/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Receptores de Hialuranos/genética , Hialuronano Sintasas , Ácido Hialurónico/genética , Microdominios de Membrana/genética , Miofibroblastos/citología , Transducción de Señal/fisiología , Proteínas Smad/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Persistent inflammation is a well-known determinant of progressive tissue fibrosis; however, the mechanisms underlying this process remain unclear. There is growing evidence indicating a role of the cytokine IL-1ß in profibrotic responses. We previously demonstrated that fibroblasts stimulated with IL-1ß increased their generation of the polysaccharide hyaluronan (HA) and increased their expression of the HA synthase enzyme (HAS-2). The aim of this study was to determine the significance of IL-1ß-induced changes in HA and HAS-2 generation. In this study, we found that stimulation of fibroblasts with IL-1ß results in the relocalization of HA associated with the cell to the outer cell membrane, where it forms HAS2- and CD44-dependent cell membrane protrusions. CD44 is concentrated within the membrane protrusions, where it co-localizes with the intracellular adhesion molecule 1. Furthermore, we have identified that these cell protrusions enhance IL-1ß-dependent fibroblast-monocyte binding through MAPK/ERK signaling. Although previous data have indicated the importance of the HA-binding protein TSG-6 in maintaining the transforming growth factor ß1-dependent HA coat, TSG-6 was not essential for the formation of the IL-1ß-dependent HA protrusions, thus identifying it as a key difference between IL-1ß- and transforming growth factor ß1-dependent HA matrices. In summary, these data suggest that IL-1ß-dependent HA generation plays a role in fibroblast immune activation, leading to sequestration of monocytes within inflamed tissue and providing a possible mechanism for perpetual inflammation.
Asunto(s)
Extensiones de la Superficie Celular/inmunología , Fibroblastos/inmunología , Receptores de Hialuranos/inmunología , Ácido Hialurónico/biosíntesis , Interleucina-1beta/inmunología , Monocitos/inmunología , Adhesión Celular/inmunología , Moléculas de Adhesión Celular/inmunología , Diferenciación Celular/inmunología , Membrana Celular/inmunología , Células Cultivadas , Fibroblastos/fisiología , Glucuronosiltransferasa/inmunología , Humanos , Hialuronano Sintasas , Molécula 1 de Adhesión Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/fisiología , Miofibroblastos/inmunología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.
Asunto(s)
Sulfatos de Condroitina/metabolismo , Glicosaminoglicanos/metabolismo , Ácido Hialurónico/metabolismo , Glomérulos Renales/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/fisiología , Factor C de Crecimiento Endotelial Vascular/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Glicocálix/metabolismo , Humanos , Glomérulos Renales/metabolismo , Microvasos/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVES: Acute Kidney Injury (AKI) and Chronic Kidney Disease (CKD) are increasingly recognised as one disease continuum, rather than distinct entities, and are associated with a huge burden to healthcare services. The leading cause of AKI worldwide is Ischaemia Reperfusion Injury (IRI), most commonly seen in clinical settings of sepsis-driven hypotension. Ischaemic Preconditioning (IPC) is a strategy aimed at reducing the deleterious effects of IRI. The objectives of this study were to demonstrate an efficacious in vivo model of Kidney IRI, and the protective influence of IPC in attenuating AKI and development of renal fibrosis. METHODS: A rat model of bilateral kidney IRI was used: Male Lewis rats (n=84) were assigned to IRI, sham or IPC. In IRI, renal pedicles were clamped for 45 minutes. IPC groups underwent pulsatile IPC prior to IRI. Kidneys were retrieved at 24 hours, 48 hours, 7 days, 14 days and 28 days, and assessed histologically. RESULTS: IRI led to marked AKI (24-48 h) and renal fibrosis development by 28 days. IPC attenuated this damage, with 66% less fibrosis. Interestingly, at 14-days, the histological appearance of both IRI and IPC kidneys was rather similar, potentially representing an important transitional point at which kidneys commit to either fibrosis or recovery. This may provide a suitable inflexion point for introduction of novel anti-fibrotic therapies. CONCLUSIONS: In conclusion, we have characterised a model of kidney injury from acute to chronic phases, allowing detailed mechanistic understanding and which can be manipulated by effective treatment strategies such as IPC.
RESUMEN
Chronic, non-healing wounds represent a significant area of unmet medical need and are a growing problem for healthcare systems around the world. They affect the quality of life for patients and are an economic burden, being difficult and time consuming to treat. They are an escalating problem across the developed world due to the increasing incidence of diabetes and the higher prevalence of ageing populations. Effective treatment options are currently lacking, and in some cases chronic wounds can persist for years. Some traditional medicines are believed to contain bioactive small molecules that induce the healing of chronic wounds by reducing excessive inflammation, thereby allowing re-epithelisation to occur. Furthermore, many small molecules found in plants are known to have antibacterial properties and, although they lack the therapeutic selectivity of antibiotics, they are certainly capable of acting as topical antiseptics when applied to infected wounds. As these molecules act through mechanisms of action distinct from those of clinically used antibiotics, they are often active against antibiotic resistant bacteria. Although there are numerous studies highlighting the effects of naturally occurring small molecules in wound-healing assays in vitro, only evidence from well conducted clinical trials can allow these molecules or the remedies that contain them to progress to the clinic. With this in mind, we review wound-healing natural remedies that have entered clinical trials over a twenty-year period to the present. We examine the bioactive small molecules likely to be in involved and, where possible, their mechanisms of action.
Asunto(s)
Antiinfecciosos Locales , Productos Biológicos , Humanos , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Calidad de Vida , Antiinfecciosos Locales/uso terapéutico , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-ß1 (TGF-ß1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-ß1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-ß1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-ß-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-ß1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-ß1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-ß1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-ß1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-ß1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-ß1. In summary, HA facilitates TGF-ß1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.
Asunto(s)
Proliferación Celular , Receptores ErbB/metabolismo , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Receptores de Hialuranos/genética , Hialuronano Sintasas , Ácido Hialurónico/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Bioactivity-guided fractionation was used to isolate two compounds, tomentosenol A (1) and torellianone A (2), from a cerumen extract from Tetragonula carbonaria. The anti-fibrotic activity of these compounds was examined using human cultured neonatal foreskin fibroblasts (NFF) and immortalised keratinocytes (HaCaTs). Tomentosenol A (1), inhibited NFF and HaCaT cell proliferation and prevented NFF and HaCaT scratch wound repopulation at 12.5-25 µM concentrations. These inhibitory effects were associated with reduced cell viability, determined by tetrazolium dye (MTT) and sulforhodamine B (SRB) assays. Compound 1 further inhibited transforming growth factor-ß1 (TGF-ß1)-stimulated, NFF-myofibroblast differentiation and soluble collagen production; and was an effective scavenger of the model oxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH·), with an EC50 value of 44.7 ± 3.1 µM. These findings reveal significant anti-fibrotic potential for cerumen-derived tomentosenol A (1).
RESUMEN
Fibroblast differentiation into myofibroblasts is a key event during normal wound repair. We have previously demonstrated an age-related defect in this process associated with impaired synthesis of hyaluronan (HA) synthase (HAS) 2 but failed to prescribe its role in a mechanistic sense. Here we demonstrate that in addition to HAS2, there is loss of EGF receptor (EGF-R) in aged cells, and both are required for normal fibroblast functionality. Analysis of molecular events revealed that in young cells, transforming growth factor (TGF)-beta1-dependent phenotypic activation uses two distinct but cooperating pathways that involve TGF-beta receptor/Smad2 activation and EGF-mediated EGF-R/extracellular signal-regulated kinase (ERK) 1/2 signaling, and the latter is compromised with in vitro aging. Pharmacological inhibition of any of the five intermediates (TGF-beta receptor, Smad2, EGF, EGF-R, and ERK1/2) attenuated TGF-beta1 induction of alpha-smooth muscle actin. We present evidence that the HA receptor CD44 co-immunoprecipitates with EGF-R after activation by TGF-beta1. This interaction is HA-dependent because disruption of HA synthesis abrogates this association and inhibits subsequent ERK1/2 signaling. In aged fibroblasts, this association is lost with resultant suppression of ERK1/2 activation. Forced overexpression of EGF-R and HAS2 in aged cells restored TGF-beta1-mediated HA-CD44/EGF-R association and alpha-smooth muscle actin induction. Taken together, these results demonstrate that HA can serve as a signal integrator by facilitating TGF-beta1-mediated CD44-EGF-R-ERK interactions and ultimately fibroblast phenotype. We propose a model to explain this novel mechanism and the functional consequence of age-dependent dysregulation.
Asunto(s)
Diferenciación Celular , Senescencia Celular , Receptores ErbB/metabolismo , Fibroblastos/citología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Transducción de Señal , Adulto , Animales , Bovinos , Diferenciación Celular/efectos de los fármacos , Separación Celular , Senescencia Celular/efectos de los fármacos , Dermis/citología , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Interstitial fibrosis, associated with extensive accumulation of extracellular matrix constituents in the cortical interstitium, is directly correlated to progression of renal disease. The earliest histological marker of this progression is the accumulation in the interstitium of fibroblasts with the phenotypic appearance of myofibroblasts. These myofibroblasts are contractile cells that express alpha smooth muscle actin and incorporate it into intracellular stress fibres. Although fibroblasts are histologically visible in normal kidneys, there are relatively few of them and proximal tubular epithelial cells predominate. In progressive disease, however, the interstitium becomes filled with myofibroblasts. In this review, we will examine the phenotype and function of fibroblasts and myofibroblasts in the cortical interstitium and the processes that may modulate them.
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Fibroblastos/patología , Riñón/patología , Miofibroblastos/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibroblastos/fisiología , Fibrosis , Humanos , Riñón/fisiopatología , Ratones , Miofibroblastos/fisiología , RatasRESUMEN
BACKGROUND: ADAM15 is a membrane-bound member of the adamalysin family that is up-regulated in areas of tissue remodelling. Previous studies have demonstrated the role of ADAM15 in mesangial cell migration, which is integral in tissue remodelling in pathology and repair. The current study was designed to identify and analyse the genomic regions upstream of ADAM15 that would regulate its transcription. METHODS: Using 5'-RACE and RT-PCR, the ADAM15 5'-UTR was extended and luciferase constructs assembled to examine the transcription start site and characterise the promoter region of this gene. RESULTS: A 145-bp proximal promoter construct showed full activity in unstimulated cells. Analysis of this region identified three potential Sp1-binding sites. Electromobility and supershift assays confirmed that Sp1 was constitutively present in MC nuclei. Mutations in each Sp1 site confirmed each was needed for full activity, while mutation of all three sites abrogated luciferase activity demonstrating that Sp1 was involved in the promoter activity of ADAM15. Methylation of this promoter fragment abolished the activity, while the methyltransferase inhibitor 5-aza-3'-deoxycytidine showed no increased activity in transfected cells, implying that the promoter was not methylated in our cells. CONCLUSION: These results demonstrate the intrinsic promoter activity of ADAM15 in quiescent MC and show the involvement of Sp1 in its regulation.
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Proteínas ADAM/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Regiones no Traducidas 5' , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Metilación de ADN , ADN Complementario/genética , Epigénesis Genética , Genes Reporteros , Humanos , Luciferasas/genética , Células Mesangiales/metabolismo , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Ratas , Elementos Reguladores de la Transcripción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción Sp1/metabolismoRESUMEN
Myofibroblasts are contractile, α-smooth muscle actin-positive cells with multiple roles in pathophysiological processes. Myofibroblasts mediate wound contractions, but their persistent presence in tissues is central to driving fibrosis, making them attractive cell targets for the development of therapeutic treatments. However, due to shared cellular markers with several other phenotypes, the specific targeting of myofibroblasts has long presented a scientific and clinical challenge. In recent years, myofibroblasts have drawn much attention among scientific research communities from multiple disciplines and specialisations. As further research uncovers the characterisations of myofibroblast formation, function, and regulation, the realisation of novel interventional routes for myofibroblasts within pathologies has emerged. The research community is approaching the means to finally target these cells, to prevent fibrosis, accelerate scarless wound healing, and attenuate associated disease-processes in clinical settings. This comprehensive review article describes the myofibroblast cell phenotype, their origins, and their diverse physiological and pathological functionality. Special attention has been given to mechanisms and molecular pathways governing myofibroblast differentiation, and updates in molecular interventions.
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Fibrosis/terapia , Miofibroblastos/metabolismo , Piel/patología , Actinas/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Cicatriz , Receptores ErbB/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Ligandos , Ratones , Fenotipo , Transducción de Señal/fisiología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Wnt/metabolismo , Cicatrización de Heridas , beta Catenina/metabolismoRESUMEN
The thyrotropin receptor (TSHR) is expressed during lineage-specific differentiation (e.g. adipogenesis) and is activated by TSH, thyroid-stimulating antibodies, and gain-of-function mutations (TSHR*). Comparison of gene expression profiles of nonmodified human preadipocytes (n = 4) with the parallel TSHR* population revealed significant up-regulation of 27 genes including hyaluronan (HA) synthases (HAS) 1 and 2. The array data were confirmed by quantitative PCR of HAS1 and HAS2 and enzyme-linked immunosorbent assay measurement of HA; all values were significantly increased (p < 0.03) in TSHR*-expressing preadipocytes (n = 10). Preadipocytes (n = 8) treated with dibutyryl (db)-cAMP display significantly increased HAS1 and HAS2 transcripts, HAS2 protein, and HA production (p < 0.02). HAS1 or HAS2 small interfering RNA treatment of db-cAMP-stimulated preadipocytes (n = 4) produced 80% knockdown in HAS1 or 61% knockdown in HAS2 transcripts (compared with scrambled), respectively; the corresponding HA production was reduced by 49 or 38%. Reporter assays using A293 cells transfected with HAS1 promoter-driven plasmids containing or not containing the proximal CRE and treated with db-cAMP revealed that it is functional. Chromatin immunoprecipitation, using a cAMP-responsive element-binding protein antibody, of db-cAMP-treated preadipocytes (n = 4) yielded products for HAS1 and HAS2 with relative fold increases of 3.3 +/- 0.8 and 2.6 +/- 0.9, respectively. HA accumulates in adipose/connective tissues of patients with thyroid dysfunction. We investigated the contributions of TSH and thyroid-stimulating antibodies and obtained small (9-24%) but significant (p < 0.02) increases in preadipocyte HA production with both ligands. Similar results were obtained with a TSHR monoclonal antibody lacking biological activity (p < 0.05). We conclude that TSHR activation is implicated in HA production in preadipocytes, which, along with thyroid hormone level variation, explains the HA overproduction in thyroid dysfunction.
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Adipocitos/metabolismo , Fibroblastos/metabolismo , Ácido Hialurónico/metabolismo , Receptores de Tirotropina/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Western Blotting , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Perfilación de la Expresión Génica , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Interferencia de ARN , Receptores de Tirotropina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiopatologíaRESUMEN
Myofibroblasts are contractile cells that are characterized by the expression of alpha-smooth muscle actin and mediate the closure of wounds and the formation of collagen-rich scars. Their presence in organs such as lungs, liver, and kidney has long been established as a marker of progressive fibrosis. The transforming growth factor beta(1)-driven differentiation of fibroblasts is a major source of myofibroblasts, and recent data have shown that hyaluronan is a major modulator of this process. This study examines this differentiation mechanism in more detail. Transforming growth factor beta(1)-dependent differentiation to the myofibroblastic phenotype was antagonized by the inhibition of hyaluronan synthesis, confirming that hyaluronan was necessary for differentiation. This response, however, was not reproduced by simply adding hyaluronan to fibroblasts, as the results implicated hyaladherins, as well as the macromolecular assembly of de novo hyaluronan, as essential in this process. We previously suggested that there is a relocalization of lipid-raft components during myofibroblastic differentiation. The present study demonstrates that the hyaluronan receptor CD44, the hyaluronidase HYAL 2, and the transforming growth factor beta(1)-receptor ALK5 all relocalized from raft to non-raft locations, which was reversed by the addition of exogenous hyaluronan. These data highlight a role for endogenous hyaluronan in the mediation of myofibroblastic differentiation. While hyaluronan synthesis was both essential and necessary for differentiation, exogenously provided hyaluronan antagonized differentiation, underscoring a pathological role for hyaluronan in such cell fate processes.
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Diferenciación Celular/efectos de los fármacos , Fibroblastos/citología , Ácido Hialurónico/metabolismo , Músculo Liso/citología , Factor de Crecimiento Transformador beta1/metabolismo , Western Blotting , Diferenciación Celular/fisiología , Línea Celular , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Receptores de Hialuranos/metabolismo , Músculo Liso/metabolismo , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.