The Novel PIAS-like Protein hZimp10 Enhances Smad Transcriptional Activity
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m508365200
ISSN1083-351X
AutoresXiaomeng Li, Gregory N. Thyssen, Jason Beliakoff, Zijie Sun,
Tópico(s)Ubiquitin and proteasome pathways
ResumoTransforming growth factor β (TGF-β) plays critical roles in the control of cell proliferation, differentiation, and apoptosis. Smad proteins are substrates of the TGF-β type I receptor and are responsible for transducing receptor signals to target genes in the nucleus. The PIAS (protein inhibitor of activated STAT) proteins were originally identified as transcriptional co-regulators of the JAK-STAT pathway. Subsequently, cross-talk between the PIAS proteins and other signaling pathways has been shown to be involved in various cellular processes. Importantly, PIAS proteins modulate TGF-β signaling by regulating the transcriptional activity of Smad3. In this study we tested whether hZimp10, a novel PIAS-like protein, acts as other PIAS proteins to regulate Smad3-mediated transcription. We show that expression of exogenous hZimp10 enhances the transcriptional activity of Smad3, which appears to be Smad4-dependent and responsive to TGF-β induction. Furthermore, knockdown of endogenous hZimp10 reduced the transcriptional activity of Smad3. A protein-protein interaction between Smad3 and Smad4 with hZimp10 was identified in glutathione S-transferase-pulldown and co-immunoprecipitation assays. The Miz domain of hZimp10 and the MH2 domains of Smad3 and Smad4 were mapped as the regions responsible for binding. Results from immunostaining assays further demonstrated that Smad3, Smad4, and hZimp10 co-localize within cell nuclei. Finally, we demonstrated that Smad3/4-mediated transcription is significantly impaired in response to TGF-β induction in Zimp10 null (zimp10-/-) embryonic fibroblasts. Taken together, these results provide the first line of evidence to demonstrate a role for Zimp10 in regulating the TGF-β/Smad signaling pathway. Transforming growth factor β (TGF-β) plays critical roles in the control of cell proliferation, differentiation, and apoptosis. Smad proteins are substrates of the TGF-β type I receptor and are responsible for transducing receptor signals to target genes in the nucleus. The PIAS (protein inhibitor of activated STAT) proteins were originally identified as transcriptional co-regulators of the JAK-STAT pathway. Subsequently, cross-talk between the PIAS proteins and other signaling pathways has been shown to be involved in various cellular processes. Importantly, PIAS proteins modulate TGF-β signaling by regulating the transcriptional activity of Smad3. In this study we tested whether hZimp10, a novel PIAS-like protein, acts as other PIAS proteins to regulate Smad3-mediated transcription. We show that expression of exogenous hZimp10 enhances the transcriptional activity of Smad3, which appears to be Smad4-dependent and responsive to TGF-β induction. Furthermore, knockdown of endogenous hZimp10 reduced the transcriptional activity of Smad3. A protein-protein interaction between Smad3 and Smad4 with hZimp10 was identified in glutathione S-transferase-pulldown and co-immunoprecipitation assays. The Miz domain of hZimp10 and the MH2 domains of Smad3 and Smad4 were mapped as the regions responsible for binding. Results from immunostaining assays further demonstrated that Smad3, Smad4, and hZimp10 co-localize within cell nuclei. Finally, we demonstrated that Smad3/4-mediated transcription is significantly impaired in response to TGF-β induction in Zimp10 null (zimp10-/-) embryonic fibroblasts. Taken together, these results provide the first line of evidence to demonstrate a role for Zimp10 in regulating the TGF-β/Smad signaling pathway. The transforming growth factor-β (TGF-β) 2The abbreviations used are: TGF, transforming growth factor; STAT, signal transducers and activators of transcription; CBP, cAMP-response element-binding protein (CREB)-binding protein; GST, glutathione S-transferase; HEK cells, human embryonic kidney cells; HA, hemagglutinin; MEF, mouse embryo fibroblast; RT, reverse transcription; shRNA, short hairpin RNA; PAI, plasminogen activator inhibitor. 2The abbreviations used are: TGF, transforming growth factor; STAT, signal transducers and activators of transcription; CBP, cAMP-response element-binding protein (CREB)-binding protein; GST, glutathione S-transferase; HEK cells, human embryonic kidney cells; HA, hemagglutinin; MEF, mouse embryo fibroblast; RT, reverse transcription; shRNA, short hairpin RNA; PAI, plasminogen activator inhibitor. family comprises a large number of structurally related polypeptide growth factors that play critical roles in cell proliferation, differentiation, motility, adhesion, and death (1Padgett R.W. Das P. Krishna S. BioEssays. 1998; 20: 382-390Crossref PubMed Scopus (91) Google Scholar). TGF-β and related factors activate signaling by binding and bringing together members of two subfamilies of transmembrane protein serine/threonine kinases, the type I (TβR-I) and type II receptors (TβR-II). Smad proteins are the substrates of TGF-β type I receptor and play a central role in transducing receptor signals to target genes in the nucleus (2Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar). The Smads can be loosely grouped into three categories. Smad2 and Smad3 are substrates and mediators of the related TGF-β and activin receptors, whereas Smad4 acts as a cofactor for the receptor-regulated Smads. Smad6 and 7, termed anti-Smads, inhibit the signaling function of the other two groups (3Kretzschmar M. Doody J. Timokhina I. Massague J. 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However, recent studies have suggested that the PIAS proteins may play a more general role in regulating chromatin structure (22Dobreva G. Dambacher J. Grosschedl R. Genes Dev. 2003; 17: 3048-3061Crossref PubMed Scopus (212) Google Scholar). An increased interest has been focused on the role of PIAS proteins in sumoylation (23Schmidt D. Muller S. Cell. Mol. Life Sci. 2003; 60: 2561-2574Crossref PubMed Scopus (222) Google Scholar). Sequence analysis has shown that the SUMO E3 ligase RING domain shares significant homology with the Miz domain of PIAS proteins (24Hochstrasser M. Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Moreover, PIASxα, -xβ, -1, and -3 have been found to interact with SUMO-1 and Ubc9 and mediate the sumoylation of p53 and steroid hormone receptors (25Jackson P.K. Genes Dev. 2001; 15: 3053-3058Crossref PubMed Scopus (200) Google Scholar, 26Kotaja N. Aittomaki S. Silvennoinen O. Palvimo J.J. Janne O.A. Mol. 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PIASy was reported to repress the transcriptional activity of Smad3, and this repressive effect was due to enhanced recruitment of HDAC1 (32Long J. Matsuura I. He D. Wang G. Shuai K. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9791-9796Crossref PubMed Scopus (102) Google Scholar). In contrast, PIAS3 showed an opposite effect, enhancing Smad3-mediated transcription (33Long J. Wang G. Matsuura I. He D. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 99-104Crossref PubMed Scopus (83) Google Scholar). The RING domain of PIAS3 can interact with the transcriptional co-activator p300/CBP and form a ternary complex with Smad3. Moreover, the SUMO-conjugating enzyme Ubc9 and PIAS proteins have been shown to enhance the sumoylation of Smad4 (34Lee P.S. Chang C. Liu D. Derynck R. J. Biol. Chem. 2003; 278: 27853-27863Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The sumoylation of Smad4 by PIAS proteins is regulated by the p38 mitogen-activated protein kinase pathway (35Ohshima T. Shimotohno K. J. Biol. Chem. 2003; 278: 50833-50842Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).hZimp10 is a novel PIAS-like protein (36Sharma M. Li X. Wang Y. Zarnegar M. Huang C.Y. Palvimo J.J. Lim B. Sun Z. EMBO J. 2003; 22: 6101-6114Crossref PubMed Scopus (92) Google Scholar). It shares a ring finger domain, termed Miz (msx-interacting zinc finger), with other PIAS proteins (37Wu L. Wu H. Ma L. Sangiorgi F. Wu N. Bell J.R. Lyons G.E. Maxson R. Mech. Dev. 1997; 65: 3-17Crossref PubMed Scopus (89) Google Scholar), which appears to be important for protein-protein interactions. A novel Drosophila gene, termed tonalli (tna), was identified recently and is the ortholog of hZimp10 (38Gutierrez L. Zurita M. Kennison J.A. Vazquez M. Development. 2003; 130: 343-354Crossref PubMed Scopus (45) Google Scholar). The protein encoded by tna genetically interacts with the chromatin remodeling complexes SWI2/SNF2 and the Mediator complex, suggesting that it may play a role in transcription. In this study, we tested whether hZimp10 affects Smad3-mediated transcription in a manner similar to that of the PIAS proteins. Using several in vivo and in vitro approaches, we demonstrated that Zimp10 interacts with Smad3/4 proteins and augments Smad-mediated transcription, which provides the first line of evidence that Zimp10 plays a critical role in the regulation of the TGF-β/Smad signaling pathway.MATERIALS AND METHODSPlasmids—The pcDNA3-FLAG-Smad3 and pcMV5-FLAG-Smad4 expression vectors were described previously (39Hayes S.A. Zarnegar M. Sharma M. Yang F. Peehl D.M. ten Dijke P. Sun Z. Cancer Res. 2001; 61: 2112-2118PubMed Google Scholar, 40Nakao A. Afrakhte M. Moren A. Nakayama T. Christian J.L. Heuchel R. Itoh S. Kawabata M. Heldin N.E. Heldin C.H. ten Dijke P. Nature. 1997; 389: 631-635Crossref PubMed Scopus (1546) Google Scholar). The HA-tagged Smad4 expression plasmid was constructed by inserting the full-length Smad4 cDNA into pcDNA3 with an N-terminal HA epitope tag (Invitrogen). Various deletion mutants of Smad3 and Smad4 were cloned into pGEX vectors (Amersham Biosciences). The pGEX4T1-Smad4 and 3TP-Luc were kindly provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York). pSV-β-gal, an SV40 driven β-galactosidase reporter plasmid (Promega, Madison, WI), was used in this study as an internal control. The pcDNA3-hZimp10, pcDNA3-FLAG-hZimp10, and pcDNA3-FLAG-hZimp7 were generated as described previously (36Sharma M. Li X. Wang Y. Zarnegar M. Huang C.Y. Palvimo J.J. Lim B. Sun Z. EMBO J. 2003; 22: 6101-6114Crossref PubMed Scopus (92) Google Scholar, 41Huang C.Y. Beliakoff J. Li X. Lee J. Li X. Sharma M. Lim B. Sun Z. Mol. Endocrinol. 2005; 19: 2915-2929Crossref PubMed Scopus (40) Google Scholar). The fragments of hZimp10, including the N terminus (amino acids 1-333), Miz domain (amino acids 728-809), and C terminus (amino acids 932-1064), were generated by PCR with appropriate primers and subcloned in-frame to the pGEX4T3 for making GST fusion proteins. The hZimp10 mutants containing double point mutations (C755G/H757A and C760G/H762A) within the Miz domain were generated by a PCR-based site-directed mutagenesis approach in the pcDNA3-FLAG vector. The pGEX4T3-PIASxα/ARIP3 plasmid was kindly provided by Dr. J Palvimo (Helsinki, Finland).Cell Cultures and Transient Transfections—A monkey kidney cell line, CV-1, a human prostate cancer cell line, PC3, a human colon cancer cell line, SW480.7, and a human embryonic kidney cell line, HEK293, were maintained in Dulbecco's modified Eagle's medium supplemented with 5 or 10% fetal bovine serum (HyClone, Denver, CO). Transient transfections were carried out using a LipofectAMINE2000 kit (Invitrogen). Approximately 1.5 × 104 cells were seeded into a 48-well plate 16 h before transfection. 300 ng of total plasmid DNA and 0.5 μl of Lipofectamine2000 per well were used in the transfection. The total amount of plasmid per well was equalized by the addition of pcDNA3 or pBluescript empty vector. Approximately 48 h after transfection, luciferase activity was measured as relative light units in a Monolight 3010 luminometer (Pharmingen) according to the manufacturer's protocol. The relative light units from individual transfections were normalized by β-galactosidase activity in the same samples. Individual transfection experiments were done in triplicate, and the results are reported as mean relative light units/β-galactosidase (±S.D.) from representative experiments.GST Pulldown Assay—Expression and purification of GST fusion proteins were performed as described previously (42Sharma M. Zarnegar M. Li X. Lim B. Sun Z. J. Biol. Chem. 2000; 275: 35200-35208Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The full-length Smad3, Smad4, and hZimp10 proteins were generated and labeled in vitro by the TnT-coupled reticulocyte lysate system (Promega). Equal amounts of GST fusion proteins coupled to glutathione-Sepharose beads were incubated with the radiolabeled proteins at 4 °C for 2 h in a modified binding buffer (20 mm Tris-HCl (pH 7.8), 180 mm KCl, 0.5 mm EDTA, 5 mm MgCl2, 50 μm ZnCl2, 10% glycerol, 0.1% Nonidet P-40, 0.05% dry nonfat milk, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). Beads were carefully washed 3 times with 500 μl of binding buffer and then analyzed by SDS-PAGE followed by autoradiography.Immunoprecipitation and Western Blotting—The HA-tagged pcDNA3-hZimp10 expression plasmid, alone or with a FLAG-tagged pcDNA3-Smad3 and/or FLAG-tagged pCMV5-Smad4 expression plasmids, was transfected into CV-1 cells. Transfected cells were cultured for 48 h and then harvested in a buffer containing 0.5% Nonidet P-40, 150 mm NaCl, 2 mm MgCl2, 50 mm HEPES-KOH (pH 7.4), 1 mm EDTA, 5% glycerol, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 25 mm NaF. Lysates were clarified by incubation on ice and centrifugation for 5 min. Four hundred μl of clarified lysate from each sample was precleared for 20 min with 10 μl of protein-A-Sepharose beads bound to 1 μg of normal mouse IgG (Pharmacia). Precleared lysates were then incubated with pre-equilibrated protein-A-Sepharose beads with either normal mouse IgG or FLAG monoclonal antibody (Sigma) at 4 °C for 3 h. The beads were washed 3 times in 500 μl of lysis buffer and eluted by boiling in SDS-PAGE sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose (Schleicher and Schuell) and blocked overnight at 4 °C in TBS-T (50 mm Tris-HCl, 150 mm NaCl, 0.08% Tween 20) with 5% lowfat milk. Membranes were probed with HA, FLAG, Smad3, Smad4, or the hZimp10 antibody at the appropriate dilutions. Anti-rabbit, mouse, or chicken IgG conjugated to horseradish peroxidase were used as secondary antibodies (Promega). Detection was performed with ECL reagents according to the manufacturer's protocol using ECL Hyperfilm (Amersham Biosciences).Immunostaining—CV-1 or PC3 cells were co-transfected with pcDNA3-hZimp10, FLAG-tagged pcDNA3-Smad3, and HA-tagged pcDNA3-Smad4 in the presence or absence of TGF-β 1 growth factor (R&D Systems, Minneapolis, MN). Specific primary antibodies and Fluorophore-conjugated secondary antibodies were used (Molecular Probes, Eugene, OR). Images were acquired using a confocal microscope.Mouse Embryonic Fibroblasts—Mice heterozygous for a neomycin-disrupted allele of the Zimp10 gene were mated, and females were sacrificed at 9.5 days post-coitus. Embryos were isolated in cold phosphate-buffered saline and then incubated in 250 μl of trypsin (0.05%) for 10 min at 37 °C with intermittent agitation. Embryos were disrupted by pipetting and then added to at least a 3× volume of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were directly plated into 6- or 48-well plates, allowed to adhere overnight, and used for assays. To determine the mouse embryo fibroblasts (MEF) genotype, embryo sacs isolated during the dissection were digested, genomic DNA was extracted, and the wild type or mutant Zimp10 alleles were PCR-amplified using specific primers.RNA Isolation and Reverse Transcription (RT)-PCR Assay— Mouse embryo fibroblasts were established as described above and serum-starved overnight. TGF-β1 was then added directly to the media to achieve a final concentration of 50 ng/ml. 5.5 h after stimulation, total RNA was isolated using RNABee (TEL-TEST, Inc., Friendswood, TX). The RT-PCR method was carried out as described previously (43Sun Z. Yergeau D.A. Tuypens T. Tavernier J. Paul C.C. Baumann M.A. Tenen D.G. Ackerman S.J. J. Biol. Chem. 1995; 270: 1462-1471Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Briefly, cDNA was synthesized from 1-5 μg of total RNA with 9 units of avian myeloblastosis virus reverse transcriptase (Promega) using 0.1 μm oligo-dT primer in a total volume of 20 μl. One μl of cDNA was added to a standard PCR mix containing 1 μm concentrations of each primer. The PCR reaction was performed on a thermal cycler using 26-30 cycles of 45 s at 95 °C, 40 s at 58 °C, and 45 s at 72 °C for glyceraldehyde-3-phosphate dehydrogenase and 30 s at 95 °C, 30 s at 52 °C, and 50 s at 72 °C for PAI-1. The final polymerization step was extended an additional 10 min at 72 °C. Primers for PAI-1 (5′-TCATCAATGACTGGGTGGAA-3′;5′-CTGCTCTTGGTCGGAAAGAC-3′) and glyceraldehyde-3-phosphate dehydrogenase (5′-CCATGGAGAAGGCTGGGG-3′; 5′-CAAAGTTGTCATGGATGACC-3′) were synthesized and used in the PCR reactions.RESULTShZimp10 Augments Smad3-mediated Transcription—PIAS proteins have been shown to regulate TGF-β/Smad3 activity. Here, we investigated a possible role for hZimp10 (36Sharma M. Li X. Wang Y. Zarnegar M. Huang C.Y. Palvimo J.J. Lim B. Sun Z. EMBO J. 2003; 22: 6101-6114Crossref PubMed Scopus (92) Google Scholar) and hZimp7 (41Huang C.Y. Beliakoff J. Li X. Lee J. Li X. Sharma M. Lim B. Sun Z. Mol. Endocrinol. 2005; 19: 2915-2929Crossref PubMed Scopus (40) Google Scholar), novel PIAS-like proteins, in regulating Smad3-mediated transcription. A plasmid containing the TGF-β-inducible luciferase reporter (3TP-Luc) was co-transfected into CV-1 cells with plasmids expressing Smad3, hZimp7, hZimp10, or PIASxα.An ∼5-fold induction of Smad3-mediated transcriptional activity was observed when cells were transfected with Smad3 (Fig. 1A). Smad3 activity was enhanced ∼2-fold in the presence of 60 ng of hZimp10, and this enhancement was dose-dependent. In contrast, co-transfection of hZimp7 and PIASxα showed no significant effect (Fig. 1A). There was no effect when only hZimp7, hZimp10, or PIASxα was transfected alone with the reporter plasmid (data not shown). These results indicate that hZimp10, but not hZimp7 or PIASxα, augments Smad3-mediated transcription.Previous studies have shown that Smad4 can form a heterodimer with Smad3, which can then translocate into the nucleus to activate the transcriptional response (44Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar, 45Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar). To test whether the enhancement of hZimp10 is mediated through the transcriptionally active Smad3/Smad4 complex, we repeated the transient transfection assays presented in Fig. 1A in the presence of a Smad4 expression vector. As shown in Fig. 1B, Smad4 increases Smad3-mediated transcription by nearly 30%, and hZimp10 further enhances Smad3/4-mediated transcription to ∼0.6-1.2-fold. Again, no enhancement was observed with hZimp7 or PIASxα. To further confirm that hZimp10 enhances the activity of the Smad3/Smad4 transcriptional complex, we repeated the above experiments in the Smad4-negative cell line SW480.7. As expected, overexpression of Smad3 showed no significant transcriptional activity on 3TP-Luc in this human colon cancer cell line (Fig. 1C). There was also no significant effect of hZimp10 on Smad3-mediated transcription. However, expression of exogenous Smad4 resulted in a dosage-dependent enhancement of Smad3-mediated transcription. In the presence of Smad4, hZimp10 further increased the activity of 3TP-luc in a dosage-dependent manner. Taken together, these data indicate that hZimp10 can enhance the activity of the Smad3/Smad4 transcriptionally active complex.Previous studies have demonstrated that TGF-β signals are transmitted through Smad proteins (2Massague J. Blain S.W. Lo R.S. Cell. 2000; 103: 295-309Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 46Heldin C.H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3316) Google Scholar). To determine whether enhancement of Smad3/4 by hZimp10 is induced by TGF-β, we repeated the transfection experiments with serum-free medium with or without TGF-β1 in HEK293 cells, which respond to TGF-β induction. As shown in Fig. 1D, there was only a slight increase in luciferase activity in cells transfected with Smad3 and Smad4 expression vectors in the absence of TGF-β1. However, co-transfection of hZimp10 with Smad3 and 4 resulted in 20-50% increased luciferase activity in cells treated with TGF-β1 (Fig. 1D). These results suggest that hZimp10 affects TGF-β-induced Smad3/4-mediated transcription.Next, we investigated the involvement of endogenous hZimp10 in regulating the transcriptional activity of the Smad3/Smad4 complex. We first generated three short hairpin RNA (shRNA) constructs for hZimp10 (47Sui G. Soohoo C. Affar E. Gay F. Shi Y. Forrester W.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5515-5520Crossref PubMed Scopus (1060) Google Scholar) and tested their knockdown effects on ectopically expressed hZimp10 in CV-1 cells (Fig. 2A). All three hZimp10 shRNA constructs reduced the expression of FLAG-tagged hZimp10 protein. There was no change in tubulin expression, confirming the specificity of the hZimp10 shRNAs. Particularly, the hZimp10 shRNA construct 2 appeared most effective in this knockdown experiment. In addition, this construct also diminished hZimp10 enhancement of Smad3/Smad4-mediated transcription (Fig. 2C). A t test showed that the hZimp10 shRNA-mediated knockdown effect is significant (p < 0.05). Using this construct, we further tested the role of endogenous hZimp10 on Smad3/4-mediated transcription in HEK293 cells. As shown in Fig. 2B, the hZimp10 shRNA2 significantly reduced the expression of the endogenous protein. This knockdown effect resulted in an ∼35 or 50% reduction in Smad3/4-mediated transcription at 15 or 45 ng of the shRNA2 construct, respectively (Fig. 2D). Taken together, the above data indicate an important role for endogenous hZimp10 in augmenting the activity of the Smad3/Smad4 transcriptional complex.FIGURE 2Knockdown of hZimp10 expression reduces Smad3/4-mediated transcription. A, CV-1 cells were transfected with 1 μg of pcDNA3-FLAG-hZimp10 (F-hZimp10) and 200 ng of different pBluescript (pBS)/U6-hZimp10-shRNA constructs (Z1, Z2, and Z3) or pBS/U6 vector only in 6-well plates. Whole cell lysates were prepared after 48 h of transfection and analyzed by Western blotting with either FLAG or tubulin antibody. B, different hZimp10 shRNA constructs or pBS/U6 vector were transfected into HEK293 cells. Cells were harvested, and cell lysates were analyzed as described in A. C, CV1 cells were transfected with different amounts of plasmids as indicated in the figure. Luciferase and β-galactosidase activities were measured in whole cell lysates as described above. The asterisk indicates that the data are significantly different by t test (p < 0.05). D, HEK293 cells were transiently transfected in 48-well plates with 100 ng of 3TP-Luc, 25 ng of pSV40-β-gal, 8 ng of pcDNA3-FLAG-Smad3, 4 ng of pCMV5-FLAG-Smad4, and 15 or 45 ng of hZimp10 shRNA. The shRNA vector backbone was used as a control. The data showing statistical significance is marked with an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Miz Domain of hZimp10 Is Involved in the Interaction with Smad3 and Smad4 Proteins—Previous reports suggest that the Miz domain plays a role in interacting with target proteins (37Wu L. Wu H. Ma L. Sangiorgi F. Wu N. Bell J.R. Lyons G.E. Maxson R. Mech. Dev. 1997; 65: 3-17Crossref PubMed Scopus (89) Google Scholar). Particularly, it has been shown that the Miz domain of PIAS3 and PIASy is responsible for interacting with the Smad3 and Smad4 proteins (32Long J. Matsuura I. He D. Wang G. Shuai K. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9791-9796Crossref PubMed Scopus (102) Google Scholar, 33Long J. Wang G. Matsuura I. He D. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 99-104Crossref PubMed Scopus (83) Google Scholar). To directly assess the involvement of the hZimp10 Miz domain in the interaction with Smad3 and Smad4, we performed in vitro GST-pulldown assays. [35S]Methionine-labeled full-length Smad3 or Smad4 bound to different GST-hZimp10 fusion proteins or GST protein alone was analyzed by SDS-PAGE and detected by autoradiography. As shown in Fig. 3A, Smad3 and Smad4 proteins bound to GST-PIASxα/ARIP3, which was used as a positive control. Importantly, a weak interaction was observed in samples containing GST-hZimp10-Miz (amino acids 728-809) but not with GST-hZimp10-N′ (amino acids 1-333), GST-hZimp10-C′ (amino acids 932-1064), or GST beads alone. Next, we used two hZimp10 Miz domain mutants that contain double point mutations, Mut1 (C755G/H757A) and Mut2 (C760G/H762A), to further assess the importance of the Miz domai
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