Elevated formation of pyridinoline cross-links by profibrotic cytokines is associated with enhanced lysyl hydroxylase 2b levels

AbstractThe hallmark of fibrosis is the excessive accumulation of collagen. The deposited collagen contains increased pyridinoline cross-link levels due to an overhydroxylation of lysine residues within the collagen telopeptides. Lysyl hydroxylase 2b (LH2b) is the only lysyl hydroxylase consistently up-regulated in several forms of fibrosis, suggesting that an enhanced LH2b level is responsible for the overhydroxylation of collagen telopeptides. The present paper reports the effect of profibrotic cytokines on the expression of collagen, lysyl hydroxylases and lysyl oxidase in normal human skin fibroblasts, as well as the effect on pyridinoline formation in the deposited matrix. All three isoforms of TGF-β induce a substantial increase in LH2b mRNA levels, also when expressed relatively to the mRNA levels of collagen type I α2 (COL1A2). The TGF-β isoforms also clearly influence the collagen cross-linking pathway, since higher levels of pyridinoline cross-links were measured. Similar stimulatory effects on LH2b/COL1A2 mRNA expression and pyridinoline formation were observed for IL-4, activin A, and TNF-α. An exception was BMP-2, which has no effect on LH2b/COL1A2 mRNA levels nor on pyridinoline formation. Our data show for the first time that two processes, i.e., up-regulation of LH2b mRNA levels and increased formation of pyridinoline cross-links, previously recognized to be inherent to fibrotic processes, are induced by various profibrotic cytokines

Mutations in the Polycomb Group Gene polyhomeotic Lead to Epithelial Instability in both the Ovary and Wing Imaginal Disc in Drosophila

Most human cancers originate from epithelial tissues and cell polarity and adhesion defects can lead to metastasis. The Polycomb-Group of chromatin factors were first characterized in Drosophila as repressors of homeotic genes during development, while studies in mammals indicate a conserved role in body plan organization, as well as an implication in other processes such as stem cell maintenance, cell proliferation, and tumorigenesis. We have analyzed the function of the Drosophila Polycomb-Group gene polyhomeotic in epithelial cells of two different organs, the ovary and the wing imaginal disc.Clonal analysis of loss and gain of function of polyhomeotic resulted in segregation between mutant and wild-type cells in both the follicular and wing imaginal disc epithelia, without excessive cell proliferation. Both basal and apical expulsion of mutant cells was observed, the former characterized by specific reorganization of cell adhesion and polarity proteins, the latter by complete cytoplasmic diffusion of these proteins. Among several candidate target genes tested, only the homeotic gene Abdominal-B was a target of PH in both ovarian and wing disc cells. Although overexpression of Abdominal-B was sufficient to cause cell segregation in the wing disc, epistatic analysis indicated that the presence of Abdominal-B is not necessary for expulsion of polyhomeotic mutant epithelial cells suggesting that additional polyhomeotic targets are implicated in this phenomenon.Our results indicate that polyhomeotic mutations have a direct effect on epithelial integrity that can be uncoupled from overproliferation. We show that cells in an epithelium expressing different levels of polyhomeotic sort out indicating differential adhesive properties between the cell populations. Interestingly, we found distinct modalities between apical and basal expulsion of ph mutant cells and further studies of this phenomenon should allow parallels to be made with the modified adhesive and polarity properties of different types of epithelial tumors

A mathematical model of the airway epithelium: Computational studies of ion and water balance

A mathematical model has been developed to investigate the time course of ion and water transport across airway epithelia. Model variables were intracellular and apical concentrations of sodium, potassium, chloride; apical, basolateral, and transepithelial membrane potentials; cell and apical volumes. Membrane transport processes included apical passive diffusion of sodium and chloride, basolateral passive diffusion of potassium, basolateral sodium-potassium active transport, basolateral sodium-potassium-chloride cotransport, water permeation across all membranes, and nonselective paracellular passive diffusion of sodium, potassium, and chloride. Initial conditions and transport parameters were derived from experimental results.To test the model, simulations were performed for epithelia in an Ussing chamber. The model predicted short-circuit currents, short-circuit ion fluxes, intracellular ion concentrations, and open circuit voltages consistent with experimental data.The model was used to investigate the behavior of airway epithelia in vivo. Simulations of the cystic fibrosis defect showed that decreased apical chloride permeability and increased apical sodium permeability both decrease water flow from the basal side toward the airway lumen. We also show that blockage of apical sodium channels could compensate completely for airway dehydration due to defective apical chloride channels of cystic fibrosis airway epithelia.Other simulations determined the role of the various ion transport mechanisms observed in this tissue. The basolateral potassium permeability maintains the apical membrane voltage and allows potassium to exit the cell. The basolateral sodium-potassium pump is necessary to maintain sodium and potassium gradients. The basolateral sodium-potassium-chloride cotransporter is essential for salt movement from the basal region to the cytoplasm to promote water flux across the basolateral membrane. The apical chloride channels allow chloride efflux, drawing water into the apical region to replace that lost by evaporation. The apical sodium channels allow sodium absorption from the apical side to the basal side of the tissue. The paracellular path permits movement of chloride, to recycle chloride which is secreted across the apical membrane, and movement of sodium, to replace sodium which is absorbed across the apical membrane. Finally, water permeation across cell membranes is vital for replacement of water lost from the periciliary fluid by evaporation.U of I OnlyETDs are only available to UIUC Users without author permissio

Impact of (Quasi-)Ballistic Transport on Operation of Complementary Metal-Oxide-Semiconductor Inverters Based on Fully-Depleted Silicon-on-Insulator and Nanowire Devices

International audienceWe have developed a numerical simulation methodology to analyze the impact of both device architecture and transport properties on static circuit performance. Firstly, to simulate ballistic transport, we have enhanced the pioneering approach of quasi-ballistic mobility proposed by Rhew and Lundstrom and introduced it into a technology computer aided design (TCAD) simulator. Secondly, we have introduced the remote Coulomb scattering (RCS) limited mobility into the effective mobility and calibrated our model using experimental mobility data. We have used our approach to simulate two ultimate architectures: fully-depleted (FD) silicon-on-insulator (SOI) metal-oxide-semiconductor field-effect transistor (MOSFET) (with either thin or thick buried oxide) and nanowire MOSFET. Finally, we have analyzed the impact of both transport properties and electrostatic control on the noise margin (NM) of complementary metal-oxide-semiconductor (CMOS) inverter. (C) 2011 The Japan Society of Applied Physic

Cooperation, cis-interactions, versatility and evolutionary plasticity of multiple cis-acting elements underlie krox20 hindbrain regulation.

Cis-regulation plays an essential role in the control of gene expression, and is particularly complex and poorly understood for developmental genes, which are subject to multiple levels of modulation. In this study, we performed a global analysis of the cis-acting elements involved in the control of the zebrafish developmental gene krox20. krox20 encodes a transcription factor required for hindbrain segmentation and patterning, a morphogenetic process highly conserved during vertebrate evolution. Chromatin accessibility analysis reveals a cis-regulatory landscape that includes 6 elements participating in the control of initiation and autoregulatory aspects of krox20 hindbrain expression. Combining transgenic reporter analyses and CRISPR/Cas9-mediated mutagenesis, we assign precise functions to each of these 6 elements and provide a comprehensive view of krox20 cis-regulation. Three important features emerged. First, cooperation between multiple cis-elements plays a major role in the regulation. Cooperation can surprisingly combine synergy and redundancy, and is not restricted to transcriptional enhancer activity (for example, 4 distinct elements cooperate through different modes to maintain autoregulation). Second, several elements are unexpectedly versatile, which allows them to be involved in different aspects of control of gene expression. Third, comparative analysis of the elements and their activities in several vertebrate species reveals that this versatility is underlain by major plasticity across evolution, despite the high conservation of the gene expression pattern. These characteristics are likely to be of broad significance for developmental genes

Pyrococcus horikoshii TET2 Peptidase Assembling Process and Associated Functional Regulation

Tetrahedral (TET) aminopeptidases are large polypeptide destruction machines present in prokaryotes and eukaryotes. Here, the rules governing their assembly into hollow 12-subunit tetrahedrons are addressed by using TET2 from Pyrococcus horikoshii (PhTET2) as a model. Point mutations allowed the capture of a stable, catalytically active precursor. Small angle x- ray scattering revealed that it is a dimer whose architecture in solution is identical to that determined by x- ray crystallography within the fully assembled TET particle. Small angle x- ray scattering also showed that the reconstituted PhTET2 dodecameric particle displayed the same quaternary structure and thermal stability as the wild-type complex. The PhTET2 assembly intermediates were characterized by analytical ultracentrifugation, native gel electrophoresis, and electron microscopy. They revealed that PhTET2 assembling is a highly ordered process in which hexamers represent the main intermediate. Peptide degradation assays demonstrated that oligomerization triggers the activity of the TET enzyme toward large polypeptidic substrates. Fractionation experiments in Pyrococcus and Halobacterium cells revealed that, in vivo, the dimeric precursor co-exists together with assembled TET complexes. Taken together, our observations explain the biological significance of TET oligomerization and suggest the existence of a functional regulation of the dimer-dodecamer equilibrium in vivo

Collaboration in <i>cis</i> between elements A and C for the control of autoregulation.

<p>Embryos carrying combinations of homozygous deletions of elements A (∆A), C (C*), D (D*), E (E*) and of heterozygous deletions of elements A (∆A/+) or C (∆C/+) were analysed for <i>krox20</i> expression by in situ hybridization at the indicated stages. The genotype (∆A/+ +/∆C) corresponds to heterozygous deletions of A and C affecting different chromosomes. Somatic deletions are indicated by the * symbol and positions of r3 and r5 are shown. Neural crest cells migrating from r5/r6 are indicated by an arrowhead.</p

Schematic of the <i>cis</i>-regulation of <i>krox20</i> expression in r3 and r5, illustrating differences between zebrafish and mouse.

<p><i>Cis</i>-acting elements are indicated by light blue boxes along the locus, with their position with respect to the site of transcription initiation underneath. The different types of activities of the elements are represented by arrows originating from the element: enhancer activities involved in the initiation of <i>krox20</i> expression are indicated by green arrows pointing toward the promoter, enhancer activities corresponding to direct autoregulation are indicated by blue arrows pointing back to the element and the potentiator activity of element C is represented by red arrows pointing toward element A. Question marks indicate that the activity is suspected, but not confirmed.</p

DNA accessibility and candidate enhancer sequences within and around the zebrafish <i>krox20</i> locus.

<p>UCSC genome browser view of the <i>krox20</i> locus showing gene positions (purple), repetitive sequences (black) and the sequences selected for enhancer activity tests (light blue), including those that showed activity (named A to F). Below are ATAC-seq data from experiments performed at the indicated stages, either on whole embryos (95% epiboly) or dissected hindbrain or posterior regions of the embryos (5s and 15s), as shown on the schematics on the right side. The seven mostly significant peaks located in non-coding sequences are highlighted in yellow. Underneath is a Vista browser view of sequence conservation between zebrafish and mouse (black) over the region.</p

Evolution of enhancer A activity in vertebrates.

<p>The orthologues of element A from 6 vertebrate species, zebrafish (zA), koi carp (kA), spotted gar (sA), <i>Xenopus laevis</i> (xA), chicken (cA) and mouse (mA) were transferred into a GFP reporter construct and the corresponding plasmids were used to generate zebrafish transgenic lines, as indicated. <i>GFP</i> expression was analysed by in situ hybridization at 8s in embryos from each line, either in wild type (WT) or <i>krox20</i> null (<i>krox20*</i>) backgrounds, the latter being obtained by injection of Cas9 protein together with guide RNAs targeting the coding sequence of Krox20’s zinc fingers. Positions of r3 and r5 are shown. A phylogenetic tree with the indication of the node time distances from the present in millions of years (MYA) is shown underneath.</p