Integrin-mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channels

Abstract

Abstract. Electrical signals elicited by integrin interaction with ECM components and theft role in neurite outgrowth were studied in two clones (N1 and N7) isolated from 41A3 murine neuroblastoma cell line. Although the two clones similarly adhered to fibronectin (FN) and vitroncctin O/N), this adhesion induced neurite outgrowth in NI but not in N7 cells. Patch clamp recordings in whole cell configuration showed that, upon adhesion to FN or VN but not to platelet factor 4 (PF4), NI cells undergo a marked (---20 mV) hyperpolarization of the resting potential (Vr, t) that occurred within the first 20 min after cell contact with ECM, and persisted for •1 h before reverting to the time zero values. This hyperpolarization was totally absent in N7 cells. A detailed analysis of the molecular mechanisms involved in N1 and N7 cell adhesion to ECM substrata was performed by using antibodies raised against the FN receptor and synthetic peptides variously competing with the FN or VN binding to integrin receptors (GRGDSP and GRGESP). Antibodies, as well as GRGDSP, abolished adhesion of N1 and N7 clones to FN and VN, revealing a similar implication of integrins in the adhesion of these clones to the ECM proteins. However, these anti-adhesive treatments, while ineffective on V,~, of N7 cells, abolished in N1 cells the FN-or VN-induced hyperpolarization and neurite outgrowth, that appeared therefore strictly associated and integrin-mediated phenomena. The nature of this association was deepened through a comparative analysis of the integrin profiles and the ion channels of N1 and N7 cells. The integrin immunoprecipitation profile resulted very similarly in the two clones, with only minor differences concerning the aV containing complexes. Both clones possessed Ca 2+ and K + delayed rectifier (KDR) channels, while only N1 cells were endowed with inward rectifier K + (Kit) channels. The latter governed the Vr~, and, unlike KDR channels, were blocked by Ba 2+ and Cs +. By moving patched cells in contact with FN-coated beads, it was shown that Km channel activation was responsible for the FN-mediated hyperpolarization of V,~. Treatment with Pertuxis toxin (PTX) abolished this hyperpolarization and neurite outgrowth, indicating that a G protein is interposed between integrins and Km channels and that the activation of these channels is required for neuritogenesis. In fact, the block of Km channels by Cs ÷ abolished both hyperpolarization and neurite outgrowth, provided that the cation was supplied during the first two hours after N1 cell contact with FN. This Cs+-sensitive commitment time for neuritogenesis coincided with the time length of the FNinduced hyperpolarization.

HERG K+ Channels Activation during β1Integrin-mediated Adhesion to Fibronectin Induces an Up-regulation of αvβ3 Integrin in the Preosteoclastic Leukemia Cell Line FLG 29.1

“…Preparation of Substrates and Coating of Culture Dishes-Heatinactivated BSA was prepared as described previously (11). Coating of culture dishes with FN or vitronectin (VN) was performed by adding the two proteins, prepared as reported in Ref.…”

“…Adhesion Assay-Adhesion assay was performed essentially according to Arcangeli et al (11). Briefly, stock cultures were radiolabeled during 36 h of exponential growth in RPMI medium containing 1 Ci/ml [methyl- 3 H]thymidine (specific activity 24 Ci/mmol).…”

“…Therefore, the FN-induced increment in I HERG could be realized by the action of a short range modulator of channel activity: a possible candidate could be envisaged in a pertussis toxin-sensitive G i protein interposed between ␤ 1 integrins and channel protein, as in neuroblastoma cells (11). And in fact, as shown in Fig.…”

“…7) through the involvement of integrin receptors, which are numerous on the osteoclast plasma membrane (8). Therefore, osteoclast precursors can be envisaged as a good model wherein studying the relationships between the ECM, integrin-mediated signals, and the induction of cell differentiation/activation.Among the various integrin-centered signaling pathways discovered so far (9, 10), we demonstrated that the modulation of V REST is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells (11)(12)(13). This modulation is sustained by the activation of a peculiar type of K ϩ channels, the HERG channels (14, 15), encoded by the ether-à -…”

“…Among the various integrin-centered signaling pathways discovered so far (9, 10), we demonstrated that the modulation of V REST is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells (11)(12)(13). This modulation is sustained by the activation of a peculiar type of K ϩ channels, the HERG channels (14, 15), encoded by the ether-à -…”