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Research Article
Rit promotes MEK-independent neurite branching in human neuroblastoma cells
DiAnna L. Hynds, Mike L. Spencer, Douglas A. Andres, Diane M. Snow
Journal of Cell Science 2003 116: 1925-1935; doi: 10.1242/jcs.00401

Summary

Rit, by sequence homology, is a member of the Ras subfamily of small guanine triphosphatases (GTPases). In PC6 cells, Rit signals through pathways both common to and different from those activated by Ras to promote cell survival and neurite outgrowth. However, the specific morphological changes induced by Rit in human cells are not known. Here, we show in a human neuronal model that Rit increases neurite outgrowth and branching through MEK-dependent and MEK-independent signaling mechanisms, respectively. Adenoviral expression of wild-type or constitutively active Rit increased neurite initiation, elongation and branching on endogenous matrix or a purified laminin-1 substratum of SH-SY5Y cells as assessed using image analysis. This outgrowth was morphologically distinct from that promoted by constitutively active Ras or Raf (evidenced by increased branching and elongation). Constitutively active Rit increased phosphorylation of ERK 1/2, but not Akt, and the MEK inhibitor PD 098059 blocked constitutively active Rit-induced neurite initiation but not elongation or branching. These results suggest that Rit plays a key role in human neuronal development and regeneration through activating both known and as yet undefined signaling pathways.

Introduction

Rit (Ras-like protein in all tissues) is a member of a recently described branch of the Ras subfamily of small guanine triphosphatases (GTPases) that includes Rit, Rin (Ras-like protein in neurons) and the Drosophila protein RIC [Ras-related protein which interacts with calmodulin (Wes et al., 1996; Shao et al., 1999)]. These proteins have GTP- binding motifs that are highly conserved but lack a known recognition signal for C-terminal lipidation, a modification necessary for correct subcellular localization and proper function of most Ras-like GTPases (Shao et al., 1999). Rit, in common with Ras, induces transformation of NIH3T3 cells and can signal to Ras-responsive elements (Shao et al., 1999; Shao and Andres, 2000). However, Rit clearly also activates pathways that are distinct from those involving Ras. For example, Rit does not activate ERK, JNK, p38 or PI3K/Akt pathways in fibroblasts (Rusyn et al., 2000).

Rit, as well as Rin, may be an important mediator of neuronal differentiation (Spencer et al., 2002a; Spencer et al., 2002b). Rin is activated in a Ras-dependent manner upon nerve growth factor (NGF) stimulation in PC6 cells, a subclone of PC12 designed to have reduced cell aggregation in culture. However, constitutively active (CA) Rin mutants do not induce neurite outgrowth (Spencer et al., 2002b). In the same cell line, Rit increases the percentage of neurite-bearing cells and promotes survival, and dominant-negative Rit can block NGF- induced outgrowth (Spencer et al., 2002a). Rit-induced outgrowth is associated with concomitant activation of ERK1/2 but not Akt. Furthermore, the patterns of expression of Rin and Rit during development are consistent with Rit playing a role in neuronal development, that is, Rin is expressed only in the mature nervous system (Lee et al., 1996; Spencer et al., 2002b), whereas Rit is expressed in primary neurons and the developing brain (Lee et al., 1996; Spencer et al., 2002a).

Previous data lead to three necessary and intriguing avenues for exploration regarding Rit. First, as PC6 cells are derived from a rat pheochromocytoma, does Rit induce similar outgrowth in human neurons, and related, does Rit activate similar signaling pathways in human cells to those in PC6 cells (i.e. ERK 1/2, but not Akt)? Second, the study by Spencer et al. was performed in PC6 cells on a substratum composed partly of the non-physiological agent, poly-L-lysine (Spencer et al., 2002a). Thus, does the availability of a physiological substratum affect Rit-induced neurite outgrowth? This question is intriguing since specific physiological extracellular matrix substrata have profound effects on neurite outgrowth (Payne et al., 1992; Gomez and Letourneau, 1994; Gomez et al., 1996; Hynds et al., 2001), and signaling pathways initiated via integrins may interact with signaling pathways involving Rit. Third, and the most promising aspect of this study, does Rit differentially effect different aspects of neurite outgrowth such as initiation, elongation and particularly arborization? This question is especially important since Rit mediates neuronal differentiation and has been shown to stimulate branching in PC6 cells (Spencer et al., 2002a). This result begs further analysis in human cells and requires full quantification. NGF through activation of Ras increases the percentage of neurite-bearing cells in PC12 cells (Hagag et al., 1986; Szeberenyi et al., 1990; Szeberényi and Erhardt, 1994) but can also influence neurite branching (Yasuda et al., 1990; Itoh et al., 1993; Scott and Davis, 1993; Struder et al., 1994; Gallo and Letourneau, 1998; Markus et al., 2002). The signaling pathways that mediate neurite branching are incompletely characterized, but may involve members of the Rho subfamily, GTPases that direct actin rearrangements (Kuhn et al., 1998; Lehmann et al., 1999; Brown et al., 2000) and affect neurite branching (Albertinazzi et al., 1998; Sebök et al., 1999; Neumann et al., 2002; Ng et al., 2002).

We chose to use the human neuroblastoma cell line SH-SY5Y to answer the questions raised above. These cells, originally subcloned from a metastatic neuroblastoma, represent a homogenous culture of sympathetic noradrenergic neurons (Ross et al., 1983) and emulate the behavior and biology of primary neurons (Ross et al., 1983; Påhlman et al., 1990; Leli et al., 1992; Påhlman et al., 1992; Choi et al., 1994; Leventhal and Feldman, 1995; Smith et al., 1995; Hynds et al., 1997; Hynds and Snow, 1999; Zeidman et al., 1999; Encinas et al., 2000; Hynds and Snow, 2001; Snow et al., 2001). Adenovirus-mediated expression of CA-Rit induced extensive neurite outgrowth and, importantly, branching in SH-SY5Y cells cultured on endogenous extracellular matrix or purified laminin-1. This outgrowth was morphologically distinct from that induced by CA-Ras. Furthermore, CA-Rit activated ERK 1/2, but not Akt, and Rit-induced neurite initiation, but not neurite elongation or branching, was blocked by treatment with the MEK inhibitor PD 098059. These data indicate that Rit is a unique effector of human neuronal development and/or regeneration, and it displays signaling patterns different from Ras.

Materials and Methods

Cell line maintenance

SH-SY5Y cells (ATCC, or a gift from Allan J. Yates, Ohio State University or Joe Springer, University of Kentucky) were routinely cultured in a 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's Nutrient Mixture F-12 (DMEM/F12, Gibco BRL, Grand Island, NY) supplemented with non-essential amino acids (NEAA, Gibco BRL, Grand Island, NY) and containing 15% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT). Cells were subcultured when confluent using 0.25% bactotrypsin (Difco Laboratories, Detroit, MI) after washing with phosphate-buffered saline (PBS, Gibco, Grand Island, NY). For all experiments, trypsinized cells were resuspended in DMEM/F12 with 15% FBS and plated as described below.

Substratum preparation for outgrowth experiments

Baked round glass coverslips (25 mm; Fisher, Pittsburgh, PA) were adhered to the bottom of pre-drilled 50 mm tissue culture plates (Fisher), using silicone aquarium sealant. Plates were allowed to cure for at least 24 hours and were sterilized by exposure to ultraviolet light for 20 minutes. Plates were either not treated or adsorbed with 500 μl of 25 μg/ml laminin-1 (Collaborative Biomedical, Bedford, MA) in PBS for 3 hours at room temperature. Plates were washed twice with PBS before use.

Adenoviral vectors

Recombinant adenoviruses were constructed for wild-type and constitutively active Rit (CA-Rit; Q79L) that co-expressed green fluorescent protein (GFP) by the Vogelstein method (He et al., 1998) as described previously (Spencer et al., 2002a). Viral stock concentrations were determined using standard plaque assays. All viruses were screened for the presence of the wild-type E1A gene by PCR in a non-permissive cell line such as PC6 cells. Adenovirus constructs expressing constitutively active Ras (R17V; kind gift from Yibin Wang, University of Maryland, MD), constitutively active Raf (deletion of amino acids 26-302), dominant-negative Raf (truncated at amino acid 257) and dominant-negative MEK (S217 deleted) have been described previously (Klesse and Parada, 1998; Klesse et al., 1999; Spencer et al., 2002a).

Neurite outgrowth quantification

SH-SY5Y cells were seeded at 20,000 cells/cm2 on uncoated or laminin-1-coated coverslips in DMEM/F12 containing 15% fetal bovine serum and incubated at 37°C in a humidified atmosphere containing 5.0% CO2. After 24 hours, cells were exposed to purified adenoviruses (400 MOI) overnight and subsequently washed and placed in serum-free medium. Cells were either incubated for 3 days and fixed with 4.0% paraformaldehyde or reinfected with adenovirus after 3 days and fixed 7 days later in 4.0% paraformaldehyde. We analyzed the number of neurites per cell, the percentage of neurite-bearing cells, the total neurite length per cell and the number of branch points per neurite in two to four separate experiments (Hynds and Snow, 1999; Hynds and Snow, 2001). We analyzed the number of neurites per cell in two ways: (1) by excluding those cells that did not extend neurites or (2) by including all cells. Each of these methods offers distinct advantages. Excluding cells that do not express neurites separates this neurites/cell measurement from the percentage of neurite-bearing cells. Including those cells that do not express neurites provides a more global measurement of neurite initiation. Phase and fluorescent digital images (five/condition in each experiment) were captured using the Attofluor ratio-imaging program, and neurite outgrowth was quantified using the KS400 imaging system from phase images of cells identified to be infected by production of GFP.

In some experiments, we compared outgrowth from cells expressing CA-Rit or GFP only to cells infected with adenovirus containing untagged mutant forms of Ras, Raf or MEK. In these experiments, average infection rates were 80% for cells infected with GFP-expressing constructs. We assumed there was similar infectivity using adenovirus containing Ras, Raf, and MEK mutants, and all analyses were performed using phase contrast images without specifically identifying infected cells. For all outgrowth experiments, a neurite was defined as a process greater than 10 μm, and the total neurite length per cell was calculated by adding the lengths of all the processes of each cell divided by cell number.

Western blotting

SH-SY5Y cells were seeded at 50,000 cells/cm2 in DMEM/F12 containing 15% fetal bovine serum in six-well plates and incubated at 37°C in a humidified atmosphere containing 5.0% CO2. Cells were incubated until 90% confluent and exposed to purified adenoviruses (400 MOI) overnight and subsequently washed and placed in serum-free medium. SDS cell lysates, normalized for protein content, were electrophoresed through 10% SDS-PAGE gels under reducing conditions. Protein bands were electrotransferred to nitrocellulose. Blots were blocked with 5.0% bovine serum albumin or non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TTBS) and incubated overnight with anti-Rit monoclonal (Gamma-1, Lexington, KY), anti-phospho-ERK monoclonal (Cell Signaling Technology, Beverly, MA), anti-ERK 1/2 polyclonal (Chemicon, Temecula, CA), anti-phospho-Akt polyclonal (R&D Systems, Minneapolis, MN) or anti-Akt monoclonal (BD Transduction Laboratories, Lexington, KY) antibodies. Blots were washed 3×10 minutes in TTBS and incubated for 2 hours in goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase (Sigma, St. Louis, MO). Immunoreactive proteins were visualized using enhanced chemiluminescence (kit from Amersham, Buckinghamshire, UK) or a colorimetric BCIP-NBT substrate (kit from Bio-Rad, Hercules, CA). Quantification of immunoreactive bands was performed by scanning densitometry using Scion software (NIH image).

Results

Rit supports neurite outgrowth in SH-SY5Y cells on endogenous matrix

We used the well-established neuronal model, SH-SY5Y neuroblastoma cells (Ross et al., 1983), to determine the effects of Rit on human neuronal differentiation. Cells plated on uncoated glass coverslips, which rapidly secrete an endogenous matrix rich in laminin (Hynds and Snow, 2001), were infected with recombinant adenovirus to co-express green fluorescent protein (GFP) and wild-type (WT-Rit) or constitutively active Rit [CA-Rit; RitQ79L (Shao et al., 1999; Shao and Andres, 2000)]. Expression of the Rit transgene was confirmed by western blotting, which showed high levels of Rit three days post-infection compared with undetectable levels in either non-infected cells or cells infected with GFP alone (Fig. 1A).

Fig. 1.
Fig. 1.

Rit supports neurite outgrowth in SH-SY5Y cells on endogenous matrix. SH-SY5Y cells were either not exposed to adenovirus or infected with adenovirus expressing only green fluorescent protein (GFP) or co-expressing GFP and wild-type (WT-Rit) or constitutively active Rit (CA-Rit). Expression of the transgene was confirmed by western blotting for Rit (A). Images of uninfected cells (B) or cells infected with GFP alone (C, and corresponding fluorescent image D) displayed basal levels of neurite outgrowth. Cells co-expressing GFP and either WT-Rit (E, and corresponding fluorescent image F) or CA-Rit (G, and corresponding fluorescent image H) displayed a qualitative increase in neurite outgrowth from SH-SY5Y cells. Bar, 50 μm.

Qualitatively, both WT- and CA-Rit induced robust neurite outgrowth in SH-SY5Y cells (Fig. 1). Compared with uninfected cells (Fig. 1B) or cells expressing only GFP (Fig. 1C,D), cells infected with WT-Rit (Fig. 1E,F), and more so with CA-Rit (Fig. 1G,H), showed a qualitative increase in neurite outgrowth. Quantification of outgrowth from Rit-expressing cells (Fig. 2) confirmed that CA-Rit significantly increased the number of neurites per cell (Fig. 2A, analysis included only those cells bearing neurites), the percentage of neurite-bearing cells (Fig. 2B), the total neurite length per cell (Fig. 2C) and the number of branch points per neurite (Fig. 2D) compared with either non-infected cells or adenovirus-infected cells expressing GFP alone (asterisks indicate significance at P<0.05 compared with both uninfected cells and cells expressing only GFP). In addition WT-Rit increased the percentage of neurite-bearing cells (Fig. 2B) and the total neurite length per cell (Fig. 2C). If all cells (including those not expressing neurites) were included in the analysis of neurites per cell, this measurement was also increased by WT-Rit (data not shown). In several measurements, cells infected with adenovirus expressing only GFP had slightly lower neurite outgrowth measurements (see Fig. 2B-D, # indicates significantly different from the control at P<0.05). However, in cells co-expressing transgenes for WT- or CA-Rit, this effect is negated, and outgrowth is promoted above the level of uninfected cells, perhaps reflecting Rit-mediated increases in cell survival (Spencer et al., 2002a). These data suggest that Rit is a potent promoter of neurite outgrowth of unstimulated SH-SY5Y cells growing on their endogenous matrix.

Fig. 2.
Fig. 2.

Quantification of the number of neurites per cell (A, excluding cells not bearing neurites), the percent of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells cultured on endogenous matrix. Wild-type (WT-Rit) and/or constitutively active (CA-Rit) Rit increases neurite initiation (A,B), elongation (C) and branching (D) in comparison with both uninfected cells (Control) and cells infected with adenovirus expressing only green fluorescent protein (GFP). Results are means±s.e.m. across experiments for four separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

Rit supports neurite outgrowth in SH-SY5Y cells on laminin-1

The extracellular matrix molecule laminin-1 supports neurite outgrowth from many types of neurons (Luckenbill-Edds, 1997; Jones et al., 2000). We, and others, have confirmed these observations in SH-SY5Y cells (Choi et al., 1994; Leventhal and Feldman, 1995; Hynds and Snow, 1999). To determine whether Rit-induced outgrowth is synergistic, additive or possibly antagonistic with outgrowth supported by laminin-1, we infected SH-SY5Y cells with adenovirus co-expressing GFP and WT- or CA-Rit and grew them on substrata coated with purified laminin-1.

WT-and CA-Rit induced robust neurite outgrowth in SH-SY5Y cells on laminin-1 (Fig. 3A) or cells which is similar to the effect seen with cells plated on endogenous matrix. Uninfected cells (Fig. 3) or cells expressing only GFP (Fig. 3B,C) displayed basal neurite outgrowth, which was more robust than when cells were plated on uncoated glass (compare to Fig. 1). Compared with this, cells infected with WT-Rit (Fig. 3D,E), and more so with CA-Rit (Fig. 3F,G), had more neurites with well-developed growth cones (indicated by arrowheads) and branching (indicated by arrows) on laminin-1. Quantification of outgrowth from Rit-expressing cells on laminin-1 (Fig. 4) confirmed that on this purified substratum, both WT-and CA-Rit significantly increased the number of neurites per cell (Fig. 4A, analyzed by excluding cells that did not express neurites), the percentage of neurite-bearing cells (Fig. 4B), the total neurite length per cell (Fig. 4C) and the number of branch points per neurite (Fig. 4D, CA-Rit only). When we included cells not extending neurites in the neurites per cell measurement, control and GFP-expressing cells were comparable, with WT-and CA-Rit-expressing cells having significantly more neurites per cell (data not shown). The slight inhibitory effects we observed with transfection with GFP only in cells plated on endogenous matrix were not evident in cells plated on laminin-1 (compare Fig. 2 with Fig. 4). Outgrowth on laminin-1 was more robust than outgrowth on endogenous matrix, particularly in regard to measures of neurite initiation and elongation (e.g. in cells expressing CA-Rit there were 1.66±0.1 neurites per cell, 89.1±14.5% neurite-bearing cells, and 81.6±24.7 μm total neurite length for cells on endogenous matrix compared with 2.06±0.1 neurites per cell, 97.2±5.9% neurite-bearing cells and 105.9±27.1 μm total neurite length for cells plated on laminin-1). Moreover, the percentage increase in the number of neurites per cell and the total neurite length per cell due solely to expression of CA-Rit was different for cells plated on laminin-1 (189% increase for the number of neurites per cell; 152% increase for total neurite length per cell) compared with cells on endogenous matrix (136% increase for the number of neurites per cell, 120% increase for total neurite length per cell). These results suggest that Rit and laminin-1 work cooperatively to maintain neurites and increase elongation. Interestingly, the increase in the number of branches per neurite due to expression of CA-Rit was consistent regardless of substratum (approximately a 50% increase over baseline for cells on either endogenous matrix or laminin-1), suggesting that the increased branching may be promoted by Rit and not laminin-1. Together, these results indicate that SH-SY5Y neurite outgrowth mediated by laminin-1 and Rit converges on a common downstream signaling molecule, which is activated by both integrins and Rit, to mediate neurite elongation but not branching.

Fig. 3.
Fig. 3.

Qualitative assessment of neurite outgrowth from SH-SY5Y cells plated on purified laminin-1. Cells were either not exposed to adenovirus (A) or exposed to adenovirus expressing only green fluorescent protein (GFP, B, and corresponding fluorescent image C), co-expressing GFP and wildtype (WT-Rit, D, and corresponding fluorescent image E) or co-expressing GFP and constitutively active Rit (CA-Rit, F, and corresponding fluorescent image G). SH-SY5Y cells expressing WT-Rit or CA-Rit have well developed growth cones (arrowheads in D,F) and display a qualitative increase in neurite outgrowth including marked increases in branching (arrows in F). Bar, 50 μm.

Fig. 4.
Fig. 4.

Quantification of the number of neurites per cell (A, excluding cells not bearing neurites), the percentage of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells cultured on laminin-1. Both wild-type (WT-Rit) and constitutively active (CA-Rit) Rit increase neurite initiation (A,B), elongation (C) and branching (D, CA-Rit only) compared with control cultures not infected with adenovirus (Control) or cultures infected with adenovirus expressing only green fluorescent protein (GFP). Results are means±s.e.m. from four separate experiments. Asterisks (*) indicate significant difference compared with Control cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

Differentiation induced by Rit is distinct from Ras or Raf

Outgrowth initiated by receptor tyrosine kinase growth factors is mediated in part through activation of the Ras/Raf/MEK/ERK cascade (Hagag et al., 1986; Szeberenyi et al., 1990; Kaplan and Stephens, 1994; Szeberényi and Erhardt, 1994). To determine if Rit signals through a distinct pathway, we compared outgrowth from three groups of SH-SH5Y cells, transfected with CA-Rit, CA-Ras or CA-Raf. All treatments induced some facets of outgrowth (Fig. 5). However, outgrowth induced by CA-Rit (Fig. 5C,H) was qualitatively different from that induced by CA-Ras (Fig. 5D,I) or CA-Raf (Fig. 5E,J), effects that were more pronounced at two weeks post-infection (Fig. 5H-J). These effects are more evident from quantification of neurite outgrowth (Fig. 6), which showed that CA-Rit increased the number of neurites per cell (Fig. 6A), the percentage of neurite-bearing cells (Fig. 6B), the total neurite length per cell (Fig. 6C) and the number of branch points per neurite (Fig. 6D). After 2 weeks (black bars), both CA-Ras and CA-Raf increased the number of neurites per cell (Fig. 6A), percentage of neurite-bearing cells (Fig. 6B) and the total neurite length per cell (Fig. 6C) compared with control values. CA-Ras also increased the total neurite length per cell after three days (Fig. 6C). If analysis of the number of neurites per cell included only cells extending neurites, the measurement was generally increased throughout each group, but the relative measurements were similar (data not shown). Importantly, neither CA-Ras nor CA-Raf affected the number of branch points per neurite (Fig. 6D). Cells transfected with CA-Rit displayed comparable or greater outgrowth, using any of these measures, including the number of branch points per neurite, than with CA-Ras or CA-Raf. These data demonstrate that Rit-induced neurite outgrowth is morphologically distinct and more pronounced than that of CA-Ras- or CA-Raf-mediated signaling.

Fig. 5.
Fig. 5.

Digital images of SH-SY5Y cells three days (3d, A-E) and two weeks (2w, F-J) after no infection (A,F) or initial infection with adenovirus expressing green fluorescent protein (GFP) only (B,G) or constitutively active (CA)-Rit (C,H), CA-Ras (D,I) or CA-Raf (E,J). Qualitatively, Rit induces greater outgrowth than Ras or Raf, and its effects on elongation and branching increase over two weeks.

Fig. 6.
Fig. 6.

Quantification of neurite outgrowth from SH-SY5Y cells expressing CA-Rit, CA-Ras or CA-Raf. CA-Rit significantly increases the number of neurites per cell (A, including cells not bearing neurites), the percentage of neurite-bearing cells (B), the length of the longest neurite per cell (C) and the number of branch points per neurite at three days (open bars) or two weeks (filled bars) after initial infection. In contrast, CA-Ras or CA-Raf increase neurite initiation (the number of neurites per cell, A; the percent of neurite-bearing cells, B) or elongation (the total neurite length per cell, C) over two weeks (CA-Ras also increased the total neurite length per cell at three days, C). Results are means±s.e.m. of pooled data from three separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

Rit activates ERK 1/2, but not Akt, to promote neurite outgrowth

Receptor tyrosine kinases support neurite outgrowth and survival through activation of ERK and Akt (Kaplan and Stephens, 1994; Encinas et al., 1999; Vaillant et al., 1999). To determine if Rit activates these same pathways to promote neurite outgrowth, we analyzed western blots of SH-SY5Y cells infected with adenovirus to induce expression of CA-Rit using antibodies specific to the phosphorylated forms of ERK 1/2 or Akt.

Constitutively active Rit activated ERK 1/2, as measured by increased immunoreactivity with antibodies specific to phosphorylated ERK (Fig. 7A), whereas ERK protein levels were comparable in all treatment conditions (Fig. 7B). The MEK inhibitor PD 098059 decreased Rit-induced ERK phosphorylation (Fig. 7A), as did dominant-negative MEK (DN-MEK; data not shown). Quantification of protein bands immunoreactive to phospho-ERK antibodies confirmed these observations (Fig. 7C). Rit did not, however, alter phosphorylation of Akt (Fig. 7D). SH-SY5Y cells expressing CA-Ras displayed increases in phospho-ERK 1/2 (Fig. 7A) and phospho-Akt (Fig. 7D). In addition, CA-Raf increased ERK phosphorylation (Fig. 7A) but not Akt phosphorylation (Fig. 7D). In quantifying phospho-Akt immunoreactivity, Ras, but not Rit, displayed increases (Fig. 7F). To determine if activation of ERK mediated Rit-induced neurite outgrowth, we performed image-analysis-based outgrowth experiments where we inhibited the activation of MEK. Inhibition of MEK by treatment with PD 098059 or expression of its dominant-negative mutant (data not shown) blocked the Rit-mediated increase in the number of neurites per cell (Fig. 8A) and the percent of neurite-bearing cells (Fig. 8B). However, neither PD 098059 nor DN-MEK (data not shown) significantly decreased the total neurite length per cell (Fig. 8C) or the number of branch points per neurite (Fig. 8D). Treatment of uninfected cells or cells expressing CA-Raf with either PD 098059 or DN-MEK (data not shown) produced similar data (i.e. the number of neurites per cell and the percent of neurite-bearing cells were reduced without significantly affecting the length of the total neurite length per cell or the number of branch points per neurite). If analysis of the number of neurites per cell included only cells extending neurites, the measurement was generally increased throughout each group, but the relative measurements were similar (data not shown). These results demonstrate that Rit-induced neurite initiation, but possibly not neurite elongation or branching, is mediated through a signaling pathway that involves ERK.

Fig. 7.
Fig. 7.

Western blots for phosphorylated ERK (A) and total ERK (B) from SH-SH5Y cells not infected (Con) or infected with adenovirus to express green fluorescent protein (GFP) or constitutively active (CA) Rit, Ras or Raf, quantified in arbitrary units compared with uninfected cells (C), demonstrate that CA-Rit, CA-Ras, and CA-Raf all increased ERK phosphorylation. Western blots for phosphorylated Akt (D), and total Akt (E), quantified in F, demonstrate that CA-Ras, but not CA-Rit, increased Akt phosphorylation. Treatment of CA-Rit infected cells with the MEK inhibitor PD 098059 (Rit, PD) inhibits Rit-induced ERK phosphorylation (A,C) but not Akt phosphorylation (F). Data shown are representative blots from an experiment performed three times (quantification includes all three experiments).

Fig. 8.
Fig. 8.

Quantification of the number of neurites per cell (A, including cells not bearing neurites), the percent of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells either not infected (Control) or infected with adenovirus to express green fluorescent protein (GFP) alone or co-express GFP and constitutively active (CA-) Rit or CA-Raf. Cells were also subjected to treatment with the MEK inhibitor PD 098059 (PD, CA-Rit + PD, CA-Raf+PD). CA-Rit increases neurite initiation (A,B), elongation (C, P=0.056) and branching (D) over Control or cultures infected with adenovirus expressing only GFP. PD 098059 blocks the CA-Rit-induced increase in the number of neurites per cell (A) and the percentage of neurite-bearing cells (B) but not the total neurite length (C) or the number of branch points per neurite (D). PD 098059 decreased basal or CA-Raf-induced neurite initiation (the number of neurites per cell, A; the percentage of neurite-bearing cells, B), but not elongation (C) or branching (D). Results are means±s.e.m. of pooled data from two separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures (not bracketed), and bracketed asterisks indicate significant differences between those respective groups at P<0.05 (ANOVA with Least Significant Difference post-hoc test).

Discussion

In this study we used adenoviral expression of the small GTPase Rit to demonstrate that Rit increases neurite outgrowth from a human neuronal model: SH-SY5Y neuroblastoma cells. The important and novel findings of this study include: (1) Rit increases neurite initiation, elongation and branching from SH-SY5Y cells on either endogenous matrix or purified laminin-1; (2) Rit acts cooperatively with laminin-1 to maintain neurites and promote neurite elongation; (3) Rit acts independently from substratum to promote neurite branching; (4) Rit-induced neurite outgrowth is morphologically distinct from outgrowths supported by Ras or Raf that do not increase branching; (5) Rit activates ERK, but not Akt, in SH-SY5Y cells; and (6) inhibition of MEK blocks Rit induced neurite initiation, but not elongation or branching. Together, these data indicate that two distinct signaling mechanisms mediate neurite outgrowth in human neuron-like cells: (1) an MEK-dependent mechanism, which may also be activated by Ras or Raf, that supports neurite initiation; and (2) an MEK-independent mechanism involving an uncharacterized signal transduction pathway that supports neurite branching.

Rit and neuronal development/regeneration

Members of the Ras and Rho subfamilies of the small GTPases are involved in mediating neuronal development and regeneration (Hagag et al., 1986; Szeberenyi et al., 1990; Qiu and Green, 1992; Kaplan and Stephens, 1994; Kuhn et al., 1998; Klesse et al., 1999; Klesse and Parada, 1999; Lehmann et al., 1999; Mazzoni et al., 1999; Sebök et al., 1999; Brown et al., 2000; Burry, 2001; Linseman et al., 2001). Rit and its most closely related proteins, Rin and RIC, constitute a branch of the Ras subfamily of small GTPases (Lee et al., 1996; Shao et al., 1999). Rin expression is restricted to postnatal and adult brain and retina (Lee et al., 1996; Spencer et al., 2002b). In contrast, Rit is temporally and spatially expressed in many tissues, including developing brain (Lee et al., 1996; Spencer et al., 2002a), which indicates a function for Rit during neuronal development (Spencer et al., 2002a) (this study). In the present study, Rit increased neurite initiation (the number of neurites per cell and the percent of neurite-bearing cells), elongation (the total neurite length per cell) and arborization (the number of branch points per neurite) in SH-SY5Y human neuron-like cells on either endogenous matrix or purified laminin-1.

Interestingly, Rit significantly increases neurite arborization, an atypical behavior for SH-SY5Y cells (Hynds et al., 1997; Hynds and Snow, 1999; Hynds and Snow, 2001). This suggests that Rit functions early in neuronal development to determine process identity (i.e. dendrite versus axon) (Ferhat et al., 1998; Higgins et al., 1988). Alternatively, Rit may function later in neuronal development during axon terminal arborization and synapse formation (Thanos and Bonhoeffer, 1987; Jhaveri et al., 1991; Kennedy and Tessier-Lavigne, 1995). The literature indicates that SH-SY5Y neurites are primarily axonal (Haque et al., 1999; Encinas et al., 2000), and our own data (data not shown) demonstrating high levels of tau in these cells confirm the results from earlier studies. The current data support previous work demonstrating that axon branching is separable from other aspects of neurite outgrowth (Weeks et al., 1991; Cogen and Cohen-Cory, 2000; Markus et al., 2002). Therefore, we hypothesize that Rit functions in neurons to increase axonal arborization during target innervation and/or synaptic plasticity events. Recently, we have confirmed that CA-Rit increases axonal branching in primary cultures of rat cortical or hippocampal neurons (M.L.S. and D.A.A., unpublished). Interestingly, Rit-induced branching could be separated from extracellular matrix influences and from activation of ERK (see below).

Rit and extracellular matrix interactions

We, and others, have previously demonstrated that laminin promotes neurite outgrowth from SH-SY5Y cells (Choi et al., 1994; Leventhal and Feldman, 1995; Hynds and Snow, 1999; Hynds and Snow, 2001). Our results presented here show that Rit may act synergistically with laminin-1 to increase the number of neurites per cell if all cells are included in the analysis (CA-Rit induced a 113% increase in cells on endogenous matrix, and a 209% increase in cells on laminin). When only cells bearing neurites were included in the analysis, CA-Rit induced a 136% increase in the number of neurites per cell for cells on the endogenous matrix, whereas the corresponding increase in the number of neurites per cell for cells on purified laminin-1 was 189%. These results support our earlier observations (Hynds and Snow, 1999) and may indicate that laminin- and Rit-activated signaling pathways act cooperatively and synergistically to promote neurite initiation. However, little synergy (approximately 10% increase over expected) was observed between Rit and laminin-1 on the percentage of neurite-bearing cells (another measure of neurite initiation). However, when CA-Rit is expressed, nearly all cells have neurites, which approaches the asymptotic limit of this parameter and probably minimizes separable differences between laminin and Rit effects. The signal transduction pathways involved in potential Rit-laminin interactions have yet to be explored. Interestingly, the percentage increase in the number of branch points per neurite due to CA-Rit is the same in cells on endogenous matrix and on purified laminin-1, suggesting that the increased branching is a unique feature of Rit-dependent signaling.

Rit and neuron survival

Ras, through activation of PI3K, promotes survival of several types of neuron and neuron-like cell lines (Hagag et al., 1986; Szeberenyi et al., 1990; Qiu and Green, 1992; Klesse and Parada, 1998; Mazzoni et al., 1999). Rit, by sequence homology, is a member of the Ras subfamily and promotes both neurite outgrowth and survival in PC6 cells (Spencer et al., 2002a). SH-SY5Y cells plated on endogenous matrix and infected with adenovirus to express GFP alone consistently displayed slightly decreased outgrowth compared with untreated controls (see Fig. 2). As has been reported, this effect may be due to toxicity mediated by GFP expression (Liu et al., 1999). Infection with WT- or CA-Rit abolishes decreases in outgrowth mediated by adenoviral constructs expressing only GFP, which may indicate that Rit has an effect on both survival and outgrowth. Interestingly, in SH-SY5Y cells plated on laminin-1, GFP did not decrease outgrowth compared with untreated controls.

The mechanism through which Rit mediates survival is unclear as CA-Rit did not increase phosphorylation (i.e. activation) of Akt in SH-SY5Y (this report) or PC6 cells (Spencer et al., 2002a). However, mechanisms that do not depend exclusively on activation of the PI3K/Akt pathway have been described (Klocker et al., 2000; Li et al., 2000; Linseman et al., 2001). The fact that we do not observe a decrease in outgrowth in SH-SY5Y cells plated on laminin-1 may indicate that this extracellular matrix molecule either further activates basal survival pathways or potentially activates different survival pathways. SH-SY5Y cells express several integrin subunits includingα 1, α3, α6 andβ 1, which can heterodimerize to form receptors activated by binding to laminin-1 (Hynds and Snow, 2001). Laminin binding to hippocampal neurons orβ 1 subunit activation activates Akt to promote neuron survival (Gary and Mattson, 2001). It is possible that laminin-1 activation of integrins leads to increased SH-SY5Y cell survival through this mechanism to eliminate GFP-mediated toxicity. An alternative and exciting prospect is that Rit activates a unique survival/differentiation pathway, as has been demonstrated in other cell types (Allsopp, 2000; Han and Holtzman, 2000; Klocker et al., 2000; Li et al., 2000; Linseman et al., 2001).

Rit mechanism of action

The signaling mechanisms through which Rit promotes neurite outgrowth are incompletely characterized. In fibroblasts, Rit promotes transformation, but does not activate known survival or proliferative signaling components such as ERK, p38 MAPK, JNK, PI3K or Akt (Rusyn et al., 2000). Rit activates ERK, but not Akt, in SH-SY5Y cells (this manuscript) and neuron-like PC6 cells (Spencer et al., 2002a) and clearly increases neurite outgrowth in both cell types. ERK activation is required for Ras-mediated neurite outgrowth (Qiu and Green, 1992; Kaplan and Stephens, 1994; Encinas et al., 1999; Klesse et al., 1999; Klesse and Parada, 1999). However, we show that neurite outgrowth induced by Rit is morphologically distinct from that induced by CA-Ras or CA-Raf and that inhibition of the ERK pathway with the MEK inhibitor PD 098059 inhibits ERK 1/2 phosphorylation and blocks some aspects of Rit-induced outgrowth (the number of neurites per cell, the percent of neurite-bearing cells) but not others (the total neurite length per cell, the number of branch points per neurite). Combining these data with those demonstrating separable effects between laminin-1- and Rit-supported outgrowth, we make the following speculations: (1) Rit-supported neurite initiation is enhanced by integrin activation and involves ERK activation; (2) Rit-supported neurite elongation is enhanced by integrin activation through a mechanism not involving ERK and (3) Rit-enhanced neurite branching is not affected by integrin engagement and does not involve ERK activation.

Some studies have demonstrated that trophic factors support neurite branching through activation of undefined pathways that do not involve activation of ERK. For example, Akt (Markus et al., 2002), cAMP (Weeks et al., 1991) and nitric oxide (Cogen and Cohen-Cory, 2000) modulate axon branching, which is differentially affected by trophic factors (Gallo and Letourneau, 1998; Szebenyi et al., 2001) and extracellular matrix molecules (Weeks et al., 1991). We propose that Rit activates a novel pathway to promote neurite outgrowth and branching (Kuo et al., 1996; Morooka and Nishida, 1998; Lehmann et al., 1999; Anneren et al., 2000; Brown et al., 2000; Ghil et al., 2000; Klocker et al., 2000; Burry, 2001). Candidate members of such a pathway may include novel protein kinases and/or additional members of the Ras superfamily of small GTPases. The last possibility is intriguing given that members of the Rho subfamily are necessary for neuronal outgrowth (Kuhn et al., 1998; Sebök et al., 1999; Brown et al., 2000; Linseman et al., 2001) and affect branching (Albertinazzi et al., 1998; Shen et al., 1998; Lehmann et al., 1999; Neumann et al., 2002). On the basis of our data, we suggest that Rit promotes neurite initiation through activation of the ERK pathway whereas it promotes neurite elongation and branching through an ERK-independent pathway, which may involve Rho family GTPase activation.

Acknowledgements

Supported in part by Kentucky Spinal Cord and Head Injury Research Trust grant #0-8 (to DLH), NIH grant EY10545 and Kentucky Spinal Cord and Head Injury Research Trust grant #7-20 (to D.M.S.), and a grant from the Kentucky Lung Cancer Research Fund (to D.A.A.). The authors thank Adina Badstibner for technical assistance and Pamela Knapp for critical review of this manuscript.

  • Accepted January 29, 2003.

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Research Article
Rit promotes MEK-independent neurite branching in human neuroblastoma cells
DiAnna L. Hynds, Mike L. Spencer, Douglas A. Andres, Diane M. Snow
Journal of Cell Science 2003 116: 1925-1935; doi: 10.1242/jcs.00401
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2020 at The Company of Biologists

Despite the challenges of 2020, we were able to bring a number of long-term projects and new ventures to fruition. While we look forward to a new year, join us as we reflect on the triumphs of the last 12 months.

Mole – The Corona Files

"This is not going to go away, 'like a miracle.' We have to do magic. And I know we can."

Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.

Cell scientist to watch – Christine Faulkner

In an interview, Christine Faulkner talks about where her interest in plant science began, how she found the transition between Australia and the UK, and shares her thoughts on virtual conferences.

Read & Publish participation extends worldwide

“The clear advantages are rapid and efficient exposure and easy access to my article around the world. I believe it is great to have this publishing option in fast-growing fields in biomedical research.”

Dr Jaceques Behmoaras (Imperial College London) shares his experience of publishing Open Access as part of our growing Read & Publish initiative. We now have over 60 institutions in 12 countries taking part – find out more and view our full list of participating institutions.

JCS and COVID-19

For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.

If you have any questions or concerns, please do not hestiate to contact the Editorial Office.