1 Department of Anatomy and Neurobiology, University of Kentucky Medical Center,
Lexington, KY 40536-0298, USA
2 Department of Molecular and Cellular Biochemistry, University of Kentucky
Medical Center, Lexington, KY 40536-0298, USA
* Author for correspondence (e-mail: dlhynd0{at}uky.edu)
Accepted 29 January 2003
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Summary |
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Key words: GTPase, Rit, SH-SY5Y human neuroblastoma cells, Axon branching, Signal transduction, Neurite outgrowth
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Introduction |
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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.
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Materials and Methods |
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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 3x10 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).
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Results |
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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.
|
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.
|
|
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.
|
|
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.
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Discussion |
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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 including
1,
3,
6 and
ß1, which can heterodimerize to form receptors activated by
binding to laminin-1 (Hynds and Snow,
2001
). Laminin binding to hippocampal neurons or
ß1 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.
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Acknowledgments |
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