Zfra affects TNF-mediated cell death by interacting with death domain protein TRADD and negatively regulates the activation of NF-κB, JNK1, p53 and WOX1 during stress response
© Hong et al; licensee BioMed Central Ltd. 2007
Received: 07 December 2006
Accepted: 13 June 2007
Published: 13 June 2007
Zfra is a 31-amino-acid zinc finger-like protein, which is known to regulate cell death by tumor necrosis factor (TNF) and overexpressed TNF receptor- or Fas-associated death domain proteins (TRADD and FADD). In addition, Zfra undergoes self-association and interacts with c-Jun N-terminal kinase 1 (JNK1) in response to stress stimuli. To further delineate the functional properties of Zfra, here we investigated Zfra regulation of the activation of p53, WOX1 (WWOX or FOR), NF-κB, and JNK1 under apoptotic stress.
Transiently overexpressed Zfra caused growth suppression and apoptotic death of many but not all types of cells. Zfra either enhanced or blocked cell death caused by TRADD, FADD, or receptor-interacting protein (RIP) in a dose-related manner. This modulation is related with Zfra binding with TRADD, NF-κB, JNK1 and WOX1, as determined by GST pull-down analysis, co-immunoprecipitation, and mapping by yeast two-hybrid analysis. Functionally, transiently overexpressed Zfra sequestered NF-κB (p65), WOX1, p53 and phospho-ERK (extracellular signal-activated kinase) in the cytoplasm, and TNF or UV light could not effectively induce nuclear translocation of these proteins. Zfra counteracted the apoptotic functions of Tyr33-phosphorylated WOX1 and Ser46-phosphorylated p53. Alteration of Ser8 to Gly abolished the apoptotic function of Zfra and its regulation of WOX1 and p53.
In response to TNF, Zfra is upregulated and modulates TNF-mediated cell death via interacting with TRADD, FADD and RIP (death-inducing signaling complex) at the receptor level, and downstream effectors NF-κB, p53, WOX1, and JNK1.
Human WWOX/FRA16D gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase, designated WWOX, FOR, or WOX1 [1–3]. This gene is located on a common fragile site ch16q23.3–24.1 [1, 2]. Loss of heterozygosity (LOH) of WWOX gene has been found in several types of cancers [[4, 5]; reviews]. WWOX/FOR/WOX1 possesses two N-terminal WW domains (containing conserved tryptophan residues), a nuclear localization sequence (NLS) between the WW domains, and a C-terminal short chain alcohol dehydrogenase/reductase (SDR) domain. WWOX mRNA may undergo alternative splicing, thereby generating at least 8 mRNAs mainly coding for proteins with altered SDR domain sequences . Several protein isoforms have been identified . Nonetheless, presence of specific protein isoforms in normal and cancerous tissues remains to be established.
WWOX/FOR/WOX1 is considered as a candidate tumor suppressor and proapoptotic protein, whereas its in vivo function is largely unknown . Under stress conditions, WOX1 undergoes phosphorylation at Tyr33 and may translocate to the mitochondria and nuclei to induce apoptosis in cultured cells and in rat eyes [3, 6–9]. Tyr33-phosphorylated or activated WOX1 binds to the proline-rich region and phospho-Ser46 of p53, and both proteins induce apoptosis synergistically [3, 6, 8]. When WOX1 is functionally suppressed by antisense mRNA, small interfering RNA (siRNA), or dominant negatives, the stability of p53 and its apoptotic function are significantly suppressed [3, 6, 8]. The proapoptotic function of WOX1 is probably associated, in part, with its interaction with p53, p73, JNK1 and other transcription factors .
To isolate WOX1-binding proteins using yeast two-hybrid cDNA library screening [3, 6, 8], we found that WOX1 interacts with a small size 31-amino-acid protein, Zfra (zinc finger-like peptide that regulates apoptosis) . Zfra belongs to the family of C2H2 type zinc finger proteins [11–13], and has a sequence homology to transcription factor forkhead protein xFKHR1 . Zinc finger proteins interact with DNA and RNA, which is essential for regulating gene transcription during cell growth and embryogenesis. Damage to the zinc fingers in DNA repair proteins may induce carcinogenesis . Zfra mRNA is expressed in many organs and tissues and most abundant in the spleen . However, it is absent in several prostate and breast cancer cell lines .
Zfra participates in the signal pathway of tumor necrosis factor (TNF or TNF-α) [[16, 17]; reviews]. Zfra appears to play a dual role in regulating the cytotoxic effects of TNF and Fas ligand (FasL) . Zfra either enhances or blocks the apoptotic functions of transiently overexpressed receptor adaptor proteins TRADD (tumor necrosis factor receptor type 1-associated death domain protein) and FADD (Fas-associated death domain-containing protein) . TRADD and FADD are recruited to the TNF receptors when cells are stimulated with TNF or Fas ligand (FasL). In response to TNF and UV light, Zfra undergoes self-binding and interacts with JNK1 . JNK1 is a downstream effector of the TNF signaling [18–20]. The functional mechanism for the action of Zfra remains to be established.
In this study, we further investigated the underlying mechanisms for the regulatory effect of Zfra on cell death caused by transiently overexpressed death domain proteins, including TRADD, FADD and RIP (receptor-interacting protein). We determined the role of a conserved phosphorylation site at serine 8 in conferring Zfra-induced apoptosis. Also, we examined whether Zfra regulates the activation of transcription factor NF-κB and tumor suppressors p53 and WOX1 in response to TNF and UV light, and discussed the biological implications of their interactions both in vitro and in vivo.
Transiently overexpressed Zfra induces apoptosis
We have previously shown that when ectopic Zfra is stably expressed in L929 fibroblasts, these cells resist the cytotoxic effects of TNF and FasL . In contrast, depending upon the concentrations used or the extent of expression, transiently expressed Zfra could either enhance or inhibit the cytotoxic function of overexpressed death domain proteins TRADD and FADD . The underlying mechanism of this regard is unknown.
Cells, including HEK-293, SK-N-SH, Molt4 and L929, express Zfra mRNA, and they are sensitive to Zfra-induced death . Breast MCF7 and MDA-MB-231 cells do not express Zfra , but were sensitive to Zfra-mediated death (Figure 1A). Prostate DU145 is a Zfra-negative cell line , and resisted death by Zfra (Figure 1A). Together, our data show that resistance to Zfra-mediated death is not related with the presence or absence of endogenous Zfra in cells.
To further determine Zfra-mediated apoptosis, Molt4 T cells were transfected with EGFP-Zfra and cultured for 48 hr. The extent of exposure of phosphatidylserine to cell surface was determined by Annexin V assay. Zfra increased the exposure of phosphatidylserine on cell surface (Figure 1C), indicating that cells have undergone apoptosis.
Involvement of conserved serine 8 (Ser8) in Zfra-induced apoptosis
Zfra regulates death domain protein-mediated cell death
Taken together from the above experiments, our data clearly show that depending upon concentrations or the extent of expression, Zfra may regulate the function of death domain proteins TRADD, FADD or RIP in a synergistic or an antagonistic manner.
Zfra physically interacts with TRADD, and binds to the N-terminal first WW domain and C-terminal SDR domain of WOX1
We also determined that Zfra physically interacted with the full-length TRADD but not RIP, and WOX1 interacted with TRADD (Figure 6). Together, these observations suggest that Zfra and WOX1 may complex with TRADD, and this complex affects TNF-mediated cell death.
TNF induces the binding of Zfra with Tyr33-phosphorylated WOX1, JNK1, and NF-κB
Zfra is able to undergo self-association and binds JNK1 . In GST pull-down assays, GST-Zfra was shown to interact with endogenous Zfra and WOX1, and TNF barely increased the binding in SK-N-SH cells (Figure 7B). In addition, TNF increased the binding of GST-Zfra with JNK1, NF-κB (p65) and p-WOX1 (Tyr33 phosphorylated), and weakly with p53 in a time-related manner (Figure 7B and 7C). Zfra could not interact with IκBα, FADD, RIP and Fas (Figure 7B and 7C). Similarly, in response to UV light (120 mJoule/cm2), there was an increased binding of GST-Zfra with endogenous Zfra, p-WOX1 and JNK1 in SK-N-SH cells, as determined by pull-down analysis (Figure 7D). UV light increased the binding of endogenous Zfra with phosphorylated JNK1 (at Thr183/Tyr185) in SK-N-SH cells, as determined by co-immunoprecipitation (Figure 7E).
Transiently overexpressed Zfra sequesters WOX1, NF-κB, p53 and p-ERK in the cytoplasm
Zfra counteracts the apoptotic function of WOX1
Similarly, L929 cells were electroporated with WOX1 and/or Zfra. These cells were cultured for 24 hr, and then processed for DNA fragmentation in agarose gel electrophoresis. WOX1-induced apoptosis was blocked by a non-apoptotic dose of Zfra (Figure 9B).
Again, L929 cells were then electroporated with apoptosis-inducing amounts of WOX1 and/or Zfra, followed by culturing for 24 and 48 hr, and then processing for cell cycle analysis by FACS. WOX1 or Zfra alone induced growth suppression and apoptosis, as evidenced by increases in the SubG1 phase and decreases in the G1 phase of the cell cycle in cell populations post transfection for 24 hr (Figure 9C; data not shown for 48 hr). However, when in combination, Zfra and WOX1 failed to synergistically increase cell death (Figure 9C). Similar results were observed by testing Zfra with JNK1 (Figure 9C). Overall, Zfra physically interacts with WOX1 and JNK1, and may counteract the apoptotic function of these proteins under stress conditions.
Phosphorylation at Tyr33 is essential for WOX1 to counteract Zfra-mediated apoptosis
We tested the function of SDR domain and a C-terminal tail (SDR/C) in WOX1 (Figure 10C). L929 cells were transfected with cDNA constructs for expressing SDR or SDR/C, followed by culturing for 24 hr in the presence or absence of a synthetic full-length Zfra peptide (10 μM). Transiently overexpressed SDR/C exerted cell death. Zfra peptide alone had no effect but enhanced cell death caused by SDR/C (Figure 10C). SDR alone could not cause cell death and Zfra and SDR, in combination, did not increase the cell death (Figure 10C). Together, these observations indicate a critical role of Tyr33 phosphorylation in WOX1 in causing cell death and interacting with Zfra. Also, when Zfra binds to the C-terminal SDR/C region, both Zfra and WOX1 may enhance cell death in a synergistic manner.
Protein expression of ECFP-SDR/C and ECFP-SDR is shown. The extent of protein expression was in more than 70% of transfected cells, as determined by fluorescence microscopy. Constructs made for the above experiments are shown (Figure 10D).
Phosphorylation of p53 at Ser46 is essential for counteracting Zfra-mediated apoptosis
Discussion and conclusion
We determined that Zfra interacts with TRADD but not FADD or RIP (Figure 12A). Zfra affects the cytotoxic function of these death domain proteins via direct and indirect manners (open arrow). Zfra physically interacts with WOX1, and that WOX1 binds TRADD. Thus, Zfra and WOX1 are likely to be recruited to the DISC during TNF signaling (Figure 12A). This assumption has yet to be validated by co-immunoprecipitation and confocal and immunoelectron microscopy.
Previously we have shown that WOX1 enhances TNF cytotoxicity . WOX1 also enhances the cytotoxic function of TRADD . Here we determined that both WOX1 and Zfra counteract with each other in regulating apoptosis. Zfra either enhances or inhibits the function of death domain proteins. Thus, the driving force for committing cells to death in response to TNF is likely coming from a balanced and counter-balanced work among Zfra, WOX1 and DISC.
In the TNF-initiated protective pathway, Zfra is shown to interact with TRADD, JNK1 and NF-κB (Figure 12B). Thus, formation of Zfra-containing regulatory complexes probably occurs in the TNF signaling cascade. In Step 1, at the membrane level, Zfra binds TRADD in the presence of TRADD, TRAF2, RIP and WOX1. In Step 2, a trimolecular complex of Zfra/JNK1/WOX1 may form when JNK1 becomes activated by the upstream activated MEK. Zfra binds and counteracts the apoptotic function of JNK1. Also, JNK1 counteracts the apoptotic function of WOX1 . In Step 3, MEK activates ERK, and that Zfra may bind and sequester ERK to the cytoplasm. In Step 4, phosphorylation of IκBα by IKK causes degradation of IκBα and release of NF-κB for nuclear translocation. Again, Zfra is able to bind and sequester NF-κB in the cytoplasm. In Step 5 and 6, p53 is a downstream effector of TNF signaling [8, 22–24]. TNF induces NF-κB activation, and then NF-κB activates p53 . The non-ankyrin C-terminus of IκBα physically interacts with cytosolic p53 to prevent degradation in vivo, and the complex dissociates in response to TNF and apoptotic stress . p53 is functionally associated with WOX1, and both proteins may induce apoptosis synergistically [3, 4, 6, 8]. Thus, an in vivo complex of IκBα/p53/WOX1 is likely (Chang et al., submitted), and that Zfra may regulate the formation of this complex. We show that ectopic Zfra blocks UV light-induced p53 nuclear translocation or activation, suggesting a negative regulation for the cell death event. Ser46-phosphorylated p53 is known to play a critical role in apoptosis [8, 25–29]. That is, Zfra may prevent cell death by blocking the apoptotic function of Ser46-phosphorylated p53. The IκBα/p53/WOX1 or p53/WOX1 may translocate to the mitochondria (Chang et al., submitted). Whether this event is blocked by Zfra is unknown.
Presence of two cysteine residues in the amino acid sequence of Zfra suggests the likely presence of a dimeric form in cells . Also, whether Zfra covalently interacts with specific proteins remains to be established. As determined by GST-pull down analysis, both TNF and UV light are able to increase self-association of Zfra, supporting the presence of dimers in vivo . Zfra is inducible under stress conditions . We show that at low levels Zfra enhances cell growth, and yet overexpressed Zfra induces cell death. Ectopic Zfra also blocked adherence-independent growth of L929 fibroblasts, suggesting that it may act as a tumor suppressor to block cancer growth and progression. Zfra is most abundant in the spleen, whereas its role in regulating immune cell differentiation and functions is unknown. However, it is reasonable to suggest that Zfra-regulated TNF signaling is likely to play a role in the lymphocyte differentiation. We also determined that absent expression of Zfra is found in several breast and prostate cancer cell lines , implying that Zfra deficiency may provide a growth advantage for cancer cells. Majority of zinc finger proteins participate in gene transcription and embryonic development [30–32]. Synthetic peptides have been shown promises in therapy to modulate the transcriptional activities of endogenous zinc finger proteins [33, 34]. Shortening and/or modification of the naturally occurring Zfra may be of therapeutic value in controlling cancer cell growth and death.
Zfra is distributed ubiquitously in cellular compartments. In response to TNF, a portion of cytosolic Zfra appears to translocate to the cell membrane area and binds TRADD. Death domain/death domain interaction is responsible for TRADD binding with p55-TNFR, FADD and RIP. Whether Zfra binds to the death domain is unknown. Sequence alignment analysis shows a common structural motif in TRADD, JNK1, NF-κB, and the N-terminal WW domain and C-terminal SDR domain of WOX1. Whether this motif interacts with Zfra remains to be established.
WWOX/WOX1 is known to interact with p53, p73, JNK1, AP-2γ, and ErbB4 [3, 6, 35–37], suggesting its role of the regulation of gene transcription. Both in vivo and in vitro studies have shown that overexpressed WWOX1/WOX1 suppresses tumor growth in nude mice, and induces apoptosis in cultured cells [3, 6, 8, 9, 38, 39]. Overexpressed WW and SDR/C domains induce cell death . Most interestingly, the C-terminal tail of WOX1 is needed to work together with SDR in causing cell death. In stark contrast, non-invasive breast and prostate cancers may have upregulated expression of WWOX/WOX1, Tyr33 phosphorylation and WWOXv2/WOX2 (and other family proteins if present) [40–42]. Downregulation of WOX1 and WOX2 promotes neurodegeneration in Alzheimer's disease, suggesting its protective role against neurodegeneration . FOR (WWOX/WOX1) is shown to protect against the effects of ionizing radiation in Drosophila . These observations support a pro-survival role of WOX1 in vivo .
In domain/domain mapping by yeast two-hybrid analysis, Zfra was shown to bind the N-terminal Tyr33-phosphorylated WW domain of WOX1. Alteration of this phosphorylation site abolishes the binding interaction. Also, it interacts with the C-terminal SDR domain of WOX1. Since Zfra is a small peptide, its simultaneous binding to these domains in WOX1 under physiologic and stress conditions is very likely. Our supporting evidence shows that stress stimuli induce phosphorylation of WOX1 at Tyr33 and Zfra at Ser8, and that the Zfra/WOX1 complex co-translocates to the mitochondria to regulate cell death via the mitochondrial pathway (Hsu et al., submitted). Also, in this study we found that Zfra enhances the apoptotic activity of SDR domain and the C-terminal tail of WOX1.
Overexpressed Zfra may nullify the apoptotic activities of WOX1 and p53. An apparent mechanism is that overexpressed Zfra sequesters transcriptional factors in the cytoplasm. For example, ectopic Zfra restricts nuclear localization of endogenous WOX1, p53, ERK, and NF-κB (p65), indicating that an upregulated level of Zfra may control the transcriptional system in cells under stress conditions.
Taken together, we have identified a molecular pathway underlying Zfra regulation of TNF cytotoxic function. Zfra interacts with TRADD and binds downstream NF-κB, p53, JNK1 and WOX1 of the TNF signal pathway, thereby either enhancing or restricting TNF-mediated cell death.
Cell lines, antibodies and chemicals
Majority of our experiments were performed using murine L929 fibroblasts, monkey kidney COS7 fibroblasts and human Molt4 T lymphocytes for ectopic gene expression. In addition, the following cell lines were cultured and tested for their sensitivity to Zfra-mediated death: 1) human neuroblastoma SK-N-SH cells, 2) human ovarian ME180 cells, 3) human breast MDA-MB-231 and MCF7 cells, 4) human embryonic kidney HEK-293 fibroblasts, 5) human prostate DU145 cells, and 6) mink lung epithelial Mv1Lu cells. Recombinant TNF-α was from PeproTech and R&D Systems. We have produced specific antibodies against the NH- and COOH-termini of WOX1, recombinant GST-WOX1 and Tyr33-phosphorylated WOX1 [3, 6, 43, 45], synthetic Zfra peptide , and recombinant GST-Zfra . The following specific antibodies were from Santa Cruz Biotechnology: p-JNK1 (phospho-JNK1 at Thr183/Tyr185), WWOX, NF-κB, IκBα, FADD, and p53. Anti-WWOX IgG, N-19 and P-20, were kind gifts of Santa Cruz Biotechnology. α-Tubulin antiserum was from Accurate Chemical, and anti-RIP from Pharmingen/BD Biosciences.
Zfra and WOX1 cDNA constructs and transient gene expression
Zfra was constructed in CMV-based mammalian expression vectors pCR3.1 (Invitrogen) and pEGFPC1 (enhanced green fluorescent protein; cloning site EcoR1; Clontech) . Murine full length WOX1, Y33R mutant, WW domains, SDR domain, and SDR domain with a C-terminal tail were constructed in pCR3.1, pEGFPC1 and pECFGC1 vectors [3, 6, 43]. Human p53 and p53(ΔS46) cDNAs were constructed in pDsRed-N1 (Clontech), as described . Where indicated, cell lines were transfected with Zfra, WOX1 or p53 constructs, or empty vectors (15–600 ng/well) by calcium phosphate (CaPO4) or electroporation [3, 6, 8, 10, 43]. In electroporation, cells were transfected with the indicated expression constructs in the presence of albumin to enhance gene expression and reduce electric shock-induced accidental cell death . Post transfection for 24–48 hr, the extent of cell growth or death was examined by crystal violet staining, cell cycle analysis by propidium iodide staining and fluorescence activated cell sorting (FACS), Annexin V assay, and DNA fragmentation analyses [3, 6, 8, 43].
The conserved phosphorylation site Ser8 in Zfra was mutated using QuikChange site-directed mutagenesis Kit (Stratagene). PCR primers for S8G-Zfra mutant were designed as follows: 1) S8G forward, GCAGAAGGTCGTCTGGCTGTAAATATTGTGA; 2) S8G reverse, TCACAATATTTACAGCCAGACGACCTTCTGC.
Soft agarose colony survival assay
Adherence-independent or transforming cell growth was performed in a soft agarose colony survival assay . Briefly, L929 cells were electroporated with expression constructs of EGFP-Zfra or EGFP alone. These cells were plated at a density of 3 × 104 cells/35-mm dish in triplicate in RPMI 1640, 10% fetal bovine serum, 0.8% agarose, and 10 mM Hepes. Dishes were incubated in a humidified CO2 incubator at 37°C for 3 weeks. The resulting colonies were stained with a soluble tetrazolium-based MTS cell proliferation reagent (Promega), and live colonies (with brown color) were counted .
Electroporation, immunoprecipitation, and GST-Zfra pull-down assays
Cells were suspended in serum-free culture medium, containing 500 μg/ml bovine albumin, and electroporated with an EGFP-Zfra or EGFP construct (20 μg DNA/3 × 106 cells; 180 volt and 50 msec; BTX ECM 830 Square Wave Electroporator; Genetronics) [3, 6, 8, 43]. Albumin enhanced both the transfection efficiency and gene expression by 3–5 folds. These cells were cultured 24–48 hr and then exposed to UV light or TNF-α for indicated times. Cytosolic and nuclear protein fractions were prepared using an NE-PER nuclear and cytosolic extraction kit (Pierce), followed by quantification, SDS-PAGE and Western blotting [3, 6, 8, 43]. Where indicated, we performed 1) immunoprecipitation using specific antibodies against Zfra, and 2) pull-down assays using glutathione-Sepharose 4B beads coated with GST (Glutathione S-Transferase) or GST-Zfra to examine the presence of Zfra-binding proteins [3, 6, 8, 10, 43].
Cytoplasmic yeast two-hybrid analysis
Ras rescue-based yeast two-hybrid analysis (CytoTrap, Stratagene) was performed as described [3, 6, 8]. Briefly, a cytosolic Sos-tagged bait protein is designed for binding to a cell membrane-anchored target protein (tagged with a myristoylation signal). This binding activates the Ras signal pathway in yeast and allows mutant yeast cdc25H to grow at 37°C using a selective agarose plate containing galactose. Without binding, yeast cells cannot grow at 37°C. We made the following bait constructs (in pSos vector): 1) a murine full-length WOX1, 2) the first WW domain and an NH2-terminal WW domain area of WOX1, 3) a COOH-terminal SDR domain area of WOX1 and a mitochondria-targeting region in this domain, and 4) a Y33R mutant in the WW domain area [3, 6, 8]. For target, a construct for Zfra was made in pMyr vector. Where indicated, full-length TRADD and RIP cDNAs were subcloned in pMyr (as target) or pSos (as bait). TRADD and RIP cDNAs were gifts of Dr. D. Goedell of Tularik/Amgen. Control constructs for the binding experiments were human MafB (in both pMyr and pSos) [3, 6, 8].
Research was supported in part by the American Heart Association, the Department of Defense (DAMD17-03-1-0736), the Guthrie Foundation for Education and Research, and the National Science Council, Taiwan, ROC (NSC96-2320-B-006-014-) (to NSC). LJH was supported by grants from the Ministry of Education (91-B-FA09-1-4) and National Science Council, Taiwan (NSC 95-2320-B-006-072-MY2).
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