©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CrmA-inhibitable Cleavage of the 70-kDa Protein Component of the U1 Small Nuclear Ribonucleoprotein during Fas- and Tumor Necrosis Factor-induced Apoptosis (*)

(Received for publication, June 5, 1995)

Muneesh Tewari (1)(§),   David R. Beidler (¶) Vishva M. Dixit (1)(**)

From theDepartment of Pathology and Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109

ABSTRACT
INTRODUCTION
Experimental Procedures
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-1beta 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.


INTRODUCTION

Apoptosis, or programmed cell death (PCD), (^1)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-1beta-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.


Experimental Procedures

Cell Culture

MCF7 cells, BJAB cells, and derived vector and CrmA stable transfectants (20) along with the CrmA mutant-transfected stable lines (24) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), L-glutamine, penicillin/streptomycin, and nonessential amino acids and additionally supplemented with G418 sulfate (Life Technologies, Inc.) to 500 µg/ml for MCF7 transfectants and 3 mg/ml for BJAB transfectants.

Treatment with Anti-Fas Antibody or TNF and Preparation of Cell Lysates

MCF7 cells or derived transfectants were plated in 100-mm dishes at a concentration of 2 10^6 cells/dish. On the 2nd day, cells were treated with TNF at 40 ng/ml for the indicated time periods. Following a PBS rinse, cells were harvested by scraping into 15 ml of PBS plus protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml aprotinin, 0.5 mg/ml antipain, and 0.5 mg/ml pepstatin), recovered by centrifugation, and lysed in 2.5 ml of sample buffer (50 mM Tris-HCl, pH 6.8, 6 M urea, 6% 2-mercaptoethanol, 3% SDS, and 0.003% bromphenol blue). In cases where nonadherent cells were present in the culture medium (e.g. at later time points), floating cells were also harvested by centrifugation and combined with the adherent cell pellet before lysis in sample buffer.

BJAB cells or derived transfectants were aliquoted at a concentration of 5 10^5/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.

Western Blotting

Immunoblotting to assess the state of U1-70 kDa was carried out similarly as described previously(23) . In brief, cell lysates (20 µl) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Schleicher and Schuell) by electroblotting. Blots were blocked at room temperature for 1 h using blocking buffer (10 mM Tris (pH 7.6), 150 mM NaCl, 0.5% Nonidet P-40, 3% bovine serum albumin). The U1-70 kDa affinity-purified antiserum was used at a 1:5000 dilution in blocking buffer and was incubated for 1 h at room temperature. The secondary reagent, a biotinylated goat anti-human IgG antibody (Southern Biotech), was used at a dilution of 1:6700 in blocking buffer and was incubated for 50 min at room temperature. The tertiary reagent, a streptavidin-horseradish peroxidase conjugate (Southern Biotech), was used at a dilution of 1:10,000 in blocking buffer and was incubated at 30 min at room temperature. Visualization of signal was by electrochemiluminescence (Amersham Corp.).

TNF, Anti-Fas Antibody, and U1-70 kDa Reactive Antiserum

Recombinant TNF (specific activity, 6.27 10^7 units/mg) was a gift from Genentech (South San Francisco, CA). Anti-Fas monoclonal antibody (clone CH-11, IgM) was obtained from PanVera (Madison, WI). The affinity-purified U1-70 kDa reactive antiserum was derived from human autoimmune sera and was a kind gift from Dr. Antony Rosen (Johns Hopkins University, Baltimore, MD).


RESULTS AND DISCUSSION

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. (^2)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 CPP32beta(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.


FOOTNOTES

*
This work was funded by National Institutes of Health Grant CA64803 (to V. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a Young Scientist MD/PhD Scholarship from the Life and Health Insurance Medical Research Fund; Fellow of the Medical Scientist Training Program.

Supported by United States Public Health Service Award HL07517.

**
Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Box 0602, Ann Arbor, MI 48109. Tel.: 313-747-2921; Fax: 313-764-4308; vishva.dixit{at}med.umich.edu.

^1
The abbreviations used are: PCD, programmed cell death; TNF-R, tumor necrosis factor receptor; ICE, interleukin-1beta-converting enzyme; PBS, phosphate-buffered saline; U1-70 kDa, 70-kDa protein component of the U1 small ribonucleoprotein; PARP, poly(ADP-ribose) polymerase.

^2
M. Tewari and V. M. Dixit, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to Antony Rosen for the generous gift of affinity-purified antisera reactive against U1-70 kDa. We thank Guy Salvesen for fruitful discussions and advice, Yongping Kuang for technical assistance, Karen O'Rourke for critical review of the manuscript, and Ian Jones for assistance in preparing the figures.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.