SEL1L expression in fetal and adult human pancreas.

<p>Representative images of pancreatic sections from 18 weeks fetus (<b>A</b>–<b>F</b>) and 54-years-old patient (<b>G</b>–<b>L</b>) immunostained for SEL1L (green; <b>A</b>, <b>D</b>, <b>G</b> and <b>J</b>), glucagon (red; <b>B</b> and <b>H</b>) and insulin (red; <b>E</b> and <b>K</b>). Dual-color immunoflurescence showed SEL1L specific immunoreactivity (<i>green, </i><b>C </b><i>and </i><b>F</b>) in the nascent acinar cells adjacent to basement membrane (<i>arrowheads</i>) and in few interspersed cells (<i>arrows</i>); a strong cytoplasmatic staining is also observed in the developing islets (<i>asterisks</i>) stained for glucagon (red, <b>C</b>) and insulin (<i>red, </i><b>F</b>); in adult human pancreas, the exocrine tissue didn’t show any SEL1L immunoreactivity (<i>green, </i><b>G </b><i>and </i><b>J</b>), while endocrine cells revealed a marked expression of SEL1L protein with a strong cytoplasmic immunoreactivity in α-cells (stained for glucagon in red, <b>I</b>) and β-cells (stained for insulin in red, <b>L</b>). Scale bar = 50 µm.</p

SEL1L expression in fetal and adult mouse pancreas.

<p>Representative images of pancreatic sections from E16.5 mouse embryos (<b>A</b>–<b>F</b>) and 8-weeks-old mice (<b>G</b>–<b>L</b>) immunostained for SEL1L (green; <b>A</b>, <b>D</b>, <b>G</b> and <b>J</b>), glucagon (red; <b>B</b> and <b>H</b>) and insulin (red; <b>E</b> and <b>K</b>). Dual-color immunoflurescence showed SEL1L specific immunoreactivity (<i>green, </i><b>C </b><i>and </i><b>F</b>) in the nascent acinar tissue and in the developing islets (<i>asterisks</i>) stained for glucagon (red, <b>C</b>) and insulin (<i>red, </i><b>F</b>). While exocrine tissue, in the adult mouse, didn’t show any SEL1L immunoreactivity (<i>green, </i><b>I </b><i>and </i><b>J</b>), endocrine cells revealed a marked expression of SEL1L protein with a strong cytoplasmic immunoreactivity in α-cells (stained for glucagon in red, <b>I</b>) and a moderate expression in β-cells (stained for insulin in red, <b>L</b>). Scale bar = 50 µm.</p

SEL1L Regulates Adhesion, Proliferation and Secretion of Insulin by Affecting Integrin Signaling

<div><p>SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (<i>i</i>) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (<i>ii</i>) neural stem cell self-renewal and lineage commitment and (<i>iii</i>) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function.</p></div

SEL1L affects cell adhesion.

<p>(<b>A</b>) MIN6 cells were transfected with: scrambled-control (<i>white bars</i>), siRNA against <i>Sel1l</i> (<i>black bars</i>) or co-transfected with <i>Sel1l-</i>siRNA and β1 integrin over-expressing construct (<i>grey bars</i>) were left to adhere for 1 hour on 96-well plate coated with Collagen-IV (<i>CollIV</i>), Fibronectin (<i>FN</i>), Laminin (<i>LN</i>) and Vitronectin (<i>VN</i>). Adherent cells were then fixed, stained and counted under a microscope. Values are relative to untransfected adherent cell and presented as means ± SD from three independent experiments. (<b>B</b>) Western blot analysis of MIN6 cells (NT) transfected with scramble-control (<i>Scr</i>), with siRNA against <i>Sel1l</i> (<i>iSel1l</i>) and co-transfected with β1 integrin over-expressing construct (<i>Scr+Itgb1</i>and <i>iSel1l+Itgb1</i>) probed for SEL1L, β1 integrin (ITGB1), phospho-ERK1/2 and total-ERK1//2. (<b>C</b>) Quantification of the immunoblot bands; values are representative of three independent experiments and are expressed as fold of expression ± SD relative to NT and normalized to total ERK1/2 as loading control. (<b>D</b>) Photomicrographs showing the effect of SEL1L down-modulation on MIN6 morphology; the <i>Sel1l</i>-interfered cells (<i>iSel1l</i>) appears round-shaped compared to controls (<i>Scr, NT, Scr+Itgb1</i>) while overexpression of β1 integrin subunit restore MIN6 polygonal spindle-like morphology (<i>iSel1l+Itgb1</i>).</p

SEL1L interacts with integrins.

<p>(<b>A</b>) Co-immunoprecipitation for integrin subunits followed by western blotting for SEL1L (<i>left panel</i>) and viceversa (<i>right panel</i>); arrows indicate 130-kDa mature β1 integrin and 110-kDa precursor. (<b>B</b>) MIN6 cells immunostained for SEL1L (<i>green</i>) and β1 integrin (<i>red</i>); a higher magnification of SEL1L/ß1 integrin co-localization to plasma membranes is shown in the inset of the right panel. Nuclei (<i>blue</i>) are counterstained with Hoechst 33258.</p

Impact of SEL1L down-modulation on insulin secretion and cell proliferation.

<p>Insulin release of MIN6 transfected (<b>A</b>) and mouse islet nucleofected (<b>B</b>) cells was quantified after 1-hour stimulation with 22.8 mM glucose. Values are normalized by DNA content and are expressed as a mean ± SD of fold increase in insulin release over basal glucose concentration from three separate experiments. MIN6 transfected cells (<b>C</b>) and mouse islet nucleofected cells (<b>D</b>) were assayed by qPCR for effective <i>Sel1l</i> down-modulation by siRNA. Values are expressed as fold expression ± SD relative to un-treated sample (value = 1) and normalized to <i>Hprt</i>. (<b>E</b>) To assess SEL1L-dependent cell proliferation, MIN6 transfected and untransfected cells were pulsed for 1 hour with BrdU, fixed and immunostained. Frequency of BrdU-positive cells were represented as percentage of total number of nuclei counted and expressed as means ± SD from three independent experiments. (<b>F</b>) Changes in the expression of the key regulators of the cell cycle progression Cyclin D1 (<i>Ccdn1</i>) and p21 (<i>Cdkn1a</i>) were validated by qPCR; values are expressed as fold expression relative to un-treated sample (value = 1) and normalized to <i>Hprt</i>.</p

Identification of pancreatic cell types expressing Netrin-4.

<p>PCR analysis (<b>A</b>) and Western blotting (<b>B</b>) on select pancreatic cell populations show significant levels of Netrin-4 expression in adult primary ductal cells, ductal cell line CAPAN-1, and fetal pancreatic cells, and low levels in pancreatic islets. Representative of n = 3. SYBR green qPCR (<b>C</b>) for Netrin-4-specific transcripts in primary microvascular endothelial cells (hMEC), fetal and adult pancreatic ductal cells, and intact adult islets. SYBR green qPCR for the endothelial-specific cell adhesion molecule VE-cadherin (<b>D</b>) showing that resident endothelia cells are positioned within fetal and adult islets. Fluorescence-activated cell sorting of a single cell suspension from isolated human fetal islets immunostained for insulin (<b>E</b>), and SYBR green qPCR analysis for insulin, Netrin-4, and VE-cadherin (<b>F</b>). Data presented in C, D, E and F are representative of n = 3, with each SYBR green qPCR reaction performed in duplicate.</p

Netrin-4 supports epithelial cell adhesion through integrin receptors and fosters the expression of islet-specific differentiation genes.

<p>(<b>A</b>) Adhesion of pancreatic epithelial cells to Netrin-1, Netrin-4, LN-1 and Collagen IV. BSA was used as negative control. (<b>B</b>) Cell adhesion to Netrin-4 in the absence (n.t., no treatment) or presence of function-blocking antibodies to select integrin subunits. Note the significant blockade of cell attachment to Netrin-4 in the presence of anti-α2, -α3, -β1, or a combination of anti-α2 and anti α3 function-blocking antibodies. (<b>C</b>) Similar results were obtained when cells were plated on a modified recombinant Netrin-4 (ΔC-Netrin-4) that lacks 155aa from its carboxy terminal domain. Data in <b>A</b> and <b>B</b> are representative of n = 4, and in C of n = 3. *p<0.001 ANOVA followed by post-test Bonferroni's multiple comparison test. (<b>D</b>) Immunoprecipitation using anti-Netrin-4, -α2, -α3, -α5, -β3 or control IgGs, followed by Western blotting for Netrin-4 revels that α2 and α3, but not α5 or β3, integrin subunits selectively interacts with Netrin-4 in live cells. Representative of n = 3. (<b>E</b>) TaqMan PCR analysis for insulin and glucagon mRNAs demonstrates that overnight culture of embryonic pancreatic cells on Netrin-4 promotes the expression of these two islet-specific differentiation genes, when compared to Collagen IV. Culture on Netrin-1, that we reported to engage integrin α3β1 as a receptor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022750#pone.0022750-Yebra1" target="_blank">[10]</a>, also revealed significantly higher levels of insulin- and glucagon-specific transcripts when compared to Collagen IV (n = 6); statistical significance of differences in insulin (p<0.001) and glucagon (p<0.005) expression between Netrins and Coll. IV overnight cultures was determined by ANOVA followed by post-test Bonferroni's multiple comparison test. (<b>F</b>) Blockade of α2, α3, β1, or α2 and α3 simultaneously, significantly reduced Netrin-mediated pro-differentiative effects on pancreatic cells (n = 4). (G) Specific immunoreactivity for the α3 integrin subunit (green fluorescence) is detected both <i>in situ</i> (G, left panel) and <i>in vitro</i> (G, right panel) in insulin-producing cells (red fluorescence, arrowheads). (H) Insulin content measured in embryonic pancreatic cells cultured on either Collagen IV, Netrin-1, or Netrin-4 (n = 4).</p

Pancreatic cell adhesion to Netrin-4 promotes cell cycle exit and fosters the expression of pro-differentiation genes.

<p>Heatmap of select genes that are either down-regulated (<b>A</b>) or up-regulated (<b>B</b> and <b>C</b>) by an 18-hours exposure of fetal pancreatic cells to Netrin-4. Data are presented as fold increase over time 0′. Note that known negative regulators of the cell cycle such as p57/kip2 and p27/kip1 are up-regulated (<b>A</b>), whereas positive regulators such as cyclins are down-regulated (<b>A</b>). Conversely, a number of genes whose function has been linked to events of cellular differentiation are all up-regulated (<b>B</b>, <b>C</b>). Changes in expression of select genes exemplified in panels <b>A</b> and <b>B</b> were validated by qPCR (<b>D</b>), where a value of 1 is equal to no change in gene expression. Complete array data have been deposited in the EBI Array Express Database (accession number pending). Data presented in <b>D</b> are representative of two independent experiments.</p