P . Lein et al. Brain Research 885 2000 220 –230
221
also significant differences in the response patterns of concentrations may represent a mechanism for controlling
PC12 cells to Mn and NGF. Mn elicits a much more rapid the rate and direction of neurite outgrowth.
outgrowth of neurites than NGF hours versus days, respectively and, unlike NGF, Mn does not support
neuronal survival [36]. Such observations suggest that
2. Materials and methods
there are likely to be differences between the Mn and NGF signaling pathways. At least one point of divergence is at
2.1. Materials the level of initiation of the signaling pathway since there
is no evidence to suggest that Mn triggers signaling All tissue culture reagents were obtained from Gibco-
through a trk-dependent mechanism. BRL Grand Island, NY with the exception of b-NGF,
Integrins represent one class of transmembrane mole- which was purchased from Harlan Bioproducts Madison,
cules that could function to transduce the Mn signal in WI. Poly-
D
-lysine, cycloheximide and actinomycin D PC12 cells. Integrins comprise a family of heterodimeric
were obtained from Sigma St. Louis, MO, and MnCl
2
ab receptors, which mediate cellular interactions with was purchased from J.T. Baker Phillipsburg, NJ. Goat
extracellular matrix ECM and cell-surface ligands antiserum anti-gp140 known as anti-ECMR was raised
[21,25]. Binding of ligand to integrin receptors activates against purified adhesion-related, 140-kDa, integral mem-
intracellular signaling cascades that may alter gene tran- brane glycoproteins from BHK cells [30] and was the
scription [21,22] and thus ECM–integrin interactions generous gift of Dr. Karen Knudsen Lankenau Medical
influence numerous aspects of neural development and Research Center, Philadelphia, PA. Polyclonal antisera
differentiation [35,45,58]. One such aspect is neurite raised against the human fibronectin receptor, a b , or the
5 1
outgrowth, and changes in cellular adhesion to the ECM human vitronectin receptors, a b
and a b , were ob-
v 3
v 5
are thought to play an important role in differentiation and tained from Gibco-BRL. Monoclonal antibody mAb to
guidance of axons [31,35,45,58]. Neuronal interactions the vitronectin receptor clone Vr147 was purchased from
with the ECM can be altered by changing integrin–ligand Chemicon Temecula, CA. mAb specific for the a
8
affinity [21,29] or by regulating either the amount or integrin subunit was generously donated by Dr. Lynn
subcellular distribution of integrins [7,8,13,19,39]. One Schnapp The Mount Sinai School of Medicine, New
factor that has been shown to modulate both the affinity York, NY. The polyclonal goat anti-bovine GFAP was
and specificity of integrins for ECM ligands is Mn. obtained from Axell Westbury, NY; the rhodamine-
Moreover, there is evidence to implicate integrins in Mn- conjugated goat anti-rabbit IgG secondary antibody was
induced neurite outgrowth in PC12 cells. Studies per- from Boehringer-Mannheim Indianapolis, IN.
formed in serum-free media indicate that serum or purified vitronectin or fibronectin are required to support Mn-
2.2. Maintenance and treatment of PC12 cell cultures induced neurite outgrowth [36]. The pentapeptide GRGDS,
which blocks RGD-mediated interactions of integrins with PC12 cells that respond to Mn with increased neurite
vitronectin or fibronectin [9,65], inhibits not only Mn- outgrowth were originally obtained from Dr. John Wagner
induced neurite outgrowth but also activation of ERK1 and Harvard University School of Medicine, and a variant
ERK2 [36,60]. These observations further suggest that the clone of PC12 cells that does not respond to Mn nrPC12
effects of Mn are mediated by a integrins since this
was obtained from Dr. John Aletta SUNY at Buffalo
v
family of integrins binds to both vitronectin and fibronectin School of Medicine, Buffalo, NY. The responsive clone
in an RGD-dependent manner [6], and their function has of PC12 cells was maintained on tissue culture plastic in
been shown to be upregulated by Mn in nonneuronal cells DMEM supplemented with 10 fetal calf serum FCS,
[9,14,17,18,50,51,68]. 5 heat-inactivated horse serum HS, penicillin 100
Although a integrins have been reported to play an
units ml, and streptomycin 100 units ml. The nrPC12
v
integral role in regulating the differentiation and migration cells were maintained in RPMI supplemented with 10
of neuronal and glial cells [3,26,36–38], their expression HS, 5 FCS, penicillin 100 units ml, and streptomycin
in PC12 cells has yet to be demonstrated. Therefore, the 100 units ml on substrates precoated with rat tail col-
validity of these interpretations depends upon demon- lagen as described in Ref. [15]. For both clones, culture
strating that PC12 cells express a integrins and that
media was changed 3 times per week, and cultures were
v
blocking a integrin function is sufficient to inhibit the
passaged at 80–90 confluence. All experiments were
v
neurite-promoting activity of Mn. The data reported herein performed on cells between passage numbers 3–20.
supports the hypothesis that Mn-induced neurite outgrowth For neurite-outgrowth studies, cells of either clone were
is mediated by a integrins and further suggests a model in subcultured onto 18-mm glass coverslips precoated with
v 5
which activation of a integrins by Mn results in increased poly-
D
-lysine 100 mg ml at a density of 2310 cells per
v
surface expression of a integrins, partly through synthesis coverslip. Cells were initially plated in maintenance media
v
of additional a integrins, which in turn serves to increase and allowed to adhere for 24 h. All experimental treat-
v
neurite outgrowth. Thus, modulation of extracellular Mn ments were initiated by rinsing cultures once with serum-
222 P
free medium and replacing the culture medium with these data, films were scanned and band density deter-
DMEM or RPMI for nrPC12 cells containing 1 FCS mined as absorption units using the MacBas software
and the experimental agents. Experiments were termi- program version 2.31, Fuji Film.
nated at various time points by fixing cultures in 4 paraformaldehyde in 0.1 M phosphate buffer for 10 min,
2.4. Immunocytochemistry then mounting cultures in Elvanol DuPont, Wilmington,
DE after rinsing with phosphate-buffered saline PBS, 5 Cells were subcultured onto poly-
D
-lysine-coated cover- mM phosphate, 150 mM NaCl, pH 7.4. Neurite outgrowth
slips as described above, and incubated for 48 h in the was analyzed by phase contrast microscopy 3200.
presence or absence of 0.1 mM MnCl . Subsequently,
2
Processes were scored as neurites if they exhibited a cultures were rinsed twice in PBS and fixed in 4
growth cone, and their length was at least equal to the paraformaldehyde in 0.1 mM phosphate buffer. The a
v
diameter of the cell body. Data are presented as the integrin subunit was localized to the surface of non-
mean6S.E.M. and were evaluated by ANOVA followed by permeabilized cells by indirect immuno fluorescence using
Fisher’s LSD test. previously described procedures [33]. Polyclonal rabbit
antiserum that specifically cross-reacts with the a subunit
v
2.3. Immunoprecipitation 1:100 was used as the primary antibody; rhodamine-
conjugated goat anti-rabbit IgG 1:450, as the secondary PC12 cells cultured in 100-mm dishes in the presence or
antibody. Immunostained cultures were mounted in Slow- absence of MnCl
were removed from the substrate by fade Molecular Probes, Eugene, OR as per the manufac-
2
gentle trituration and rinsed sequentially with ice-cold PBS turer’s instructions and analyzed by confocal laser micro-
and labeling buffer 5 mM NaPO , 150 mM NaCl, 1 mM scopy BioRad 1024 confocal with a krypton–argon laser
4
CaCl , and 1 mM MgCl . Pelleted cells were resuspended linked to a Nikon Optiphot microscope. Images were
2 2
and incubated in labeling buffer supplemented with 10 obtained using a 340 lens with a numerical aperture of
mg ml biotin–X-NHS Calbiochem, Cambridge, MA for 1.3.
1 h at 48C. Biotinylated cells were extracted for 1 h at 48C in extraction buffer 1 Triton X-100, 5 mM NaPO , 150
4
mM NaCl, 1 mM CaCl , 1 mM MgCl , and 1 mM PMSF 3. Results
2 2
then incubated with Protein-A complexed to sepharose beads Sigma, St. Louis, MO to reduce background.
3.1. Mn-induced neurite outgrowth is mediated by b1 Pre-cleared extracts were incubated overnight at 48C with
integrins 100 ml Protein-A beads plus relevant antisera 1–10 mg
ml in immunoprecipitation buffer 0.1 Triton X-100, 5 PC12 cells were plated onto polylysine-coated glass
mM NaPO , 150 mM NaCl, 1 mM CaCl , 1 mM MgCl , coverslips and maintained in culture medium containing
4 2
2
and 1 mM PMSF. Pellets were washed 6 times with 15 serum for 24 h prior to the initiation of experimental
immunoprecipitation buffer to remove unbound antibody, treatments. As reported in earlier studies [36], the addition
and boiled for 5 min in reducing SDS–PAGE sample of Mn to the culture medium elicits a rapid morphological
buffer to strip the biotinylated proteins from the Protein-A change in PC12 cells characterized by a flattening of the
sepharose beads. Samples were separated by 5 SDS– cells and outgrowth of processes Fig. 1A,B. The pro-
PAGE, and electroblotted onto nitrocellulose. Blots were cesses formed by PC12 cells in response to Mn express
blocked for 20 min in 3 BSA in TBS-T 50 mM Tris, pH peripherin and GAP-43, and thus represent true neurites
7.6, 150 mM NaCl, and 1 Tween-20, and then incu- and not simply cytoplasmic extensions [36]. Also con-
bated overnight in streptavidin–HRP 1:300, Amersham– sistent with previous data [36], we observed that the
Pharmacia Biotech, Piscataway, NJ in TBS-T. Blots were neurite promoting activity of Mn occurs independently of
rinsed extensively in TBS-T prior to processing for NGF and is both time- and concentration-dependent data
enhanced chemiluminescence using the ECL kit Amer- not shown. When exposed to Mn in culture medium
sham–Pharmacia Biotech according to the manufacturer’s containing 1 serum, significant neurite outgrowth mea-
instructions. Data were captured on Kodak X-OMAT film. sured as the percentage of cells that extend neurites 20
For those experiments in which relative amounts of a is observed within 24 h at concentrations of Mn as low as
specific integrin subunit were compared between two 10 mM. Neurite outgrowth increases with increasing Mn
experimental conditions responding PC12 versus nrPC12 concentrations reaching a maximal 24-h response 55 at
and responding PC12 1 2 Mn, the protein concentration 100 mM Mn. The extent of neurite outgrowth also in-
of PC12 extracts was determined using the BioRad Protein creases with time such that maximal effects 82 are
Assay BioRad, Hercules, CA. The volume of extract observed after 72 h of exposure to Mn at concentrations of
used for subsequent immunoprecipitation studies was 10–20 mM. The EC
at 72 h is approximately 1.0 mM.
50
adjusted such that equivalent amounts of protein were Since these initial studies indicated that 100 mM total Mn
processed for each experimental condition. To quantify in culture medium containing 1 serum consistently
P . Lein et al. Brain Research 885 2000 220 –230
223
dent mechanism. First, PC12 cells do not extend neurites in response to Mn unless grown on substrates treated with
vitronectin, fibronectin, or culture medium containing serum. Second, the morphological effects of Mn are
blocked by the pentapeptide GRGDS, an analog of the RGD binding sequence recognized by various integrins
[9,65], but not by GRGES, the biologically inactive sequence. To further examine the role of integrins in
Mn-induced morphological differentiation, we tested the ability of function-blocking antibodies specific for integ-
rins to inhibit the neurite-promoting effects of Mn. Anti- ECMR serum has been shown to selectively interfere with
the function of b
integrins in both nonneuronal and
1
neuronal tissues [1,34,38,52], and in PC12 cells, this antiserum immunoprecipitates b
integrins [55,56]. As
1
indicated in Figs. 1 and 2, addition of anti-ECMR 0.2 almost completely blocks neurite outgrowth in response to
Mn at both 24 and 48 h as measured by the percentage of cells with neurites Fig. 2A and the number of neurites per
cell Fig. 2B. Isotype-matched control serum added to the culture medium at the same concentration has no effect on
process growth. The attachment of PC12 cells to coverslips precoated with poly-
D
-lysine is not affected by anti-ECMR Fig. 2C, and the effects of anti-ECMR on Mn-induced
neurite outgrowth are reversible after removal of the antibody not shown.
3.2. a Integrins play a significant role in Mn-induced
v
neurite outgrowth We next wanted to determine the identity of the a
integrin subunits mediating the Mn response. The a
v
represented a likely candidate since previous studies of PC12 cells suggested that Mn-induced neurite outgrowth is
mediated by b integrins that recognize the RGD ligand
1
motif [36]. In addition, anti-ECMR was shown to immuno- precipitate the a
integrin subunit from oligodendrocyte
v
precursor cells [38]. To determine if a b is expressed by
v 1
PC12 cells, and if it is recognized by anti-ECMR, PC12 cell extracts were immunoprecipitated with anti-ECMR or
with antibodies specific for the a integrin subunit. As
v
indicated in Fig. 3, anti-ECMR immunoprecipitates two
Fig. 1. Mn induces neurite outgrowth in PC12 cells grown in the absence
strong bands with molecular weights of approximately 150
of NGF and this activity is significantly inhibited by function-blocking
and 125 kDa lane 1. Polyclonal antibody to the a
v
antisera specific for b1 integrins. A Phase contrast photomicrographs of
integrin subunit immunoprecipitates two bands; one strong
PC12 cells grown in the absence of Mn exhibit a rounded morphology
band at approximately 150 000 and a weaker band at
with very few neuritic extensions. B PC12 cells exposed to Mn 0.1
115 000 Da Fig. 3, lane 2. As illustrated in lane 3, the
mM for 48 h typically appear flattened with multiple neurites exhibiting growth cones. C The addition of antisera specific for b1 integrins
higher molecular weight protein also appears to be precipi-
anti-ECMR, 0.2 significantly reduces Mn-induced neurite outgrowth
tated by the monoclonal antibody mAb specific for a .
v
but does not interfere with cell spreading. Bar, 50 mm.
Western blotting of these immunoprecipitates with the a
v
mAb confirmed that the 150 000-Da band present in all resulted in robust neurite outgrowth after a 24 or 48 h
three lanes is the a integrin subunit data not shown.
v
exposure, this exposure paradigm was used for the bio- A functional role for a
integrins in the response of
v
chemical and morphometric studies described below. PC12 cells to Mn is indicated by observations that
Several observations from earlier studies [36] suggest polyclonal antibodies specific for the a integrin subunit
v
that Mn induces neurite outgrowth via an integrin-depen- significantly inhibit the neurite-promoting activity of Mn
224 P
Fig. 3. Immunoprecipitation
of integrin
subunits from
surface biotinylated PC12 cells. Anti-ECMR serum lane 1 immunoprecipitates
integrin subunits whose electrophoretic properties are similar to those immunoprecipitated by polyclonal a
integrin antiserum lane 2 and
v
anti-a mAb lane 3.
v
the presence of a antibodies did not interfere with cell
v
adhesion Fig. 4C. However, the extent of inhibition exerted by a -specific antibodies is not as robust as that
v
observed in the presence of anti-ECMR 90–95. There are at least two possible interpretations. First, there may be
significant differences in the function-blocking capabilities of the anti-ECMR and a antibodies. Second, it is possible
v
that multiple b integrins mediate the morphological
1
response to Mn. In addition to the a integrins, plausible
v
candidates include the fibronectin receptor, a b , which
5 1
also binds the RGD sequence, and a b which has been
8 1
shown to function as a receptor for vitronectin and fibronectin [40,47]. Immunoblot analyses of PC12 cell
extracts suggest that these cells express a b , but not a b
Fig. 2. Mn-induced neurite outgrowth is inhibited by an antiserum to the
5 1
8 1
data not shown, and antibody blocking experiments
b1 integrin subunit anti-ECMR. Addition of anti-ECMR serum 0.2, column c to PC12 cell cultures exposed to Mn 100 mM significantly
indicate that a b functions in Mn-induced neurite out-
5 1
inhibited the percentage of cells with neurites A and the number of
growth Fig. 4A,B. Simultaneous exposure to both a and
v
neurites per cell B. Exposure to similar concentrations of an antiserum
a antibodies causes a significantly greater inhibition of
5
to an intracellular antigen GFAP, column d had no effect on Mn-
neurite outgrowth than either antibody alone, and these
induced neurite outgrowth. Data are expressed as mean6S.E.M. of three
effects are independent of changes in cell adhesion.
trials n50 per experimental condition per trial. Indicates a significant difference at P,0.01 ANOVA; the lower case letters over the bars
Although these data suggest that each of these integrins is
indicate which data sets are significantly different from the set repre-
important in the response of PC12 cells to Mn, the
sented by the bars. C Anti-ECMR serum does not alter adhesion of
remainder of our study focuses on the a integrins since
v
PC12 cells to the culture substratum. Cell adhesion was evaluated by
these appear to play a more significant role in axonal
counting the number of cells per culture following fixation with 4
outgrowth and synaptogenesis than a integrins in many
paraformaldehyde. Data are expressed as the percentage of adherent cells
5
relative to control cultures n53 coverslips per experimental condition.
neuronal cell types [35,41,42,45,58]. To further examine the importance of a
integrins in
v
Fig. 4. When assessed 48 h after the addition of Mn, a Mn-induced neurite outgrowth, we screened PC12 cell
v
antibodies caused a 45 reduction in the percent of cells clones for aberrant Mn responses and identified a stable
with neurites Fig. 4A, and a 55 reduction in the number clone nrPC12 cells that does not extend neurites when
of neurites per cell Fig. 4B. The addition of an isotype- exposed to Mn Fig. 5. The morphology of nrPC12 cells
matched control antibody at the same concentration had no exposed for 24 or 48 h to Mn at concentrations ranging
effect on Mn-induced neurite outgrowth Fig. 4A,B, and from 10 mM to 1 mM does not differ significantly from
P . Lein et al. Brain Research 885 2000 220 –230
225
Fig. 5. Characterization of a stable clone of PC12 cells nrPC12 that does not respond to the neurite-promoting effects of Mn. Exposure of
nrPC12 cells to varying concentrations of Mn 10 mM to 1 mM fails to elicit neurite outgrowth as measured by the percentage of cells with
neurites A and the number of neurites per cell B. However, nrPC12 cells did respond to NGF 100 ng ml with significant neuritic outgrowth.
Data are expressed as the mean6S.E.M. of three separate trials n50 per experimental condition per trial.
in response to Mn not because they are deficient in the cytoskeletal and membrane elements necessary to sustain
neurite outgrowth but rather because they lack the factors necessary to trigger neurite outgrowth in response to Mn.
Immunoprecipitation studies indicate that nrPC12 cells express negligible levels of the a integrin subunit on their
v
surface Fig. 6A and immunocytochemical analyses fail to
Fig. 4. Anti-a and a integrin sera inhibit neurite outgrowth in response
detect a data not shown. These data support the
v 5
v
to Mn. The addition of polyclonal antibodies to a and or a integrin
v 5
hypothesis that a integrins are necessary to trigger the
v
subunits 1:100, columns c, d, e to PC12 cell cultures exposed to Mn
morphological response of PC12 cells to Mn.
100 mM significantly reduces the percentage of cells with neurites A and the number of neurites per cell B. Addition of isotype-matched
3.3. Mn increases surface expression of a integrins
antisera specific for GFAP column f at a similar concentration had no
v
effect on neurite outgrowth. Data are expressed as the mean6S.E.M. of three trials n50 per condition per trial. Indicates a significant
One mechanism by which Mn could modulate the
difference at P,0.01 ANOVA, Fisher’s LSD test; the lower case letters
function of a integrins is by altering their level of
v
over the bars indicate which data sets are significantly different from the
expression in the plasma membrane. To determine if Mn
set represented by the bars. C The addition of a and a antibodies did
v 5
upregulates surface expression of a integrins, PC12 cells
not alter the adhesion of PC12 cells to the culture substratum as
v
determined by counting the number of cells per culture. Data are
grown in the absence or presence of Mn were immuno-
expressed as a percentage of adherent cells relative to control cultures
precipitated with a antibodies subsequent to biotinylation
v
n53 coverslips per condition.
of surface proteins. SDS–PAGE analysis of these immuno- precipitates indicates that Mn causes an |100-fold increase
that of control cells grown in the absence of Mn. However, in a levels 24 31065670 absorbance units in Mn-treated
v
a 24- or 48-h exposure to NGF 100 ng ml elicits cells as compared to 222642 in control cells, n54 on the
significant neurite outgrowth from nrPC12 cells Fig. 5. surface of these cells within 24 h of exposure Fig. 6B.
These data suggest that nrPC12 cells fail to extend neurites Similarly, comparative analysis of fluorescent intensity in
226 P
Fig. 6. SDS–PAGE of proteins immunoprecipitated from surface biotinylated PC12 cells with polyclonal a antibodies. A a Integrin
v v
expression by the nrPC12 cell subclone lane 1 is significantly less than in Mn-responsive PC12 cells lane 2. B Comparison of the amount of
a immunoprecipitated in responding PC12 cells not exposed to Mn lane
v
1 versus comparable cultures exposed to Mn 100 mM for 24 h lane 2 suggests that Mn upregulates a expression.
v
Fig. 8. Mn-induced neurite outgrowth requires de novo mRNA and protein synthesis. Addition of actinomycin D ACT-D, 0.1 mg ml or
cycloheximide CHX, 1.0 mg ml to cultures exposed to Mn 0.1 mM significantly inhibits the percentage of cells with neurites A, and the
number of neurites per cell B. Data are expressed as the mean6S.E.M. n100 per experimental condition. Indicates a significant difference at
P,0.01 ANOVA, Fisher’s LSD test; the lower case letters over the bars indicate which data sets are significantly different from the set repre-
sented by the bars.
confocal images of surface immunoreactivity for a in
v
non-permeabilized PC12 cells support the hypothesis that Mn specifically increases surface levels of a
in PC12
v
cells Fig. 7. This upregulated surface expression may result from de novo protein synthesis of a
integrin
v
subunits since addition to the culture medium of the RNA synthesis inhibitor, actinomycin D, or the protein synthesis
inhibitor, cycloheximide, markedly reduces neurite out- growth in the presence of Mn Fig. 8.
Fig. 7. Mn alters the amount and distribution of a immunoreactivity on
v
the surface of PC12 cells. PC12 cells grown for 48 h in the absence A or
4. Discussion
