BMC Cell Biology - Latest Articles
The latest research articles published by BMC Cell Biology

Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line w...
by Taila HartleyMadura SivaElida LaiTracy TeodoroLiling ZhangAllen Volchuk
25 Jul 2010 at 6:00pm
Background: Cells respond to endoplasmic reticulum stress (ER) stress by activating the unfolded protein response. To study the ER stress response in pancreatic beta-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y), which prevents normal proinsulin folding causing ER stress and eventual beta-cell apoptosis. A double-stable pancreatic beta-cell line (pTet-ON INS-1) with inducible expression of insulin 2 (C96Y) fused to EGFP was generated to study the ER stress response. Results: Expression of Ins 2 (C96Y)-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6) and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3) following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival. Conclusions: The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in beta-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.
Peptide aptamers as new tools to modulate clathrin-mediated internalisation -...
by Rochana WickramasinghePaul Ko FerrignoChristian Roghi
22 Jul 2010 at 6:00pm
Background: Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo. Results: Here we report the isolation of a peptide aptamer named swiggle that interacts with the very short (21 amino acid long) intracellular domain of membrane type 1-metalloproteinase (MT1-MMP), a key cell surface protease involved in numerous and crucial physiological and pathological cellular events. Expression of swiggle in mammalian cells was found to increase the cell surface expression of MT1-MMP by impairing its internalisation. Swiggle interacts with the LLY573 internalisation motif of MT1-MMP intracellular domain, thus disrupting the interaction with the mu2 subunit of the AP2 internalisation complex required for endocytosis of the protease. Interestingly, swiggle-mediated inhibition of MT1-MMP clathrin-mediated internalisation was also found to promote MT1-MMP-mediated cell migration. Conclusions: Taken together, our results provide further evidence that peptide aptamers can be used to dissect molecular events mediated by individual protein domains, in contrast to the pleiotropic effects of RNA interference techniques.
Maged1, a new regulator of skeletal myogenic differentiation and muscle regen...
by Tuan NguyenMathieu BertrandChristiane SterpinYounes AchouriOlivier De Backer
19 Jul 2010 at 6:00pm
Background: In normal adult skeletal muscle, cell turnover is very slow. However, after an acute lesion or in chronic pathological conditions, such as primary myopathies, muscle stem cells, called satellite cells, are induced to proliferate, then withdraw definitively from the cell cycle and fuse to reconstitute functional myofibers. Results: We show that Maged1 is expressed at very low levels in normal adult muscle but is strongly induced after injury, during the early phase of myoblast differentiation. By comparing in vitro differentiation of myoblasts derived from wild-type or Maged1 knockout mice, we observed that Maged1 deficiency results in reduced levels of p21CIP1/WAP1, defective cell cycle exit and impaired myotube maturation. In vivo, this defect results in delayed regeneration of injured muscle. Conclusions: These data demonstrate for the first time that Maged1 is an important factor required for proper skeletal myoblast differentiation and muscle healing.
Hydrogen peroxide stimulates nuclear import of the POU homeodomain protein Oc...
by Peixiang WangTianru Jin
15 Jul 2010 at 6:00pm
Background: The ubiquitously expressed POU homeodomain protein Oct-1 serves as a sensor for stress induced by irradiation. We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP). The caudal homeobox gene Cdx-2 is a transactivator of proglucagon (gcg) and pro-insulin genes. Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression. Results: We show in this study that inducing oxidative stress by hydrogen peroxide (H2O2) increased nuclear Oct-1 content in both pancreatic and cell lines, as well as in a battery of other cells. This increase was then attributed to accelerated nuclear import of Oct-1, assessed by Fluorescence Recovery After Photobleaching (FRAP) using green fluorescence protein (EGFP) tagged Oct-1 molecule. H2O2 treatment was then shown to stimulate the activities of DNA-dependent protein kinase (DNA-PK) and c-jun N-terminal kinase (JNK). Finally, increased Oct-1 nuclear content upon H2O2 treatment in a pancreatic cell line was associated with reduced Cdx-2 and gcg mRNA expression. Conclusion: These observations suggest that Oct-1 functions as a sensor for both metabolic and stress/survival signaling pathways via altering its nuclear-cytoplasmic shuttling.
Characterization of Diaphanous-related formin FMNL2 in human tissues
by Maria GardbergKati TalvinenKatja KaipioKristiina IljinCaroline KampfMathias UhlenOlli Carpen
14 Jul 2010 at 6:00pm
Background: Diaphanous-related formins govern actin-based processes involved in many cellular functions, such as cell movement and invasion. Possible connections to developmental processes and cellular changes associated with malignant phenotype make them interesting study targets. In spite of this, very little is known of the tissue distribution and cellular location of any mammalian formin. Here we have carried out a comprehensive analysis of the formin family member formin -like 2 (FMNL2) in human tissues. Results: An FMNL2 antibody was raised and characterized. The affinity-purified FMNL2 antibody was validated by Western blotting, Northern blotting, a peptide competition assay and siRNA experiments. Bioinformatics-based mRNA profiling indicated that FMNL2 is widely expressed in human tissues. The highest mRNA levels were seen in central and peripheral nervous systems. Immunohistochemical analysis of 26 different human tissues showed that FMNL2 is widely expressed, in agreement with the mRNA profile. The widest expression was detected in the central nervous system, since both neurons and glial cells expressed FMNL2. Strong expression was also seen in many epithelia. However, the expression in different cell types was not ubiquitous. Many mesenchymal cell types showed weak immunoreactivity and cells lacking expression were seen in many tissues. The subcellular location of FMNL2 was cytoplasmic, and in some tissues a strong perinuclear dot was detected. In cultured cells FMNL2 showed mostly a cytoplasmic localization with perinuclear accumulation consistent with the Golgi apparatus. Furthermore, FMNL2 co-localized with F-actin to the tips of cellular protrusions in WM164 human melanoma cells. This finding is in line with FMNL2's proposed function in the formation of actin filaments in cellular protrusions, during amoeboid cellular migration. Conclusion: FMNL2 is expressed in multiple human tissues, not only in the central nervous system. The expression is especially strong in gastrointestinal and mammary epithelia, lymphatic tissues, placenta, and in the reproductive tract. In cultured melanoma cells, FMNL2 co-localizes with F-actin dots at the tips of cellular protrusions.
The individual-cell-based cryo-chip for the cryopreservation, manipulation an...
by Mordechai DeutschElena AfrimzonYaniv NamerYana ShafranMaria SobolevNaomi ZurgilAssaf DeutschSteffen HowitzMartin GreunerMichael ThaeleHeiko ZimmermannIna MeiserFriederike Ehrhart
6 Jul 2010 at 6:00pm
Background: Cryopreservation is the only widely applicable method of storing vital cells for nearly unlimited periods of time. Successful cryopreservation is essential for reproductive medicine, stem cell research, cord blood storage and related biomedical areas. The methods currently used to retrieve a specific cell or a group of individual cells with specific biological properties after cryopreservation are quite complicated and inefficient. Results: The present study suggests a new approach in cryopreservation, utilizing the Individual Cell-based Cryo-Chip (i3C). The i3C is made of materials having appropriate durability for cryopreservation conditions. The core of this approach is an array of picowells, each picowell designed to maintain an individual cell during the severe conditions of the freezing - thawing cycle and accompanying treatments. More than 97% of cells were found to retain their position in the picowells throughout the entire freezing - thawing cycle and medium exchange. Thus the comparison between pre-freezing and post-thawing data can be achieved at an individual cell resolution. The intactness of cells undergoing slow freezing and thawing, while residing in the i3C, was found to be similar to that obtained with micro-vials. However, in a fast freezing protocol, the i3C was found to be far superior. Conclusions: The results of the present study offer new opportunities for cryopreservation. Using the present methodology, the cryopreservation of individual identifiable cells, and their observation and retrieval, at an individual cell resolution become possible for the first time. This approach facilitates the correlation between cell characteristics before and after the freezing - thawing cycle. Thus, it is expected to significantly enhance current cryopreservation procedures for successful regenerative and reproductive medicine.
Arecoline induced disruption of expression and localization of the tight junc...
by Sarbani GiriKevin PoindexterShyam SundarGary Firestone
5 Jul 2010 at 6:00pm
Background: Approximately 600 million people chew Betel nut, making this practice the fourth most popular oral habit in the world. Arecoline, the major alkaloid present in betel nut is one of the causative agents for precancerous lesions and several cancers of mouth among those who chew betel nut. Arecoline can be detected in the human embryonic tissue and is correlated to low birth weight of newborns whose mothers chew betel nut during pregnancy, suggesting that arecoline can induce many systemic effects. However, few reports exist as to the effects of arecoline in human tissues other than oral cancer cell lines. Furthermore, in any system, virtually nothing is known about the cellular effects of arecoline treatment on membrane associated signaling components of human cancer cells. Results: Using the human Ishikawa endometrial cancer cell line, we investigated the effects of arecoline on expression, localization and functional connections between the ZO-1 tight junction protein and the HER2 EGF receptor family member. Treatment of Ishikawa cells with arecoline coordinately down-regulated expression of both ZO-1 and HER2 protein and transcripts in a dose dependent manner. Biochemical fractionation of cells as well as indirect immunofluorescence revealed that arecoline disrupted the localization of ZO-1 to the junctional complex at the cell periphery. Compared to control transfected cells, ectopic expression of exogenous HER2 prevented the arecoline mediated down-regulation of ZO-1 expression and restored the localization of ZO-1 to the cell periphery. Furthermore, treatment with dexamethasone, a synthetic glucocorticoid reported to up-regulate expression of HER2 in Ishikawa cells, precluded arecoline from down-regulating ZO-1 expression and disrupting ZO-1 localization. Conclusion: Arecoline is known to induce precancerous lesions and cancer in the oral cavity of betel nut users. The arecoline down-regulation of ZO-1 expression and subcellular distribution suggests that arecoline potentially disrupts cell-cell interactions mediated by ZO-1, which may play a role in arecoline-mediated carcinogenesis. Furthermore, our study has uncovered the dependency of ZO-1 localization and expression on HER2 expression, which has therefore established a new cellular link between HER2 mediated signaling and apical junction formation involving ZO-1.
The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an ...
by Huamei ForsmanClaes Dahlgren
5 Jul 2010 at 6:00pm
Background: The molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP2-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca2+. Results: The transient rise in intracellular Ca2+ induced by agonists for FPR1 or FPR2 in human neutrophils occurred also in the presence of a chelator of Ca2+ (EGTA). PBP10 inhibited not only FPR2-induced oxidase activity, but also the transient rise in intracellular Ca2+. Conclusions: Ca2+ signaling mediated via FPR2 follows the same route as FPR1, which involves initial emptying of the intracellular stores. PBP10 inhibits selectively the signals generated by FPR2, both with respect to NADPH-oxidase activity and the transient rise in intracellular Ca2+ induced by agonist exposure.
Monoclonal antibody 4C5 prevents activation of MMP2 and MMP9 by disrupting th...
by Dimitris StellasAvraam El HamidiehEvangelia Patsavoudi
4 Jul 2010 at 6:00pm
Background: Heat shock protein 90 (HSP90) is a molecular chaperone that is considered a new target for the treatment of cancer. Increasing data reveal an extracellular chaperoning activity for HSP90. Here we investigate the interaction of the secreted isoforms of HSP90 with matrix metalloproteinases (MMP) MMP2 and MMP9. Moreover we examine the role of a monoclonal antibody (mAb) against HSP90, mAb 4C5, regarding these interactions and its value as a potential inhibitor of human breast cancer cell invasion and metastasis. Results: Our results showed that both HSP90alpha and HSP90beta are secreted by MDAMB453 human breast cancer cells and interact with MMP2 and MMP9. MAb 4C5, while not affecting secretion of the inactive MMPs, inhibits their activation by disrupting their interaction extracellularly with both isoforms of HSP90. The in vivo studies revealed that mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells into the lungs of SCID mice. Conclusion: Both isoforms of HSP90 are secreted by MDAMB453 cells and interact with the MMP2 and MMP9. MAb 4C5 prevents MMP2 and MMP9 activation, by disrupting their interaction with HSP90. Finally mAb 4C5 significantly inhibits the metastatic deposit formation of MDAMB453 cells, by preventing their extravasation and infiltration in the lung tissue and therefore it could be used as a potential therapeutic agent for cancer metastasis.
Mouse lung contains endothelial progenitors with high capacity to form blood...
by Judith SchniedermannMoritz RenneckeKerstin ButtlerGeorg RichterAnna-Maria StadtlerSusanne NorgallMuhammad BadarBernhard BarleonTobias MayJorg WiltingHerbert Weich
30 Jun 2010 at 6:00pm
Background: Postnatal endothelial progenitor cells (EPCs) have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. Results: In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels.Mouse lung microvascular endothelial cells (MLMVECs) were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony). These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs) in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. Conclusion: The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

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