Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells

Role of fibronectin deposition in cystogenesis of Madin-Darby canine kidney cells.BackgroundMadin-Darby canine kidney (MDCK) cells cultured within collagen I gel exhibit clonal growth and form spherical multicellular cysts. The cyst-lining epithelial cells are polarized with the basolateral surface in contact with the collagen gel and the apical surface facing the lumen. To understand whether MDCK cysts construct the basal lamina, we characterized the composition of the extracellular matrix deposited by MDCK cysts. The cyst-lining cells produced an apparently incomplete basal lamina containing a discontinuous laminin substratum. In addition, the basal cell surface of the cyst was surrounded by a thick layer of fibronectin. This study was conducted to delineate the role of fibronectin deposition in cystogenesis.MethodsMDCK cells cultured in collagen gel were employed. We first used Arg-Gly-Asp (RGD) peptides containing disintegrin rhodostomin to disturb the interaction between fibronectin and the cell surface integrin. We then established several stable transfectants expressing the fibronectin antisense RNA and with which to directly examine the role of fibronectin in cystogenesis.ResultsRhodostomin markedly decreased the growth rates of the MDCK cyst, suggesting the importance of a normal interaction between fibronectin and integrins. The stable transfectants overexpressing the fibronectin antisense RNA exhibited relatively lower levels of fibronectin and markedly lower cyst growth rates than the control clone. The lower growth rate was correlated with an increase in collagen gel-induced apoptosis.ConclusionsThe results indicate that the deposition of fibronectin underlying the cyst-lining epithelium serves to prevent apoptosis induced by three-dimensional collagen gel cultures, and hence facilitates cyst growth of MDCK cells

Deltex1 Is a Target of the Transcription Factor NFAT that Promotes T Cell Anergy

SummaryThe molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 β (Gadd45β) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy

Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology

The syndecan Sds2 and the innate immunity inhibitor Sarm1 function together and in distinct pathways to promote proper neuronal morphogenesis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Do Corrective Effects Last? Results from a Longitudinal Experiment on Beliefs toward Immigration in the U.S.

Replication data and code for all analyses in the main text and supplementary file for: "Do Corrective Effects Last? Results from a Longitudinal Experiment on Beliefs toward Immigration in the U.S.

Role of α3β1 integrin in tubulogenesis of Madin-Darby canine kidney cells

Role of α3β1 integrin in tubulogenesis of Madin-Darby canine kidney cells.BackgroundWe isolated several Madin-Darby canine kidney (MDCK) subclones that exhibit different degrees of branching tubulogenesis in lower concentrations of collagen gel. The M634 clone formed cell aggregates in 0.3% collagen gel, but developed branching tubules vigorously in 0.1% collagen gel. In contrast, the Y224 clone formed cysts in 0.3% collagen gel and displayed fewer branching structures in 0.1% collagen gel. Morphologically, M634 cells exhibited higher levels of cell scattering as well as collagen-induced cell migration than Y224. We conducted this study to delineate the underlying mechanism of branching tubulogenesis in M634 cells.MethodsComponents of the focal contact machinery were analyzed in both cell lines, including the extracellular matrix glycoproteins fibronectin, laminin, and vitronectin; cytoskeleton-associated elements α-actinin, talin, and vinculin; and receptors for extracellular matrix and α2, α3, α5, αv, β1, and β3 integrins. Furthermore, we established several stable transfectants of α3 integrin antisense RNA in M634 cells to examine the role of α3β1 integrin in branching morphogenesis directly.ResultsThere were no obvious differences in levels of the focal adhesion complex proteins between M634 and Y224 cells, except that the content of the α3 and β1 integrins were 1.2- and 0.6-fold higher in M634 cells, respectively. The expression of α3 integrin antisense RNA significantly lowered the levels of α3 integrin mRNA and protein. The potential of cell scattering, migration, and branching tubulogenesis in M634 cells was inhibited according to the decrease in α3 integrin expression.ConclusionOur data indicate that expression of α3β1 integrin regulates cell scattering, migration, and branching tubulogenesis of MDCK cells, possibly via adhesion to or serving as a signaling molecule for type I collagen

Establishing F1A-CreER^T2 Mice to Trace <i>Fgf1</i> Expression in Adult Mouse Cardiomyocytes

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo