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

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

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

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

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

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

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

Laminins protect from inflammation.

<p>(<b>A–B</b>) Histological views of colon tissue (stained with PAS) and expression of LMα5 and LMα1 on cryosections of colon from parental, Tg-<i>lama5</i> or Tg-<i>lama1</i> mice untreated or treated with DSS. This showed that both chains were ectopically expressed in the glandular crypt region in the transgenic animals (arrows) and that LMα1 expression was further extended by DSS treatment. (<b>C</b>) Inflammatory scores (mean +/− SEM; n = 5) assessed on the Swiss-roll of the colon and rectum of transgenic <i>lama5</i> and <i>lama1</i> animals (black columns) as compared to controls (grey columns), all treated with DSS. (<b>D</b>) Levels of pro-inflammatory cytokines (mean +/− SEM; n = 6) measured by ELISA in protein extracts from distal colon of DSS-treated parental (grey columns) and DSS-Tg-<i>lama1</i> or DSS-Tg-<i>lama5</i> (black columns) mice. Data were normalized to the mean of parental values as levels of cytokines turned out to be mouse strain-dependent. Statistical differences were compared to parental mice. e: epithelial cells; lp: <i>lamina propria</i>; mm: <i>muscularis mucosae</i>. Nuclei are visualized with DAPI. *<i>p</i><0.05, **<i>p</i><0.01. Scale bars: 50 µm.</p

Important role of p53 in regulating LM expression in IBD and colitis-associated cancer.

<p>We propose the following scenario where inflammation triggers nuclear accumulation of p53 that transactivates expression of LMα1 and its deposition to the BM concomitantly to that of LMα5. Overexpression of LMα5 in response to inflammation is a p53 independent mechanism. In a chronically inflamed microenvironment highly expressed LMα1 and LMα5 may provide physical barrier function resulting in attenuated inflammation as demonstrated in transgenic mice. Yet, high LMα1 and LMα5 in a carcinogenic setting subsequent to chronic colitis may contribute to a pro-tumorigenic microenvironment.</p