Comparative analysis of human and mouse transcriptomes of Th17 cell priming

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models

ZO-1 is required for cytokinesis in NCI-H460 cells on fibronectin.

<p>(A) Confocal sections depicting the localization of GFP-ZO-1 during cell division. Numbers indicate minutes and arrow indicates the furrow. (B, D) Control or ZO-1 silenced NCI-H460 cells in cytokinesis stained with antibodies against tubulin and Plk-1 (B) or phalloidin (D). DAPI was used to visualize nuclear morphology. Arrows point to midbodies and arrowheads to ruffles. (C) Quantification of midbody structures that show normal or abnormal localization in control or ZO-1 silenced NCI-H460 cells (n = 142 cells, ***, p<0.005). (E) STED super-resolution micrographs of ZO-1 or control silenced cells stained with Atto647-phalloidin and α-tubulin antibody (12G10) followed by Anti-Mouse IgG - Mega 520 together with a regular confocal channel to observe dapi stained nuclei. The lower panel shows surface rendered images of the cells shown in the top panels and additional side view examples of other representative cells. Cells in panels A, B, and D were on 5 µg/ml fibronectin, while cells in panel E were on uncoated precision cover glasses for high performance microscopes. Arrows point to midbodies. All scale bars are 10 µm.</p

Model for the role of ZO-1, α5-integrin and PKCε in cytokinesis of epithelial NCI-H460 cells on fibronectin.

<p>During normal cytokinesis (left panel), PKCε-dependent phosphorylation of ZO-1 is required for interaction between α5-integrin and ZO-1. This interaction allows α5-integrin to mediate adhesion to fibronectin in the cleavage furrow, and this adhesion is required for successful cytokinesis. In addition, the ZO-1/α5-integrin complex contributes to actin assembly in the furrow. Cells that lack ZO-1 (right panel) fail to accumulate α5-integrin in the furrow which correlates with impaired cytokinesis. In addition, actin structures in the cleavage furrow of cells lacking ZO-1 are disorganized.</p

A ZO-1/α5β1-Integrin Complex Regulates Cytokinesis Downstream of PKCε in NCI-H460 Cells Plated on Fibronectin

<div><p>Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.</p></div

ZO-1 PDZ2 binds PI(4,5)P2 and α5-integrin.

<p>(A) NCI-H460 cells in cytokinesis expressing low level of PI(4,5)P2 binding PH-PLCδ-GFP and stained as indicated. Arrows indicate furrow. (B, D) ELISA assay for interaction between recombinant WT (B, D) or K253A (D) GST-ZO-1 PDZ1-3 or PDZ2 domains and α5-integrin peptides: α5 WT (PPATSDA) and α5 PPAA (AAATSDA). (C) Surface plasmon resonance measurements of interaction between GST-ZO-1-PDZ1-3, with (blue dotted line) or without pre-incubation (red line) with α5 WT peptide, and PI(4,5)P2. Binding of GST-ZO-1-PDZ1-3 to PI(4,5)P2 was corrected for non-specific binding. (E) NCI-H460 cells in cytokinesis expressing GFP-ZO-1-WT or lipid-binding deficient GFP-ZO-1K253A and stained for α5-integrin. Cells in panel A were growing on fibronectin coated coverslips, while cells in panel E were on uncoated coverslips. Arrows indicate the furrow. All scale bars are 10 µm.</p

ZO-1- α5-integrin complex formation is required for cytokinesis.

<p>(A) Time lapse imaging (1 frame/10 min for 4 h) of control and α5-integrin silenced NCI-H460 cells. (B) Micrograph of α5-integrin silenced NCI-H460 cells stained as indicated. (C) NCI-H460 cell in cytokinesis stained as indicated. (D) α5-integrin silenced NCI-H460 cells expressing siRNA resistant WT or PPAA (unable to bind ZO-1) α5-integrin in cytokinesis stained as indicated. All cells were plated on fibronectin and dapi was used to visualize nuclear morphology. (E) TIRF (red) and epifluorescence (green) microscopy of ZO-1 or control silenced NCI-H460 cells stained for active β1-integrin during cytokinesis. All cells were growing on 5 µg/ml fibronectin. All arrows indicate the cleavage furrow. All scale bars are 10 µm.</p

PKCε and ZO-1 phosphorylation are necessary for cytokinesis.

<p>(A) Immunostaining of NCI-H460 cells treated with or without 2 µM PKC inhibitor Calphostin C stained as indicated. (B) Immunostaining of control or PKCε silenced NCI-H460 cells in cytokinesis stained as indicated. (C) Time-lapse imaging of control and PKCε silenced NCI-H460 cells (1 frame/min for 30 minutes). (D) Immunostaining of NCI-H460 cells expressing WT or phosphomutants S168A or S168D ZO-1-Flag in cytokinesis stained as indicated. DAPI was used to visualize nuclear morphology. All cells were growing on 5 µg/ml fibronectin. Arrows indicate the furrow. Arrowheads indicate nuclei (B) or FLAG-ZO-1 localization (D). All scale bars are 10 µm.</p

Comparative analysis of human and mouse transcriptomes of Th17 cell priming

Uncontrolled Th17 cell activity is associated with cancer and autoimmune and inflammatory diseases. To validate the potential relevance of mouse models of targeting the Th17 pathway in human diseases we used RNA sequencing to compare the expression of coding and non-coding transcripts during the priming of Th17 cell differentiation in both human and mouse. In addition to already known targets, several transcripts not previously linked to Th17 cell polarization were found in both species. Moreover, a considerable number of human-specific long non-coding RNAs were identified that responded to cytokines stimulating Th17 cell differentiation. We integrated our transcriptomics data with known disease-associated polymorphisms and show that conserved regulation pinpoints genes that are relevant to Th17 cell-mediated human diseases and that can be modelled in mouse. Substantial differences observed in non-coding transcriptomes between the two species as well as increased overlap between Th17 cell-specific gene expression and disease-associated polymorphisms underline the need of parallel analysis of human and mouse models. Comprehensive analysis of genes regulated during Th17 cell priming and their classification to conserved and non-conserved between human and mouse facilitates translational research, pointing out which candidate targets identified in human are worth studying by using in vivo mouse models.Peer reviewe