Endothelium-derived Netrin-4 supports pancreatic epithelial cell adhesion and differentiation through integrins α2β1 and α3β1

Abstract

Background: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified.

Methodology/principal findings: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1 and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated.

Conclusions/significance: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins.

Figure 1. Expression of Netrin-4 in the developing pancreas.

Three-color immunofluorescent staining of fetal pancreas showing expression of Netrin-4 (B) in ductal cells, identified by the ductal marker CA19-9 (A). Arrowheads in A, B, and D points to Netrin-4 immunoreactivity in basal membranes of ductal cells. Insulin-expressing cells (C, G) do not show Netrin-4-specifc immunoreactivity, although they appear to be infiltrated by strings of Netrin-4-postive cells (F, arrowheads) that co-express the endothelial marker PECAM-1 (E, arrowheads). Images presented in A–D and E–H were acquired from consecutive sections. (I, J) negative controls using isotype matched IgGs. (*) Lumen of ductal structures.

Figure 2. Identification of pancreatic cell types expressing Netrin-4.

PCR analysis (A) and Western blotting (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 (C) 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 (D) 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 (E), and SYBR green qPCR analysis for insulin, Netrin-4, and VE-cadherin (F). Data presented in C, D, E and F are representative of n = 3, with each SYBR green qPCR reaction performed in duplicate.

Figure 3. Netrin-4 supports epithelial cell adhesion through integrin receptors and fosters the expression of islet-specific differentiation genes.

(A) Adhesion of pancreatic epithelial cells to Netrin-1, Netrin-4, LN-1 and Collagen IV. BSA was used as negative control. (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. (C) 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 A and 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. (D) 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. (E) 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 , 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. (F) 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 in situ (G, left panel) and in vitro (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).

Figure 4. Knock-down of α2 and α3 integrin subunits by siRNA affects pancreatic cell adhesion to Netrin-4 and expression of islet-specific differentiation gene expression.

(A) Western blot analysis of pancreatic cells transfected with siRNA specific for α2 and α3 integrin subunits effectively knocked-down α2 and α3 protein expression. Down-regulation of α2 and α3 integrin subunits in pancreatic cells resulted in a significant reduction of adhesion to Collagen IV (B) Laminin-5 (C), and to Netrin-4 (D), as well as a decrease in glucagon and/or insulin gene expression when cultured overnight on Netrin-4 (E). Data presented are representative of three independent experiments. Data presented in B, C and D are representative of n = 3, *p<0.001 and **p<0.01 as determined by ANOVA followed by post-test Bonferroni's multiple comparison test.

Figure 5. Pancreatic cell adhesion to Netrin-4 promotes cell cycle exit and fosters the expression of pro-differentiation genes.

Heatmap of select genes that are either down-regulated (A) or up-regulated (B and C) 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 (A), whereas positive regulators such as cyclins are down-regulated (A). Conversely, a number of genes whose function has been linked to events of cellular differentiation are all up-regulated (B, C). Changes in expression of select genes exemplified in panels A and B were validated by qPCR (D), 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 D are representative of two independent experiments.