RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-ß1 (TGF-ß1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-ß1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-ß type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-ß type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-ß1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-ß type II receptor knockout mice. Taken together, data from this study suggest that TGF-ß1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.
Assuntos
Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Humanos , Lactente , Fator de Crescimento Transformador beta1/metabolismo , Camundongos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Bleomicina/efeitos adversos , Fibroblastos/metabolismo , Camundongos KnockoutRESUMO
Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-ß) receptor II knockout mice indicating a role for TGF-ß signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-ß1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-ß signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-ß) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-ß-induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.
Assuntos
Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Peroxissomos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Metabolismo dos Lipídeos , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Biológicos , Oxirredução , PPAR alfa/agonistas , PPAR alfa/metabolismo , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Smad/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Alveolar epithelial cell injury has been proposed as a causative factor for the onset and progression of pulmonary fibrosis. However, the role of type II alveolar epithelial cells (AECs) in the epithelial mesenchymal transition (EMT) is controversial. AIMS: The present study performed in rats instilled with bleomycin investigated a) the expressions of the insulin growth factor (IGF-1) and insulin growth factor binding protein 5 (IGFBP-5) and transforming growth factor (TGF-ß1) in the type II AECs, b) the role of type II AECs in EMT and extracellular matrix (ECM) formation and, c) the effect of pioglitazone on all the above parameters. METHODS: Male Wistar rats were divided into three Groups: Group I (saline control), Group II (Bleomycin, given as a single intratracheal instillation, 7U/kg) and Group III (Bleomycin+Pioglitazone (40mg/kg/day orally, starting 7days post bleomycin instilled as in Group II). From lung tissues, the protein expressions of IGF-1, IGFBP-5, TGF-ß1, surfactant protein C (SP-C, as a marker for type II AECs) and α-smooth muscle actin (α-SMA, as a marker for EMT), were determined on day 7 in Groups I and II and on days 14, 21 and 35 in all the three groups. RESULTS: IGFBP-5 and IGF-1 expressions were reduced significantly and TGF-ß1 expression increased significantly in type II AECs in Group II from day 7 till day 35 as compared to Group I. An increase in SP-C and α-SMA expression and their co-localization were seen in the type II AECs undergoing EMT from day 7 till day 35. A concomitant remodeling and laying down of ECM was observed also. In Group III, with pioglitazone, there was a reversal with significant up-regulation in IGFBP-5 and IGF-1 expressions and down-regulation of TGF-ß1 in the type II AECs along with a significant decrease in the solid area fraction, EMT and ECM in the lung tissue. CONCLUSIONS: IGFBP-5, IGF-1 and TGF-ß1 in the type II AECs play a key role in lung injury caused by bleomycin and pioglitazone attenuates the lung injury/fibrosis by restoring IGFBP-5 and IGF-1 and decreasing TGF-ß1 expressions in the type II AECs.
Assuntos
Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Fator de Crescimento Insulin-Like I/biossíntese , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta1/biossíntese , Actinas/metabolismo , Animais , Bleomicina , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Masculino , Microscopia de Fluorescência , Pioglitazona , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/prevenção & controle , Proteína C Associada a Surfactante Pulmonar/metabolismo , Ratos Wistar , Tiazolidinedionas/farmacologia , Fatores de TempoRESUMO
ABSTRACT: Trauma hemorrhagic shock (THS) is a major cause of death and disability worldwide. It is the leading cause of death with or without sepsis in approximately 50% of patients. In THS, there is an incidence of cellular apoptosis, which contributes majorly to cellular dysfunction, organ failure, and mortality. The Akt (protein kinase B) isoform, Akt1, and glycogen synthase kinase 3ß (Akt1-GSK3ß) signaling pathway controls cell survival and apoptosis. Deleterious consequences of alteration of this signaling system might lead to inflammation, cytokine storm, and other diseases. Hence, in the present study, we investigated the role of this signaling system by measuring the phosphorylation levels of Akt1-GSK3ß. Here, we demonstrated that the downregulation of pAkt1 and upregulation of pGSK3ß in THS were significantly associated with the severity of the shock, apoptosis of immune cells, altered glucose metabolism, inflammation, cytokine storm, hemostasis, and acidosis, causing mortality with or without sepsis. For the first time, this study shows that a dysregulated pAkt1-GSK3ß pathway causes contrasting cell fates in THS, leading to trauma pathology. Hence, the delineation and the implications of this signaling system may provide a new important target for the treatment of THS. In addition, Akt activation may become a potential strategy for increasing the survival rate following THS.
Assuntos
Glicogênio Sintase Quinase 3 beta , Proteínas Proto-Oncogênicas c-akt , Choque Hemorrágico , Ferimentos e Lesões , Humanos , Síndrome da Liberação de Citocina/etiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sepse/etiologia , Choque Hemorrágico/tratamento farmacológico , Choque Hemorrágico/etiologia , Choque Hemorrágico/metabolismo , Transdução de Sinais , Ferimentos e Lesões/complicaçõesRESUMO
Carbenoxolone (CBX) is a semi-synthetic plant derivative with pleiotropic pharmacological properties like anti-microbial and anti-inflammatory activities. Though approved for treatment of gastric ulcers, its use is limited due to adverse effects such as cytotoxicity. Bovine serum albumin (BSA) is a natural, non-toxic protein with high water-solubility and low immunogenicity, and is widely used as a nanocarrier for targeted drug delivery. In the present study, controlled release BSA-CBX nanoparticles (NPs) were synthesized by desolvation method to reduce drug cytotoxicity. These NPs showed desirable physicochemical properties such as particle size (â¼240 nm), polydispersity index (0.08), zeta potential (-7.12 mV), drug encapsulation efficiency (72 %), and were stable for at least 3 months at room temperature. The drug was released from the BSA-CBX NPs in a biphasic manner in vitro following non-fickian diffusion. Computational analysis determined that the binding between BSA and CBX occurred through van der Waals forces, hydrophobic interactions, and hydrogen bonds with 93 % steric stability. Further, the cytotoxic assays demonstrated â¼1.8-4.9-fold reduction in cytotoxicity using three human cell lines (A549, MCF-7, and U-87). Subsequently, this novel CBX formulation with BSA as an efficient carrier can potentially be used for diverse biomedical applications.