Disruption of E-cadherin by matrix metalloproteinase directly mediates epithelial-mesenchymal transition downstream of transforming growth factor-beta1 in renal tubular epithelial cells.
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ABSTRACT:
SUBMITTER: Zheng G
PROVIDER: S-EPMC2716958 | biostudies-literature | 2009 Aug
REPOSITORIES: biostudies-literature
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Project description:As a rich source of pro-fibrogenic growth factors and matrix metalloproteinases (MMPs), macrophages are well-placed to play an important role in renal fibrosis. However, the exact underlying mechanisms and the extent of macrophage involvement are unclear. Tubular cell epithelial-mesenchymal transition (EMT) is an important contributor to renal fibrosis and MMPs to induction of tubular cell EMT. The aim of this study was to investigate the contribution of macrophages and MMPs to induction of tubular cell EMT. The murine C1.1 tubular epithelial cell line and primary tubular epithelial cells were cultured in activated macrophage-conditioned medium (AMCM) derived from lipopolysaccharide-activated J774 macrophages. MMP-9, but not MMP-2 activity was detected in AMCM. AMCM-induced tubular cell EMT in C1.1 cells was inhibited by broad-spectrum MMP inhibitor (GM6001), MMP-2/9 inhibitor, and in AMCM after MMP-9 removal by monoclonal Ab against MMP-9. AMCM-induced EMT in primary tubular epithelial cells was inhibited by MMP-2/9 inhibitor. MMP-9 induced tubular cell EMT in both C1.1 cells and primary tubular epithelial cells. Furthermore, MMP-9 induced tubular cell EMT in C1.1 cells to an extent similar to transforming growth factor-beta. Transforming growth factor-beta-induced tubular cell EMT in C1.1 cells was inhibited by MMP-2/9 inhibitor. Our in vitro study provides evidence that MMPs, specifically MMP-9, secreted by effector macrophages can induce tubular cell EMT and thereby contribute to renal fibrosis.
| S-EPMC2832147 | biostudies-literature
Project description:Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin.
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Project description:BackgroundSex differences in idiopathic pulmonary fibrosis (IPF) suggest a protective role for estrogen (E2); however, mechanistic studies in animal models have produced mixed results. Reports using cell lines have investigated molecular interactions between transforming growth factor beta1 (TGF-β1) and estrogen receptor (ESR) pathways in breast, prostate, and skin cells, but no such interactions have been described in human lung cells. To address this gap in the literature, we investigated a role for E2 in modulating TGF-β1-induced signaling mechanisms and identified novel pathways impacted by estrogen in bronchial epithelial cells.MethodsWe investigated a role for E2 in modulating TGF-β1-induced epithelial to mesenchymal transition (EMT) in bronchial epithelial cells (BEAS-2Bs) and characterized the effect of TGF-β1 on ESR mRNA and protein expression in BEAS-2Bs. We also quantified mRNA expression of ESRs in lung tissue from individuals with IPF and identified potential downstream targets of E2 signaling in BEAS-2Bs using RNA-Seq and gene set enrichment analysis.ResultsE2 negligibly modulated TGF-β1-induced EMT; however, we report the novel observation that TGF-β1 repressed ESR expression, most notably estrogen receptor alpha (ESR1). Results of the RNA-Seq analysis showed that TGF-β1 and E2 inversely modulated the expression of several genes involved in processes such as extracellular matrix (ECM) turnover, airway smooth muscle cell contraction, and calcium flux regulation. We also report that E2 specifically modulated the expression of genes involved in chromatin remodeling pathways and that this regulation was absent in the presence of TGF-β1.ConclusionsCollectively, these results suggest that E2 influences unexplored pathways that may be relevant to pulmonary disease and highlights potential roles for E2 in the lung that may contribute to sex-specific differences.
| S-EPMC6117929 | biostudies-literature
Project description:PurposeCockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292).Materials and methodsmRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting.ResultsGCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration.ConclusionGCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.
| S-EPMC6240571 | biostudies-literature
Project description:Fibroblasts can be directly reprogrammed to induced renal tubular epithelial cells (iRECs) using four transcription factors. These engineered cells may be used for disease modeling, cell replacement therapy or drug and toxicity testing. Direct reprogramming induces drastic changes in the transcriptional landscape, protein expression, morphological and functional properties of cells. However, how the metabolome is changed by reprogramming and to what degree it resembles the target cell type remains unknown. Using untargeted gas chromatography-mass spectrometry (GC-MS) and targeted liquid chromatography-MS, we characterized the metabolome of mouse embryonic fibroblasts (MEFs), iRECs, mIMCD-3 cells, and whole kidneys. Metabolic fingerprinting can distinguish each cell type reliably, revealing iRECs are most similar to mIMCD-3 cells and clearly separate from MEFs used for reprogramming. Treatment with the cytotoxic drug cisplatin induced typical changes in the metabolic profile of iRECs commonly occurring in acute renal injury. Interestingly, metabolites in the medium of iRECs, but not of mIMCD-3 cells or fibroblast could distinguish treated and non-treated cells by cluster analysis. In conclusion, direct reprogramming of fibroblasts into renal tubular epithelial cells strongly influences the metabolome of engineered cells, suggesting that metabolic profiling may aid in establishing iRECs as in vitro models for nephrotoxicity testing in the future.
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Project description:BackgroundMatrix Metalloproteinase-9 (MMP-9) has been shown to play a key role in mediating inflammation and tissue damage in inflammatory bowel disease (IBD). In patients with IBD, the intestinal tight junction (TJ) barrier is compromised as characterized by an increase in intestinal permeability. MMP-9 is elevated in intestinal tissue, serum and stool of patients with IBD. Previous studies from our laboratory showed that MMP-9 causes an increase in intestinal epithelial TJ permeability and that the MMP-9 induced increase in intestinal permeability is an important pathogenic factor contributing to the development of intestinal inflammation in IBD. However, the intracellular mechanisms that mediate the MMP-9 modulation of intestinal barrier function remain unclear.AimsThe main aim of this study was to further elucidate the molecular mechanisms involved in MMP-9 induced increase in intestinal epithelial TJ permeability using Caco-2 monolayers as an in-vitro model system.ResultsMMP-9 induced increase in Caco-2 TJ permeability was associated with activation and cytoplasmic-to-nuclear translocation of NF-κB p65. Knocking-down NF-κB p65 by siRNA transfection prevented the MMP-9 induced expression of the NF-κB target gene IL-8, myosin light chain kinase (MLCK) protein expression, and subsequently prevented the increase in Caco-2 TJ permeability. In addition, the effect of MMP-9 on Caco-2 intestinal epithelial TJ barrier function was not mediated by apoptosis or necrosis.ConclusionOur data show that the MMP-9 induced disruption of Caco-2 intestinal epithelial TJ barrier function is regulated by NF-κB pathway activation of MLCK.
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Project description:Transforming growth factor-beta1 (TGF-beta) can be tumor suppressive, but it can also enhance tumor progression by stimulating the complex process of epithelial-to-mesenchymal transdifferentiaion (EMT). The signaling pathway(s) that regulate EMT in response to TGF-beta are not well understood. We demonstrate the acquisition of a fibroblastoid morphology, increased N-cadherin expression, loss of junctional E-cadherin localization, and increased cellular motility as markers for TGF-beta-induced EMT. The expression of a dominant-negative Smad3 or the expression of Smad7 to levels that block growth inhibition and transcriptional responses to TGF-beta do not inhibit mesenchymal differentiation of mammary epithelial cells. In contrast, we show that TGF-beta rapidly activates RhoA in epithelial cells, and that blocking RhoA or its downstream target p160(ROCK), by the expression of dominant-negative mutants, inhibited TGF-beta-mediated EMT. The data suggest that TGF-beta rapidly activates RhoA-dependent signaling pathways to induce stress fiber formation and mesenchymal characteristics.
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Project description:Matrix metalloproteinases (MMPs) degrade and modify the extracellular matrix (ECM) as well as cell-ECM and cell-cell contacts, facilitating detachment of epithelial cells from the surrounding tissue. MMPs play key functions in embryonic development and mammary gland branching morphogenesis, but they are also upregulated in breast cancer, where they stimulate tumorigenesis, cancer cell invasion and metastasis. MMPs have been investigated as potential targets for cancer therapy, but clinical trials using broad-spectrum MMP inhibitors yielded disappointing results, due in part to lack of specificity toward individual MMPs and specific stages of tumor development. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells take on the characteristics of invasive mesenchymal cells, and activation of EMT has been implicated in tumor progression. Recent findings have implicated MMPs as promoters and mediators of developmental and pathogenic EMT processes in the breast. In this review, we will summarize recent studies showing how MMPs activate EMT in mammary gland development and in breast cancer, and how MMPs mediate breast cancer cell motility, invasion, and EMT-driven breast cancer progression. We also suggest approaches to inhibit these MMP-mediated malignant processes for therapeutic benefit.
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Project description:Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors was determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test. We found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p-c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p-c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD. KEY MESSAGES: • ROS is triggered when reflux occurs. • ROS regulates the transcription factor c-Jun via the ERK pathway. • The increase in MMP-7 that induces LPRD is induced via the ROS/ERK/c-Jun pathway. • This study revealed for the first time the expression mechanism of MMP-7, which is one of the causes of LPRD.
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Project description:Renal tubular epithelial cells (TECs) undergoing partial epithelial-mesenchymal transition (pEMT) during renal fibrosis has been recognized as a featuring and detrimental event. However, the mechanism for redirecting the cell fate of pEMT remains unclear. Here we mapped the temporal expression trajectories of a series of EMT-related molecules in renal fibrosis. It revealed a unique expression profile of N-cadherin of initial rising and late dropdown, which is distinct from that of other mesenchymal markers. The transcription factor Foxk1, which serves as a negative regulator of the N-cadherin gene, was induced by TGF-β1 but was tightly regulated in the presence of JNK-associated leucine zipper protein (JLP). The loss of JLP resulted in Foxk1 induction, leading to N-cadherin downregulation and compromised cell viability. We propose a novel axis consisting of JLP/Foxk1/N-cadherin in shaping the EMT program and suggest JLP as the checkpoint of the EMT continuum during renal fibrosis progression.
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