Omi/HtrA2 protease mediates cisplatin-induced cell death in renal cells.
ABSTRACT Omi/HtrA2 is a mitochondrial proapoptotic serine protease that is able to induce both caspase-dependent and caspase-independent cell death. After apoptotic stimuli, Omi is released to the cytoplasm where it binds and cleaves inhibitor of apoptosis proteins. In this report, we investigated the role of Omi in renal cell death following cisplatin treatment. Using primary mouse proximal tubule cells, as well as established renal cell lines, we show that the level of Omi protein is upregulated after treatment with cisplatin. This upregulation is followed by the release of Omi from mitochondria to the cytoplasm and degradation of XIAP. Reducing the endogenous level of Omi protein using RNA interference renders renal cells resistant to cisplatin-induced cell death. Furthermore, we show that the proteolytic activity of Omi is necessary and essential for cisplatin-induced cell death in this system. When renal cells are treated with Omi's specific inhibitor, ucf-101, they become significantly resistant to cisplatin-induced cell death. Ucf-101 was also able to minimize cisplatin-induced nephrotoxic injury in animals. Our results demonstrate that Omi is a major mediator of cisplatin-induced cell death in renal cells and suggest a way to limit renal injury by specifically inhibiting its proteolytic activity.
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ABSTRACT: Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death.Journal of Biological Chemistry 04/2003; 278(13):11489-94. · 4.65 Impact Factor
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ABSTRACT: Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183-10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.Molecular and Cellular Biology 07/1998; 18(6):3300-9. · 5.37 Impact Factor
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ABSTRACT: We report here the identification of a novel protein, Smac, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway. Smac promotes caspase-9 activation by binding to inhibitor of apoptosis proteins, IAPs, and removing their inhibitory activity. Smac is normally a mitochondrial protein but is released into the cytosol when cells undergo apoptosis. Mitochondrial import and cleavage of its signal peptide are required for Smac to gain its apoptotic activity. Overexpression of Smac increases cells' sensitivity to apoptotic stimuli. Smac is the second mitochondrial protein, along with cytochrome c, that promotes apoptosis by activating caspases.Cell 08/2000; 102(1):33-42. · 31.96 Impact Factor
Omi/HtrA2 protease mediates cisplatin-
induced cell death in renal cells
Lucia Cilenti*, George A. Kyriazis*, Mangala M. Soundarapandian*, Valerie
Stratico*, Adam Yerkes*, Kwon Moo Park~, Alice M. Sheridan~, Emad S.
Alnemri#, Joseph V. Bonventre~, and Antonis S. Zervos*‡
*Biomolecular Science Center, Burnett College of Biomedical Science, University of
Central Florida (UCF), 12722 Research Parkway, Orlando, FL 32826
#Center of Apoptosis Research and Department of Microbiology and Immunology,
Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107
~Renal Division, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA
Running head: Omi/HtrA2 in renal cell death
Antonis S. Zervos
Biomolecular Science Center/
Burnett College of Biomedical Science
University of Central Florida
12722 Research Parkway
Orlando, FL 32826
Tel: (407) 882-2263
Fax: (407) 384-2062
Articles in PresS. Am J Physiol Renal Physiol (September 28, 2004). doi:10.1152/ajprenal.00154.2004
Copyright © 2004 by the American Physiological Society.
Omi/HtrA2 is a mitochondrial pro-apoptotic serine protease that is able to induce both caspase-dependent
and caspase-independent cell death. Following apoptotic stimuli, Omi is released to the cytoplasm, where
it binds and cleaves inhibitor of apoptosis proteins (IAPs). In this report we investigated the role of Omi
in renal cell death following cisplatin treatment. Using primary mouse proximal tubule (MPT) cells, as
well as established renal cell lines, we show that the level of Omi protein is up-regulated after treatment
with cisplatin. This up-regulation is followed by the release of Omi from mitochondria to the cytoplasm
and degradation of XIAP. Reducing the endogenous level of Omi protein, using RNA interference,
renders renal cells resistant to cisplatin-induced cell death. Furthermore, we show that the proteolytic
activity of Omi is necessary and essential for cisplatin-induced cell death in this system. When renal cells
are treated with Omi’s specific inhibitor, ucf-101, they become significantly resistant to cisplatin-induced
cell death. Ucf-101 was also able to minimize cisplatin-induced nephrotoxic injury in animals. Our
results demonstrate that Omi is a major mediator of cisplatin-induced cell death in renal cells and suggest
a way to limit renal injury by specifically inhibiting its proteolytic activity.
Keywords: renal cell death, Omi/HtrA2, cisplatin, serine protease, ucf-101
Omi/HtrA2 is a mitochondrial serine protease originally isolated through its interaction with Mxi2 (11),
an alternatively spliced form of the p38 stress-activated kinase (10). Omi is expressed ubiquitously and
the amount of protein increases when cells are exposed to heat shock or treated with tunicamycin (14).
Omi is the mammalian homolog of the prokaryotic HtrA proteins. HtrAs are chaperones that are essential
for bacterial survival after heat shock or oxidative stress (6,23,34,43). In addition to Omi, the family of
eukaryotic HtrAs includes two other members: HtrA1/L56 (18,59) and HtrA3/PRSP (31). Both HtrA1
and HtrA3 are secreted proteins whose normal function is not known. Down-regulation or absence of
HtrA1 has been reported in some human metastatic melanoma tumors (1).
Recent studies identified Omi as a nuclear encoded mitochondrial serine protease that is released into the
cytoplasm upon induction of apoptosis (16,30,46,52,53). In the cytoplasm, Omi activates caspase-9 by
interacting with and degrading inhibitor of apoptosis proteins (IAPs) (44,58). The interaction of Omi
with IAPs is mediated via an AVPS amino-terminal sequence of mature Omi protein that is similar to the
corresponding motif in other known IAP-binding proteins including Reaper (54), Hid (55) and Grim (4)
in Drosophila, as well as the mammalian proteins Smac/Diablo (9) and caspase-9 (45). Furthermore,
Omi can promote apoptosis in a caspase-independent pathway through its ability to function as a protease
(53). Omi protein can be defined by three different and functionally distinct domains. An amino
terminal domain (aa 1-133) carries the mitochondrial targeting sequence and is immediately cleaved after
Omi enters mitochondria (6,23,34,43). The second domain of Omi is a catalytic domain and shows the
highest homology with the bacterial HtrAs as well as with the other two eukaryotic homologs (11,31). A
PDZ domain at the carboxyl terminus of Omi represents the third domain of the protein and has a unique
binding specificity (20). Structural and biochemical studies defined the role of this PDZ domain as a
regulator of the proteolytic activity of the enzyme (26,15). The proteolytic activity of Omi is essential for
its normal function to promote both caspase-dependent and caspase-independent cell death (16,53) .
Recently, we described the isolation and characterization of a specific non-peptidyl inhibitor of Omi’s
proteolytic activity(5). This inhibitor, we call ucf-101, can enter mammalian cells where it co-localizes
with endogenous Omi and inhibits its activity (5). The ability of ucf-101 to specifically inhibit the
proteolytic activity of Omi in vitro, as well as in vivo, makes this compound a very useful reagent to
delineate the function of Omi in cell injury and apoptosis.
In the present study we explored the potential role of Omi in renal tubular cells following cisplatin-
induced cell death. Cisplatin [cis-diammine dichloroplatinum(II)] is a chemotherapeutic drug used to
treat several solid tumors including testicular, lung, head, neck, and cervical cancers (32,37). Because
cisplatin has an inherent dose-dependent toxicity, its use is limited by its nephrotoxicity. The mechanism
by which cisplatin causes renal injury is not clear; it can induce apoptosis as well as necrosis and
proximal tubules are particularly sensitive (7,29). Understanding the mechanism of cisplatin-induced
renal cell death could lead to the development of drugs that can be used to protect patients undergoing
chemotherapy from developing acute renal failure (ARF).
Our results demonstrate that Omi is expressed throughout the mouse kidney and its subcellular
localization is regulated by cisplatin treatment of renal cells. Furthermore, using RNA interference, to
reduce the amount of endogenous Omi protein, or the specific inhibitor ucf-101 afforded significant
protection on renal cells, both in vitro and in vivo, from the cytotoxic effects of cisplatin.
MATERIALS AND METHODS
HEK-293 cells were grown using DMEM (GIBCO, Carlsbad, CA) supplemented with 10% FBS
(Hyclone Logan, UT), 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, 50 U/ml
penicillin, and 50 mg/ml streptomycin (GIBCO). HK-2 cells were grown in DMEM media supplemented
with 10% FBS (Hyclone), 15 mM Hepes, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin
(GIBCO), 0.4 µg/ml hydrocortisone, 5 µg/ml insulin, and 5 µg/ml apo-transferrin (Sigma, St. Louis,
Isolation of mouse kidney proximal tubular (MPT) cells
Mouse proximal tubular (MPT) cells were isolated from collagenase-digested fragments derived from the
cortices of kidneys of C57BL6 mice as previously described (28,42). Briefly, kidneys were dissected to
obtain cortical tissue, which was digested with a solution of collagenase (Worthington Biochemical
Corporation, Lakewood, NJ) and soybean trypsin inhibitor (GIBCO) at 37˚C for 45 minutes. Cells were
grown in serum-free mixture (1:1) of Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12
containing 15 mM Hepes, 2 mM L-glutamine, 5 µg/ml insulin, 50 nM hydrocortisone, 5 µg/ml apo-
transferrin, 50 U/ml penicillin, and 50 µg/ml streptomycin. The medium was replaced every two days
and MPT cells were used after they reached confluence, between the 7th and 10th day after culture.
Confocal microscopy of mouse proximal tubular cells
For immunofluorescence, MPT cells were grown for 7 days on microscope glass cover slides. Adherent
cells were washed in PBS, fixed in 4% paraformaldehyde and made permeable using ice-cold acetone.
Non-specific binding was blocked with 2% BSA in PBS and, subsequently, cells were stained using