(Received for publication, June 5, 1995)
From the
Fas and the type I tumor necrosis factor receptor (TNF-R) are
two cell surface receptors that, when stimulated with ligand or
cross-linking antibody, trigger apoptotic cell death by a mechanism
that has yet to be elucidated. The CrmA protein is a serpin family
protease inhibitor that can inhibit interleukin-1 converting
enzyme (ICE) and ICE-like proteases. We showed previously that
expression of CrmA potently blocks apoptosis induced by activation of
either Fas or TNF-R, implicating protease involvement in these death
pathways (Tewari, M., and Dixit, V. M.(1995) J. Biol. Chem. 270, 3255-3260). Here we report that the 70-kDa component of
the U1 small ribonucleoprotein (U1-70 kDa) is a proteolytic
substrate rapidly cleaved during both Fas- and TNF-R-induced apoptosis.
This cleavage was inhibited by expression of CrmA but not by expression
of an inactive point mutant of CrmA, confirming the involvement of an
ICE-like protease. These data for the first time identify U1-70
kDa as a death substrate cleaved during Fas- and TNF-R-induced
apoptosis and emphasize the importance of protease activation in the
cell death pathway.
Apoptosis, or programmed cell death (PCD), ()is of
pivotal importance to a variety of biological processes, including
regulation of the immune system, embryonic development, and maintenance
of tissue homeostasis(1) . A wide variety of triggers for
apoptosis have been identified, which induce cell death by poorly
defined mechanisms that may or may not be in common with different
stimuli. Our laboratory has a special interest in apoptosis induced by
two cell surface cytokine receptors: the Fas antigen and the type I
tumor necrosis factor receptor (TNF-R). Fas, in particular, has been
shown to be a significant mediator of apoptosis in vivo, as in lpr mice, a point mutation in the Fas antigen gene attenuates
T-cell apoptosis resulting in a fatal, lupus-like autoimmune
disease(2) . Additionally, cytotoxic T-lymphocytes activate Fas
on target cells, inducing
cytolysis(3, 4, 5, 6) . More
recently, Fas has also been shown to be responsible for
activation-induced death of T-cells (7, 8, 9) , which likely plays an important
role in the pathogenesis of a number of diseases, including
AIDS(10) . TNF has been long recognized for its ability to
induce PCD of tumor cells in culture and has been investigated as a
therapeutic agent in this capacity(11) . Fas and TNF-R both
induce apoptosis when cross-linked by their respective ligands or by
specific agonist monoclonal
antibodies(11, 12, 13, 14) .
Although the molecular mechanism of Fas- and TNF-induced apoptosis,
as well as that of all other forms of apoptosis, remains to be
elucidated, there is strong evidence that an intracellular protease is
part of the cell death machinery. Initial support for this hypothesis
came from the genetic analysis of programmed cell death in the
nematode Caenorhabditis elegans, where a gene designated ced-3 was found to be obligatory for PCD(15) .
Sequence analysis of ced-3 revealed significant homology to
the mammalian protein interleukin-1-converting enzyme (ICE), an
Asp-specific cysteine protease(16) . The crystal structure of
ICE revealed that catalytically important residues were conserved in
the Ced-3 protein(17, 18) , suggesting that Ced-3
itself was a cysteine protease. These findings also raised the
possibility that in mammalian cells a cysteine protease homologous to
Ced-3, perhaps ICE, is a component of the death machinery. We tested
this hypothesis by expressing, in cells sensitive to Fas- or
TNF-induced apoptosis, the cowpox virus crmA gene, which
encodes a serpin that inhibits the enzymatic activity of
ICE(19) . CrmA was found to be a potent inhibitor of both TNF-
and Fas-induced apoptosis(20) , suggesting that ICE or a
related CrmA-inhibitable protease is a component of the death pathway
activated by these receptors. These findings were recently confirmed by
two other groups(21, 22) .
Although the CrmA experiments implicated protease involvement in Fas- and TNF-R-induced cell death, they did not identify specific substrates cleaved during this process. Casciola-Rosen et al.(23) recently demonstrated that the 70-kDa protein component of the U1 small ribonucleoprotein (U1-70 kDa) was cleaved during apoptosis induced by UVB irradiation or nutrient deprivation of HeLa cells(23) . The cleavage appeared to be mediated by an enzyme with a chemical inhibitor profile characteristic of an ICE-like protease but was distinct from ICE, since purified ICE did not cleave U1-70 kDa(23) .
These studies prompted us to ask whether cleavage of U1-70 kDa occurred during Fas- and/or TNF-R-induced apoptosis and if CrmA could block this event. We now show that proteolytic cleavage of U1-70 kDa occurs remarkably early during Fas- and TNF-R-induced apoptosis and is potently blocked by native CrmA but not by a CrmA point mutant that is incapable of inhibiting ICE. Taken together, these data identify U1-70 kDa as a death substrate in the Fas- and TNF-induced apoptosis pathways and suggest that the cleavage may be generated by a CrmA-inhibitable, ICE-like protease.
BJAB cells or derived transfectants
were aliquoted at a concentration of 5 10
/ml into
six-well dishes, with 4 ml in each well. The following day, cells were
treated with anti-Fas antibody (250 ng/ml) for the indicated time
periods, harvested by centrifugation, washed once with PBS plus
protease inhibitors, and lysed in 2 ml of sample buffer.
To determine whether cleavage of U1-70 kDa is an event characteristic of either Fas- or TNF-R-induced apoptosis, we examined the BJAB human lymphoma cell line and the MCF7 human breast carcinoma cell line. The BJAB and MCF7 cells have been documented by confocal and electron microscopy to undergo genuine apoptotic cell death in response to agonist anti-Fas antibody or recombinant TNF, respectively(20) .
BJAB cells were treated with anti-Fas
antibody or TNF for various time periods, and cell lysates were
prepared and analyzed for the integrity of U1-70 kDa by
immunoblotting. Treatment of Fas rapidly (as early as 1 h) induced
U1-70 kDa cleavage to a 40-kDa form (Fig.1A), similar in size to the cleavage product
described by Casciola-Rosen et al.(23) in serum
deprivation-induced and UVB irradiation-induced apoptosis.
Figure 1: Activation of either Fas or TNF receptor induces U1-70 kDa cleavage. A, BJAB cells were either left untreated (UnRx) or treated with agonist anti-Fas monoclonal antibody (250 ng/ml) for the indicated time periods, and cell lysates were prepared and analyzed by Western blotting using a U1-70 kDa reactive antiserum as described under ``Experimental Procedures.'' The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis. B, MCF7 cells were either left untreated (UnRx) or treated with TNF (40 ng/ml) for the indicated time periods, and cell lysates were similarly analyzed. The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis.
Activation of TNF receptors stimulated U1-70 kDa cleavage in
MCF7 cells, though at a slower time course than that observed with
anti-Fas in BJAB cells (Fig.1B). Importantly, however,
this delayed time course of cleavage paralleled the slower time course
of cell death seen in response to TNF. Ligation of Fas on BJAB cells
induces cell death within hours, whereas TNF requires at least
overnight exposure. ()In both cases U1-70 kDa cleavage
was detectable at time points at which no gross morphological changes
were discernible, indicating that this cleavage may represent an early
event in the apoptotic cascade.
The fact that U1-70 kDa
cleavage was seen in response to both TNF and Fas indicated that these
receptors activated a protease capable of cleaving U1-70 kDa
protein. Since we had previously shown that the cowpox virus-encoded
inhibitor of ICE and ICE-like proteases, CrmA, inhibits cell death
induced by these receptors, we examined the effect of CrmA on this
cleavage event. MCF7 and BJAB stable cell lines were selected that had
been transfected with either vector alone, CrmA, or mutant CrmA
expression plasmids, and comparable levels of protein expression were
confirmed by Western analysis(24) . The mutant CrmA plasmid
encodes a point mutant (Thr
Arg change) in the
reactive site loop. This alteration disables its protease-inhibitory
capacity without affecting overall tertiary structure (24) and
thus serves as an important control.
MCF7 and BJAB transfectants were treated with TNF or anti-Fas antibody, respectively, for varying time periods, after which the cells were lysed and the integrity of U1-70 kDa was assessed by immunoblotting. In the case of Fas-induced apoptosis, the non-expressing BJAB lines (clones V4, V1, and CrmA5) showed kinetics of U1-70 kDa cleavage (Fig.2A) similar to that observed previously with the parental cell line. However, expression of wild-type CrmA (clones CrmA2 and CrmA3) markedly inhibited the cleavage (Fig.2B), whereas mutant CrmA (clones 12 and 17) was inactive (Fig.2C), allowing the cleavage to occur unabated.
Figure 2: CrmA, but not mutant CrmA, blocks Fas-induced cleavage of U1-70 kDa. A, clonal BJAB cell lines not expressing CrmA (V4, V1, CrmA5) were either left untreated (UnRx) or treated with agonist anti-Fas antibody (250 ng/ml) for the indicated time periods, and lysates were prepared and analyzed by Western blotting using a U1-70 kDa reactive antiserum as described under ``Experimental Procedures.'' The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis. B, clonal BJAB transfectants expressing CrmA (CrmA2, CrmA3) were either left untreated (UnRx) or treated with agonist anti-Fas antibody (250 ng/ml) for the indicated time periods and similarly analyzed. The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis. C, clonal BJAB transfectants expressing mutant CrmA (mutant CrmA/clone #12, mutant CrmA/clone #17) were either left untreated (UnRx) or treated with agonist anti-Fas antibody (250 ng/ml) for the indicated time periods and similarly analyzed. The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis.
In the case of TNF-induced PCD, non-expressing MCF7 cell lines (clones V4 and CrmA2) showed obvious cleavage (Fig.3A), whereas expression of CrmA (clones CrmA3 and CrmA4) potently inhibited this proteolytic event (Fig.3B). Mutant CrmA (clones 1 and 2), however, was ineffective (Fig.3C).
Figure 3: CrmA, but not mutant CrmA, blocks TNF-R-induced cleavage of U1-70 kDa. A, clonal MCF7 transfectants not expressing CrmA (V4, CrmA2) were either left untreated or treated with TNF (40 ng/ml) for the indicated time periods, and lysates were prepared and analyzed by Western blotting using a U1-70 kDa reactive antiserum as described under ``Experimental Procedures.'' The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis. B, clonal MCF7 transfectants expressing CrmA (CrmA3, CrmA4) were either left untreated (UnRx) or treated with TNF (40 ng/ml) for the indicated time periods and similarly analyzed. The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis. C, clonal MCF7 transfectants expressing mutant CrmA (clone #1, clone #2) were either left untreated (UnRx) or treated with TNF (40 ng/ml) for the indicated time periods and similarly analyzed. The arrow designated ``b'' indicates a background protein that cross-reacts with the antiserum but is not cleaved during apoptosis.
In summary, we have shown that CrmA inhibits cleavage of U1-70 kDa during Fas- and TNF-R-induced cell death and, furthermore, that the protease-inhibitory activity of CrmA is required for this effect. These data lend additional support to the contention that Fas and TNF-R induce cell death by a common mechanism, since both appear to activate a CrmA-inhibitable protease and cause U1-70 kDa cleavage.
The finding that CrmA, a protease inhibitor and antidote to Fas- and TNF-induced cell death, inhibits the cleavage of U1-70 kDa suggests that the protease responsible may be an obligatory component of the death pathway. An alternative possibility is that CrmA inhibits an ICE-like protease upstream of the one that actually cleaves U1-70 kDa. This possibility is also tantalizing, since it would imply that there exists an amplifiable cascade of at least two ICE-like proteases in the cell death pathway.
It is important to note that
the time course of U1-70 kDa cleavage parallels that of another
proteolytic event recently reported to occur during Fas- or
TNF-R-mediated apoptosis, the cleavage of poly(ADP-ribose) polymerase
(PARP)(24) . This event, which is also CrmA-inhibitable, has
been proposed by us to be catalyzed by the protease Yama, also known as
CPP32(24) . Yama is an ICE/Ced-3 family member that
cleaves PARP in vitro to a characteristic 85-kDa apoptotic
fragment. It will be of importance to determine if U1-70 kDa too
is a substrate for Yama and, if so, whether the cleavage product
generated is identical to that seen in Fas- and TNF-induced cell death.
The contribution of U1-70 kDa cleavage to apoptosis is not clear at this juncture. Casciola-Rosen et al.(23) have suggested that the cleavage may play a role in regulating RNA splicing during apoptosis. As several substrates have now been identified that are specifically cleaved during apoptosis, it is likely that U1-70 kDa cleavage may be one of multiple proteolytic events resulting in the irreversible molecular inactivation of the cell's biochemical machinery. Thus, cleavage of U1-70 kDa, together with that of other identified death substrates such as fodrin (28) and PARP(24, 25, 26, 27) , may act in concert to yield the apoptotic phenotype. Characterizing the ICE-like protease(s) that cleave U1-70 kDa as well as the other death substrates will be of paramount importance in the elucidation of the components of the death pathway.