Abnormal Wnt and PI3Kinase Signaling in the Malformed Intestine of lama5 Deficient Mice

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here

Glycated Polyelectrolyte Multilayer Films: Differential Adhesion of Primary versus Tumor Cells

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Inhibition of Sox2 Expression in the Adult Neural Stem Cell Niche In Vivo by Monocationic-based siRNA Delivery

RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ). This brain region contains the neural stem and progenitor cells populations that express the stem cell marker, SOX2. Temporally and spatially controlled Sox2 knockdown was achieved using the monocationic lipid vector, IC10. siRNA/IC10 complexes were stable over time and smaller (<40 nm) than jetSi complexes (≈400 nm). Immunocytochemistry showed that siRNA/IC10 complexes efficiently target both the progenitor and stem cell populations in the adult SVZ. Injection of the complexes into the lateral brain ventricle resulted in specific knockdown of Sox2 in the SVZ. Furthermore, IC10-mediated transient in vivo knockdown of Sox2-modulated expression of several genes implicated in NSC maintenance. Taken together, these data show that IC10 cationic lipid formulation can efficiently vectorize siRNA in a specific area of the adult mouse brain, achieving spatially and temporally defined loss of function

Conjugating Phosphospermines to siRNAs for Improved Stability in Serum, Intracellular Delivery and RNAi-Mediated Gene Silencing

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered <i>in vivo</i>. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand. Cationic siRNAs have been previously shown to display gene silencing activity in human cell line (Nothisen et al. <i>J. Am. Chem. Soc.</i> <b>2009</b>). We have improved the synthesis, purification and characterization of oligospermine-oligoribonucleotide conjugates which provide cationic siRNAs with enhanced biological activity. We show data supporting their carrier-free intracellular delivery in a molecular, soluble state. Additional results on the relationship between global charge, uptake and silencing activity confirm the requirement for an overall positive charge of the conjugated siRNA in order to enter cells. Importantly, conjugated siRNAs made of natural phosphodiester nucleotides are protected from nuclease degradation by the oligophosphospermine moiety, operate through the RNAi mechanism and mediate specific gene silencing at submicromolar concentration in the presence of serum

Schematic of the role of laminin-511 in intestinal tissue homeostasis.

<p>Laminin-511 is deposited in the intestine by both epithelial and mesenchymal cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037710#pone.0037710-Lefebvre1" target="_blank">[11]</a>. Its absence (KO model or RNA knockdown experiments) or presence (<i>in vitro</i> assays) leads to activation or inactivation of Wnt and PI3Kinase signaling pathways promoting multiple cellular responses that impact on survival, migration and differentiation.</p

Deregulated LMα5 expression in the intestine of patients with tufting enteropathy and collagenous colitis.

<p>LMα5 detection on small intestine (A) and colon (B) from patients with tufting enteropathy and collagenous colitis reveal an abnormal location of this chain as compared to controls. While α5 is detected mostly at the villus compartment in control specimen, it is found also in the crypt region (inset) in tufting enteropathy specimen. In a specimen of collagenous colitis, the staining is stronger all over the crypt-villus axis. Arrows point to the BM region.</p

Laminin-511 controls survival and epithelial cell migration.

<p>(A) In a survival assay, m-IC_Cl2 intestinal cells were cultured with H₂O₂ on laminin-111 (LM-111), laminin-511 coated-dishes or on laminin-511 (LM-511) with wortmannin (Wm). Survival rates, as ratios normalized to plastic, were determined by a MTS assay. Note a better cell survival rate on laminin-511 as compared to laminin-111, which is abolished upon treatment with Wm (mean +/− SEM, n = 5) (*** p<0.001). Immunofluorescence pictures (right) show more caspase-3-positive cells (arrows) on uncoated dishes (control) as compared to laminin-511. (B) Both migration velocity and cumulative migration distance of cells are significantly enhanced when m-IC_Cl2 cells are seeded on laminin-511 (LM-511; +/− wortmannin: Wm) versus laminin-111 (LM-111) or uncoated dishes (control) (mean +/− SEM, n = 5) (*** p<0.001). (C) Chemotactic migration of m-IC_Cl2 cells was visualized by phase contrast microscopy and cell counting on uncoated dishes (control), laminin-111 (LM-111), and laminin-511 (LM-511) in the presence or absence of wortmannin (Wm). Note that laminin-511 stimulated significantly cell migration independently of the PI3K/Akt pathway. In both assays, wortmannin did not affect laminin-511 enhanced migration. The dotted line represents the starting point of migration. Data (n≥5) are given as mean +/− SEM;** p<0.01; *** p<0.001.</p

Presence of laminin-511 inhibits TOPflash activity.

<p>(A) <i>In situ</i> hybridization of Msx1 on embryonic control and KO intestines showing that Msx1 is stimulated in knockout endodermal cells (arrow); e: endoderm; m: mesenchyme. (B) Gene expression ratios determined by RT-qPCR of Pitx2 and Sfrp2 between intestinal E15.5 knockout and control tissues, and on isolated mesenchymal or endodermal compartments confirm the increase of both molecules in the absence of laminin α5; for further details see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037710#pone-0037710-g002" target="_blank">figure 2</a> (mean +/− SEM, n = 7–9; * p<0.02). (C) HEK293 cells and lentiviral <i>lama5</i> shRNA m-IC_Cl2 infected cells seeded on plastic, laminin-111, cell-derived laminin-511 (LM-511C) or on recombinant laminin-511(LM-511R) were transfected with TOPflash or the negative FOPflash vector. The graphs represent the average relative luciferase activity normalized to luciferase Renilla activity; this ratio was then normalized to that obtained on plastic (n = 5, n = 3 for HEK293 on laminin-111, n = 1 for laminin-511(R); in duplicate; mean +/− SEM). For each cell line, TOPflash activity on laminin-111 does not statistically differ to that observed on plastic. Note that the TOPflash activity is statistically inhibited when cells are grown on laminin-511 as compared to laminin-111 (* p<0.05; *** p<0.001).</p

Cell survival and activation of Akt in epithelial cells adhering to laminin-511.

<p>(A) m-IC_Cl2 epithelial cells and (B) embryonic smooth muscle cells (SMC) were plated on uncoated dishes +/− EGF and insulin, and on dishes containing laminin-511 (LM-511) or laminin-111 (LM-111). The PI3K inhibitor wortmannin was added where indicated. Cell lysates were analyzed by western blotting for phosphorylated Akt (P-Akt), Akt (pan-Akt) and actin. Note that activation of Akt is detectable in epithelial cells cultured on laminin-511 but not on laminin-111 (see quantification of the representative gel). No activation occurred in smooth muscle cells in the presence of laminin-511. In parallel, epithelial (A) and smooth muscle cells (B) were photographed by phase contrast microscopy on laminin-511 matrix (LM-511) and on laminin-111 (LM-111) with or without wortmannin (Wm). Cell spreading on laminin-511 (left pictures) versus laminin-111 (right pictures) was confirmed by flattening of the cells and reorganization of the cytoskeleton as probed with TRITC-phalloidin to visualize F-actin. (C) Representative immunoblots showing the expression of Akt (pan-Akt), Akt2 and Phospho-Akt (P-Akt) in E15.5 control (WT) and knockout (KO) intestines and quantification of two independent experiments as ratio between KO (grey bars) and WT (black bars) intestines. Data were normalized using actin.</p

Muscle differentiation genes are regulated by laminin α5 chain.

<p>(A) Semi-quantitative RT-PCR experiments were performed on E15.5 control and knockout intestines for genes belonging to the muscle compartment. Data are presented as fold changes between knockout (grey bars) and wild-type (black bars) (mean +/− SEM, n = 5 to n = 9) (* p<0.05). (B) Immunostaining of MyoD1 on E15.5 control and KO intestines or on derived-cultured mesenchymal cells shows that MyoD1 is induced in knockout mesenchymal cells (arrows). Nuclei are stained with DAPI (blue). (C) Expression of Hlx1 by RT-qPCR showing that its expression is enhanced in <i>lama5</i> deficient versus wild-type intestines (mean +/− SEM, n = 6) (* p<0.05). Quantitative RT-PCR was performed on separated endodermal and mesenchymal compartments. The diagram shows the relative expression of Hlx1 between mesenchyme (mes) and endoderm (endo) with value 1 representing the total amount in wild-type intestines. Expression of Hlx1 is increased specifically in the mesenchymal compartment of LMα5^−/− intestines. (D) Effect of <i>lama5</i> siRNA on mesenchyme-derived target gene expression: Hlx1 (a, c) and MyoD1 (b, d). Embryonic mesenchymal cells (panels a and b) and adult intestinal smooth muscle cells (panels c and d) were cultured in the presence of control- and <i>lama5</i>-siRNA, respectively. <i>Lama5</i>-siRNA decreases LMα5 gene (up to 68%) and protein expression (in green) in both embryonic and adult cells. Note that <i>lama5</i>-siRNA upregulates Hlx1 gene expression and MyoD protein expression. After 72 h, gene expression was analyzed by RT-qPCR upon normalization to GAPDH and is expressed as relative fold-change (mean +/− SEM; n = 3) compared to control-siRNA. Arrows point at MyoD positive cells. Nuclei are stained with DAPI.</p