IRAS, the human homologue of Nischarin, prolongs survival of transfected PC12 cells

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Letter to the Editor
IRAS, the human homologue of Nischarin, prolongs
survival of transfected PC12 cells
Cell Death and Differentiation (2003) 10, 933–935. doi:10.1038/sj.cdd.4401275
Dear Editor,
IRAS was discovered only recently
1
and is a highly unique
protein with little structural similarity to known proteins listed in
sequence databases. Nischarin was cloned as the mouse
homologue of IRAS.
2
The amino-acid sequence of Nischarin
is about 80% homologous with IRAS, with a main difference
being that Nischarin lacks the N-terminal 244 amino acids of
IRAS.
1,2
Nischarin was reported
2
to interact with the
cytoplasmic tail of the integrin a5subunit of the fibronectin
receptor, and to inhibit cell migration and lamellipodia
formation in transfected 3T3 cells. The human protein IRAS
(hIRAS) was also found to interact with insulin receptor
substrates (IRS) in HEK 293 cells and to enhance IRS4-
dependent insulin activation of extracellularly regulated
kinase, ERK1/2.
3
Herein, we report a new cellular role for
IRAS, which is to allow PC12 cells to experience markedly
less caspase-3 enzyme activity induced by apoptotic stimuli
such as serum deprivation, thapsigargin, or staurosporine
treatment. This decrease in apoptotic effector enzyme activity
was accompanied by a decrease in the proportion of apoptotic
cells presumably due to an increase in the delay to initiate
apoptosis.
IRAS and Nischarin have been shown to exist in multiple
tissues and cell lines.
1-4
Rat IRAS is endogenously expressed
in PC12 cells (Genbank EST 106159) as a 210 kDa
immunoreactive protein (Figure 1a), which is similar in size
to the full-length form of Nischarin already detected in
neuronal mouse cells.
2
Three clonal sublines (IE10, 6E7,
and 7D5) were selected by transfecting hIRAS cDNA into
PC12 cells, isolating single cells, and propagating under
geneticin selection. In addition, two empty-vector clonal
sublines (pcDNA3.1 and 7D5a) were isolated in parallel by
the same procedure. Cytologically, the cell lines were
indistinguishable under phase contrast microscopy. Transfec-
tion of hIRAS into PC12 cells led to the appearance of the
167 kDa human protein that was predominantly localized in
the cytosol of cells (Figure 1a). The exact relation between the
210 kDa rat IRAS (rIRAS) and the 167 kDa hIRAS protein is
not fully known, since the rat homologue has only been
partially sequenced (EST 106159). Based on rat and human
tissue Northern blot analysis,
4
it has been suggested that
alternative splicing may be responsible for two molecular
weight forms of this protein. If we assume that the two proteins
play the same role in PC12 cells, then hIRAS-expressing PC12
cells constitute a moderate overexpression model of IRAS.
IRAS and Nischarin exhibit 100% amino-acid homology
over an amino-acid sequence domain identified to bind a5-
integrin.
2
The integrins, particularly the fibronectin receptor,
have been linked to cell survival pathways. Overexpression of
the a5 subunit protects cell lines against apoptotic stimuli, in
part by modulating the expression of the antiapoptotic protein
Bcl2 by activating the PI3kinase/Akt pathway.
5,6
In this
context, we hypothesized that IRAS might also modulate the
survival of cells. An increase in caspase-3 activity is an index
of apoptosis. We therefore measured the activities of
caspase-3-like proteases after serum deprivation and com-
pared the hIRAS-expressing and -nonexpressing cell lines.
No differences in caspase-3 basal activities could be
observed between the five cell lines grown in 15% serum-
containing medium. However, by 3 h of serum starvation,
caspase-3-like activities had increased dramatically in the
control cells, attaining their plateau by 6 h (5.9-fold increase;
Figure 1b). By comparison, caspase-3 activities in hIRAS-
transfected cells (i.e., IE10 clone) were increased four-fold by
6 h of serum starvation, but remained statistically less than
those of control cells (Figure 1b) at least until 24 h in serum-
free medium. As shown in Figure 1c, after 6 and 24 h of serum
deprivation, higher levels of enzymatic activity were found in
both control cell lines (pcDNA3.1 and 7D5a) compared to all
hIRAS-expressing cell lines (IE10, 7D5 and 6E7 lines). Assay
of Annexin V-FITC binding followed by flow cytometric
measurements was next performed to evaluate quantitatively
the apoptotic population in hIRAS-expressing and -nonex-
pressing cell lines. As shown in Figure 1d, about 60% of
control cells (pcDNA3.1 and 7D5a clones) appeared apoptotic
(Annexin V-FITC positive, lower right quadrant) after 48 h in
serum-free medium. Under the same conditions, hIRAS-
expressing sublines (IE10, 7D5, and 6E7) showed a marked
reduction in apoptotic populations (Figure 1d). Thus, reduced
caspase-3 activation in serum-deprived hIRAS-expressing
cells seemed to correlate with a reduction in the apoptotic cell
population. Next, cell growth rate and survival in serum-
containing medium or in serum-free medium, respectively,
were assessed microscopically. In this set of experiments, the
number of viable cells were recorded by counting trypan blue
excluding cells with a hemocytometer after different periods of
time. The growth curves indicated that hIRAS-expressing
cells behave similarly as control cells in serum-containing
medium (data not shown). However, serum deprivation
resulted in the death of 45% control cells within 24 h versus
that of 0% hIRAS-expressing IE10 cells (Figure 1e). After 72 h
serum deprivation, all the hIRAS-expressing clones (IE10,
7D5, and 6E7) showed a greater proportion of viable cells as
assessed by trypan blue exclusion than control cell lines
(pcDNA3.1 and 7D5a) (data not shown). Thus, another main
Cell Death and Differentiation (2003) 10, 933–935
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Figure 1 (a) Western blotting of hIRAS-transfected PC12 cell membrane and cytosolic fractions. Immunorevelation was performed using 1201 antiserum specific for
human IRAS
1
with enhanced chemiluminescence. Memb, membranes; cyt, cytosol. (b) Effect of hIRAS transfection on caspase-3 activity in serum-deprived cells. PC12
cells transfected with empty vector (pcDNA3.1) and hIRAS-transfected cells (IE10 subclone) were grown in complete (open symbols) or serum-free (closed symbols)
medium. At the indicated times, cell lysates were prepared and used to determine caspase enzymatic activities by measuring the release of the para-nitroaniline
chromophore from peptide substrate (DEVD) selective for caspase-3-like protease. Background levels were determined by parallel incubations of samples in the
presence of caspase-3-like inhibitor, Ac-DEVD-CHO (10 mM) and subtracted for each individual value. Enzymatic activity is expressed in arbitrary units as O.D/mg protein
for each sample. Data are the mean7S.E.M. of triplicate determinations from three experiments. *Po0.05 compared to hIRAS-transfected cells. (c) PC12 cells either
expressing hIRAS (IE10, 7D5, and 6E7 sublines) or non-expressing hIRAS (pcDNA3.1 and 7D5a sublines) were grown in serum-free medium for 6 and 24 h. Caspase-3
activities were measured as in (b) Results are expressed as percent activity recorded in pcDNA3.1 cells for each experiment. Mean7S.E.M. of three to five independent
experiments. *Po0.03 as compared to pcDNA3.1 cell line. (d) hIRAS-mediated protection of apoptosis measured by Annexin V-FITC staining and flow cytometry. Upper
two panels, control cells or hIRAS-expressing cells were grown for 48 h in serum-free medium before harvesting and analysis. Percentage values represent late
apoptosis (upper right quadrant) and early apoptosis (lower right quadrant) populations of cells. In each case, a representative experiment out of three is shown. Lower
panel: results are mean7S.E.M. of three independent experiments. The late and early apoptotic cells were taken together for this purpose. (e) Survival curves of empty
vector and hIRAS-transfected cells. PC12 cells stably transfected with empty vector (pcDNA3.1 subclone) or hIRAS (IE10 subclone) were recovered after 24, 48, 72, and
96 h in serum-free medium and viable cells excluding trypan blue were counted. Results are expressed as percent of control at time 0 h for each cell line. Data shown are
mean7S.E.M. of triplicate determinations from three to six independent experiments. *Po0.05 as compared to pcDNA3.1 cells. (f) Upper graph: Effect of hIRAS
transfection on staurosporine-induced caspase-3 activation. PC12 cells not expressing hIRAS (pcDNA3.1 and 7D5a sublines) or expressing hIRAS (IE10 and 7D5
sublines) were grown in complete medium (15% serum) in the absence (open bars) or presence (closed bars) of staurosporine (1 mM) for 6 h. Results are expressed as
the mean7S.E.M. of three independent experiments each performed in triplicate. Lower graph: Effect of hIRAS transfection on thapsigargin-induced caspase-3
activation. PC12 cells transfected with empty vector (pcDNA3.1 subline) and IRAS-transfected cells (IE10 subline) were grown in 5% serum-containing medium with
thapsigargin (100 nM) for 6 h. Results are expressed as the mean7S.E.M. of two independent experiments each performed in sextuplicate. *Po0.01 as compared to
pcDNA3.1 cells.
Letter to the Editor
934
Cell Death and Differentiation

difference between control cells and hIRAS-expressing cell
lines is prolonged survival of the latter in serum-free
conditions, which may be related to the decrease in
caspase-3 activity and apoptotic cell population observed
previously. When serum is readded after 24 h serum depriva-
tion, we observed that hIRAS-expressing cell lines proved
able to proliferate again in marked contrast with control cells
that continued to die (56715% and 250767% of trypan blue
excluding cells after 72 h in serum-containing medium for
control and hIRAS-expressing cells, respectively; 100% refers
to the viable cells recorded after 24 h serum deprivation in
each cell line).
In an attempt to generalize that transfection of hIRAS
delays PC12 cells apoptosis, treatments were next performed
with two different cytotoxic agents, thapsigargin and staur-
osporine. Thapsigargin, a Ca
2 þ
-ATPase inhibitor that in-
creases intracellular calcium by inhibiting the uptake of
calcium into the endoplasmic reticulum, is known to induce
apoptosis in many cell types including PC12 cells.
7
Staur-
osporine, a protein kinase inhibitor, also induces apoptosis in
PC12 cells.
8
Staurosporine treatment of PC12 cells induces a
sustained elevation of intracellular calcium and accumulation
of reactive oxygen species.
8
Both of these apoptotic stimuli
are known to trigger caspase-3 activation.
7,8
. Thapsigargin
was applied to the cells in 5% serum-containing medium, and
then caspase-3 activities were recorded. As shown in
Figure 1f, thapsigargin (100 nM) enhanced caspase-3 activity
in control cells (pcDNA3.1 clone). In contrast, no enhance-
ment by thapsigargin of caspase-3 activity was observed in
hIRAS-expressing cells (IE10 clone). Staurosporine (1 mM)
was added to complete medium (containing 15% serum), thus
excluding any impact of low serum concentration on caspase-
3 activity. In this paradigm, the sublines stably expressing
hIRAS (1E10 and 7D5) showed lower caspase-3 activation
after 6 h treatment with 1 mM staurosporine compared to
control cells (pcDNA3.1 and 7D5a clones) (Figure 1f).
Caspase-3 activity after 6 h was increased 3.6- and 8.8-fold
in control sublines (pcDNA3.1 and 7D5a, respectively) versus
2.3- and 2.8-fold in hIRAS-expressing cells (IE10 and 7D5
clones respectively). We thus have confirmed the activation of
this apoptosis effector enzyme by these two drugs, and in the
process shown that hIRAS also inhibits the increase in
caspase-3 activity in these conditions.
In summation, IRAS appears to impinge on a mechanism
central to apoptosis induced by distinct stimuli and which lies
upstream of the caspase-3 activation resulting at least in a
delayed cell death. According to the results of Figure 1e, it
seems that a continued proapoptotic stimulus (96 h serum
deprivation, for example) eventually overrides the IRAS block
of apoptosis. As readdition of serum at an earlier time point of
apoptosis triggering (after 24 h serum deprivation) allowed the
hIRAS-expressing cells to divide again, we speculate that
hIRAS lowers the probability of initiation of the apoptotic
program rather than prolonging each individual apoptotic
event as described for caspase-3 inhibitors.
9
Thus, hIRAS
blocks apoptosis before destruction of membranes and loss of
phosphatidylserine asymmetry and seems to protect the cells
from the loss of the proliferative potential. Intriguingly, it
appears from the literature that expression of the a5 integrin
subunit delays serum-deprivation-induced as well as staur-
osporine-induced apoptosis in a manner very similar to the
one obtained by the transfection of hIRAS.
10
The mechanism of action of IRAS remains unresolved at
this time and is currently under investigation. Hypotheses
have been tested according to the proteins that are known to
interact with IRAS.
2,3
IRAS may be implicated either in the
insulin or the integrin dependent survival pathway, both of
which imply PI3-kinase activation.
11,6
However, our assays to
inhibit the protective effect of IRAS with LY294002 or
wortmannin (two PI3-kinase inhibitors) were unsuccessful;
although a little enhancement of caspase-3 activity in serum-
deprived hIRAS-expressing clones was recorded, no sig-
nificant increase in the proportion of apoptotic cells could be
reached with these inhibitors (data not shown). Likewise,
PD98059, an inhibitor of the ERK1/2 pathway, was unable to
induce an increase in hIRAS-expressing apoptotic cells (data
not shown).
Our study has thus identified a new cellular role for the
recently cloned human protein IRAS. We demonstrated that
hIRAS expression in PC12 cells results in protection against
apoptosis over a 2–3-day period of time. Although the
mechanism of action of IRAS is not resolved, it may represent
a previously unknown class of protein modulating the fine
regulation of cell death and growth.
Acknowledgements
We thank Dr. Peter Eickelman (Solvay Pharmaceuticals, Hannover,
Germany) for advice with the transfection protocol, and Mary Elise Lutrick
(University of Mississippi Medical Center) for carefully selecting and
maintaining the sublines. This work was partially supported by a grant from
Solvay Pharma (Hanover, Germany) and Grant MH49248 from the
National Institute of Mental Health (USA) for J Piletz’s group.
M Dontenwill
1*
, G Pascal
2
, JE Piletz
3
, M Chen
3
,
J Baldwin
3
, P Ronde
´
1
, L Dupuy
2
, D Urosevic
2
,
H Greney
2
, K Takeda
1
and P Bousquet
2
1
Pharmacologie et Physicochimie des Interactions Cellulaires et Mole
´
culaires,
UMR 7034 CNRS, Faculte
´
de Pharmacie, Universite
´
Louis Pasteur de
Strasbourg, Illkirch, France
2
Laboratoire de Neurobiologie et Pharmacologie Cardiovasculaire, Faculte
´
de
Medecine, Strasbourg, France
3
Department of Psychiatry, Division of Neurobiology and Behavior Research,
University of Mississippi Medical Center, Jackson, MS, USA
* Corresponding author: M Dontenwill, Pharmacologie et Physicochimie des
Interactions Cellulaires et Mole
´
culaires, UMR CNRS 7034, Faculte
´
de
Pharmacie, Universite
´
Louis Pasteur de Strasbourg, Illkirch, France. Tel: þ 33
3 90244267; Fax: þ 33 3 90244313; E-mail: mdontenwill@aspirine.u-
strasbg.fr
1. Piletz et al. (2000). DNA Cell Biol 19: 319–329
2. Alahari et al. (2000). J. Cell. Biol. 151: 1141–1154
3. Sano et al. (2002). J. Biol. Chem. 277: 19439–19447
4. Ivanov et al. (1998). J. Auton. Nerv. Sys. 7: 98–110
5. Matter and Ruoslahti (2001). J Biol. Chem. 276: 27757–27763
6. Lee and Juliano (2000). Mol. Biol. Cell 11: 1973–1987
7. Takadera and Ohyashiki (1998). Biochim. Biophys. Acta 1401: 63–71
8. Kruman et al. (1998). J. Neurosci. Res. 51: 293–308
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10. O’Brien et al. (1996). Exp. Cell. Res. 224: 208–213
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Letter to the Editor
935
Cell Death and Differentiation