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• W2031774227 abstract "The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21 WAF1 (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells. Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter. This activation does not require the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids (152–193) of Sp1 that are important for the interaction with human TAF130. Because transforming growth factor-β enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined. PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-β-responsive promoter, p3TP-lux. Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus. However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region. Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA. These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter. The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21 WAF1 (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells. Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter. This activation does not require the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids (152–193) of Sp1 that are important for the interaction with human TAF130. Because transforming growth factor-β enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined. PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-β-responsive promoter, p3TP-lux. Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus. However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region. Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA. These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter. phorbol 12-myristate 13-acetate TATA-binding protein hepatocyte growth factor transforming growth factor β phosphate-buffered saline transcription factor TBP-associated factor N-tris(hydroxymethyl)methylglycine polymerase chain reaction The p21 protein (WAF1 or CIP1) mediates p53-induced growth arrest by complexing with G1 cyclin-dependent kinases and inhibiting them from phosphorylating the retinoblastoma protein (1El-Deiry W.S. Tokino T. Velculescu V.E. Lev D. Parsons R. Trent J. Lin D. Mercer W. Kinzler K. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7935) Google Scholar,2Harper J.W. Adami G. Wei N. Keyomarse K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5239) Google Scholar). Additionally, WAF1 complexes directly with the proliferating cell nuclear antigen, suggesting a role for this protein in the regulation of DNA synthesis. WAF1 may have a direct role in controlling apoptotic cell death as genetic disruption of the WAF1 gene increases the apoptotic response to DNA damage (3Waldman T. Lengaver C. Kinzler K. Vogelstein B. Nature. 1996; 381: 713-716Crossref PubMed Scopus (719) Google Scholar). Increases in WAF1 protein can be seen during skeletal muscle and hematopoietic differentiation (4Steinman R.A. Hoffman B. Iro A. Guillouf C. Leibermann D. El-Houseini M. Oncogene. 1994; 9: 3389-3396PubMed Google Scholar), suggesting that this protein is important in the terminal cell cycle arrest in the G0 phase of the cycle. The levels of WAF1 protein are controlled both transcriptionally and post-transcriptionally. After DNA damage of fibroblasts, p53 protein increases and binds to two upstream promoter sites increasing the transcriptional activation of the WAF1 promoter, while in comparison tumor necrosis factor-α addition to hematopoietic cells modulates WAF1 levels through posttranslational mechanisms (5Shiohara M. Akashi M. Gombart A. Yang R. Koeffler H.P. J. Cell. Physiol. 1996; 166: 568-576Crossref PubMed Scopus (53) Google Scholar, 6Viziri H. West M. Allsopp R. Davison T. Wu Y. Arrowsmith C. Poirier G. Benchimol S. EMBO J. 1997; 16: 6018-6033Crossref PubMed Scopus (333) Google Scholar). In hematopoietic cells that are deleted for p53, PMA1 induces the transcription of WAF1 in a p53-independent manner. This PMA-mediated transcriptional stimulation is regulated by one of several GC-rich regions adjacent to the TATA box that bind the Sp1 and Sp3 transcription factors (7Biggs J.R. Kudlow J. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Mutation of this site reduces both basal and PMA-induced transcription. Because transcription of a reporter plasmid containing only Sp1 binding sites and a TATA box was induced by PMA, it was suggested that PMA may regulate Sp1 levels directly. However, analysis of Sp1 binding to DNA by gel mobility shift and DNase I footprint experiments indicated that Sp1 was bound to the WAF1/CIP1 promoter both before and after PMA treatment. The importance of these Sp1 binding sites to p53-independent transcription of WAF1 is demonstrated by the observations that these sequences are essential for the induction of WAF1 by TGFβ during normal keratinocyte differentiation (8Datto M. Yu Y. Wang X. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) and for the induction of WAF1 in colon cancer cells by butyrate (9Nakano K. Mizuno T. Sowa Y. Orita T. Yoshino T. Okuyama Y. Fujita T. Ohtani-Fujita N. Matsukawa Y. Tokino T. Yamagishi H. Oka T. Nomura H. Sakai T. J. Biol. Chem. 1997; 272: 22199-22206Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), as well as the observation that Sp1 bound to these sites interacts with the progesterone receptor during progesterone induction of WAF1 (10Owen G. Richer J. Tung L. Takimoto G. Horwitz K. J. Biol. Chem. 1998; 273: 10696-10701Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Nerve growth factor induces both WAF1 and cyclin D1 gene transcription in PC12 cells by activating the Sp1 transcription factor (11Billon N. van Grunsuen L. Rudkin B. Oncogene. 1996; 13: 2047-2054PubMed Google Scholar). Likewise, in osteosarcoma cells, Sp1 plays a role in histone deactylase inhibitor activation of WAF1 transcription (12Sowa Y. Orita T. Minamikawa S. Nakano K. Mizuno T. Nomura H. Sakai T. Biochem. Biophys. Res. Commun. 1997; 241: 142-150Crossref PubMed Scopus (287) Google Scholar). Thus, Sp1 binding to the WAF1 promoter is an essential element in the control of transcription of this gene. It is possible that the regulation of Sp1 through this diverse set of agents occurs by modulation of the basal transcription machinery or other enhancer-binding transcription-activating proteins. Recently, we have shown that the addition of PMA to hematopoietic cells stimulates the phosphorylation of the amino-terminal tail of TBP, suggesting that direct modulation of TBP by PMA could control transcription (13Biggs J.R. Ahn N.G. Kraft A. Cell Growth Diff. 1998; 9: 667-676PubMed Google Scholar). Others (14Naar A. Beaurang P. Zhou S. Abraham S. Solomon W. Tjian R. Nature. 1999; 398: 828-832Crossref PubMed Scopus (373) Google Scholar) have demonstrated that PMA increases an essential protein in the basal transcription complex. Studies of the WAF1 promoter transfected into HepG2 cells demonstrated that overexpression of Smad3 could stimulate promoter activity (15Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar). Smad3 is phosphorylated by TGFβ-mediated activation of the type I receptor and dimerizes with Smad4 (16Zhang Y. Feng X.H. Wu R. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (759) Google Scholar); this phosphorylated heterodimer then translocates to the nucleus, where it binds to enhancer elements and activates transcription. A dominant-negative mutant Smad4 inhibits induction of the WAF1 promoter by TGFβ, suggesting that Smad proteins play an important role in modulating the activity of this promoter (17Hunt K.K. Fleming J. Abramian A. Zhang L. Evans D. Chiao P.J. Cancer Res. 1998; 58: 5656-5661PubMed Google Scholar). In HepG2 cells overexpression of Smad3 also stimulated the activity of a GAL4-Sp1 construct (15Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar), suggesting an interaction between Sp1 and Smad3 in regulating the WAF1 promoter. Because Smad 3 binds to the p300 co-activator and to the transcription factor c-Jun as well as forming a dimer with Smad4, it was proposed that Smad3 might activate transcription of WAF1 by forming a bridge between p300 and other upstream transcriptional activators (18Pouponnet C. Jayanaman L. Massague J. J. Biol. Chem. 1998; 273: 22865-22868Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Google Scholar, 20Zhang Y. Feng X.H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (686) Google Scholar). To evaluate the complex mechanism of transcriptional regulation of the WAF1 promoter, we have used K562 cells that respond to PMA treatment with megakaryocytic differentiation (21Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1195-1947Crossref Scopus (203) Google Scholar). We find that PMA-induced regulation of Sp1 is mediated by 43 amino acids of Sp1 protein necessary for binding to the TBP-associated factor, human TAF130. This PMA activation is inhibited by E1A, but not by an E1A mutant incapable of binding to p300, suggesting that p300 plays a role in PMA-induced WAF1 transcription. Additionally, in K562 cells PMA activated both GAL4-Smad and p3TP-lux, a TGFβ-responsive promoter. PMA induced the phosphorylation of Smad3 and caused the translocation of Smad3 to the nucleus. However, in K562 cells Smad3 transfection did not increase WAF1 transcription or PMA-induced WAF1 transcription. Additionally, EVI-1, a protein that binds Smad3 and inhibits its activity (22Kurokawa M. Mitani K. Irie K. Matsuyama T. Takahashi T. Chiba S. Yazaki Y. Matsumoto K. Hirai H. Nature. 1998; 394: 92-96Crossref PubMed Scopus (301) Google Scholar), had no effect on PMA-induced WAF1 transcription. Taken together, these results suggest that PMA regulation of the WAF1 promoter occurs through core transcription factors, but does not involve the Smad pathway. K562 human leukemic cells obtained from ATCC (Rockville, MD) were passaged in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated bovine calf serum and antibiotics at 37 °C in 5% CO2. For selection of stably transfected cell lines carrying the pMEP vector, cells were passaged after transfection in media containing 200 μg/ml hygromycin B for approximately 3 weeks. For transfection, 20 million K562 cells were resuspended in 0.8 ml of PBS and transferred to an electroporation cuvette (Bio-Rad). 20–50 μg of plasmid DNA was added, and the cells were pulsed two times at 25 V, 250 microfarads. The cells were then divided into aliquots and allowed to recover for 5–6 h in the incubator. Cells were then treated with CdSO4, PMA, or other reagents as indicated. PMA was obtained from Calbiochem. When using TGFβ-neutralizing antibodies (R&D systems), transfected cells were pretreated for 1 h with antibodies before addition of PMA or TGFβ. The GAL4-Smad3 construct, a gift from Y. Zhang and R. Derynck, was transferred from pSG424 to pBluescript SK using flanking HindIII and XbaI sites, and then into pMEP using KpnI and NotI sites. To make GAL4-Smad3A, a BglII/NotI fragment from the Smad3A expression vector (a gift from X. Liu and H. Lodish) containing COOH-terminal coding sequence and three serine to alanine mutations was used to replace the equivalent sequence in wild-type GAL4-Smad3. To make GAL4-Sp1 fusions, PCR primers were designed to amplify the appropriate regions of Sp1 using a full-length Sp1 cDNA as template. The PCR primers were designed with a BamHI site on 5′ primers and an XbaI site on 3′ primers to allow cloning of PCR products into pSG424, which contains the GAL4 DNA binding domain (amino acids 1–147), and giving in-frame fusions between GAL4 and Sp1. The sequences encoding the GAL4-Sp1 fusions were cloned into pBluescript (Stratagene) using HindIII and XbaI sites, and then cloned into pMEP using KpnI andNotI sites. GAL4 luciferase reporter plasmids were a gift from A. Patterson and J. Kudlow. Cell pellets were lysed in 0.5% Nonidet P-40, 50 mm Tris, pH 7.5, 150 mm NaCl, 20 mm EDTA, 20 mm NaF, 1 mm vanadate, 10 mm benzamide, 40 mm glycerol phosphate, and protease inhibitors for 30 min at 4 °C. Debris was removed by spinning for 10 min in a microcentrifuge at 4 °C. Equal amounts of cell lysate were run on SDS-polyacrylamide gels and transfered to nitrocellulose. After blocking in 10 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Tween 20, and 5% bovine serum albumin, the filter was incubated in blocking solution plus anti-GAL4 antibodies (Upstate Biotechnology, Inc.) for 2–4 h. The filter was washed in Tris-buffered saline plus 0.5% Nonidet P-40 for 30 min, then incubated with anti-rabbit horseradish peroxidase antibodies (Amersham Pharmacia Biotech). After further washing, antibody bound to the filter was visualized using ECL detection reagents (Amersham Pharmacia Biotech). For luciferase assays, cells were lysed for 10 min at room temperature in 25 mm Tris-phosphate, pH 7.8, 2 mm dithiothreitol, 2 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 10% glycerol, and 1% Triton X-100. After a 30-s spin in a microcentrifuge to remove debris, equal amounts of protein were brough to a final sample volume of 100 μl. Just prior to measurement of light intensity, 100 μl of luciferin buffer was added to each sample. The luciferin buffer contained 20 mm Tricine, 1.07 mm magnesium carbonate, 2.67 mmMgSO4, 0.1 mm EDTA, 33.3 mmdithiothreitol, 530 μm ATP, 270 μm coenzyme A, and 470 μm luciferin. K562 cells transfected with vectors expressing FLAG-Smad3 or FLAG-Smad3A were incubated 5 h in medium containing either 100 μCi/ml [35S]methionine or 200 μCi/ml [32P]orthophosphate. PMA was added at various times before the cells were lysed in the buffer described under “Western Blots.” 1 μg of anti-FLAG antibody (Sigma M2 monoclonal) bound to 10 μl of protein A/G-agarose (Calbiochem) was added to each sample, followed by an overnight incubation on a rocker at 4 °C. Immunoprecipitates were then washed three times in lysis buffer, followed by heating at 96 °C for 5 min in SDS buffer for SDS-polyacrylamide gel electrophoresis. K562 cells transfected with FLAG-Smad3 were stained as in Ref. 21Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1195-1947Crossref Scopus (203) Google Scholar; briefly, 104 cells in PBS plus 1% bovine serum albumin were centrifuged onto glass slides. The cells were then fixed in neutral buffered 10% formalin (Sigma) for 5 min at room temperature and washed three times in PBS. Slides were blocked with PBS containing 1% bovine serum albumin and 0.5% Tween 20 (PBS-TB), incubated 1–4 h at room temperature with 4 μg/ml anti-FLAG antibody (Sigma M2 monoclonal) in PBS-TB, washed three times for 5 min at room temperature with PBS-TB, then incubated 1–2 h at room temperature with 3 μg/ml anti-mouse IgG-fluorescein isothiocyanate conjugate (Sigma). After three final washes in PBS-TB, coverslips were mounted on the slides with Fluoromount G (Southern Biotechnology Associates). Previous studies (15Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar) have shown that PMA induction of the WAF1 promoter is mediated through Sp1 binding sites, but the mechanism by which Sp1 regulates PMA-induced transcriptional activation remains unclear. The Sp1 transcription factor contains A and B activation domains and within each domain there are Ser/Thr- and glutamine-rich regions (23Kadonaga J. Courey A.J. Ladika J. Tjian R. Science. 1988; 242: 1566-1570Crossref PubMed Scopus (284) Google Scholar). Since PMA regulates the activity of a number of transcription factors, e.g. c-Jun, c-Ets, through phosphorylation, one possibility to explain the transcriptional regulation of Sp1 by PMA is an induced modification of the Ser/Thr region of this protein. Alternatively, other portions of the molecule might be essential for this induction and thus point to the mechanism of PMA regulation of Sp1. To examine whether specific portions of the Sp1 molecule are necessary for PMA-induced regulation of Sp1, the entire A domain (amino acids 83–232) or portions of that domain were cloned as GAL4 fusions under the control of the metallothionein promoter. This expression vector (pMEP) was chosen because the addition of PMA does not affect the level of expression of this promoter. Thus, K562 cells treated with CdSO4 alone or CdSO4 plus PMA contain the same amount of GAL4-Sp1 fusion protein (Fig. 1 A). When GAL4-Sp1 fusion proteins containing either Sp1 domain A or Sp1 domain B were transfected into K562 cells along with a GAL4 driven luciferase reporter; both regions of the protein were identically stimulated by PMA. For simplicity, only the construct containing domain A was used for further experiments. Although the removal of the Ser/Thr region decreases the level of basal transcription, deletion of this domain does not affect the level of activation of GAL4/Sp1 transcription by PMA (Fig. 1 B). 41 amino acids in the glutamine-rich region are sufficient for PMA-induced regulation (Fig. 1 B). These 41 amino acids contain the region of Sp1 shown to interact with the basal transcription factor hTAFII130, a component of the TFIID complex (24Gill G. Pascal E. Tsing Z.H. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 192-196Crossref PubMed Scopus (470) Google Scholar). p300 interacts with TAFs and TBP and forms a bridge between the basal transcription machinery and specific transcription factors (25Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This interaction is inhibited by the E1A protein, which binds directly to p300 (26Yang J.Y. Ogryzko V. Nishikawa J. Howard B. Nakatani Y. Nature. 1996; : 319-324Crossref PubMed Scopus (1317) Google Scholar). The TGFβ induction of the WAF1 promoter is blocked by the adenovirus protein E1A (27Datto M.B. Hu P. Kowalik T.F. Yingling J. Wang X.F. Mol. Cell. Biol. 1997; 17: 2030-2037Crossref PubMed Google Scholar), and the ability of E1A to block induction is dependent on its ability to bind to the coactivator p300. For this reason, the role of p300 in regulating PMA-induced activation by Sp1 was investigated. The E1A protein was transfected along with the Sp1(152–193)-Gal4 construct and the GAL4/luciferase reporter plasmid into K562 cells and the cells treated with PMA. The presence of E1A blocked the activation of this fusion protein by PMA (Fig. 1 C). In comparison, an E1A protein lacking the p300 binding domain (E1A2–36) had no inhibitory effect. Identical results were demonstrated using the WAF1 promoter (bases −154 to +16) containing multiple Sp1 binding sites (data not shown). These results suggest a role for p300 in the PMA-induced regulation of the WAF1 promoter. Possible direct contact between Sp1 (or Sp1-associated proteins) and p300 may be necessary for PMA induction of this promoter. Since both TGFβ and PMA activate the WAF1 promoter through Sp1 sites (7Biggs J.R. Kudlow J. Kraft A.S. J. Biol. Chem. 1996; 271: 901-906Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 8Datto M. Yu Y. Wang X. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) and E1A blocks the stimulatory effects of both of these activators, the possibility that they shared a further common mechanism of action was investigated. Recent studies have suggested that the TGFβ-induced interaction between Sp1 and p300 may be mediated by the Smad3 protein. Smad3 has been shown to bind to p300 (18Pouponnet C. Jayanaman L. Massague J. J. Biol. Chem. 1998; 273: 22865-22868Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 19Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Google Scholar), and exogenous Smad3 stimulates both the WAF1 promoter and a GAL4-Sp1 fusion protein in HepG2 cells (15Moustakas A. Kardassis D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6733-6738Crossref PubMed Scopus (321) Google Scholar). These observations led to the proposal that Smad3, when phosphorylated and activated by TGFβ, mediates TGFβ stimulation of the WAF1 promoter by interacting with both Sp1 and p300. This model would account for the fact that TGFβ mediates WAF1 promoter induction through Sp1 binding sites, but does not affect the binding of Sp1 to these sites (8Datto M. Yu Y. Wang X. J. Biol. Chem. 1995; 270: 28623-28628Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). If PMA were to activate the Smad signal-transduction pathway, a similar mechanism, in which phosphorylated and activated Smad3 forms a bridge between Sp1 and p300, might explain PMA induction of the WAF1 promoter. To determine whether PMA treatment activates the Smad signal-transduction pathway in K562 cells, the cells were co-transfected with a vector expressing a GAL4-Smad3 fusion protein and a luciferase reporter containing GAL4 DNA binding sites. The GAL4-Smad3 fusion gene was placed in the pMEP expression vector described above. Fig. 2 shows that PMA treatment causes a substantial increase in the transcriptional activity of the GAL4-Smad3 fusion protein. This response is enhanced by cotransfection with a Smad4 expression vector, a protein that dimerizes with Smad3 and enhances its activity, and inhibited by cotransfection with the Evi-1 oncoprotein, a protein that binds to Smad3 and inhibits its activity (data not shown). Previous studies of Smad3 activation by TGFβ have demonstrated that three carboxyl-terminal serines on Smad3 are phosphorylated in response to treatment with TGFβ, and that these serines are necessary for Smad3 functions, including transcriptional activation (19Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Google Scholar, 28Liu X. Sun Y. Constantinescu S. Karam E. Weinberg R. Lodish H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94 (669–10, 674): 10Google Scholar). Mutation of these serines to alanines creates a dominant-negative Smad3, which inhibits the Smad pathway response to TGFβ. This inhibition is believed to occur through stable interactions between the dominant-negative Smad and the TGFβ receptor (28Liu X. Sun Y. Constantinescu S. Karam E. Weinberg R. Lodish H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94 (669–10, 674): 10Google Scholar). When a plasmid expressing a GAL4-Smad3A protein, which contains the three serine to alanine mutations, was transfected into K562 cells, the response to PMA was greatly reduced when compared with the wild-type GAL4-Smad3 fusion protein (Fig. 2). This result shows that the carboxyl-terminal serines are important for the Smad3 response to PMA. These results with GAL4-Smad3 fusion proteins provide strong evidence that transcriptional activation by Smad3 is mediated by PMA. To confirm PMA activation of the Smad signal-transduction pathway using a known Smad-regulated promoter, the p3TP-lux reporter plasmid was transfected into K562 cells. The p3TP-lux plasmid promoter consists of three 32-base pair phorbol ester response elements and a 94-base pair sequence from the plasminogen activator inhibitor 1 (PAI-1) gene promoter. p3TP-lux is synergistically induced by overexpression of Smads (29Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Miyamozo K. ten Dijke P. EMBO J. 1997; 16: 5353-53362Crossref PubMed Scopus (916) Google Scholar). As shown in Fig. 3, p3TP-lux is strongly stimulated by PMA treatment of K562 cells. In comparison, TGFβ or activin A treatment of K562 cells induces significantly less p3TP-lux activity (data not shown). Transfection of the wild-type Smad3 greatly enhances the PMA-induced stimulation of the 3TP-lux promoter, whereas the COOH-terminal phosphorylation mutant of Smad3 had no effect. In comparison, the Evi-1 protein, an inhibitor of Smad3, does block PMA induction of p3TP-lux. Taken together, these data suggest that PMA regulates the p3TP-lux promoter at least in part through Smad3. The results shown in Figs. 2 and 3 indicate that PMA regulates Smad-dependent promoters in K562 cells. However, PMA-induced stimulation of these promoters could be occurring through the secretion of TGFβ or the up-regulation of TGFβ receptors. Both proteins are known to be up-regulated by PMA in other hematopoietic cells (30Taipale J. Matikain S. Hurme M. Keski-Oja J. Cell Growth Diff. 1994; 5: 1309-1319PubMed Google Scholar). To examine the role of the TGFβ protein in PMA-induced activation of the p3TP-lux construct, K562 cells were pretreated with either anti-TGFβ antibodies (shown to neutralize TGFβ) or control antibodies, then treated with either PMA or TGFβ. As shown in Fig. 4 A, although the addition of TGFβ to K562 cells had only a small effect on p3TP-lux promoter activity, the neutralizing antibodies inhibit TGFβ activation of p3TP-lux, while the nonspecific antibodies had no effect. In comparison, the TGFβ antibodies had no effect on PMA stimulation of the p3TP-lux promoter (Fig. 4 B). To evaluate the role of the TGFβ receptor in PMA-induced stimulation, K562 cells were transfected with a dominant-negative type 1 receptor missing the Smad 3 binding site (31DeCaestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (254) Google Scholar) and the p3TP-lux promoter construct. As shown in Fig. 5, the dominant-negative receptor blocked TGFβ activation of p3TP-lux (Fig. 5 A), but not PMA activation (Fig. 5 B). Therefore, neither secretion of TGFβ nor modulation of the TGFβ receptor is required for PMA-induced activation of p3TP-lux promoter, suggesting that PMA may modulate Smad3 directly.FIG. 5A dominant-negative TGFβ receptor inhibits TGFβ but not PMA induction of p3TP-lux. K562 cells were transfected with p3TP-lux and either empty pcDNA3 vector or vector expressing a dominant-negative TGFβ receptor. After 24 h, transfected cells were treated with either 10 ng/ml TGFβ (A) or 200 nm PMA (B). After an additional 16 h, cells were lysed and assayed for luciferase activity.View Large Image Figure ViewerDownload (PPT) Recent work (31DeCaestecker M.P. Parks W.T. Frank C.J. Castagnino P. Bottaro D.P. Roberts A.B. Lechleider R.J. Genes Dev. 1998; 12: 1587-1592Crossref PubMed Scopus (254) Google Scholar) suggests that Smad2, which is highly homologous to Smad3, can be activated by the ERK pathway independently of the TGFβ receptor family. Since PMA regulates the activity of the ERK pathway in K562 cells (21Whalen A.M. Galasinski S.C. Shapiro P.S. Nahreini T.S. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1195-1947Crossref Scopus (203) Google Scholar), the ability of PMA to modify the phosphorylation and cellular location of Smad3 was examined. K562 cells were stably transfected with vectors expressing either Flag-tagged wild-type Smad3 or mutant Smad3A (with the carboxyl-terminal serines mutated to alanines). Analysis of [35S]methionine immunoprecipitates (Fig. 6) demonstrates that PMA treatment of these K562 cell lines induces the appearance of a diffuse, higher molecular weight band running just above Smad3 in both wild-type Smad3 and Smad3A cell lines. To verify that this band represents phosphorylated Smad3, these two cell lines were labeled with [32P]orthophosphate and the experiment repeated. As shown in Fig. 6, PMA induces similar phosphorylation on both wild-type Smad3 and Smad3A. This result indicates that PMA-induced phosphorylation is occurring at a site(s) other than the carboxyl-terminal serines. Because Smad2 is phosphorylated in an ERK-dependent manner and PMA regulates ERK in K562 cells, the effect of the MEK kinase inhibitor PD98059 on PMA-induced Smad phosphorylation was examined. This inhibitor was added to [32P]orthophosphate-labeled K562 cells expressing FLAG-Smad3 prior to PMA stimulation. Immunoprecipitation with the Flag antibody demonstrates that PMA-induced phosphorylation of wild-type and mutant Smad is partially blocked by this inhibitor (Fig. 7 A), suggesting that PMA activates ERKs to phosphorylate Smad3. The observation that PMA activation of Smad3 is associated with ERK-mediated phosphorylation suggests that ERK activity may be necessary for Smad3 transcriptional activation. To determine whether ERK activity is necessary for PMA activation of Smad3 in K562 cells, cells were transfected either with p3TP-lux or" @default.
• W2031774227 created "2016-06-24" @default.
• W2031774227 creator A5071541538 @default.
• W2031774227 creator A5086393033 @default.
• W2031774227 date "1999-12-01" @default.
• W2031774227 modified "2023-10-03" @default.
• W2031774227 title "The Role of the Smad3 Protein in Phorbol Ester-induced Promoter Expression" @default.
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