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Molecular Mechanism of hTid-1, the Human Homolog of Drosophila Tumor Suppressor l(2)Tid, in the Regulation of NF-B Activity and Suppression of Tumor Growth

Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas,¹ Università Cattolica del Sacro Cuore, Istituto di Neurochirurgia, Rome, Italy,² Aaron Diamond AIDS Research Center, The Rockefeller University, New York, New York³

Received 26 March 2004/ Returned for modification 3 May 2004/ Accepted 30 September 2004

	ABSTRACT

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hTid-1, a human homolog of the Drosophila tumor suppressor l(2)Tid and a novel DnaJ protein, regulates the activity of nuclear factor B (NF-B), but its mechanism is not established. We report here that hTid-1 strongly associated with the cytoplasmic protein complex of NF-B-IB through direct interaction with IB/ß and the IKK/ß subunits of the IB kinase complex. These interactions resulted in suppression of the IKK activity in a J-domain-dependent fashion and led to the cytoplasmic retention and enhanced stability of IB. Overexpression of hTid-1 by using recombinant baculovirus or adenovirus led to inhibition of cell proliferation and induction of apoptosis of human osteosarcoma cells regardless of the p53 expression status. Adherent cultured cells transduced with Ad.hTid-1 detached from the dish surface. Morphological changes consistent with apoptosis and cell death were evident 48 h after Ad.EGFP-hTid-1 transduction. In contrast, cells transduced with Ad.EGFP or Ad.EGFP-hTd-1N100, a mutant that has the N-terminal J domain deletion and that lost suppressive activity on IKK, continued to proliferate. Similar data were obtained with A375 human melanoma cells. Ad.EGFP or Ad.EGFP-hTd-1N100 ex vivo-transduced A375 cells injected subcutaneously into nude mice produced growing tumors, whereas Ad.EGFP-hTid-1-transduced cells did not. Collectively, the data suggest that hTid-1 represses the activity of NF-B through physical and functional interactions with the IKK complex and IB and, in doing so, it modulates cell growth and death.

	INTRODUCTION

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hTid-1 is a human homologue of the Drosophila tumor suppressor Tid56 encoded by the lethal(2)tumorous imaginal discs, or l(2)tid, gene (26). Loss of the l(2)tid gene causes malignancy of the anlagen of the adult organs, the imaginal discs in Drosophila (26). hTid-1 was initially identified as a cellular target for the viral oncogenic protein E7 derived from human papillomavirus type 16 (HPV16) (36). hTid-1 also serves as the intracellular target for the viral transforming protein Tax from human T-cell leukemia virus type 1 (HTLV-1) and represses the Tax-induced transactivation of nuclear factor B (NF-B) (7, 8). Subsequent studies demonstrated that hTid-1 interacts with the viral nuclear protein UL9 from herpes simplex virus type 1 (HSV-1) in enhancing the binding of UL9 to the viral genome to facilitate viral replication (14) and that hTid-1 forms a protein complex with Jak2 tyrosine kinase, adversely affecting its kinase activity (35).

The htid1 gene encodes two spliced variants of hTid-1, hTid-1_L, and hTid-1_S (37). The full-length hTid-1_L (long) comprises 480 amino acids, whereas hTid-1_S (short), generated via alternative splicing, produces a predicted protein of 453 amino acids that lacks the C-terminal 33 amino acids of hTid-1_L but contains an additional 6 amino acids (KRSTGN) (37). Aside from differences at the C termini, both hTid-1 variants share identical structural motifs, including an N-terminal mitochondria-processing signal peptide, an N-terminal conserved signature J domain, and a central cysteine-rich motif. hTid-1 was classified as a member of the DnaJ protein family and a molecular cochaperone based on its signature J domain and ability to interact with the heat shock protein 70 chaperone (Hsp70) (25, 27). Currently, the biological functions of these two forms of hTid-1 are unclear. The two variants were reported to exhibit opposing effects on induction of apoptosis in the human osteosarcoma cell line U2OS in response to tumor necrosis factor-alpha (TNF-) and mitomycin C (37), but the mechanism by which hTid-1-mediated apoptosis and antiapoptosis is unknown. Whether hTid-1 functions as a human tumor suppressor is also unknown. The early reports that hTid-1 serves as an intracellular target for viral oncogenic proteins Tax and E7 suggest a role for hTid-1 as a tumor suppressor protein. Indeed, exogenous expression of hTid-1 in human lung adenocarcinoma cells suppressed their ability to form colonies in soft agar (7), supporting the assignment of hTid-1 as a suppressor of transformation. Moreover, a recent report shows that altered expression of hTid-1 is associated with primary human skin cancers and medulloblastoma (5).

To elucidate the intracellular mechanisms of hTid-1 in mediating suppression of transformation, we have assessed its activity on various signaling pathways and demonstrated that hTid-1 can repress NF-B activity induced by TNF-, Tax, and IB kinase (IKKß) by suppressing serine phosphorylation of IB, the inhibitor of NF-B (8). NF-B is a protein family of transcriptional factors expressed virtually in all tissues and is evolutionally conserved in humans and Drosophila. In complex with members of IB family, NF-B plays a key role in regulating the expression of genes involved in immunity, inflammation, the antiapoptotic response, and oncogenesis (16, 30). NF-B is composed of homo- and heterodimers of NF-B/Rel family proteins with a predominant form as a p65/p50 heterodimer. In resting cells, NF-B is sequestered by its inhibitor IB proteins, such as IB and IBß, in the cytoplasm through formation of an inactive NF-B-IB protein complex (16). Upon stimulation of cells by a number of extracellular inducers, IB is rapidly phosphorylated, ubiquitinated, and degraded in the proteasome. Consequently, NF-B is released from the protein complex, moves to the nucleus, and there activates the expression of various genes.

Regulation of NF-B activity is controlled at multiple steps. The IB kinase (IKK) complex and its substrate IB proteins are central elements in regulating NF-B activity (20, 24, 38, 44). IB kinases are components of a 700-kDa protein complex that mediates specific serine phosphorylation of IB (44). This kinase complex consists of three essential components: two catalytic subunits, IKK and IKKß, and one regulatory subunit, IKK (28, 34, 41, 44). In response to certain cytokines, growth factors, and viral proteins such as Tax, signals transmitted from particular upstream cascades converge at the IKK complex (9, 12, 15, 31, 33). The activated IKK phosphorylates IB and IBß at their N-terminal serine residues, targeting them for degradation in the proteasome. Recent studies showed that the IKK activity can be regulated by molecular chaperone proteins (1, 3, 6, 13, 43). In TNF--induced activation of NF-B, recruitment of the IKK complex to the plasma membrane upon stimulation is a crucial step for the enhanced IKK activation. This recruitment process involves two additional components within the IKK complex: CDC37 and a molecular chaperone Hsp90 that are required for TNF--induced assembly, activation and trafficking of IKK in response to TNF receptor 1 (6). IKK, an essential modulator of the IKK complex, was found to associate with Hsp70 by a high-affinity interaction (1). Although hTid-1 is a cellular partner for Hsp70 and potentially functions as a cochaperone, it is not clear if hTid-1 physically interacts with components of the IKK complex to exert its repressive activity on NF-B.

Constitutive NF-B activity is found in a variety of human tumors, including prostate, ovarian, and melanoma tumors (4, 21-23, 42). The viral oncoprotein Tax-mediated transformation of primary T cells exclusively relies on its activation of NF-B (32). Increased activity of NF-B can stimulate tumor cell growth, angiogenesis and metastasis by enhancing the expressions of interleukin-8, matrix metalloproteinases 2 and 9, basic fibroblast growth factor, and vascular endothelial growth factor (4, 21-23, 42). Conversely, suppression of NF-B activity has been shown to downregulate the expression of these proteins, resulting in growth arrest of tumors, increased apoptosis, and decreased metastatic potential (4, 21-23, 42). Some tumor suppressors can induce growth arrest and apoptosis through inhibition of NF-B. For example, PTEN, one of the most frequently mutated and deleted genes in various human tumors, inhibits Akt activation and its downstream IKK activity and represses the nuclear transactivation potential of NF-B (17). The tumor suppressor promyelocytic leukemia protein represses NF-B activation by interacting with RelA/p65 to prevent its binding to the cognate enhancer (40). Promyelocytic leukemia protein potentiates apoptosis in the TNF--resistant cell line U2OS, and this proapoptotic activity is associated with its inhibition on NF-B.

To address the molecular basis of hTid-1's regulation of NF-B activity, we assessed the interaction of hTid-1 with components of the NF-B signaling cascade. We found that hTid-1 regulated NF-B activity through a strong physical and functional association with IB and IKK complex. Overexpression of hTid-1 induced growth arrest and death in several tumor cell lines and suppressed tumor growth of human melanoma in nude mice. We conclude that hTid-1 may play a significant role in the regulation of cell growth and death, in part by regulating NF-B activity.

	MATERIALS AND METHODS

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Cell cultures and antibodies. HEK293 (human embryonic kidney cells), SAOS-2, U2OS, HOS (human osteosarcoma cells), and A375 cells (human melanoma cells) were grown in Dulbecco minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics. Jurkat T cells were cultured in RPMI medium with 10% FCS and antibiotics. SF9 insect cells were grown in Grace's insect supplemented medium containing 5% FCS and antibiotics at 28°C. Monoclonal antibody against hTid-1 was kindly provided by Karl Munger (Harvard Medical School, Boston, Mass.). Antibodies for IB, IBß, p65, IKK/ß, and hemagglutinin (HA) were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-FLAG epitope antibody was purchased from Zymed (South San Francisco, Calif.).

Expression plasmids. The methods constructing mammalian expression plasmids for FLAG-, HA-, glutathione S-transferase (GST), or green fluorescent protein (GFP)-tagged hTid-1_L, IKKß, dnIKKß (dominant-negative mutant of IKKß), dnJNK1 (dominant-negative mutant of JNK1), Hsp70, IB, IBß, and HTLV-1 Tax were described previously (7, 8). A cDNA construct (pCMV-hTid-1_S) encoding hTid-1_S was kindly provided by Karl Munger, and this cDNA was further modified by attaching a C-terminal FLAG tag (hTid-1_S-FLAG). The cDNA fragments for the p65 subunit of NF-B, IKK, and IKK were obtained by high-fidelity PCR with human lymph node cDNAs as templates. They were subcloned in the pCEF vector backbone to generate epitope-tagged fusion gene constructs as indicated in the figures. Various hTid-1 mutants (N50, N100, N150, C140, C180, and Cys) were created by PCR and constructed in the pCEF vector backbone containing the human elongation factor 1 promoter to drive expression of interest genes.

Recombinant baculo- and adenoviruses. A modified version of the baculoviral and adenoviral expression systems was developed. This system combines the advantages of site-specific transposition technology from pFastBac1 (Invitrogen, Carlsbad, Calif.) and a tripromoter derived from pTriEX-1 (Novagen, San Diego, Calif.) for expression of a given gene in mammalian and insect cells and bacteria. Briefly, a BsmBI/SphI fragment from pTriEX-1 was blunt-ended and ligated with pFastBac1 pretreated with HindIII (blunt ended) and EcoRV to generate a new transfer vector named pBCAG that contained promoters for expression of foreign genes in both mammalian cells (driven by the CAG promoter, a hybrid promoter containing cytomegalovirus [CMV] enhancer and chicken ß-actin promoter) and insect cells (controlled by the p10 promoter) in addition to transposase recognition sites (Tn7R and Tn7L). DNA fragments corresponding to hTid-1_L-FLAG, hTid-1_S-FLAG, p65-GST, IB-GST, IBß-GST, Tax-GST, and GST-tagged subunits of the IB kinase complex, including GST-IKK, GST-IKKß, and GST-IKK, were constructed in pBCAG, and these transfer plasmids were used to transform DH10B-Bac competent bacteria (Invitrogen) containing a bacmid- and a plasmid-expressing transposase. Colonies containing recombinant bacmids were selected by Blue-gal and antibiotic screening, and the recombinant bacmids were prepared for transfection of SF9 cells by using Cellfectin (Invitrogen, Carlsbad, Calif.). At 4 days after transfection, the culture media were collected (P1 viral stock). P1 stocks were further amplified twice to generate P2 and P3 viral stocks. The high-titer P3 viral stocks were used for protein expression and in vitro protein purification.

Recombinant adenoviruses were generated by using the same strategy. The same pBCAG constructs were used to transform AD-294 competent bacteria (Qbiogene, Carlsbad, Calif.) containing preexisting Admid and transposase vector, and the recombinant adenoviruses including Ad.EGFP, Ad.EGFP-hTid-1 (the full-length of hTid-1) and Ad.EGFP-hTid-1N100 were generated and purified according to the manufacturer's recommended protocol. For baculovirus-mediated transduction, 10⁵ cells were transduced with 100 µl (10⁸ viral particles) of the P3 viral stocks plus 900 µl of complete culture medium for 6 h at 37°C in a 5% CO₂ incubator. The viruses were removed posttransduction, and fresh complete medium were added to the cell culture. Under these conditions, the transduction efficiency in SAOS-2 and U2OS cells was nearly 100% as assessed with Bac.EGFP to visualize GFP-positive cells. For adenovirus-mediated transduction, cells in log-phase growth in six-well plates or in 150-mm dishes were transduced with the purified recombinant adenoviruses at a multiplicity of infection of 25 to 50 at 37°C in a 5% CO₂ incubator for 6 h. The adenoviruses were removed posttransduction, and complete medium was added to the culture.

Transfection, immunoprecipitation, and GST pull-down. HEK293 cells (10⁶ cells/well) of a six-well plate in log-phase growth were transiently transfected with expression plasmids as indicated in the figures by using PolyFect reagent (Qiagen, Valencia, Calif.). At 24 h later, the cells were harvested and lysed in 1 ml of the lysis buffer containing 1% Triton X-100, 20 mM Tris-Cl (pH 8.0), and 150 mM NaCl plus protease inhibitors (50 µg of aprotinin and 5 µg of leupeptin/ml) and Na₃VO₄ (1 mM). A total of 50 µl of the total cellular protein extracts was saved for measuring protein expression, and the rest was incubated with 30 µl of glutathione-Sepharose 4B beads (GSB; Amersham, Sunnyvale, Calif.) at room temperature for 2 h. After incubation, the beads were washed three times with the lysis buffer described above but without protease inhibitors, and the precipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis.

NF-B reporter assay and in vitro kinase assay. HEK293 cells (2 x 10⁵) in log-phase growth in a 12-well plate were transiently transfected with different expression plasmids, and at 20 h after transfection the cells were analyzed for luciferase activity by using a kit purchased from Promega (Madison, Wis.). The in vitro kinase assay for IKK activity was performed as previously described (8).

MTT and soft-agar assays. Methods for assessing the in vitro proliferation of cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for examining anchorage-independent growth of tumor cells in soft agar were described previously (22, 23, 29).

In vivo tumorigenicity assay. Male athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility (Frederick, Md.). The mice were housed and maintained under specific-pathogen-free conditions. The facilities were approved by the American Association for Accreditation of Laboratory Animal Care and met all current regulations and standards of the U.S. Department of Agriculture, the U.S. Department of Health and Human Services, and the National Institutes of Health. The mice were used in accordance with institutional guidelines when they were 8 to 10 weeks old.

A375SM cells were transduced ex vivo with recombinant adenoviruses expressing enhanced GFP (EGFP) or EGFP-tagged fusion proteins, including the full length of hTid-1 and the N100 mutant at a multiplicity of infection of 20. After overnight incubation at 37°C in a 5% CO₂ incubator, cell viability (>95%) was determined by trypan blue dye exclusion. A375 melanoma cells (first set of experiments, 5 x 10⁵ cells/mouse; second set of experiments, 10⁶ cells/mouse) were injected subcutaneously into nude mice as described in detail previously (21).

Fluorescence imaging. Fluorescent fusion constructs were transfected into HEK cells with PolyFect reagent (Qiagen). Expression and subcellular localization of the fluorescent fusion proteins in living cells were analyzed by conventional fluorescence microscopy.

hTid-1 RNAi. The hTid1 interfering RNA (RNAi) construct was generated by using the lentivirus vector pLL3.7, kindly provided by Luk Van Parijs (Massachusetts Institute of Technology, Boston, Mass.). The target sense sequence of hTid-1 is 5'-GCGGCTCCCAGCATAGCTACT-3', and the shRNA of hTid-1 was engineered in the pLL3.7 vector to express RNAi for knocking down the endogenous hTid-1. The recombinant lentivirus expressing hTid-1 RNAi was generated by cotransfection of pLL3.7/hTid-1 RNAi plasmid with the packaging plasmids (Invitrogen, Carlsbad, Calif.) into 293T packaging cells, and the viruses in the culture supernatants were harvested 48 h posttransfection and were used to transduce human melanoma cells A375SM and MeWo.

	RESULTS

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hTid-1 interacts with the NF-B-IB complex through direct binding to IB or IBß. In the first set of experiments, we determined whether hTid-1 interacts with factors involved in the NF-B signaling cascade by transient cotransfection of HEK293 cells with the FLAG-tagged hTid-1 and the GST fusion genes encoding IB, IBß, and the p65 subunit of NF-B. In a GST pull-down assay, two spliced variants of FLAG-tagged hTid-1 proteins were reproducibly detected in the IB-GST and IBß-GST precipitates (Fig. 1A, lanes 2, 3, 6, and 7) but not in the GST precipitate (Fig. 1A, lanes 1 and 5). Coprecipitation of hTid-1_S-FLAG with p65-GST was also detected but at a lower efficiency (Fig. 1A, lane 8).

View larger version (59K): [in this window] [in a new window]   FIG. 1. hTid-1 associates with the cytoplasmic NF-B-IB protein complex through direct binding to IB and IBß. (A) hTid-1 interacts with IB and IBß in cotransfected HEK293 cells. Transient cotransfections were performed as described in Materials and Methods with hTid-1-FLAG and GST fusion constructs including IB-GST, IBß-GST, and p65-GST (1 µg each). The GST pull-down precipitates were analyzed by anti-FLAG immunoblot (top panel). The membrane was then stripped and reblotted with anti-GST (bottom panel). Total cellular extracts were analyzed with anti-FLAG immunoblot to detect hTid-1-FLAG protein levels (middle panel). (B) hTid-1 coprecipitates with IB proteins expressed in SF9 insect cells. In the top panel, a GST pull-down was analyzed by anti-FLAG immunoblotting. The GST fusion proteins in the GST pull-down were detected by using anti-GST antibody (bottom panel), and the total lysates were examined for hTid-1 expression levels with anti-FLAG (middle panel). (C) Protein complex formation of endogenous hTid-1 and IB proteins. Endogenously expressed hTid-1 appeared in the anti-IB and anti-IBß immunoprecipitates but not in the nonspecific immunoprecipitates in Jurkat T, SAOS-2, and HEK293 cells. The immunoprecipitates were analyzed with anti-hTid-1 immunoblot (upper panel), and the total protein lysates were assessed for endogenous hTid-1 protein levels (lower panel). (D) hTid-1 binds to IB proteins with high affinity. SF9 cells (2 x 106) were coinfected with recombinant baculoviruses coexpressing hTid-1S-FLAG and IB-GST or IBß-GST (100 µl of P3 virus stocks). The GST pull-down precipitates were washed extensively with 1% Triton X-100 lysis buffer (lanes 1 and 4), 1 M NaCl (lanes 2 and 5) or 2 M urea (lanes 3 and 6) in addition to 1% Triton X-100. The precipitates were then analyzed by immunoblotting with anti-FLAG antibody (top panel), and the membrane was stripped and reblotted with anti-GST (bottom panel). The hTid-1S-FLAG expression levels are indicated in the middle panel. (E) Endogenous hTid-1 and p65 proteins were coprecipitated with IB-GST and IBß-GST proteins. HEK cells (2 x 106 cells) were transfected with GST, IB-GST, or IBß-GST. The GST pull-down precipitates were evaluated with anti-hTid-1 antibody (left panel). The membrane was stripped and reblotted with anti-p65 antibody (middle panel); the GST fusion protein levels are indicated in the right panel. (F) hTid-1 is associated with the p65-IB protein complex. HA-tagged IB and p65, with or without hTid-1L-GST, were cotransfected into HEK293 cells. The GST pull-down was analyzed by anti-HA blotting (upper panel), and the IB-HA and p65-HA expression levels in total lysates were assessed by anti-HA blotting (lower panel). The results are representative ones of three independent experiments.

The finding that hTid-1 interacts with IB proteins indicates that hTid-1 may be associated with the NF-B-IB complex. To examine this possibility, we assessed the coprecipitation of endogenously expressed hTid-1 with NF-B and IB. As shown in Fig. 1E, endogenous hTid-1 in HEK293 cells coprecipitated with exogenously expressed GST-tagged IB and IBß fusions, but not with GST (Fig. 1E, left panel). Endogenous p65 protein could also be detected (middle panel) in the same IB-GST and IBß-GST precipitates. In HEK293 cells cotransfected with p65-HA and IB-HA in the presence of hTid-1-GST, a GST pull-down assay showed that both the p65-HA and IB-HA proteins were coprecipitated by GST-tagged hTid-1 (Fig. 1F). Collectively, these results suggest that hTid-1 associates with the cytoplasmic protein complex of NF-B-IB through direct interaction with IB and IBß.

hTid-1 associates with the IKK complex and represses kinase activity. To investigate the possibility that hTid-1 interacts with the IKK complex, HEK293 cells were cotransfected with FLAG-tagged hTid-1 and GST-tagged IKK, IKKß, and IKK. In a GST-IKK pull-down assay, significant amounts of both variants of hTid-1 were detected (Fig. 2A, lanes 1, 4, 7, and 10). In addition, hTid-1_S coprecipitated, though weakly, with IKKß and IKK (Fig. 2A, lanes 8, 9, 11, and 12). When both hTid-1 and IKK subunits were overexpressed in HEK cells, the formation of an hTid-1-IKK complex did not appear to be affected by a 20-min treatment with 20 ng of TNF-/ml (Fig. 2A); however, at lower expression levels of both hTid-1 and IKK, the hTid-1-IKK complex dissociated within 20 min of stimulation with 50 ng of TNF-/ml; it began to regenerate 40 min later (Fig. 2B). This dynamic interaction could also be detected by using endogenously expressed proteins as seen in Fig. 2C. These results imply that the interaction of hTid-1 with IKK complex may be a dynamic response to TNF- stimulation. To determine whether hTid-1 interacts directly with the IKK subunits, we expressed and purified the GST-tagged IKK proteins and FLAG-tagged hTid-1 in SF9 insect cells. As shown in Fig. 2D, both splicing variants of hTid-1 were coprecipitated by GST-IKK and GST-IKKß and less potently by GST-IKK. This result indicates that hTid-1 binds directly to the IKK complex.

View larger version (51K): [in this window] [in a new window]   FIG. 2. Both hTid-1 variants bind to the IKK complex and repress kinase activity. (A) hTid-1 also associates with IKK/ß. Transient cotransfection of FLAG-tagged hTid-1 with GST-tagged subunits of the IKK complex (1 µg each) was performed by using a method similar to that described in Fig. 1. The transfected cells were stimulated with or without TNF- (20 ng/ml, 20 min). The GST pull-down precipitates were analyzed with anti-FLAG immunoblot (top panel). The amount of GST fusion proteins is indicated at the middle panel, whereas the hTid-1-FLAG protein levels are shown at the bottom panel. (B) Time course of the interaction of hTid-1 and IKK upon cell stimulation with TNF-. Cellular lysates from hTid-1-IKK (0.2 µg each)-cotransfected HEK cells at different times of TNF- stimulation (50 ng/ml) were assessed for the coprecipitation of both coexpressed proteins. In the top panel, hTid-1 proteins were detected by a GST pull-down assay with an anti-FLAG antibody. The expression levels of GST-IKK and hTid-1-FLAG are shown in the middle and bottom panels, respectively. (C) Interaction of hTid-1 and IKK with endogenously expressed proteins. Total lysates from HEK293 cells stimulated with or without TNF- (50 ng/ml) were immunoprecipitated by nonspecific antibody (anti-FLAG) or by anti-IKK. The immunoprecipitates were then analyzed by anti-hTid-1 blotting (top panel). The endogenous proteins of hTid-1 and IKK from total lysates are shown in the middle and bottom panels, respectively. (D) hTid-1 binds directly to the subunits of IKK and IKKß. Coprecipitation of hTid-1-FLAG by GST tagged three subunits of the IKK complex expressed in SF9 insect cells. The top panel shows that the hTid-1-FLAG protein was coprecipitated by GST-tagged IKK subunits, the middle panel shows the hTid-1-FLAG expression level from the total lysates, and the GST-tagged IKK subunits from the GST pull-down is shown in the bottom panel.

View larger version (36K): [in this window] [in a new window]   FIG. 3. hTid-1 downregulates TNF--mediated activation of NF-B and represses the kinase activity of IKK. Both variants of hTid-1 suppress TNF- (A)- and IKKß (B)-induced activation of NF-B by the NF-B reporter assay, as described previously (8). The results shown are averages ± the SD; experiments were repeated three times with similar patterns. (C) Both hTid-1 variants inhibit serine phosphorylation of IB by IKKß. The in vitro kinase assay was performed as described in Materials and Methods. The top panel shows the in vitro phosphorylated GST-IB; the protein levels of FLAG-IKKß in the total cellular extracts (middle panel) were detected by anti-IKK immunoblotting, and the hTid-1 protein expression shown in the bottom panel was analyzed with anti-FLAG blot. KA, kinase assay. (D) Kinase-active IKKß phosphorylates hTid-1. Transient cotransfection of FLAG-IKKß and hTid-1-FLAG was performed in HEK293 cells. The FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody plus protein A beads. In vitro kinase assay was performed by using conditions similar to those described above with the exception that GST-IB protein was not added to the kinase reaction. The phosphorylated IKKß and hTid-1 proteins are indicated (p, phosphorylated protein). (E) hTid-1 is not phosphorylated by JNK2. Transient cotransfection of hTid-1L-FLAG and JNK2-HA in HEK293 cells were performed, and the total lysates were coimmunoprecipitated by both anti-FLAG and anti-HA. The in vitro kinase assay was done by the same condition as the IKK kinase reaction. Expressions of hTid-1L-FLAG and JNK2 from total lysates are indicated. (F) Phosphorylation of hTid-1 by IKKß by using recombinant proteins expressed in SF9 insect cells. (G) hTid-1 suppresses IKKß activity in SF9 insect cells. The fixed amount of Bac.GST-IKKß (100 µl) and various amounts of Bac.hTid-1L-FLAG (0, 10, 50, or 200 µl) were used to coinfect SF9 cells (5 x 106 cells). An in vitro kinase assay was performed to detect GST-IB phosphorylation (top panel). Protein expression levels of hTid-1-FLAG and GST-IKKß are shown in the middle and bottom panels, respectively.

The N terminus of hTid-1 mediates interaction with IB.

View larger version (39K): [in this window] [in a new window]   FIG. 4. An N-terminal domain of hTid-1 is required for interaction with IB and is important for hTid-1's repression of NF-B activity. (A) Schematics of the wild-type and deletion mutants of hTid-1. The N-terminal conserved signature J domain and the central Cys-rich domain are indicated. The in vivo interaction of wild-type hTid-1 and its mutant proteins with GST-tagged IB (B) or IBß (C) were evaluated by using transient cotransfections in HEK cells as described in Fig. 1A. The presence of FLAG-tagged hTid-1 proteins in the GST pull-down was analyzed by anti-FLAG immunoblotting (B, top panel), and the membrane was stripped and reblotted with anti-GST (B, bottom panel). The expression of various hTid-1 proteins in whole-cell extracts was detected with anti-FLAG immunoblot (B, middle panel). Only hTid-1 proteins coprecipitated with IBß-GST are shown in panel C. (D) The in vivo binding of GST-tagged hTid-1 or the N100 mutant with IBß-HA. In the top panel, proteins in the GST pull-down were analyzed with anti-HA antibody. The middle panel shows the protein levels of HA-tagged IBß; GST fusion proteins were detected by anti-GST immunoblotting (bottom panel). (E) The N-terminal domain of hTid-1 is important for the NF-B repressive activity of the hTid-1. The repressive activity of the hTid-1 mutants was assessed by using the NF-B reporter assay. The results shown are averages ± the SD; experiments were repeated three times with similar patterns. (F) In vitro kinase assay was performed by cotransfection of the kinase active IKKß with hTid-1 or with the N100 mutant in HEK cells, and the kinase activity was examined as previously described (8).

hTid-1 retains IB in the cytoplasm and prevents IB degradation induced by the IKK activator.
We used the fluorescence imaging technique to determine whether hTid-1 codistributes with free IB in resting cells. De novo-synthesized free IB can translocate from cytoplasm to the nucleus (2, 38). As shown in Fig. 5A, bright nuclear fluorescent signals were found in HEK293 cells expressing IB-EGFP (panel 1). This nucleus-imported, newly generated free IB was detected in roughly 30% of the transfected cells. Translocation of free IB proteins to the nucleus was blocked by coexpression of the p65 subunit of NF-B (Fig. 5A). Likewise, hTid-1 also efficiently blocked the translocation of free IB to the nucleus by retaining IB in the cytoplasm. Virtually all green fluorescent signals of IB-EGFP were retained in the cytoplasm with coexpression of wild-type hTid-1 (Fig. 5A) and various mutants (Fig. 5B2, 3, 6, 7, and 8), the exceptions being N100 and N150. Cotransfection of either N100 or N150 with IB-EGFP resulted in redistribution of the fluorescent signals to the perinuclear clusters and plasma membrane, and it induced cytoplasmic aggregation of IB-EGFP (Fig. 5B4 and 5) (aggregation can be better seen in Fig. 5C). The rounding of cells depicted in panels 4 and 5 probably resulted from protein aggregation of IB-EGFP. The effects observed appeared to be specific for IB-EGFP, since the expression and subcellular distribution of EGFP was not altered by cotransfection with wild-type hTid-1 or the N100 mutant (Fig. 5D). Very bright fluorescent signals (Fig. 5B and C) and accumulated proteins of IB-EGFP (Fig. 5E) were seen in the cells cotransfected with N100, probably due to the protein aggregation, but not the increased expression levels of IB-EGFP. Although it is unclear how the N100 and N150 mutants induce IB redistribution and aggregation, our observations suggest that hTid-1 sequesters IB in the cytoplasm where it forms an inactive complex with NF-B.

View larger version (65K): [in this window] [in a new window]   FIG. 5. hTid-1 retains IB in the cytoplasm. (A) hTid-1 blocks the nuclear import of free IB. EGFP-tagged IB (1 µg) was cotransfected with empty vector, p65-HA, hTid-1L-FLAG, or hTid-1S-FLAG (1 µg each) in HEK293 cells. The fluorescent signal was examined with a fluorescence microscope 24 h after transfection. (B) Analysis of the subcellular distribution and expression of IB-EGFP-cotransfected wild-type hTid-1 and various hTid-1 mutants in HEK cells as indicated in the figure. (C) Panel B4 was enlarged for better viewing and showed protein aggregation and plasma and nuclear membrane attachment of IB-EGFP-cotransfected with the N100 mutant. The results represent at least three independent experiments. (D) hTid-1 has no effect on EGFP subcellular distribution. EGFP was cotransfected with hTid-1-FLAG or the N100 mutant in HEK cells. The EGFP fluorescence intensity and cellular distribution were not altered by coexpression with hTid-1. (E) Anti-GFP immunoblot to detect IB-EGFP levels from total lysates in the HEK293 cells that were cotransfected with hTid-1L-FLAG or N100.

View larger version (45K): [in this window] [in a new window]   FIG. 6. hTid-1 protects IB from degradation induced by an IKK activator, Tax. (A) Visualization of EGFP-tagged IB degradation induced by Tax. IB-EGFP or IBß-EGFP (1 µg) was cotransfected with an empty vector or with Tax1-HA (1 µg) in HEK293 cells. The fluorescence images were taken 24 h after transfection. (B) Immunoblot assay of IB-induced degradation by Tax. GST-tagged IB or IBß was cotransfected with Grb2-HA (a negative control) or with Tax-HA in HEK293 cells. The total protein extracts were analyzed with immunoblot with anti-GST (upper panel). Expression of the HA-tagged Grb2 and Tax is shown in the lower panel. (C) A Tax binding-minus mutant of hTid-1 (hTid-1Cys) protects the hTid-1-IB complex from dissociation induced by Tax. Wild-type hTid-1-FLAG or hTid-1Cys-FLAG was cotransfected with GST-tagged IB or IBß, together with Grb2-HA or Tax-HA, into HEK cells. Protein precipitates from a GST pull-down were analyzed by anti-FLAG immunoblotting (top panel), and the total cellular protein extracts were examined with anti-FLAG blot to determine FLAG-tagged hTid-1 expression (middle panel). Protein levels of HA-tagged Grb2 and Tax are shown in the bottom panel.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Overexpression of hTid-1 induces cell growth arrest and death. (A) Expression of the hTid-1 variants by using recombinant baculoviruses to transduce SAOS-2 and U2OS cells. The osteosarcoma cells were transduced with baculoviruses expressing EGFP (control), hTid-1L-FLAG, or hTid-1S-FLAG. At 40 h after transduction, the total protein lysates were collected for analysis of the protein expression of hTid-1 with anti-FLAG immunoblot. Lanes 1 to 4, SAOS-2 cells; lanes 5 to 8, U2OS cells. Lanes 1 and 5, mock treatment; lanes 2 and 6, Bac.EGFP; lanes 3 and 7, Bac.hTid-1L-FLAG; lanes 4 and 8, Bac.hTid-1S-FLAG. (B) Growth rate of SAOS-2 cells (105 cells) transduced with Bac.hTid-1L-FLAG, Bac.hTid-1S-FLAG, or Bac.hTid-1N100 mutant at indicated times. (C) Growth rate of U2OS cells transduced with the recombinant baculoviruses expressing two spliced variants of hTid-1 at the indicated times. The results are representative of at least three independent experiments.

View larger version (59K): [in this window] [in a new window]   FIG. 8. Adenovirus-mediated gene transfer of hTid-1 leads to cell death in a J domain-dependent manner. Recombinant adenoviruses expressing EGFP (control) and EGFP-tagged hTid-1 were used to transduce SAOS-2 (A) and U2OS (B) cells. At the indicated times, fluorescent images were recorded. (C) HOS cells were transduced with recombinant adenoviruses expressing EGFP-tagged full-length hTid-1 and the hTid-1 N100 mutant. The fluorescent images were taken at different times, as indicated in the figure. The results represent at least three independent experiments. (D) Digitally enlarged fluorescent images show the subcellular distribution of EGFP-hTid-1 and EGFP-hTid-1N100 in transduced U2OS cells. (E) In vitro cytotoxicity assay to assess the viability of SAOS-2 cells transduced with the recombinant adenoviruses expressing EGFP (control), EGFP-hTid-1, and EGFP-hTid-1N100. At 3 days after transduction, viable cells were counted with a microscope, and dead cells were excluded by trypan blue staining.

Overexpression of hTid-1 induces cell growth arrest and death. Since NF-B regulates gene expression involved in oncogenesis, we next evaluated the effect of hTid-1 on tumor cell growth and survival. To avoid potential variation generated by clonal selection, we constructed recombinant baculoviruses expressing wild-type and mutant hTid-1. The human osteosarcoma cell lines SAOS-2 (p53^–) and U2OS (p53⁺) were chosen because their p53 status differed. Nearly 100% of these cells were efficiently transduced by the recombinant baculoviruses as demonstrated by fluorescence microscopy of Bac.EGFP-transduced cells (data not shown). The expression levels were severalfold higher in SAOS-2 cells than in U2OS cells when equal virus titers were applied (Fig. 7A). The growth rate of the cells transduced with Bac.EGFP was near that of mock treated SAOS-2 cells (Fig. 7B), indicating that the baculoviruses alone did not induce significant cytotoxicity. However, the growth and viability of SAOS-2 cells transduced with the recombinant baculovirus overexpressing hTid-1_L or hTid-1_S was significantly impaired, whereas the N100 mutant did not affect growth in vitro (Fig. 7B).

Transduction of U2OS cells with recombinant baculovirus expressing both hTid-1 variants reduced in vitro growth rate within 2 days. Thereafter, the cells continued to proliferate (Fig. 7C). This growth pattern is consistent with baculovirus-mediated gene expression in mammalian cells in that the peak expression is reached 48 to 72 h after transduction and then the expression level decreases gradually over time. The less dramatic effect of hTid-1 on cell growth in the transduced U2OS cells may be due to the reduced expression level of the transgene, as seen in Fig. 7A.

To confirm that tumor cell growth arrest and death is induced by recombinant baculoviruses expressing hTid-1, we constructed recombinant adenoviruses that expressed high levels of EGFP-tagged hTid-1. Ad.hTid-1_L EGFP efficiently transduced SAOS-2 and U2OS cells and induced cell death 3 to 4 days after transduction (Fig. 8A and B). The transduced cells were condensed, detached, and floating from the bottom of tissue culture dishes, a finding consistent with apoptosis. In Ad.EGFP-hTid-1_L-transduced HOS cells, apoptotic cells were seen at 3 days posttransduction. The cells transduced with Ad.EGFP-hTid-1N100 did not exhibit growth arrest or cell death (Fig. 8C). These results clearly indicate that overexpression of hTid-1 can induce growth arrest and cell death in a p53-independent and J domain-dependent manner.

Full-length hTid-1 and N100 had different subcellular distribution patterns. The former distributed evenly in the cytoplasm with a perinuclear cluster pattern, whereas the latter was heavily localized in perinuclear clusters (Fig. 8C). Similar patterns were seen in U2OS cells (Fig. 8D). The in vitro cytotoxicity assay showed that the viability of SAOS-2 cells transduced with Ad.EGFP-hTid-1_L was reduced, whereas Ad.EGFP-hTid-1N100 and the control Ad.EGFP did not significantly affect the viability of SAOS-2 cells (Fig. 8E). Cellular fractionation analysis showed that both the full-length and N100 hTid-1 were present in the cytoplasm and mitochondria (data not shown). Whether the N100 mutant that lacks the mitochondrial processing signal can translocate to the mitochondria remains unknown. One possibility is that this mutant could still form a protein complex with mitochondrial proteins or with its endogenous form of hTid-1 for targeting to the mitochondria.

Adenovirus-mediated gene transfer of hTid-1 into human melanoma cells inhibits proliferation in vitro and tumorigenicity in nude mice. Altered expression of hTid-1 has been reported in human skin cancers (5). We examined the effect of hTid-1 on human melanoma in vitro proliferation and tumorigenicity in nude mice. Cells of the human melanoma line A375SM have a high basal level of IKK activity, probably because of constitutive activation of the IKK complex. Transduction of these cells with recombinant adenoviruses overexpressing hTid-1, but not N100, led to reduced activity of IKK (Fig. 9A). MTT assay (22, 23, 29) of in vitro cell proliferation of transduced A375SM cells indicated overexpression of the full-length hTid-1, but not the N100 mutant, inhibited cell proliferation in culture (Fig. 9B). Exogenous expression of hTid-1 but not N100 suppressed the colony-forming ability of transduced melanoma cells in soft agar (Fig. 9C).

View larger version (44K): [in this window] [in a new window]   FIG. 9. Adenovirus-mediated gene transfer of hTid-1 into human melanoma cells suppresses proliferation. (A) Endogenous IKK activity in A375SM cells transduced with recombinant adenoviruses expressing EGFP, EGFP-hTid-1, and EGFP-hTid-1N100 was assessed by the in vitro kinase assay. (B) Overexpression of hTid-1, not the N100 mutant, inhibits cell proliferation of A375 cells. Melanoma cells (3 x 103/per well) transduced with recombinant adenoviruses were plated into 96-well plates and incubated at 37°C. The number of viable cells at different times was determined by MTT assay. (C) Wild-type hTid-1, not the N100 mutant, suppresses colony-forming ability of A375 cells in soft agar. The adenovirus-transduced cells (5 x 105/per well) were plated into six-well plates in 0.6% agar at 37°C in a 5% CO2 incubator (29). Ten days after plating, the cells were stained and recorded. The results are average ± the SD in triplicate. (D) The photo shows mice injected with ex vivo transduced human melanoma at the end of the 6-week observation period.

Short interfering RNA directed against hTid-1 enhances TNF--induced activation of NF-B. To determine the hTid-1 knockdown effect, we constructed recombinant lentivirus-mediated expression of hTid-1 RNAi in the lentivirus vector pLL3.7 (32a), in which the U6 promoter drives the expression of hTid-1 RNAi and the CMV promoter controls the expression of the fluorescent marker protein EGFP. The human melanoma cells were efficiently transduced with the lentivirus, and >95% of the cells were transduced by visualizing the green fluorescence by fluorescence imaging analysis (data not shown). The high transduction efficiency and long-term expression of the hTid-1 RNAi could be advantageous in reducing clonal selection variation. As shown in Fig. 10A, reduced levels of total endogenous hTid-1 in the A375SM and MeWo cells transduced with hTid-1 RNAi, but not with the control lentivirus pLL3.7, were observed. The protein levels of actin and IKKß from the same total protein preparations were unchanged, indicating that the hTid-1 RNAi specifically suppressed the endogenous hTid-1 expression. We next evaluated the effect of hTid-1 knockdown on the NF-B activity by using an NF-B reporter assay. The NF-B basal activity was higher in cells expressing hTid-1 RNAi than in the control cells (Fig. 10B). When stimulated with TNF- or cotransfected with FLAG-IKKß, the hTid-1 RNAi-expressing cells exhibited a higher level of the NF-B activity than did the control cells at the same stimulation condition (Fig. 10B and C). We further observed an antiapoptotic effect of hTid-1RNAi-expressing melanoma cells in resisting TNF--cycloheximide-induced apoptosis (Fig. 10D), a finding consistent with results demonstrated in U2OS cells (13a). However, unlike the hTid-1 RNAi effect on U20S cells, the MeWo cells expressing Tid-1 RNAi exhibited a similar growth pattern as the control cells and the hTid-1 RNAi expressing A375SM cells showed a slightly retarded growth compared to the control cells (Fig. 10E and F). Taken together, these results support the role of hTid-1 as a negative regulator of the IKK complex.

View larger version (38K): [in this window] [in a new window]   FIG. 10. Expression of hTid-1 RNAi enhances the NF-B activity. (A) A375SM and MeWo cells were transduced with the lentivirus hTid-1 RNAi or with the control virus pLL3.7. At 3 days after transduction, the total lysates were analyzed with immunoblot with antibodies for hTid-1 (top panel), actin (middle panel), and IKKß (bottom panel). (B) The stably transduced cells were transfected with NF-B luciferase reporter plasmid (1 µg/each). At 2 days after transfection, the cells were stimulated with or without TNF- (20 ng/ml) for 5 h. The reporter assay was performed as described in Materials and Methods. (C) The stably transduced cells were transfected with the NF-B reporter (1 µg) alone or in combination with pCEF/FLAG-IKKß (0.4 µg), and the reporter activity was determined 2 days posttransfection. (D) The transduced cells were treated with a combination of TNF- (20 ng/ml) and cycloheximide (30 µg/ml) for 5 h. The viable cells were measured as described previously. The growth rates of the transduced A375SM and MeWo cells expressing hTid-1 RNAi or transduced with the control lentivirus pLL3.7 are shown in panels E and F, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The NF-B regulatory activity of hTid-1 is dependent on the physical association of the hTid-1 proteins with the NF-B-IB and IKK complexes. Exogenous and endogenous hTid-1 and IB coprecipitated in transfected cell lines. Fluorescence imaging analysis demonstrated that the nuclear translocation of newly synthesized IB was blocked by hTid-1. Significantly, two hTid-1 mutants, N100 and N150, which could not interact with IB, lost the ability to repress NF-B activity and instead facilitated IB protein aggregation and dislocation. It is unclear how these two mutants induce the protein aggregation of IB-EGFP. It is possible that they inhibit the function of the endogenous hTid-1 leading to inappropriate folding of IB, causing protein aggregation and dislocation. Regardless, hTid-1-mediated cytoplasmic retention and enhanced stability of IB could maximize the inhibitory activity of IB on NF-B to stabilize the NF-B-IB complex by preventing its undesirable dissociation in resting cells.

We also demonstrated that hTid-1 associated with the IKK complex through an interaction with the IKK/ß subunit. These three subunits are integral components of the IKK complex in the forms of homodimers and heterodimers and are indispensable for intact activity of the complex (24, 34, 41, 44). Dominant-negative mutants of IKK and IKK can diminish the kinase activity of IKKß, a subunit of the IKK complex with a high intrinsic kinase activity. IKKß was chosen to evaluate the effect of hTid-1 on the IKK complex due to its high intrinsic kinase activity in the absence of extracellular stimulation. Our results indicate that hTid-1 suppressed autophosphorylation of IKKß. Further, by utilizing an RNA interference approach, we demonstrated that reduction of the endogenous protein level of both hTid-1 isoforms enhanced TNF- and FLAG-IKKß-mediated activation of NF-B and protected the hTid-1 RNAi-expressing cells from TNF--induced apoptosis. These evidences are in support of a role for hTid-1 in the IKK complex.

How does hTid-1 stabilize the NF-B-IB protein complex? Based on the structural and functional analyses, hTid-1 may regulate NF-B activity at several levels. First, through physical and functional interactions with the IKK complex, hTid-1 may help to maintain the stability of the kinase complex and, when overexpressed, suppress the activity of IKK. This action may be particularly important to ensure that transmission of activation signals passing through the complex is controlled, thus avoiding excessive stimulation of cells which can have an adverse effect on cell survival. Conversely, to transmit the activation signal through the IKK complex, the activated IKK could phosphorylate hTid-1 and modulate its activity. Indeed, we demonstrate that hTid-1 is phosphorylated by the kinase-active IKK, although this phosphorylation is less efficient than IKK-mediated phosphorylation of IB. It remains to be determined how this phosphorylation is related to the hTid-1 modulation of NF-B. Second, hTid-1 may block access of the IKK complex to IB. The binding of hTid-1 to IBs could mask their serine phosphorylation sites, thus decreasing phosphorylation and slowing IB turnover. Consistent with this, we found that hTid-1 inhibits in vitro phosphorylation of IB and prolongs its half-life (8). Third, by serving as an alternative substrate for activated IKK, overexpressed hTid-1 could compete for IKK phosphorylation, protecting IB from phosphorylation and subsequent degradation. Fourth, the enhanced stability of IB proteins may be related to hTid-1's cochaperone activity. The fluorescence imaging data implicate hTid-1 in maintaining proper protein folding and cytoplasmic distribution of IB (10, 11, 19). This potential chaperone activity of hTid-1 requires the J domain of hTid-1, indicating the possible involvement of Hsp70. In light of increasing evidence for molecular chaperones in the regulation of the NF-B signaling complexes (1, 3, 6, 13, 43), it is tempting to speculate that the activity of hTid-1 is regulated in cooperation with its associated Hsp70.

The growth and death of cells is modulated by multiple signaling events. NF-B is one of the key molecular switches, providing a survival mechanism in response to stress signals by upregulating antiapoptotic molecules such as Bcl-2. Altered expression and