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. 2010 Oct-Dec;4(4):541-50.
doi: 10.4161/cam.4.4.12923.

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

Rachel Cohen-Kupiec  1 , Shiri Weinstein, Gal Kantor, Dan Peer, Miguel Weil
Affiliations

IKAP/hELP1 down-regulation in neuroblastoma cells causes enhanced cell adhesion mediated by contactin overexpression

Rachel Cohen-Kupiec et al. Cell Adh Migr. 2010 Oct-Dec.

Abstract

A splicing mutation in the IKBKAP gene encoding the IKAP/hELP1 (IKAP) protein was found to be the major cause of Familial Dysautonomia (FD). This mutation affects both the normal development and survival of sensory and sympathetic neurons of the peripheral nervous system (PNS). To understand the FD phenotype it is important to study the specific role played by IKAP in developing and mature PNS neurons. We used the neuroblastoma SHSY5Y cell line, originated from neural crest adrenal tumor, and simulated the FD phenotype by reducing IKAP expression with retroviral constructs. We observed that IKAP – down - regulated cells formed cell clusters compared to control cells under regular culture conditions. We examined the ability of these cells to differentiate into mature neurons in the presence of laminin, an essential extracellular matrix for developing PNS neurons. We found that the cells showed reduced attachment to laminin, morphological changes and increased cell-to-cell adhesion resulting in cell aggregates. We identified Contactin as the adhesion molecule responsible for this phenotype. We show that Contactin expression is related to IKAP expression, suggesting that IKAP regulates Contactin levels for appropriate cell-cell adhesion that could modulate neuronal growth of PNS neurons during development.

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Figures

Figure 1
Figure 1
IKAP downregulation. (A) Graphic summary of qRT-PCR showing the relative expression of IKBAP mRNA in control and IKAP shRNA infected cells (IKAP-DR) compared to SHSY5Y non-infected cells. (B) Western blot analysis showing the reduced IKAP protein expression in IKAP-DR cells compared to control, beta actin expression was used for normalization. (C–E) Immunofluorescence staining of control [C and D (higher magnification)] and of IKAP-DR cells (E), IKAP is stained in green, nuclei are stained in blue (Hoechst) and the neuronal marker Rab3a is stained in red. (F) control and IKAP-DR cells morphology when grown on regular plates with selective growth medium. Arrow heads show the spheres of IKAP-DR cells.
Figure 2
Figure 2
Attachment and spreading assays. (A) Calcein stained control or IKAP-DR cells plated on laminin-coated plates without retinoic acid (NO RA) or with 10 µM retinoic acid (RA). (B) Graphic summary describing the averaged percentage of cells of three different experiments, that attached to the laminin coated plates in the indicated growth conditions. (C) Graphic summary of cell death indicated as percent of plated control or IKAP-DR cells. (D) Cell spreading described as the average cell area of a single cell of each strain in the indicated growth conditions. Statistical significance in (B–D) was determined using one-way ANOVA program, values are depicted at the bottom left corner of the figure. (E) Western blot analysis showing the expression of β-integrin1 in control and IKAP-DR cells grown on laminin without or with RA addition. Beta tubulin expression was used for normalization.
Figure 3
Figure 3
Differentiation on laminin. (A and C) control and IKAP-DR cells stained with calcein, respectively. (B and D) control and IKAP-DR cells nuclei staining. Arrows show the area in the inserts (X4). (E and G) Calcein-stained differentiated control and IKAP-DR cells respectively, disrupted by thorough pipetting after differentiation as described in Materials and Methods and grown for three more days in serum-free medium with BDNF. (F and H) nuclei staining of control and IKAP-DR cells shown in (E and G). Size bars in (E and F) apply to all pictures in this figure.
Figure 4
Figure 4
(A) Expression of Contactin-1 and Tenascin R as measured by RT PCR of mRNA extracted from control and IKAP-DR cells grown in non differentiation (NDF) or differentiation (DF) conditions. GAPDH expression was used for normalization. (B) FACS analysis of control cells grown in NDF (blue) and DF (green) conditions, stained with anti-CD56 (NCAM) antibody or IgG2a isotype as a reference for control cells in NDF (black) and DF (orange) conditions. (C) Same as in (B), but for IKAP-DR cells: isotype in black (NDF) and orange (DF) and anti-CD56 in blue (NDF) and green (DF). (D) FACS analysis of control cells stained with anti-Contactin1 in NDF (blue) and DF (green) conditions or control cells stained with only secondary antibody as a reference in NDF (black) and DF (orange) conditions. (E) same as in (D) but for IKAP-DR cells: secondary antibody staining in black (NDF) and orange (DF) and anti-Contactin1 in blue (NDF) and green (DF).
Figure 5
Figure 5
Control cells stained with calcein or Hoechst stain and grown on laminin with no added antibody (A and D), with goat IgG (B and E) or with anti-Contactin1 antibody (C and F), compared to IKAP-DR cells grown with no added antibody (G and J), with goat IgG (H and K) or with anti-Contactin1 antibody (I and L). In (M), a graphic summary of the average number of cells in a cluster for each of the cell lines and growth conditions is shown. ***p value < 0.001.
Figure 6
Figure 6
(A) Western blot analyses showing the expression of IKAP and of Contactin1 proteins in IKAP-DR or control cells grown in non-differentiation (NDF), in differentiation (DF) or with retinoic acid only (RA) conditions. For both analyses, GAPDH expression was used for normalization. In (B) the ratio between the expression levels of IKAP and GAPDH (upper graph) or Contactin1 (lower graph) and GAPDH is shown.
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