(Received for publication, January 10, 1995)
From the
Several recent studies have implicated proteases as important
triggers of apoptosis. Thus far, substrates that are cleaved during
apoptosis have been elusive. In this report we demonstrate that
cleavage of -fodrin (non-erythroid spectrin) accompanies
apoptosis, induced by activation via the CD3/T cell receptor complex in
a murine T cell hybridoma, ligation of the Fas (CD95) molecule on a
human T cell lymphoma line and other Fas-expressing cells, or treatment
of cells with staurosporine, dexamethasone, or synthetic ceramide.
Furthermore, inhibition of activation-induced apoptosis by pretreatment
of T hybridoma cells with antisense oligonucleotides directed against c-myc also inhibited fodrin proteolysis, confirming
that this cleavage process is tightly coupled to apoptosis. Fodrin
cleavage during apoptosis may have implications for the membrane
blebbing seen during this process.
Apoptosis(1) , a mode of cell death characterized by a series of distinct morphological changes, largely involving the nucleus and the plasma membrane, is currently the subject of intense study. The majority of programmed cell deaths from insects to mammals exhibit strikingly consistent morphological features, typical of apoptosis, suggesting that a molecular machinery for death has been well conserved through evolution(2) . This machinery, which is still largely uncharacterized, executes a series of changes in the cell resulting in condensation of the nucleus(1) , cleavage of chromatin(3) , disruption of the nuclear lamin network(4, 5) , and plasma membrane changes that result in recognition and engulfment of apoptotic cells by neighboring phagocytes(6) .
Recent studies have revealed important
physiological triggers for apoptosis. These include the Fas
(CD95/Apo-1) cell surface molecule, which is a member of the tumor
necrosis factor/nerve growth factor receptor
family(7, 8) . Some of the intracellular signals
generated distal to engagement of these ``death receptors''
have now been partly characterized(9) . ()Much
attention has also focused on gene products found to act as
intracellular regulators of the cell death machinery; these include
Bcl-2, Abl, Myc, Ras, and p53 (see (10) and (11) for
recent reviews). It is notable also that mutated or dysregulated forms
of all of these genes are found in association with many cancers,
underscoring the importance of the cell death machinery as a regulator
of cell growth in multicellular organisms(12) . However,
although we are rapidly uncovering information about the triggers and
regulators of this machinery, the death machinery itself still remains
a largely unknown quantity.
Several recent studies have implicated
proteases as important players in
apoptosis(13, 14, 15, 16, 17, 18, 19, 20, 21, 22) .
It has also been suggested that activation of a protease may be a
central control point in apoptosis, much like activation of the
p34/cyclin B complex is essential for
mitosis(23) . In this scenario, upon activation, this putative
cytoplasmic regulator acts on several substrates simultaneously, rather
than initiating a linear cascade of events. However, although several
proteases have been reported to become activated during apoptosis, the
only proteins to date that have been reported to become cleaved during
this process are poly(A)DP-ribose polymerase (15) and the
protein component of the U1 small nuclear
ribonucleoprotein(22) , although there is also a possibility
that nuclear lamins become cleaved(5) , rather than
depolymerized(4) , during this process.
In this report, we present evidence that fodrin (non-erythroid spectrin), a major component of the cortical cytoskeleton that has binding sites for several proteins, becomes cleaved during apoptosis induced by ligation of the CD3/T cell receptor complex, Fas ligation, or treatment of cells with staurosporine, glucocorticoid, or synthetic ceramide. Fodrin cleavage was detected during apoptosis of a variety of cell lines of murine and human origin and was inhibited under conditions where apoptosis was inhibited. These data provide further support for protease activation as a general feature of apoptosis and, given the localization of fodrin to the cortical cytoskeleton, suggest that membrane blebbing in apoptosis may be at least partly due to disruption of the fodrin network in these cells.
A rabbit polyclonal
antibody against the peptide GMMPR, which matches the N-terminal
sequence of the calpain-catalyzed proteolytic fragment of the fodrin
subunit, was generated as described previously(24) .
Anti-fodrin monoclonal antibody (mAb) (
)1622 was purchased
from Chemicon International Inc. (Temecula, CA), anti-Fas mAb CH-11 was
purchased from Kamiya Biomedical Co. (Thousand Oaks, CA), anti-CD3 mAb
(145-2C11) was purified as described(25) , N-acetylsphingosine (C
-ceramide) was purchased
from Biomol (Plymouth Meeting, PA), and phosphothioate-derivatized
antisense oligonucleotides directed against murine c-myc (CACGTTGAGGGGCAT), as well as a same base composition scrambled
control (AGTGGCGGAGACTCT), were purchased from Quality Controlled
Biochemicals Inc. (Hopkinton, MA). Staurosporine, dexamethasone, and
all other chemicals were purchased from Sigma, unless otherwise
indicated.
Previous studies have shown that activation of A1.1 cells, a T cell hybridoma line, via the T cell receptor/CD3 complex results in apoptosis of these cells(25) . This form of apoptosis requires c-myc expression since it can be inhibited by antisense oligonucleotides targeted against c-myc mRNA(28) , as well as a dominant negative inhibitor of Myc/Max heterodimerization, MaxRx(29) . Recent studies also indicate that apoptosis in this context is due to activation-induced up-regulation of Fas and Fas ligand on these cells, which interact in a cell-autonomous manner to generate a death signal(30) . Protease inhibitors, particularly inhibitors of calpain activity, have been demonstrated to have inhibitory effects on activation-induced cell death of T cell hybridomas(14) , as well as death of mature T cells(31) , suggesting the involvement of a protease in this process. However, protein substrates that are cleaved during this type of apoptosis have yet to be reported.
Since there is indirect
evidence that calpain becomes activated during apoptosis, we wished to
determine whether a known intracellular substrate for calpain, the
fodrin subunit, became cleaved during activation-induced cell
death. Thus, A1.1 cells were induced to die by culturing in wells
coated with anti-CD3 mAb, and cell lysates were made and then separated
by SDS-polyacrylamide gel electrophoresis, followed by probing for
-fodrin expression. Fig. 1illustrates that A1.1 cells
underwent apoptosis following stimulation on anti-CD3 mAb-coated plates
or during culture with dexamethasone. Furthermore, cells were prevented
from undergoing anti-CD3 mAb-induced, but not dexamethasone-induced,
apoptosis by preincubation with antisense oligonucleotides targeted to c-myc (see ``Materials and
Methods''), as described previously (28) .
Figure 1:
Apoptosis of A1.1 cells induced by
CD3 ligation and dexamethasone treatment. Cells of the A1.1 murine T
cell hybridoma line were induced to undergo apoptosis by exposure to
plate-immobilized anti-CD3 mAb or 1 µM dexamethasone (Dex). A, morphology of A1.1 cells either untreated
or exposed to anti-CD3 mAb for 7 h, as indicated. Magnification,
400. B, light scattering properties of A1.1 cells, as
assessed by flow cytometry, after exposure to either immobilized
anti-CD3 mAb or dexamethasone (1 µM) for 7 h, in the
presence or absence of antisense (AS) oligonucleotides
directed against c-myc (3.5 µM), or a scrambled
nonsense (NS) control oligonucleotide (3.5 µM).
Apoptotic cells appear as a well defined cell peak with decreased
forward scatter (FSC) properties, roughly equating with
decreased cell size, as indicated. The percentages of apoptotic cells
in each culture are indicated in parentheses. 5000 cells were analyzed
in each condition. C, cell death, as assessed by the uptake of
PI dye in A1.1 cell cultures exposed to the same treatments as
indicated in B. 5000 cells were analyzed in each
condition.
Lysates made
from these cultures were probed for -fodrin expression by
immunoblotting. As shown in Fig. 2, cleaved fodrin was detected
in lysates made from apoptotic cells, yielding a major detectable
fragment of
150 kDa. In contrast, in cell populations with few
apoptotic cells, a band of
240 kDa, corresponding to intact
-fodrin, was the major band in evidence (Fig. 2A).
Cleavage of fodrin during anti-CD3 mAb-induced apoptosis was largely
inhibited by preincubation of cells with antisense oligonucleotides
targeted to c-myc (Fig. 2B), whereas
dexamethasone-induced fodrin cleavage was unaffected by similar
concentrations of the same oligonucleotide (Fig. 2C).
Control oligonucleotides had no effect on either apoptosis or fodrin
cleavage ( Fig. 1and Fig. 2). These results are in
agreement with the differential inhibitory effects of myc antisense oligonucleotides on these different forms of
apoptosis(28) .
Figure 2:
Cleavage of fodrin during anti-CD3 and
dexamethasone-induced apoptosis of A1.1 cells and inhibition of this
cleavage under conditions in which apoptosis is inhibited. Lysates were
prepared from control A1.1 cells and from A1.1 cells induced to undergo
apoptosis as indicated in Fig. 1legend. Proteins were
solubilized as described under ``Materials and
Methods'' and separated by SDS-polyacrylamide gel electrophoresis
followed by Western blotting. Blots were then probed with an antibody
to the fodrin subunit. A, A1.1 cells were incubated for
7 h in either medium alone (untreated), or in the presence of
immobilized anti-CD3 mAb or 1 µM dexamethasone, as
indicated. B, A1.1 cells were incubated for 7 h in either
medium alone (untreated), c-myc antisense
oligonucleotide (3.5 µM), or a nonsense control
oligonucleotide (3.5 µM), in the presence or absence of
plate-immobilized anti-CD3 mAb, as indicated. C, A1.1 cells
were incubated for 7 h in either medium alone (untreated), c-myc antisense oligonucleotide (3.5
µM), or a nonsense control oligonucleotide (3.5
µM), in the presence or absence of 1 µM dexamethasone, as indicated.
Since it has been demonstrated recently that
anti-CD3 mAb-induced apoptosis of T cell hybridoma cells is due to a
Fas and Fas ligand interaction(30) , then it follows that
direct ligation of Fas on a cell line constitutively expressing this
molecule should also result in proteolysis of fodrin. Thus, CEM cells,
which are a T cell leukemia line that expresses Fas, were induced to
undergo apoptosis in the presence of anti-Fas IgM (Fig. 3, A and B) and fodrin expression in these cells was examined
as before (Fig. 3C). In addition, to test whether
diverse apoptosis-inducing stimuli stimulated fodrin cleavage, CEM
cells were also treated with the cell-permeable synthetic ceramide N-acetylsphingosine (C-ceramide) or with
staurosporine, both of which are potent inducers of apoptosis in many
cell types(32, 33) . These experiments revealed that,
once again, the fodrin
subunit was cleaved in association with
apoptosis, whether induced by Fas ligation, treatment with
C
-ceramide, or staurosporine. Interestingly, in CEM
cultures that had undergone extensive apoptosis, fodrin was cleaved to
a single detectable fragment of
120 kDa, whereas in cultures
containing fewer apoptotic cells, a larger fragment of
150 kDa was
also observed. It is plausible that the 120-kDa fragment is a further
breakdown product of the 150-kDa fodrin fragment.
Figure 3:
Cleavage of fodrin during Fas, ceramide
and staurosporine-induced apoptosis of CEM cells. Human T cell leukemia
CEM cells were induced to undergo apoptosis by exposure to either
anti-Fas mAb (250 ng/ml), C-ceramide (50 µM),
or staurosporine (0.5 µM) for 16 h, as indicated.
Western-blotted protein extracts made from these cells were then probed
with an antibody to the fodrin
subunit. A, morphology of
CEM cells undergoing apoptosis in response to the indicated stimuli.
Note the abundance of cells with with many blebs in treated cultures.
Magnification,
200. B, light scattering properties of
CEM cells, as assessed by flow cytometry, after 16 h of exposure to the
indicated stimuli. The percentages of apoptotic cells in each culture
are indicated in parentheses. 5000 cells were analyzed in each
condition. Results are from a representative experiment. C,
detection of
-fodrin in cell lysates made from CEM cells treated
as in B.
To further extend
these findings, we also induced apoptosis in murine P815 cells that had
been trans-infected with human Fas (Fig. 4, A and B) and, once again, found that fodrin became cleaved in
association with apoptosis induced either by Fas ligation or
staurosporine treatment (Fig. 5A), confirming the
widespread nature of this observation. Furthermore, probing of cell
extracts made from apoptotic and control cell populations of
P815 and CEM cells with an antibody that specifically
recognizes the peptide (GMMPR) that forms the N-terminal sequence of
the of calpain I-generated fragment of fodrin(24) , revealed
that this antibody also recognized the fodrin fragments generated in
apoptotic cells (Fig. 5, A and B). These data
suggest that proteolysis of fodrin during apoptosis may be a
consequence of calpain activation.
Figure 4:
Induction of apoptosis in P815 cells by Fas ligation and staurosporine-treatment. Murine
P815
cells were induced to undergo apoptosis by exposure
to 250 ng/ml anti-Fas mAb or 0.5 µM staurosporine. A, morphology of a group of four P815
cells
prior to and after several hours of treatment with anti-Fas mAb.
Magnification,
400. (B) Light scattering properties of
P815
cells, as assessed by flow cytometry, 7 h after
exposure to the indicated stimuli. The percentages of apoptotic cells
in each culture are indicated in parentheses. 5000 cells were
analyzed in each condition.
Figure 5:
Fodrin proteolysis during P815 and CEM cell apoptosis is catalyzed by calpain. Murine
P815
cells were induced to undergo apoptosis as described
in Fig. 4legend and CEM cells as described in Fig. 3legend. Western-blotted protein extracts made from these
cells were probed with an antibody to the fodrin
subunit,
followed by stripping of the blots and reprobing with an antibody
specific for the peptide GMMPR, which matches the N-terminal sequence
of the calpain-catalyzed proteolytic fragment of the fodrin
subunit. A, detection of
-fodrin in cell lysates made
from P815
cells treated as in Fig. 4B. B, detection of
-fodrin in cell lysates made from CEM
cells treated as in Fig. 3B.
Fodrin, a multifunctional protein
with spectrin-like properties, is a major component of the cortical
cytoskeleton of most eukaryotic cells (34) . Fodrin, like
spectrin, is a rod-shaped protein consisting of and
subunits, which form heterodimers aligned in a side-to-side manner.
Fodrin heterodimers then further associate end-on-end to form
tetramers, which cross-link actin filaments at their ends(35) .
Fodrin possesses binding sites for many proteins, including
actin(35) , calmodulin(36) , CD45(37) , and
possibly also for members of the phospholipid-binding annexin family of
proteins(38) , consistent with its perceived role in the
organization of receptor domains and vesicle traffic at the plasma
membrane(35) . Proteolysis of fodrin by calcium-dependent
proteases has been observed during several processes, notably, in the
context of the present study, during ischemia-induced cell death in the
rodent forebrain(24) .
The direct consequences of fodrin proteolysis are still unknown. However, given its role as a major component of the plasma membrane-associated cytoskeleton, and since dramatic plasma membrane blebbing and reorganization are a consistent feature of apoptosis, it is conceivable that fodrin proteolysis during apoptosis may contribute to these rearrangements. Fodrin is also cleaved during platelet activation(39) , a process that results in exposure of phosphatidylserine on the outer leaflet of the plasma membrane of these cells. Such an event also occurs during apoptosis and seems to trigger recognition of apoptotic cells by phagocytes(6) . Thus it is plausible that fodrin proteolysis may be linked to the externalization of phosphatidylserine in both processes. These possibilities are currently under investigation.