RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

<div><p>Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.</p></div

RabGEF1 regulates TrkA-mediated downstream signaling pathways in NGF-differentiated PC12 cells.

<p>A) Enhancement of ERK and JNK activation in PC12 cells transfected with RabGEF1-AS expression construct in response to NGF stimulation. Cell lysates were analyzed by Western blotting and probed with antibody against phospho-ERK or phospho-JNK. B) Reduced ERK5 activation in PC12 cells transfected with RabGEF1-AS expression construct in response to NGF stimulation. Cell lysates were analyzed by Western blotting and probed with antibody against phospho-ERK5. C) Western blot analysis of expression of p27^Kip1, PCNA (PC10) and p21^Waf1/Cip1 in CMV and RabGEF1-AS PC12 transfectants stimulated by NGF (50 ng/ml).</p

RabGEF1 binds to NMDA receptor subunit NR2B and negatively regulates NMDA-mediated NOS1 activation in NGF-differentiated PC12 cells.

<p>A) Binding of RabGEF1 to NMDA receptor NR2B subunit (NMDAR2B) in PC12 cells and mouse hippocampus (HP). Cell lysates were immunoprecipitated (IP) with anti-NR2B antibody or anti-RabGEF1 antibody and then immunoblotted (IB) with anti-RabGEF1 antibody or anti-NR2B antibody, respectively. Rabbit IgG was used as the negative control and the positive control was represented by total cell lysate. B) Binding of RabGEF1 to SynGAP in PC12 cells stimulated by NGF for 0, 0.5 or 1.0 hour. Cell lysates were immunoprecipitated with anti-RabGEF1 antibody and immunoblotted with anti-SynGAP antibody, anti-Rabaptin-5, anti-NR2B antibody, or anti-RabGEF1 antibody, respectively. C) Activation of NOS1 activity following NMDA receptor stimulation in PC12 cells transfected with CMV or RabGEF1-AS expression construct. Transfectants were differentiated by NGF stimulation (50 ng/ml) for 3 days and then stimulated with NMDA (750 μM) for 10 min. Production of ³H-citrulline was assayed using ³H-arginine as the substrate, and NOS1 activity was calculated as % citrulline conversion in the reaction in relation to total counts. Data shown were pooled from three separate independent experiments (n = number of samples). ** p<0.01 by unpaired <i>t</i>-test.</p

Reduced RabGEF1 expression decreases NGF-induced TrkA phosphorylation but not TrkA internalization.

<p>A) CMV and AS stable transfectants were stimulated by NGF (50 ng/ml) for 30 min, 1 hour, 3 hours, or 5 hours. Total cell lysates were analyzed by Western blot using anti-phospho-TrkA and anti-TrkA antibody. B) CMV and AS stable transfectants were incubated with biotinylated anti-rat TrkA IgG antibody for the indicated times. Surface TrkA was assessed by streptavidin (SA)-APC fluorescence and analyzed by flow cytometry. Percentage TrkA internalization was calculated by subtracting mean fluorescence intensity at 2.5 min, 15 min, 30 min, or 60 min from mean fluorescence intensity at 0 min and dividing this number by mean fluorescence intensity at 0 min (x 100%). Data shown were pooled from four separate experiments.</p

Reduced RabGEF1 expression enhances neurite outgrowth in NGF-differentiated PC12 cells.

<p>A) Phase-contrast photomicrographs showing morphological characteristics of PC12 cells transfected with RabGEF1-AS construct or control empty CMV vector. PC12-CMV control and PC12-AS transfectants were grown on collagen-coated plates and stimulated with NGF (50 ng/ml) for 1, 4, 7, 9, or 12 days. B)—D) AS transfectants undergoing differentiation induced by NGF exhibited increased total neurite length, longest neurite length, and tip end numbers at 1 day (CMV: n = 84; AS: n = 73), 2 days (CMV: n = 87; AS: n = 96), 3 days (CMV: n = 113; AS: n = 84) or 4 days (CMV: n = 103; AS: n = 86) after stimulation, where n = number of cells counted. Data shown were pooled from three separate experiments. * p<0.05 by unpaired <i>t</i>-test, ** p<0.01 by unpaired <i>t</i>-test.</p

Reduced RabGEF1 expression enhances cell proliferation and altered cell cycle progression in NGF-differentiated PC12 cells.

<p>A) Enhanced cell proliferation in PC12 AS transfectants stimulated by NGF. PC12 transfectants were cultured overnight in 0.5% FCS and then incubated with NGF for 24 hours. Cells were then pulsed with 10 μCi/ml of ³H-thymidine for 4 hours and ³H-thymidine incorporation was counted. n = 6 for both CMV and AS samples. * p<0.05 by unpaired <i>t</i>-test, ** p<0.01 by unpaired <i>t</i>-test. B) Flow cytometric analysis of BrdU-labeled PC12 transfectants. CMV and AS transfectants were treated with NGF (50 ng/ml) for 6 days. Cells were then pulsed with 10 μM BrdU for 1 hour and then stained with anti-BrdU-FITC and PI. Samples were analyzed by flow cytometry. Data shown are representative of three independent experiments.</p

RabGEF1 differentially regulates activation of Ras, Rac1, and Cdc42 in NGF-differentiated PC12 cells.

<p>A) NGF-induced Ras activation was reduced in RabGEF1-AS transfectants. CMV and AS transfectants were stimulated by NGF (50 ng/ml) for the indicated times. Activated Ras-GTP was affinity-precipitated with GST fusion protein containing the Ras binding domain of Raf-1 and analyzed by Western blotting using anti-Ras antibody. B) Enhanced Rac1 activation in RabGEF1-AS transfectants stimulated by NGF (50 ng/ml). Activated Rac1-GTP was affinity-precipitated with GST fusion protein containing the p21-binding domain of Pak1 and analyzed by Western blotting using anti-Rac1 antibody. C) Enhanced Cdc42 activation in RabGEF1-AS tranfectants stimulated by NGF. Activated Cdc42-GTP was affinity-precipitated using GST fusion protein containing the p21-binding domain of Pak1 and analyzed by Western blotting using anti-Cdc42 antibody. D) Kinetics of Rho activation in PC 12 transfectants stimulated by NGF. Activated Rho-GTP was affinity-precipitated with GST fusion protein containing the Rho-binding domain of Rhotekin and analyzed by Western blotting using anti-Rho antibody. Blot images were scanned and specific signals were quantified by UN-SCAN-IT gel Version 5.3. Data shown are representative of three independent experiments.</p

Models illustrating the interaction of RabGEF1 with NGF-induced TrkA-mediated signaling pathways (differentiation) and with NMDA-dependent signaling pathways (neuronal plasticity).

<p>Models illustrating the interaction of RabGEF1 with NGF-induced TrkA-mediated signaling pathways (differentiation) and with NMDA-dependent signaling pathways (neuronal plasticity).</p

Expression of RabGEF1 in PC12 cells stimulated with NGF.

<p>A) Analysis of RabGEF1 mRNA expression in PC12 cells stimulated with NGF. PC12 cells were stimulated with NGF (50 ng/ml) for the indicated time periods and RNA was extracted and analyzed using Northern blot analysis. The bottom panel shows equal loading of RNA in each sample. B) Analysis of RabGEF1 protein expression in PC12 cells transfected with empty CMV vector or construct expressing antisense (AS) RabGEF1 mRNA. Stable PC12 transfectants expressing CMV or RabGEF1-AS construct were stimulated with NGF (50 ng/ml). Cells were lysed and RabGEF1 expression was assessed by Western blot analysis. The bottom panel shows equal loading of protein by probing the blots with anti-GAPDH antibody. Blot images were scanned and specific signals were quantified by UN-SCAN-IT gel Version 5.3. Data shown are representative of four independent experiments.</p

RabGEF1 binds directly to Ras, Rac1 and Cdc42.

<p>Binding of RabGEF1 to Ras and Rac1/Cdc42, but not Rho. Cell lysates derived from PC12 cells stimulated by NGF for 0 or 15 min were subjected to “pull down” (affinity-precipitation) assays using GST-Raf1-RBD fusion protein, GST-Pak1-PBD fusion protein, or GST-Rhotekin-RBD fusion protein. Proteins bound to the beads were analyzed by Western blotting using anti-RabGEF1 antibody. Data shown are representative of three independent experiments. IB: Immunoblotting.</p