Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells

Abstract

The Rho family of small GTPases has been implicated in cytoskeletal reorganization and subsequent morphological changes in various cell types. Among them, Rac and Cdc42 have been shown to be involved in neurite outgrowth in neuronal cells. In this study, we examined the role of RhoG, another member of Rho family GTPases, in nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Expression of wild-type RhoG in PC12 cells induced neurite outgrowth in the absence of NGF, and the morphology of wild-type RhoG-expressing cells was similar to that of NGF-differentiated cells. Constitutively active RhoG-transfected cells extended short neurites but developed large lamellipodial or filopodial structures at the tips of neurites. RhoG-induced neurite outgrowth was inhibited by coexpression with dominant-negative Rac1 or Cdc42. In addition, expression of constitutively active RhoG elevated endogenous Rac1 and Cdc42 activities. We also found that the NGF-induced neurite outgrowth was enhanced by expression of wild-type RhoG whereas expression of dominant-negative RhoG suppressed the neurite outgrowth. Furthermore, constitutively active Ras-induced neurite outgrowth was also suppressed by dominant-negative RhoG. Taken together, these results suggest that RhoG is a key regulator in NGF-induced neurite outgrowth, acting downstream of Ras and upstream of Rac1 and Cdc42 in PC12 cells.

FIG. 1

Effect of expression of wild-type RhoG and various RhoG mutants on PC12 cell morphology. (A) PC12 cells were transiently cotransfected with an expression vector encoding GFP and an empty vector (vector; a and b) or an expression vector encoding Myc epitope-tagged wild-type RhoG (RhoGwt; c and d), RhoG^V12 (e to h), or RhoG^A37 (i and j). At 48 h after transfection, cells were observed under the phase-contrast microscope (a, c, e, g, and i). Transfected cells were identified by the fluorescence of GFP (b, d, f, h, and j). Arrows (e and g), large lamellipodial (e) and filopodial (g) structures at the tips of neurites. The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of neurite outgrowth induced by various RhoG mutants. At 48 h after transfection, cells were stained with an anti-Myc antibody, and positively stained cells were assessed. Cells with neurites were defined as cells that possessed at least one neurite more than 1 cell body diameter in length, and results are percentages of the total number of transfected cells. At least 100 cells were assessed in each experiment, and data are the means ± standard errors of triplicate experiments.

FIG. 1

Effect of expression of wild-type RhoG and various RhoG mutants on PC12 cell morphology. (A) PC12 cells were transiently cotransfected with an expression vector encoding GFP and an empty vector (vector; a and b) or an expression vector encoding Myc epitope-tagged wild-type RhoG (RhoGwt; c and d), RhoG^V12 (e to h), or RhoG^A37 (i and j). At 48 h after transfection, cells were observed under the phase-contrast microscope (a, c, e, g, and i). Transfected cells were identified by the fluorescence of GFP (b, d, f, h, and j). Arrows (e and g), large lamellipodial (e) and filopodial (g) structures at the tips of neurites. The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of neurite outgrowth induced by various RhoG mutants. At 48 h after transfection, cells were stained with an anti-Myc antibody, and positively stained cells were assessed. Cells with neurites were defined as cells that possessed at least one neurite more than 1 cell body diameter in length, and results are percentages of the total number of transfected cells. At least 100 cells were assessed in each experiment, and data are the means ± standard errors of triplicate experiments.

FIG. 2

Inhibition of RhoG-induced neurite outgrowth by Rac1^N17 and Cdc42^N17. (A) PC12 cells were cotransfected with an expression vector encoding Myc-tagged wild-type RhoG and an empty vector (a) or a vector encoding HA-tagged Rac1^N17 (b) or Cdc42^N17 (c). At 48 h after transfection, cells were fixed and stained with an anti-Myc antibody. Expression of HA-tagged Rac1^N17 and Cdc42^N17 was also detected with an anti-HA antibody (data not shown). The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of the effect of Rac1^N17 and Cdc42^N17 on RhoG-induced neurite outgrowth. At 48 h after transfection, cells were costained with anti-Myc and anti-HA antibodies, and positively stained cells were assessed as described in the legend to Fig. 1. Cells transfected with GFP were used as a control. Data are the means ± standard errors of triplicate experiments. RhoGwt, wild-type RhoG.

FIG. 2

Inhibition of RhoG-induced neurite outgrowth by Rac1^N17 and Cdc42^N17. (A) PC12 cells were cotransfected with an expression vector encoding Myc-tagged wild-type RhoG and an empty vector (a) or a vector encoding HA-tagged Rac1^N17 (b) or Cdc42^N17 (c). At 48 h after transfection, cells were fixed and stained with an anti-Myc antibody. Expression of HA-tagged Rac1^N17 and Cdc42^N17 was also detected with an anti-HA antibody (data not shown). The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of the effect of Rac1^N17 and Cdc42^N17 on RhoG-induced neurite outgrowth. At 48 h after transfection, cells were costained with anti-Myc and anti-HA antibodies, and positively stained cells were assessed as described in the legend to Fig. 1. Cells transfected with GFP were used as a control. Data are the means ± standard errors of triplicate experiments. RhoGwt, wild-type RhoG.

FIG. 3

RhoG^V12 activates endogenous Rac1 and Cdc42 in PC12 cells. PC12 cells were transiently transfected with an empty expression vector (vector) or an expression vector encoding RhoG^V12 or RhoG^V12A37. At 36 h after transfection, cells were serum starved for 12 h. The cell lysates were incubated with GST-CRIB, and the amounts of GTP-bound Rac1 and Cdc42 were determined by immunoblotting using a monoclonal antibody against Rac1 (A, top) and a rabbit polyclonal antibody against Cdc42 (B, top), respectively. Total amounts of Rac1 (A, middle) and Cdc42 (B, middle) in cell lysates and expression of Myc-tagged RhoG^V12 and RhoG^V12A37 (bottom) are also shown. In this experiment, about 20 to 30% of total cells were transfected with RhoG^V12 or RhoG^V12A37. The anti-Rac1 antibody used in this experiment is cross-reactive with expressed Myc-tagged RhoG^V12 but not with RhoG^V12A37 (A, arrow). The results shown are representative of three independent experiments that yielded similar results.

FIG. 4

Effects of Rac1 and Cdc42 expression on PC12 cell morphology. (A) PC12 cells were transiently transfected with an expression vector encoding Myc-tagged Rac1^V12 (a and b) or HA-tagged Cdc42^V12 (c and d) or were cotransfected with Myc-tagged Rac1^V12 and HA-tagged Cdc42^V12 (e and f) or with Myc-tagged wild-type Rac1 (Rac1wt) and HA-tagged wild-type Cdc42 (Cdc42wt) (g and h). At 48 h after transfection, cells were fixed and stained with anti-Myc (a, e, and g) and anti-HA (c, f, and h) antibodies or with Alexa 488 phalloidin to visualize filamentous actin (b and d). The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of effects of Rac1 and Cdc42 expression on neurite outgrowth. At 48 h after transfection, cells were costained with anti-Myc and anti-HA antibodies, and positively stained cells were assessed as described in the legend to Fig. 1. Cells transfected with GFP were used as a control. Data are the means ± standard errors of triplicate experiments.

FIG. 5

mRNA level of endogenous RhoG in PC12 cells. (A) Total RNA (15 μg) isolated from serum-starved PC12 cells treated with 50 ng of NGF/ml for the indicated times or from cells growing in serum-containing medium was subjected to Northern blot analysis with a cDNA probe for RhoG, as described in Materials and Methods. The same membrane was rehybridized with a GAPDH cDNA probe. Arrows, hybridized bands for RhoG and GAPDH. The positions of 18S and 28S rRNAs are indicated. (B) Quantification of changes of the RhoG mRNA level and the percentages of cells with neurites after NGF stimulation. The level of endogenous RhoG mRNA (a) is normalized to that of GAPDH mRNA and expressed as fold increases over the value for serum-starved cells at 0 min. Cells with neurites (b) were defined as the cells that possessed at least one neurite more than 1 cell body diameter in length. At least 100 cells were assessed in each experiment, and data are the means ± standard errors of triplicate experiments.

FIG. 6

Effect of expression of various RhoG mutants on NGF-induced neurite outgrowth. (A) PC12 cells were transfected with an expression vector encoding Myc-tagged wild-type RhoG (RhoGwt; a and b), RhoG^V12 (c and d), RhoG^N17 (e and f), or RhoG^A37 (g and h) and then treated with 50 ng of NGF/ml for 48 h. Cells were fixed and stained with an anti-Myc antibody (a, c, e, and g) to identify transfected cells. The morphology of the cells was visualized by filamentous actin staining with Alexa 488 phalloidin (b, d, f, and h). The results shown are representative of three independent experiments. Bar, 25 μm. (B) Length distribution of NGF-induced neurites in various RhoG mutant-expressing PC12 cells. PC12 cells were transfected with an empty vector (vector) or vectors encoding various RhoG mutants. At 48 h after transfection, cells were stained with an anti-Myc antibody, and positively stained cells were assessed. In this experiment, a vector encoding GFP was cotransfected to visualize tips of neurites. Cells with neurites exceeding 1-, 3-, or 5 times the length of the cell body were scored as a percentage of the total number of transfected cells. At least 100 cells were assessed in each experiment, and data are the means ± standard errors of triplicate experiments.

FIG. 6

Effect of expression of various RhoG mutants on NGF-induced neurite outgrowth. (A) PC12 cells were transfected with an expression vector encoding Myc-tagged wild-type RhoG (RhoGwt; a and b), RhoG^V12 (c and d), RhoG^N17 (e and f), or RhoG^A37 (g and h) and then treated with 50 ng of NGF/ml for 48 h. Cells were fixed and stained with an anti-Myc antibody (a, c, e, and g) to identify transfected cells. The morphology of the cells was visualized by filamentous actin staining with Alexa 488 phalloidin (b, d, f, and h). The results shown are representative of three independent experiments. Bar, 25 μm. (B) Length distribution of NGF-induced neurites in various RhoG mutant-expressing PC12 cells. PC12 cells were transfected with an empty vector (vector) or vectors encoding various RhoG mutants. At 48 h after transfection, cells were stained with an anti-Myc antibody, and positively stained cells were assessed. In this experiment, a vector encoding GFP was cotransfected to visualize tips of neurites. Cells with neurites exceeding 1-, 3-, or 5 times the length of the cell body were scored as a percentage of the total number of transfected cells. At least 100 cells were assessed in each experiment, and data are the means ± standard errors of triplicate experiments.

FIG. 7

Effect of RhoG^N17 on Ras^V12-induced neurite outgrowth. (A) PC12 cells were cotransfected with an expression vector encoding HA-tagged Ras^V12 and an empty vector (a and b) or a vector encoding Myc-tagged RhoG^N17 (c and d). At 48 h after transfection, cells were fixed and costained with anti-HA (a and c) and anti-Myc (b and d) antibodies. The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of the effect of RhoG^N17 on Ras^V12-induced neurite outgrowth. At 48 h after transfection, cells were costained with anti-Myc and anti-HA antibodies, and positively stained cells were assessed as described in the legend to Fig. 1. GFP-transfected cells were used as a control. Data are the means ± standard errors of triplicate experiments.

FIG. 7

Effect of RhoG^N17 on Ras^V12-induced neurite outgrowth. (A) PC12 cells were cotransfected with an expression vector encoding HA-tagged Ras^V12 and an empty vector (a and b) or a vector encoding Myc-tagged RhoG^N17 (c and d). At 48 h after transfection, cells were fixed and costained with anti-HA (a and c) and anti-Myc (b and d) antibodies. The results shown are representative of three independent experiments. Bar, 25 μm. (B) Quantification of the effect of RhoG^N17 on Ras^V12-induced neurite outgrowth. At 48 h after transfection, cells were costained with anti-Myc and anti-HA antibodies, and positively stained cells were assessed as described in the legend to Fig. 1. GFP-transfected cells were used as a control. Data are the means ± standard errors of triplicate experiments.