Epithelial Cell Extracellular Matrix Research Articles

Recovery of ΔF508-CFTR Function by Citrate.

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells.

The HDAC2/SP1/miR-205 feedback loop contributes to tubular epithelial cell extracellular matrix production in diabetic kidney disease.

Extracellular matrix (ECM) accumulation is considered an important pathological feature of diabetic kidney disease (DKD). Histone deacetylase (HDAC) inhibitors protect against kidney injury. However, the potential mechanisms of HDACs in DKD are still largely unknown. Here, we describe a novel feedback loop composed of HDAC2 and miR-205 that regulates ECM production in tubular epithelial cells in individuals with DKD. We found that HDAC2 mRNA expression in peripheral blood was markedly higher in patients with DKD than in patients with diabetes. Nuclear HDAC2 protein expression was increased in TGFβ1-stimulated tubular epithelial cells and db/db mice. We also found that miR-205 was regulated by HDAC2 and down-regulated in TGFβ1-treated HK2 cells and db/db mice. In addition, HDAC2 reduced histone H3K9 acetylation in the miR-205 promoter region to inhibit its promoter activity and subsequently suppressed miR-205 expression through an SP1-mediated pathway. Furthermore, miR-205 directly targeted HDAC2 and inhibited HDAC2 expression. Intriguingly, miR-205 also regulated its own transcription by inhibiting HDAC2 and increasing histone H3K9 acetylation in its promoter, forming a feedback regulatory loop. Additionally, the miR-205 agonist attenuated ECM production in HK2 cells and renal interstitial fibrosis in db/db mice. In conclusion, the HDAC2/SP1/miR-205 feedback loop may be crucial for the pathogenesis of DKD.

Ameloblastin Binds to Phospholipid Bilayers via a Helix-Forming Motif within the Sequence Encoded by Exon 5.

Ameloblastin (Ambn), the most abundant non-amelogenin enamel protein, is intrinsically disordered and has the potential to interact with other enamel proteins and with cell membranes. Here, through multiple biophysical methods, we investigated the interactions between Ambn and large unilamellar vesicles (LUVs), whose lipid compositions mimicked cell membranes involved in epithelial cell-extracellular matrix adhesion. Using a series of Ambn Trp/Phe variants and Ambn mutants, we further showed that Ambn binds to LUVs through a highly conserved motif within the sequence encoded by exon 5. Synthetic peptides derived from different regions of Ambn confirmed that the sequence encoded by exon 5 is involved in LUV binding. Sequence analysis of Ambn across different species showed that the N-terminus of this sequence contains a highly conserved motif with a propensity to form an amphipathic helix. Mutations in the helix-forming sequence resulted in a loss of peptide binding to LUVs. Our in vitro data suggest that Ambn binds the lipid membrane directly through a conserved helical motif and have implications for biological events such as Ambn-cell interactions, Ambn signaling, and Ambn secretion via secretory vesicles.

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens.

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10(-10) M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H2O2 and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.

A microRNA-30e/mitochondrial uncoupling protein 2 axis mediates TGF-β1-induced tubular epithelial cell extracellular matrix production and kidney fibrosis

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-β1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-β1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3′-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-β1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-β1-induced tubular-cell epithelial–mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-β1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-β1-induced epithelial–mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

Abstract 5185: Evaluation of biomarker candidate genes for prostate cancer metastasis

Abstract Prostate cancer is the most frequently diagnosed malignancy in men and although effective treatment is possible for early stage disease, advanced prostate cancer has a very poor prognosis. Thus, identification of biomarkers that can accurately and reliably diagnose prostate cancer in its early stages is urgently needed. MatLyLu and Dunning G are rat prostate cancer cell lines which have different metastatic capabilities. Using microarray analysis we previously identified a set of metastasis-associated genes that are over-expressed in MatLyLu. Genes found to be differentially expressed include several previously associated with the metastatic process, but not previously reported in prostate cancer. We also have found differential expression of genes that code for several proteoglycans involved in epithelial cell-extracellular matrix interaction, cell adhesion molecules, genes related to motility, growth factor receptors, and tumor suppressor genes. We are currently validating the expression of these candidate genes in human prostate cancer cell lines with different metastatic abilities (LNCaP, PC3, PWR-1E) and in clinical samples of prostate cancer at different disease stages using quantitative real time PCR (qRT-PCR) and immunohistochemistry. A better understanding of the molecular events leading to the metastatic prostate cancer phenotype will help to further identify genes or gene-products involved in this process, which may prove useful as biomarkers for diagnosis and/or prognosis of the disease. Project code: 110745921483. Supported by Colciencias Grant # RC No.462-2008 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5185. doi:10.1158/1538-7445.AM2011-5185

Dumpy interacts with a large number of genes in the developing wing of Drosophila melanogaster

The complex Drosophila dumpy gene encodes a gigantic protein located in the apical extracellular matrix of epithelial cells. It has been shown to interact with several proteins notably during embryonic tracheal development. Here we examine Dumpy's interactions in vivo with mutations in 20 genes previously recovered in a screen for recessive lethals that generate blisters when somatic clones are produced by mitotic crossing over during wing development. Primarily using double mutants, we looked for both dominant effects of the wing blister mutants and the effects of blister mutant clones on dumpy expression. Sixteen of the mutants either suppressed or enhanced dumpy mutant phenotypes indicating the large Dumpy protein is a very important component of the epithelial extracellular matrix in the wing. Dumpy also interacts strongly with held out wings, which is involved in RNA localization and possibly alternative splicing.

Epilysin (MMP-28) is deposited to the basolateral extracellular matrix of epithelial cells

Epilysin (MMP-28) is a conserved member of the matrix metalloproteinase (MMP) family. It is expressed in various normal tissues, and induced in wounds and in developing and regenerating nerves. Epilysin induces TGF-β mediated epithelial to mesenchymal transition, but its other functions are largely unknown. We have characterized the localization of both catalytically active and mutated inactive, overexpressed epilysin in established epithelial cell lines. We found that epilysin was localized abundantly to the basolateral side of the cells and associated with the extracellular matrix (ECM) as verified by immunoblotting and confocal microscopy. Overexpression of epilysin in MDCK cells resulted in a drastic reduction of basolateral ECM, as observed by the disappearance of collagen type IV, laminin and fibronectin. Cultivation of epilysin expressing MDCK cells in defined serum free medium resulted in the restoration of these proteins to the ECM. The levels of fibronectin and collagen IV were, however, reduced in epilysin expressing cells under the serum free conditions, and degradation fragments of collagen IV were detected supporting the activation of proteolysis by epilysin. Epilysin was observed in its unprocessed 50 kDa active form in the ECM of MDCK cells under serum free conditions whereas in cells cultured in serum containing it was processed to the 48 kDa form. Current results indicate that epilysin associates with the basolateral ECM of cultured epithelial cells, where it plausibly plays a role in the regulation of matrix composition and turnover.

Epithelial Cell-Extracellular Matrix Interactions and Stem Cells in Airway Epithelial Regeneration

In healthy subjects, the respiratory epithelium forms a continuous lining to the airways and to the environment, and plays a unique role as a barrier against external deleterious agents to protect the airways from the insults. In respiratory diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), chronic bronchitis, or asthma, the airway epithelium is frequently remodeled and injured, leading to the impairment of its defense functions. The rapid restoration of the epithelial barrier is crucial for these patients. The complete regeneration of the airway epithelium is a complex phenomenon, including not only the epithelial wound repair but also the epithelial differentiation to reconstitute a fully well differentiated and functional epithelium. The regeneration implies two partners: the epithelial stem/progenitor cells and factors able to regulate this process. Among these factors, epithelial cells-extracellular matrix (ECM) interactions play a crucial role. The secretion of a provisional ECM, the cell-ECM relationships through epithelial receptors, and the remodeling of the ECM by proteases (mainly matrix metalloproteinases) contribute not only to airway epithelial repair by modulating epithelial cell migration and proliferation, but also to the differentiation of repairing cells leading to the complete restoration of the wounded epithelium. A better characterization of resident stem cells and of effectors of the regeneration process is an essential prerequisite to propose new regenerative therapeutics to patients suffering from infectious/inflammatory respiratory diseases.

Integrin-αvβ6, a Putative Receptor for Foot-and-Mouth Disease Virus, Is Constitutively Expressed in Ruminant Airways

Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.

Differential effects of transforming growth factors on localization of adhesion complex proteins following corneal epithelial cell wounding

PURPOSE. The differential effects of transforming growth factor (TGF) alpha, beta1 and beta2 on the de novo localization of heparan sulfate proteoglycan, collagen type VII and laminin-1 to the adhesion complex were analyzed using an in vitro model of corneal epithelial cell wound healing. METHODS. Bovine corneal explants were maintained in culture media containing either no growth factor or 1, 5, or 10 ng/ml TGFalpha, TGFbeta1 or TGFbeta2. After 24 or 48 hours in culture, cryostat sections of explants were processed for immuno-fluorescence microscopy using antibodies directed against heparan sulfate proteoglycan, collagen type VII or laminin-1. RESULTS. A comparison of antibody labeling patterns and relative fluorescence intensity of antibody labeling to controls suggested that TGFalpha inhibits the spatial polarization of proteins into the reforming adhesion complex during early stages of wound healing. Both TGFbeta1 and beta2 enhanced the linear localization of the three proteins to the site of the reforming adhesion complex. However, in our model TGFbeta isoforms did not have identical functions. TGFbeta2 accelerated the temporal localization of collagen type VII to the adhesion complex, an effect which was not observed with TGFbeta1. CONCLUSIONS. TGFbeta, but not TGFalpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

Treatment with chemopreventive agents, difluoromethylornithine and retinyl acetate, results in altered mammary extracellular matrix.

The effect of treatment with D-L-2-difluoromethylornithine (DFMO) plus retinyl acetate (RA) on the promotion stage of 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis was evaluated in female Sprague-Dawley rats. Combined treatment was more effective than single agent treatment in decreasing cancer incidence and multiplicity and in prolonging cancer latency. Increased efficacy was associated with reduced morphological complexity of the gland and increased mammary extracellular matrix. Using ovarian hormones to model mitogen stimulation in the mammary gland, DFMO plus RA treatment reduced mammary gland complexity in the absence of an effect on bromodeoxyuridine (BrDU) labeling index measured immunohistochemically. Morphological and biochemical evaluation of these glands revealed increased levels of extracellular matrix in rats treated with chemopreventive agents. Tenascin expression and fibronectin levels were elevated and laminin levels were decreased. The fact that matrix degrading proteinase activity was also increased indicated that tissue remodeling was modulated by these chemopreventive agents. These data provide evidence of alterations in epithelial cell-extracellular matrix interactions that could account in part for the chemopreventive effects of DFMO plus RA.

The National epidermolysis Bullosa Registry.

Since its inception in 1986, the NEBR has proved to be an excellent example of how a relatively small allocation of federal research funds for the development of a registry of cases of a single rare disease can have a major impact on the rapid expansion in the depth of knowledge of not only the disease itself but of a number of associated biologic principles, including keratinization and epithelial cell-extracellular matrix interactions. At present, the NEBR is generating extensive clinical, laboratory, and demographic data, both from cross-sectional and longitudinal perspectives, as well as establishing a centralized cell and tissue bank that will serve the scientific community at large as a valuable resource for future basic research on this oftentimes devastating genetic disease.

Basally located epithelial cell surface component identified by a novel monoclonal antibody technique

Tubular aggregates of glandular epithelial cells (gland fragments) were isolated from human endometrium by collagenase digestion of surrounding stroma, thus exposing the basal surfaces of the cells. Using these aggregates as immunogen, monoclonal antibodies could be derived that recognized basally located antigens. One such antibody, G71, is described, that binds to a basal epithelial cell antigen present in a variety of human epithelia. Epitope-bearing molecules in the range M r 60 000–180 000 are present in two of the tissues studied, amnion and endometrium. The epitope is associated with areas of epithelial cell-extracellular matrix contact.