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The molecular mechanisms underlying the transition from nonalcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) are incompletely understood. During the development of NAFLD, Perilipin 5 (PLIN5) can regulate lipid metabolism by suppressing lipolysis and preventing lipotoxicity. Other reports suggest that the lack of PLIN5 decreases hepatic injury, indicating a protective role in NAFLD pathology. To better understand the role of PLIN5 in liver disease, we established mouse models of NAFLD and NAFLD-induced HCC, in which wild-type and Plin5 null mice were exposed to a single dose of acetone or 7,12-dimethylbenz[a]anthracene (DMBA) in acetone, followed by a 30-week high-fat diet supplemented with glucose/fructose. In the NAFLD model, RNA-seq revealed significant changes in genes related to lipid metabolism and immune response. At the intermediate level, pathways such as AMP-activated protein kinase (AMPK), signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase (JNK), and protein kinase B (AKT) were blunted in Plin5-deficient mice (Plin5-/-) compared to wild-type mice (WT). In the NAFLD-HCC model, only WT mice developed liver tumors, while Plin5-/- mice were resistant to tumorigenesis. Furthermore, only 32 differentially expressed genes associated with NALFD progession were identified in Plin5 null mice. The markers of mitochondrial function and immune response, such as the peroxisome proliferator-activated receptor-γ, coactivator 1-α (PGC-1α) and phosphorylated STAT3, were decreased. Lipidomic analysis revealed differential levels of some sphingomyelins between WT and Plin5-/- mice. Interestingly, these changes were not detected in the HCC model, indicating a possible shift in the metabolism of sphingomelins during carcinogenesis.
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In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.
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Células Endoteliales , Células Estrelladas Hepáticas , Ratones , Animales , Cirrosis Hepática/metabolismo , HepatocitosRESUMEN
Experimental bile duct ligation (BDL) in rodents causes cholestatic liver injury characterized by structural and functional alterations that include periportal biliary fibrosis. These changes are time-dependent and based on excess accumulation of bile acids in the liver. This in turn causes damage of hepatocytes and functional loss, leading to recruitment of inflammatory cells. Liver resident pro-fibrogenic cells facilitate extracellular matrix synthesis and remodeling. The proliferation of bile duct epithelial cells provokes a ductular reaction characterized by bile duct hyperplasia. Experimental BDL surgery is technically simple and quick to perform and reliably generates progressive liver damage with a predictable kinetics. The cellular, structural, and functional alterations induced in this model are similar to that in humans suffering from diverse forms of cholestasis including primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). Therefore, this extrahepatic biliary obstruction model is used in many laboratories worldwide. Nevertheless, BDL can result in significant variations and high mortality rates when surgery is carried out by untrained or inexperienced personnel. Here we present a detailed protocol to achieve a robust experimental obstructive cholestasis in mice.
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Colestasis , Humanos , Ratones , Animales , Colestasis/etiología , Colestasis/patología , Hígado/patología , Conductos Biliares/cirugía , Conductos Biliares/patología , Hepatocitos/patología , Cirrosis Hepática/patologíaRESUMEN
The cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-ß receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.
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Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on interleukine 3 (IL-3) and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The observation of cellular transformation was accompanied by the loss of Cdkn2a and Arf expression, which are both described as critical cell cycle regulators. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by sustained SCF supplementation strongly arguing for an involvement of KIT activation in the regulation of Cdkn2a/Arf expression. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.
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Mastocitos , Peritoneo , Animales , Humanos , Ratones , Línea Celular , Tejido Conectivo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Factor de Células Madre/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factores de Ribosilacion-ADP/metabolismoRESUMEN
Misidentification, cross-contamination and genetic drift of continuous animal cell lines are persistent problems in biomedical research, leading to erroneous results and inconsistent or invalidated studies. The establishment of immortalized hepatic stellate cell line Col-GFP HSC was reported in PLoS One in the year 2013. In the present study a multi loci short tandem repeat signature for this cell line was established that allows for unique cell line authentication.
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Células Estrelladas Hepáticas , Repeticiones de Microsatélite , Animales , Línea Celular , Macrófagos del HígadoRESUMEN
Immortalized hepatic stellate cells (HSCs) established from mouse, rat, and humans are valuable in vitro models for the biomedical investigation of liver biology. These cell lines are homogenous, thereby providing consistent and reproducible results. They grow more robustly than primary HSCs and provide an unlimited supply of proteins or nucleic acids for biochemical studies. Moreover, they can overcome ethical concerns associated with the use of animal and human tissue and allow for fostering of the 3R principle of replacement, reduction, and refinement proposed in 1959 by William M. S. Russell and Rex L. Burch. Nevertheless, working with continuous cell lines also has some disadvantages. In particular, there are ample examples in which genetic drift and cell misidentification has led to invalid data. Therefore, many journals and granting agencies now recommend proper cell line authentication. We herein describe the genetic characterization of the rat HSC line HSC-T6, which was introduced as a new in vitro model for the study of retinoid metabolism. The consensus chromosome markers, outlined primarily through multicolor spectral karyotyping (SKY), demonstrate that apart from the large derivative chromosome 1 (RNO1), at least two additional chromosomes (RNO4 and RNO7) are found to be in three copies in all metaphases. Additionally, we have defined a short tandem repeat (STR) profile for HSC-T6, including 31 species-specific markers. The typical features of these cells have been further determined by electron microscopy, Western blotting, and Rhodamine-Phalloidin staining. Finally, we have analyzed the transcriptome of HSC-T6 cells by mRNA sequencing (mRNA-Seq) using next generation sequencing (NGS).
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Autenticación de Línea Celular , Células Estrelladas Hepáticas , Animales , Línea Celular , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Ratones , ARN Mensajero/metabolismo , RatasRESUMEN
Lipocalin 2 (LCN2) mediates key roles in innate immune responses. It has affinity for many lipophilic ligands and binds various siderophores, thereby limiting bacterial growth by iron sequestration. Furthermore, LCN2 protects against obesity and metabolic syndrome by interfering with the composition of gut microbiota. Consequently, complete or hepatocyte-specific ablation of the Lcn2 gene is associated with higher susceptibility to bacterial infections. In the present study, we comparatively profiled microbiota in fecal samples of wild type and Lcn2 null mice and show, in contrast to previous reports, that the quantity of DNA in feces of Lcn2 null mice is significantly lower than that in wild type mice (p < 0.001). By using the hypervariable V4 region of the 16S rDNA gene and Next-Generation Sequencing methods, we found a statistically significant change in 16 taxonomic units in Lcn2-/- mice, including eight gender-specific deviations. In particular, members of Clostridium, Escherichia, Helicobacter, Lactococcus, Prevotellaceae_UCG-001 and Staphylococcus appeared to expand in the intestinal tract of knockout mice. Interestingly, the proportion of Escherichia (200-fold) and Staphylococcus (10-fold) as well as the abundance of intestinal bacteria encoding the LCN2-sensitive siderphore enterobactin (entA) was significantly increased in male Lcn2 null mice (743-fold, p < 0.001). This was accompanied by significant higher immune cell infiltration in the ileum as demonstrated by increased immunoreactivity against the pan-leukocyte protein CD45, the lymphocyte transcription factor MUM-1/IRF4, and the macrophage antigen CD68/Macrosialin. In addition, we found a higher expression of mucosal mast cell proteases indicating a higher number of those innate immune cells. Finally, the ileum of Lcn2 null mice displayed a high abundance of segmented filamentous bacteria, which are intimately associated with the mucosal cell layer, provoking epithelial antimicrobial responses and affecting T-helper cell polarization.
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Bacterias/clasificación , Disbiosis/microbiología , Lipocalina 2/genética , Mutación con Pérdida de Función , Análisis de Secuencia de ADN/métodos , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , ADN Bacteriano/genética , ADN Ribosómico/genética , Modelos Animales de Enfermedad , Disbiosis/genética , Disbiosis/inmunología , Heces/microbiología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Ratones , Ratones Noqueados , Filogenia , ARN Ribosómico 16S/genética , Factores SexualesRESUMEN
Transforming growth factor-ß1 (TGF-ß1) is a pleiotropic factor sensed by most cells. It regulates a broad spectrum of cellular responses including hematopoiesis. In order to process TGF-ß1-responses in time and space in an appropriate manner, there is a tight regulation of its signaling at diverse steps. The downstream signaling is mediated by type I and type II receptors and modulated by the 'accessory' receptor Endoglin also termed cluster of differentiation 105 (CD105). Endoglin was initially identified on pre-B leukemia cells but has received most attention due to its high expression on activated endothelial cells. In turn, Endoglin has been figured out as the causative factor for diseases associated with vascular dysfunction like hereditary hemorrhagic telangiectasia-1 (HHT-1), pre-eclampsia, and intrauterine growth restriction (IUPR). Because HHT patients often show signs of inflammation at vascular lesions, and loss of Endoglin in the myeloid lineage leads to spontaneous inflammation, it is speculated that Endoglin impacts inflammatory processes. In line, Endoglin is expressed on progenitor/precursor cells during hematopoiesis as well as on mature, differentiated cells of the innate and adaptive immune system. However, so far only pro-monocytes and macrophages have been in the focus of research, although Endoglin has been identified in many other immune system cell subsets. These findings imply a functional role of Endoglin in the maturation and function of immune cells. Aside the functional relevance of Endoglin in endothelial cells, CD105 is differentially expressed during hematopoiesis, arguing for a role of this receptor in the development of individual cell lineages. In addition, Endoglin expression is present on mature immune cells of the innate (i.e., macrophages and mast cells) and the adaptive (i.e., T-cells) immune system, further suggesting Endoglin as a factor that shapes immune responses. In this review, we summarize current knowledge on Endoglin expression and function in hematopoietic precursors and mature hematopoietic cells of different lineages.
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Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Diferenciación Celular , Endoglina/genética , Endoglina/metabolismo , Hematopoyesis , Inflamación/etiología , Inflamación/metabolismo , Animales , Diferenciación Celular/genética , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Hematopoyesis/genética , HumanosRESUMEN
INTRODUCTION: Hepatic fibrosis is the excessive synthesis and deposition of extracellular matrix including collagen in the tissue. Chronic liver insult leads to progressive parenchymal damage, portal hypertension, and cirrhosis. Determination of hepatic collagen by invasive liver biopsy is the gold standard to estimate severity and stage of fibrosis. However, this procedure is associated with pain, carries the risk of infection and bleeding, and is afflicted with a high degree of sampling error. Therefore, there is urgent need for serological collagen-derived markers to assess collagen synthesis/turnover. AREAS COVERED: Biochemical properties of collagens, cellular sources of hepatic collagen synthesis, and regulatory aspects in collagen expression. Markers are discussed suitable to estimate hepatic collagen synthesis and/or turnover. Discussed studies were identified through a PubMed search done in May 2020 and the authors' topic knowledge. EXPERT OPINION: Hepatic fibrosis is mainly characterized by accumulation of collagen-rich scar tissue. Although traditionally performed liver biopsy is still standard in estimating hepatic fibrosis, there is evidence that noninvasive diagnostic scores and collagen-derived neo-epitopes provide clinical useful information. These noninvasive tests are less expensive than liver biopsy, better tolerated, safer, and more acceptable to patients. Therefore, these tests will lead to dramatic changes in diagnosis.
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Biomarcadores , Colágeno/metabolismo , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/metabolismo , Animales , Biopsia , Toma de Decisiones Clínicas , Colágeno/sangre , Colágeno/genética , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Cirrosis Hepática/etiología , Pronóstico , Índice de Severidad de la EnfermedadRESUMEN
Chemoresistance is a multifactorial and complex phenomenon, leading to re-adjustment of several intracellular signaling pathways and expression patterns which compromises the efficacy of cancer drug chemo-therapy. Via comparative analysis of sensitive and doxorubicin-resistant 4T1 cells, here we show that Lipocalin 2 (LCN2) is downregulated at the mRNA and protein level in resistant cells. The pro-inflammatory cytokine, IL-1ß was found to be a potent inducer of LCN2 expression most likely involving STAT3 activation. Upregulation in both sensitive and resistant 4T1 cells argues against complete silencing of the LCN2 gene. Coinciding with LCN2 downregulation, we observed an increased activation of bone morphogenetic protein (BMP)-signaling in resistant cells, as evidenced by higher Smad1/5/9 phosphorylation and Id1 target gene expression. Blockade of the BMP-pathway by Dorsomorphin increased the expression of LCN2. Conversely, BMP2, which is known to be a pro-tumorigenic ligand in breast cancer, potently inhibited LCN2 expression at both the mRNA and protein level in resistant cells. These findings indicate that in doxorubicin-resistant 4T1 cells, LCN2 expression is negatively regulated by BMP signaling.
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Proteína Morfogenética Ósea 2/metabolismo , Lipocalina 2/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Proteína Smad1/metabolismoRESUMEN
Mast cells (MCs) are immune cells of the myeloid lineage that are present in the connective tissue throughout the body and in mucosa tissue. They originate from hematopoietic stem cells in the bone marrow and circulate as MC progenitors in the blood. After migration to various tissues, they differentiate into their mature form, which is characterized by a phenotype containing large granules enriched in a variety of bioactive compounds, including histamine and heparin. These cells can be activated in a receptor-dependent and -independent manner. Particularly, the activation of the high-affinity immunoglobulin E (IgE) receptor, also known as FcεRI, that is expressed on the surface of MCs provoke specific signaling cascades that leads to intracellular calcium influx, activation of different transcription factors, degranulation, and cytokine production. Therefore, MCs modulate many aspects in physiological and pathological conditions, including wound healing, defense against pathogens, immune tolerance, allergy, anaphylaxis, autoimmune defects, inflammation, and infectious and other disorders. In the liver, MCs are mainly associated with connective tissue located in the surrounding of the hepatic arteries, veins, and bile ducts. Recent work has demonstrated a significant increase in MC number during hepatic injury, suggesting an important role of these cells in liver disease and progression. In the present review, we summarize aspects of MC function and mediators in experimental liver injury, their interaction with other hepatic cell types, and their contribution to the pathogenesis of fibrosis.
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Susceptibilidad a Enfermedades , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Animales , Biomarcadores , Comunicación Celular , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Humanos , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación , Hígado/inmunología , Hígado/patología , Cirrosis Hepática/patología , Cirrosis Hepática/terapia , Hepatopatías/etiología , Hepatopatías/metabolismo , Hepatopatías/patología , Mastocitos/efectos de los fármacos , Transducción de SeñalRESUMEN
Lipocalin 2 (LCN2) is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.
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The vast majority of colorectal cancer-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFß signaling activity, and reduced expression of the miR-371â¼373 cluster compared with nonmetastatic cultures. TGFß receptor 2 (TGFBR2) and aldehyde dehydrogenase A1 (ALDH1A1) were identified as important target genes of the miR-371â¼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371â¼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced in vitro self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity in vivo following stable overexpression of the miR-371â¼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371â¼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371â¼373 and ID1. Altogether, our results establish the miR-371â¼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization.Significance: These findings establish the miR-371â¼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg Cancer Res; 78(14); 3793-808. ©2018 AACR.
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Neoplasias del Colon/genética , Neoplasias del Colon/patología , Proteína 1 Inhibidora de la Diferenciación/genética , MicroARNs/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Transducción de Señal/genética , Animales , Línea Celular Tumoral , Autorrenovación de las Células/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/genética , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/patologíaRESUMEN
We have identified a phenomenon occurring in the usage of proposed "specific" Mitogen-activated protein kinase (MAPK) inhibitors. We found that especially inhibitors of p38 potentiate the activation of other MAPKs in various cell types. This finding will have tremendous impact on the interpretation of all former studies using MAPK inhibitors.
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ITIH5 has been proposed being a novel tumor suppressor in various tumor entities including breast cancer. Recently, ITIH5 was furthermore identified as metastasis suppressor gene in pancreatic carcinoma. In this study we aimed to specify the impact of ITIH5 on metastasis in breast cancer. Therefore, DNA methylation of ITIH5 promoter regions was assessed in breast cancer metastases using the TCGA portal and methylation-specific PCR (MSP). We reveal that the ITIH5 upstream promoter region is particularly responsible for ITIH5 gene inactivation predicting shorter survival of patients. Notably, methylation of this upstream ITIH5 promoter region was associated with disease progression, for example, abundantly found in distant metastases. In vitro, stably ITIH5-overexpressing MDA-MB-231 breast cancer clones were used to analyze cell invasion and to identify novel ITIH5-downstream targets. Indeed, ITIH5 re-expression suppresses invasive growth of MDA-MB-231 breast cancer cells while modulating expression of genes involved in metastasis including Endoglin (ENG), an accessory TGF-ß receptor, which was furthermore co-expressed with ITIH5 in primary breast tumors. By performing in vitro stimulation of TGF-ß signaling using TGF-ß1 and BMP-2 we show that ITIH5 triggered a TGF-ß superfamily signaling switch contributing to downregulation of targets like Id1, known to endorse metastasis. Moreover, ITIH5 predicts longer overall survival (OS) only in those breast tumors that feature high ENG expression or inversely regulated ID1 suggesting a clinical and functional impact of an ITIH5-ENG axis for breast cancer progression. Hence, we provide evidence that ITIH5 may represent a novel modulator of TGF-ß superfamily signaling involved in suppressing breast cancer metastasis.
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Neoplasias de la Mama/genética , Endoglina/genética , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Metilación de ADN/genética , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Regiones Promotoras Genéticas/genética , RiesgoRESUMEN
BACKGROUND & AIMS: Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor-ß (TGF-ß) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF-ß type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC-specific endoglin deletion. METHODS: Eng expression was measured in human and murine samples of liver injury. After generating GFAPCre(+) EngΔHSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Engflox/flox HSCs were infected with Cre-expressing virus to deplete Endoglin and fibrogenic responses were analysed. RESULTS: Endoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L-Eng splice variant. Comparing GFAPCre(-) Engf/f to GFAPCre(+) EngΔHSC mice in toxic liver injury, livers of GFAPCre(+) EngΔHSC mice showed 39.9% (P < .01) higher Hydroxyproline content compared to GFAPCre(-) Engf/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P < .05) more Collagen deposition in livers of GFAPCre(+) EngΔHSC mice. Similar results were obtained in mice subjected to cholestatic injury. CONCLUSION: Endoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α-SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF-ß signalling.
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Endoglina/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Modelos Animales de Enfermedad , Endoglina/genética , Fibronectinas/metabolismo , Humanos , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores Protectores , Transducción de SeñalRESUMEN
BACKGROUND: Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive. METHODS: ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement. RESULTS: ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards ß1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility. CONCLUSIONS: Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Metilación de ADN , Proteínas Quinasas Asociadas a Muerte Celular/genética , Neoplasias Pulmonares/secundario , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Animales , Línea Celular Tumoral , Epigénesis Genética , Matriz Extracelular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Ratones , Trasplante de Neoplasias , Pronóstico , Análisis de SupervivenciaRESUMEN
SCOPE: Regulatory T cells (Treg) play a pivotal role in immune regulation. For proper immune function, also trace elements such as zinc, and anti-inflammatory cytokines, including transforming growth factor beta 1 (TGF-ß1) and interleukin (IL)-10 are indispensable. Hence, in this study the influence of TGF-ß1, IL-10, and zinc supplementation on Treg cells differentiation was investigated. METHODS AND RESULTS: A synergistic effect of a combined zinc and TGF-ß1 treatment on Foxp3 expression in peripheral blood mononuclear cells and mixed lymphocyte cultures (MLC) was found by performing Western blot analysis. Additionally, combined treatment causes elevated Smad 2/3 phosphorylation, which plays an important role in Foxp3 expression. This is due to a TGF-ß1-mediated increase of intracellular-free zinc measured by zinc probes Fluozin3-AM and ZinPyr-1. Moreover, zinc as well as TGF-ß1 treatment caused significantly reduced interferon (IFN)-γ secretion in MLC. CONCLUSION: Combined zinc and TGF-ß1 treatment provoked an increased Treg cell induction due to a triggered intracellular zinc signal, which in association with an increased Smad 2/3 activation leads to a boosted Foxp3 expression and resulting in an ameliorated allogeneic reaction in MLC. Thus, zinc can be used as a favorable additive to elevate the induction of Treg cells in adverse immune reactions.
Asunto(s)
Linfocitos T Reguladores/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Zinc/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Suplementos Dietéticos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-10/farmacología , Transducción de Señal , Proteínas Smad/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Zinc/metabolismoRESUMEN
The endoplasmic reticulum (ER) is primarily recognized as the site of synthesis and folding of secreted membrane-bound and certain organelle-targeted proteins. Optimum protein folding requires several factors, including ATP, Ca2+ and an oxidizing environment to allow disulphide-bond formation. ER is highly sensitive to stress that perturb cellular energy levels, the redox state or the Ca2+ concentration. Such stresses reduce the protein folding capacity of the ER, resulting in the accumulation and aggregation of unfolded proteins, a condition referred to as unfolded protein response (UPR). Matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover. They exhibit a similar type of organization and share a closely related primary structure, including 38 conserved cysteine residues. Since CCN1/CYR61 overexpression in hepatic stellate cells (HSC) induces ER stress-related apoptosis, we endeavored to investigate whether the adenovirus mediated gene transfer of other members of CCN proteins incurs ER stress in primary HSC and hepatocytes. We found Ad5-CMV-CCN2, Ad5-CMV-CCN3 and Ad5-CMV-CCN4 to induce ER stress and UPR comparable to Ad5-CMV-CCN1. UPR is a pro-survival response to reduce accumulation of unfolded proteins and restore normal ER functioning. If, however protein aggregation is persistent and the stress cannot be resolved, signaling switches from pro-survival to pro-apoptosis. The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. Adenoviral gene transfer induced massive amounts of matricellular proteins proving to effectively mitigate liver fibrosis if targeted cell specific in HSC and myofibroblasts.