p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells

Abstract

Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.

Figure 1

NGF reduces GTP-RhoA levels in PC12 cells. (A) PC12 cells were treated with 100 ng/ml NGF for the indicated times at 37℃ and lysed. The cell lysates were incubated with the GST-Rhotekin-RBD domain for GTP-RhoA detection. Bound proteins were analyzed by Western blotting. (B) Wild-type (WT)-RhoA, constitutively active (CA)-RhoA (V14), or dominant negative (DN)-RhoA (N19) cDNAs were co-transfected with a green fluorescence protein (GFP) plasmid and incubated in serum-containing media for 24 h. The transfected PC12 cells were split and incubated for 12 h in serum-starved media, and the cells were then treated with 100 ng/ml NGF for 2 days to determine induction of neurites. GFP-positive cells were counted under a microscope (Zeiss, Gottingen, Germany). Transfected HA-RhoA was identified using a Western blot (lower panel). (C) PC12 cells were pre-treated without (Control) or with 0.33 µM Tat-C3 toxin and 0.33 µM Tat-peptide for 1 h and then treated with100 ng/ml NGF for 2 days. Modified RhoA by Tat-C3 in PC12 cells was determined by Western blotting with anti-RhoA antibody (right panel). (D) PC12 cells were preincubated for 1 h with 10 µM lysophosphatidic acid (LPA) and then treated with 100 ng/ml NGF for 2 days, and the cells with neurites were counted. (E) PC12 cells were pre-incubated with 10 µM Y27632, a ROCK inhibitor, for 30 min, then treated with 100 ng/ml NGF for 2 days, and cells harboring neurites were counted. Data are presented as means ± S.E. of three independent experiments (^*P < 0.05; ^**P < 0.01; ^***P < 0.001, compared with control using student's t-test). (F) PC12 cells were incubated for 12 h in serum-starved media, then treated with 100 ng/ml NGF for the indicated times, and then washed out. The cells were incubated with fresh serum-starved media for up to 4 days to induce neurite outgrowth.

Figure 2

p190RhoGAP and Src are implicated in neurite outgrowth of PC12 cells in response to NGF. (A) PC12 cells were transiently co-transfected with GFP and control (MOCK), WT- or DN-p190RhoGAP (lacking the GTP-binding domain) plasmid and incubated in serum-containing media for 24 h. The transfected PC12 cells were serum-starved for 12 h and then treated with 100 ng/ml NGF for 2 days. Cells with neurites were assessed. Expression of transfected p190RhoGAP was identified using a Western blot in the upper panel. Data are represented as means ± S.E. of three independent experiments (^*P < 0.05, compared with control using Student's t-test). (B) PC12 cells were pretreated with 20 µM SU6656 (a Src family kinase inhibitor), 20 µM PP2 (an inhibitor of the Src family of tyrosine kinases), or 20 µM PP3 (a control compound for PP2) for 30 min, and 100 ng/ml NGF was then administered for 2 days. The cells with neurites were counted. Data are represented as means ± S.E. of three independent experiments (^**P < 0.01; ^***P < 0.001, compared with control using Student's t-test). (C) PC12 cells were transiently co-transfected with GFP and MOCK, WT-c-Src, WT-p190RhoGAP, or WT-c-Src plus WT-p190RhoGAP plasmid and incubated in serum-containing media for 24 h. The transfected PC12 cells were serum-starved for 12 h and treated with 100 ng/ml NGF for 2 days. GFP-positive cells with neurites were counted. Expression of transfected Src and p190RhoGAP was identified in the right panel. Data were presented as means ± S.E. of three independent experiments (^**P < 0.01, compared with control using Student's t-test). (D) Either control (MOCK) or WT-c-Src plasmid and WT-p190RhoGAP were transiently overexpressed in PC12 cells and incubated in serum-containing media for 24 h. After serum starvation, the cells were treated with 100 ng/ml NGF for 4 h. GTP-loaded RhoA was analyzed using the GST-pull down assay as described in methods. The amount of RhoA pulled down with Rhotekin-RBD or total lysate was visualized by immunoblotting using anti-RhoA antibody. Data are representative of two independent experiments. (E) PC12 cells were treated with 100 ng/ml NGF for indicated times and lysed. p190RhoGAP was immunoprecipitated from cell lysates with anti-phospho-tyrosine antibody and was analyzed by Western blot using anti-p190RhoGAP antibody to assess tyrosine phosphorylation. Data are representative of three independent experiments demonstrating the same patterns.

Figure 3

Rap1 is involved in neurite outgrowth of PC12 cells in response to NGF. (A) Active Rap1 was measured in PC12 cells in response to NGF using a pull-down assay with the His-RalGDS-RBD domain, and the level of the GTP-bound Rap1 was visualized by immunoblotting using anti-Rap1 antibody. Data are representative of three independent experiments. (B) PC12 cells were transiently co-transfected with GFP and control (MOCK), WT-, CA (V14) -, or DN (N19)-Rap1 plasmid and incubated with serum-containing media for 24 h. The transfected PC12 cells were split and incubated for 12 h with serum-starved media. The cells were treated with 100 ng/ml NGF for 2 days for induction of neurites, and cells with neurites were determined. Expression of transfected HA-Rap1 was identified with Western blot using anti-HA antibody (upper panel). Data are represented as means ± S.E. of three independent experiments (^***P < 0.001, compared with control using Student's t-test). (C) PC12 cells were transfected with control plasmid (MOCK) or DN-Rap1 and incubated in serum-containing media for 24 h. The cells were incubated in serum-starved media for 12 h and then treated with 100 ng/ml NGF for 24 h. GTP-RhoA was analyzed using the GST-pull down assay. The amount of RhoA pulled down with Rhotekin-RBD or total lysate was visualized by immunoblotting. Data are representative of three independent experiments with similar results.

Figure 4

ARAP3 is a mediator of RhoA inactivation. (A) The interaction of Rap1 and RhoA in response to NGF was examined. Serum-starved PC12 cells were treated with 100 ng/ml NGF for various times as indicated and lysed. The lysates were immunoprecipitated with anti-Rap1 antibody. The interaction was analyzed by Western blot using anti-RhoA antibody. Data are representative of three independent experiments with similar results. (B) PC12 cells were transiently co-transfected with GFP and control (MOCK), WT-ARAP3 or DN (double mutations in both the RhoGAP and ArfGAP (R982A/C504A) domains)-ARAP3 plasmid and incubated in serum-containing media for 24 h. The transfected PC12 cells were serum-starved for 12 h and then treated with 100 ng/ml NGF for 2 days. Cells with neurites were assessed. Expression of GFP-ARAP3 was identified with a Western blot using anti-GFP antibody in the upper panel. Data are represented as means ± S.E. of three independent experiments (^*, P < 0.05, compared with control using Student's t-test). (C) PC12 cells were transfected with control (MOCK), WT-ARAP3, or DN-ARAP3 plasmid and incubated in serum-containing media for 24 h. The cells were serum-starved for 12 h and then treated with 100 ng/ml NGF for 24 h. GTP-loaded RhoA was analyzed by the GST-pull down assay. The amount of RhoA pulled-down with GST-Rhotekin-RBD or total lysate was visualized by immunoblotting using anti-RhoA antibody. Data are representative of two independent experiments with similar results.