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

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

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