FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2070750236> ?p ?o ?g. }

• W2070750236 endingPage "5287" @default.
• W2070750236 startingPage "5278" @default.
• W2070750236 abstract "GADD45β regulates cell growth, differentiation, and cell death following cellular exposure to diverse stimuli, including DNA damage and transforming growth factor-β (TGFβ). We examined how cells transduce the TGFβ signal from the cell surface to the gadd45β genomic locus and describe how GADD45β contributes to TGFβ biology. Following an alignment of gadd45β genomic sequences from multiple organisms, we discovered a novel TGFβ-responsive enhancer encompassing the third intron of the gadd45β gene. Using three different experimental approaches, we found that SMAD3 and SMAD4, but not SMAD2, mediate transcription from this enhancer. Three lines of evidence support our conclusions. First, overexpression of SMAD3 and SMAD4 activated the transcriptional activity from this enhancer. Second, silencing of SMAD protein levels using short interfering RNAs revealed that TGFβ-induced activation of the endogenous gadd45β gene required SMAD3 and SMAD4 but not SMAD2. In contrast, we found that the regulation of plasminogen activator inhibitor type I depended upon all three SMAD proteins. Last, SMAD3 and SMAD4 reconstitution in SMAD-deficient cancer cells restored TGFβ induction of gadd45β. Finally, we assessed the function of GADD45β within the TGFβ response and found that GADD45β-deficient cells arrested in G2 following TGFβ treatment. These data support a role for SMAD3 and SMAD4 in activating gadd45β through its third intron to facilitate G2 progression following TGFβ treatment. GADD45β regulates cell growth, differentiation, and cell death following cellular exposure to diverse stimuli, including DNA damage and transforming growth factor-β (TGFβ). We examined how cells transduce the TGFβ signal from the cell surface to the gadd45β genomic locus and describe how GADD45β contributes to TGFβ biology. Following an alignment of gadd45β genomic sequences from multiple organisms, we discovered a novel TGFβ-responsive enhancer encompassing the third intron of the gadd45β gene. Using three different experimental approaches, we found that SMAD3 and SMAD4, but not SMAD2, mediate transcription from this enhancer. Three lines of evidence support our conclusions. First, overexpression of SMAD3 and SMAD4 activated the transcriptional activity from this enhancer. Second, silencing of SMAD protein levels using short interfering RNAs revealed that TGFβ-induced activation of the endogenous gadd45β gene required SMAD3 and SMAD4 but not SMAD2. In contrast, we found that the regulation of plasminogen activator inhibitor type I depended upon all three SMAD proteins. Last, SMAD3 and SMAD4 reconstitution in SMAD-deficient cancer cells restored TGFβ induction of gadd45β. Finally, we assessed the function of GADD45β within the TGFβ response and found that GADD45β-deficient cells arrested in G2 following TGFβ treatment. These data support a role for SMAD3 and SMAD4 in activating gadd45β through its third intron to facilitate G2 progression following TGFβ treatment. Normal epithelial cells are in constant communication with their surrounding environment, largely through the detection, interpretation, and response to extracellular signaling molecules. The TGFβ 1The abbreviations used are: TGFtransforming growth factorHMEChuman mammary epithelial cell(s)siRNAshort interfering RNAsiScrscrambled siRNAMES4-morpholineethanesulfonic acidPAI1plasminogen activator inhibitor-1. superfamily of growth factors comprises 42 such signaling molecules in humans, many of which play fundamental roles in development and adult tissue homeostasis. The epithelial response to members of this family is highly varied and includes such diverse cellular processes as proliferation, movement, differentiation, and apoptosis. Indeed, cells harboring mutations within the signal transduction proteins or the TGFβ target genes either fail to respond or respond inappropriately to the TGFβ signal, often leading to developmental problems, oncogenesis, fibrotic disease, metastasis, and autoimmune disorders. Greater understanding of how cells interpret the TGFβ signal will facilitate the prevention, detection, and treatment of various human diseases. transforming growth factor human mammary epithelial cell(s) short interfering RNA scrambled siRNA 4-morpholineethanesulfonic acid plasminogen activator inhibitor-1. The central elements of TGFβ signal transduction are now known (1Massague J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1653) Google Scholar, 2Shi Y. Massague J. Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4836) Google Scholar). TGFβ activates the serine/threonine kinase activity of a multimeric receptor complex. Activation of this complex initiates a cascade of intracellular events that culminate in altered gene expression. The SMAD proteins form the foundation of this signaling network, since they are the only proteins directly phosphorylated by the receptor complex. However, these transcription factors are by no means sufficient to impart a TGFβ response. To specifically target a gene for transcriptional regulation, the SMADs require assistance by accessory factors. Consequently, the presence and activity of these accessory factors is are important to the TGFβ transcriptional program as are the SMAD proteins. By designing the system in such a way, cell-specific responses to TGFβ can be achieved. Further, the logic of the TGFβ signaling network explains how the cell integrates multiple signals to generate highly specific phenotypic responses. In an attempt to better understand how TGFβ regulates gene transcription and how those gene products contribute to TGFβ biology, we have partially defined the TGFβ transcriptional profile in normal human mammary epithelial cells (HMEC). cDNA microarray expression analysis of TGFβ-treated HMEC revealed a set of genes involved in cellular proliferation, differentiation, and apoptosis. One of these genes, gadd45β/hMyD118, is regulated by TGFβ in multiple cell types, thus suggesting that this gene is of central importance to the TGFβ response. GADD45β and two similar small acidic nuclear proteins, GADD45α and GADD45γ, make up the GADD45 family (3Liebermann D.A. Hoffman B. Leukemia. 2002; 16: 527-541Crossref PubMed Scopus (74) Google Scholar). All three proteins regulate diverse cellular mechanisms including cell growth, DNA repair, differentiation, and apoptosis, four phenotypes that are also controlled by TGFβ signaling. Aside from sequence similarity, these genes share transcriptional regulation by DNA damage insult and growth factors. gadd45β is, however, the only member of this family regulated by TGFβ (4Zhang W. Bae I. Krishnaraju K. Azam N. Fan W. Smith K. Hoffman B. Liebermann D.A. Oncogene. 1999; 18: 4899-4907Crossref PubMed Scopus (131) Google Scholar, 5Takekawa M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (151) Google Scholar). gadd45β was first discovered as a transcript rapidly induced by either TGFβ treatment or the onset of terminal differentiation in M1 murine myeloid cells (6Selvakumaran M. Lin H.K. Sjin R.T. Reed J.C. Liebermann D.A. Hoffman B. Mol. Cell. Biol. 1994; 14: 2352-2360Crossref PubMed Google Scholar, 7Abdollahi A. Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1991; 6: 165-167PubMed Google Scholar). Subsequent studies employing antisense-mediated silencing established GADD45β as an important regulator of the G2/M checkpoint following genotoxic stress (8Vairapandi M. Balliet A.G. Hoffman B. Liebermann D.A. J. Cell. Physiol. 2002; 192: 327-338Crossref PubMed Scopus (264) Google Scholar) and apoptosis during M1 myeloid cell terminal differentiation (6Selvakumaran M. Lin H.K. Sjin R.T. Reed J.C. Liebermann D.A. Hoffman B. Mol. Cell. Biol. 1994; 14: 2352-2360Crossref PubMed Google Scholar, 9Zhan Q. Lord K.A. Alamo Jr., I. Hollander M.C. Carrier F. Ron D. Kohn K.W. Hoffman B. Liebermann D.A. Fornace Jr., A.J. Mol. Cell. Biol. 1994; 14: 2361-2371Crossref PubMed Scopus (468) Google Scholar). Human GADD45β, which was first identified in a complex containing the p38-activating kinase MTK1 (MEKK4), is now a well established regulator of p38 activity and consequently p38-regulated biology (5Takekawa M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (151) Google Scholar, 10Takekawa M. Saito H. Cell. 1998; 95: 521-530Abstract Full Text Full Text PDF PubMed Scopus (650) Google Scholar, 11Yoo J. Ghiassi M. Jirmanova L. Balliet A.G. Hoffman B. Fornace Jr., A.J. Liebermann D.A. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2003; 278: 43001-43007Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). TGFβ activates p38 kinase activity and induces apoptosis in normal murine hepatocytes, but not in hepatocytes derived from gadd45β knockout mice (11Yoo J. Ghiassi M. Jirmanova L. Balliet A.G. Hoffman B. Fornace Jr., A.J. Liebermann D.A. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2003; 278: 43001-43007Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). An initial characterization of the molecular mechanism by which TGFβ induces gadd45β transcription has recently been reported. First, reconstitution of SMAD4 expression in SMAD4-null pancreatic cell lines restored gadd45β induction by TGFβ (5Takekawa M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (151) Google Scholar). The nature of the TGFβ-SMAD-gadd45β link appears to be direct; exogenously expressed SMAD2 and SMAD4 or SMAD3 and SMAD4 induce gadd45β proximal promoter activity 3–4-fold (11Yoo J. Ghiassi M. Jirmanova L. Balliet A.G. Hoffman B. Fornace Jr., A.J. Liebermann D.A. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2003; 278: 43001-43007Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). However, the relative importance and function of each SMAD protein to the transcriptional activation of the endogenous gadd45β gene is not known. Utilizing RNA interference and reconstitution of SMAD3 and SMAD4 protein expression in SMAD-deficient cell lines, we exclude SMAD2 and include SMAD3 and SMAD4 as transcription factors involved in the TGFβ induction of gadd45β. Additionally, through a genomics-based approach, we identified a SMAD-dependent TGFβ-responsive enhancer encompassing the third intron of gadd45β. The importance of this enhancer is indicated by a 3-fold greater transcriptional induction following TGFβ treatment than transcriptional effects mediated by 5′ promoter sequences. Finally, using a cell system that does not undergo TGFβ-induced apoptosis but does respond to TGFβ by gadd45β transcriptional induction, we establish an apoptosis-independent role for GADD45β as an important mediator of G2/M progression following TGFβ treatment. Cell Culture and Drug Treatments—The following cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2.0 μml-glutamine, 1.0 μm sodium pyruvate, penicillin, and streptomycin and split every third day or at 80% confluence: Mv1Lu (CCL64), HaCaT, HeLa, 293, and 10T1/2. HT29 adenocarcinoma colon cells were cultured in McCoy's medium supplemented with 10% fetal bovine serum. The HepG2 and JAR cell lines were cultured in minimal essential medium and RPMI supplemented with 10% fetal bovine serum, respectively. We obtained all of the cell lines from ATCC except for the HaCaT immortalized keratinocyte cell line, which was a kind gift from D. Grossman (University of Utah, Salt Lake City, UT), the Mv1Lu cells were a kind gift from D. Ayer (University of Utah), and the JAR cells were a kind gift from E. Adashi (University of Utah). Human mammary epithelial cells (HMEC) were obtained from BioWhittaker (Walkersville, MD) and cultured in complete mammary epithelial growth medium. HMEC were seeded at passage 7 or 8 and harvested at no greater than 80% confluence for all experiments. For treatments with TGFβ (isoform type 1; Peprotech, Rocky Hill, NJ), we found little to no difference with respect to gene transcription if the cells had been previously serum-starved. The vehicle control for TGFβ comprised 4 mm HCl, 1 mg/ml bovine serum albumin. Cyclohexamide and actinomycin D (Calbiochem) were used at 10 and 5 μg/ml, respectively, and treated as described in Fig. 2. RNA Interference—siRNAs were designed to specifically target either smad2, smad3, or smad4 in accordance with the guidelines developed by Tuschl et al. (12Elbashir S.M. Harborth J. Weber K. Tuschl T. Methods. 2002; 26: 199-213Crossref PubMed Scopus (1030) Google Scholar). Because the sequence of mink smad2 and smad3 cDNAs is unknown, siRNAs were designed against the human sequences. The human-designed smad2 and smad3 siRNAs efficiently and specifically silenced mink SMAD2 and SMAD3 protein expression, thus indicating that these sequences are conserved in mink. We designed the smad4 siRNA-A and siRNA-B against the mink sequence, and consequently they do not silence human smad4 (data not shown). The control siRNA (scrambled siRNA; siScr) specifically recognizes human smad4 and thus does not affect mink SMAD2, SMAD3, or SMAD4 expression. The sequences of the chemically synthesized and high pressure liquid chromatography-purified RNA oligomers are as follows (sense strand shown): Smad2 5′-UCUUUGUGCAGAGCCCCAAtt; Smad3 5′-ACCUAUCCCCGAAUCCGAUtt; Smad4-A 5′-GGACGAAUAUGUUCAUGACtt; Smad4-B 5′-UUGGAUUCUUUAAUAACAGtt; siScr 5′-GGAUGAAUAUGUGCAUGACtt. To silence gadd45β expression, three siRNAs were designed (sense strand shown): siGadd45β-A (5′-GUU GAU GAA UGU GGA CCC Att), siGadd45β-B (AUC CAC UUC ACG CUC AUC Ctt), and siGadd45β-C (CUU GGU UGG UCC UUG UCU Gtt). Of these three siRNAs, siGadd45β-A was the most efficacious and was used to generate the data seen in Fig. 8. All RNA oligomers were reconstituted and annealed following the protocol of Tuschl et al. (12Elbashir S.M. Harborth J. Weber K. Tuschl T. Methods. 2002; 26: 199-213Crossref PubMed Scopus (1030) Google Scholar). Mv1Lu cells were plated 24 h prior to transfection and transfected at 70% confluence. All siRNAs were transfected using 18 μl of LipofectAMINE 2000/10-cm2 plate according to manufacturer's guidelines (Invitrogen). For the SMAD silencing experiments, total RNA or protein was isolated 40–48 h after transfection. In time course experiments, we found that maximal silencing occurred 36 h after transfection for all three SMAD proteins (data not shown). For Gadd45β silencing, 3 h after the start of Gadd45β siRNA transfection, cells were treated with vehicle or TGFβ for an additional 2 h prior to RNA isolation or 12 h prior to flow cytometry. Plasmids and Genomic Alignments—We electronically cloned the human, murine, and rat gadd45β genomic loci from publicly available sequence databases. Approximately 8 kb of the genomic loci, starting at 5000 kb upstream of the transcriptional start site, were aligned using the MAVID alignment algorithm (13Bray N. Pachter L. Nucleic Acids Res. 2003; 31: 3525-3526Crossref PubMed Scopus (52) Google Scholar, 14Bray N. Dubchak I. Pachter L. Genome Res. 2003; 13: 97-102Crossref PubMed Scopus (365) Google Scholar). The portion of this piece of genomic DNA showing conservation among all three species is shown in Fig. 7A. The G45β-1 (–1470 bp, +362 bp), G45β-2 (–972 bp, +362 bp), G45β-3 (–476 bp, +362 bp), G45β-A (–1535 bp, –1042 bp), G45β-B (–572 bp, –79 bp), and G45β-C (+941 bp, +1428 bp) reporter constructs were created as follows. The indicated region of the human gadd45β genomic locus was PCR-amplified from HMEC genomic DNA and cloned into the pCR2.1-TOPO vector (Invitrogen). These DNAs were then subcloned into pGL3basic, sequence-verified, and utilized in subsequent dual luciferase assays. J. Massague generously provided the 3TPLux reporter construct (Memorial Sloan-Kettering Cancer Center, New York). The murine Smad7 cDNA (generously provided by R. Derynk, University of California, San Francisco, CA) was subcloned into pcDNA3.1. Similarly, the FLAG-tagged Smad expression vectors used in the reporter experiments were created by subcloning the cDNAs from constructs provided by D. Satterwhite into pCMV2-FLAG (University of Utah, Salt Lake City, UT). For luciferase assays, all reporters were co-transfected with an SV40-Renilla luciferase reporter plasmid that was used to normalize transfection efficiencies. For retroviral infections, we PCR-amplified the smad3 or smad4 open reading frames from HMEC cDNA and then cloned them into the pBabe retroviral vector. D. Ayer generously provided the GFP-pBabe vector (University of Utah, Salt Lake City, UT). Luciferase Assays—Fugene 6 (Roche Applied Science) was used to transfect HaCaT cells as instructed by the manufacturer. We seeded cells at a density of 80,000 cells/well in 24-well plates and transfected them the next day. Transfections were performed using 0.6 μg of DNA (including either 0.1 μg of normalization vector and 0.5 μg of reporter vector or 0.1 μg of normalization vector, 0.2 μg of reporter vector, and 0.3 μg of expression vector) and harvested 20 h after the start of transfection. For TGFβ treatment, medium containing either TGFβ (200 pm) or an equal volume of vehicle was added to cells 3 h after the start of transfection. Luciferase values were analyzed using a dual luciferase assay system (Promega). Dividing the firefly luciferase activity from each well by the Renilla luciferase activity from the same well normalized transfection efficiencies. Data in each experiment are presented as the mean ± S.D. of triplicates from a representative experiment. All experiments were performed at least three times, producing qualitatively similar results. Retroviral Transduction—Expression of the GFP, SMAD3, or SMAD4 retroviral constructs was verified by Western blot in a transient assay prior to virus production. To produce the retrovirus, Phoenix helper cells were seeded in 60-mm2 plates 24 h prior to transfection with LipofectAMINE 2000. 24 h after transfection began, we split the cells 1:3 to 10-cm2 plates. 48 h after the cells had been split, virus-containing medium was removed from the Phoenix cells, filtered (0.22 μm; low protein binding filter), and added to a 6-well plate containing the HT29 or JAR target cells (at 60% confluence). We added Polybrene (4 μg/ml) to the virus immediately before transduction of the target cells to facilitate infection. 24 h after infection, the target cells were split to 10-cm2 plates and placed under selection with 750 ng/ml puromycin for 10 days. Quantitative Reverse Transcription-PCR—Trizol (Invitrogen) was used to isolate the total RNA from the HT29 and JAR retroviral polyclonal stables according to the manufacturer's guidelines. cDNA was synthesized from 2 μg of total RNA using Superscript III (Invitrogen). Real time PCR was performed using the Roche Light Cycler instrument and software, version 3.5 (Roche Applied Science). Intron-spanning primers (Gadd45β, forward (5′-CGGTGGAGGAGCTTTTGGTG-3′) and reverse (5′-CACCCGCACGATGTTGATGT-3′); 18 S rRNA, forward (5′-GGTGAAATTCTTGGACCGGC-3′) and reverse (5′-GACTTTGGTTTCCCGGAAGC-3′)) were designed to amplify 200-bp products in order to minimize contamination from genomic DNA. PCR was performed in duplicate (or triplicate for 18 S rRNA) with a master mix consisting of cDNA template, buffer (500 mm Tris, pH 8.3, 2.5 mg/ml bovine serum albumin, 30 mm MgCl2), dNTPs (2 mm), TaqStart antibody (Clontech), Biolase DNA polymerase (Bioline), gene-specific forward and reverse primers (10 μm), and SYBR Green I (Molecular Probes, Inc., Eugene, OR). The PCR conditions are as follows: 35 cycles of amplification with 1-s denaturation at 95 °C and 5-s annealing at 57 °C for Gadd45β and 53 °C for 18 S rRNA. A template-free negative control was included in each experiment. We determined the copy number by comparing gene amplification with the amplification of standard samples that contained 103 to 107 copies of the gene or 105 to 109 for 18 S rRNA. The relative expression level of each gene was calculated by averaging the replicates and then dividing the average copy number of Gadd45β by the average copy number of 18 S rRNA. S.E. of the ratios was calculated using a confidence interval. Northern and Western Blotting—Total RNA was isolated using Trizol following the manufacturer's protocol (Invitrogen). Where indicated, total RNA isolation was followed by poly(A) RNA selection using a PolyATtract™ mRNA Isolation kit (Promega). Total RNA or poly(A) RNA was fractionated through formaldehyde-containing agarose gels and transferred onto N+Hybond nylon membranes (Amersham Biosciences). Labeled probes were generated using the Rediprime II random prime labeling system (Amersham Biosciences) supplemented with [32P]dCTP (ICN). To generate Northern blot probes, we PCR-amplified gene-specific sequences from human, mink, or murine cDNA. Mink gadd45β was PCR-amplified using the following degenerate primers: 5′-CTGCARATYCACTTCACSCT and 3′-GGRAYCCAYTGGTTDTTGC. Hybridizations with 32P-labeled probes were carried out using ULTRA-hyb buffer (Ambion) as recommended by the manufacturer. For Western blotting, protein lysates were harvested in a buffer containing 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm CaCl2. 1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm benzamidine, 1 mg/ml pepstatin A, and 1 mg/ml phenanthroline. The resulting whole cell lysates were centrifuged at 20,800 × g for 10 min at 4 °C. Following protein quantitation using the DC Protein Assay (Bio-Rad), equal amounts of protein were fractionated through Tris-glycine 4–12% gradient Nu-PAGE gels using the MES buffer system (Invitrogen). The following antibodies were used to detect the SMAD proteins in both Mv1Lu and human cell lines: SMAD3 (catalog no. 51-1500; Zymed Laboratories Inc.), SMAD2 (catalog no. S66220; Transduction Laboratories), SMAD4 (catalog no. sc-7966; Santa Cruz Biotechnology), and β-catenin (catalog no. 610153; Transduction Laboratories). Immune complexes were visualized using a secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) and Western Lighting chemiluminescence reagent (PerkinElmer Life Sciences). gadd45β Is a Primary TGFβ-responsive Gene in Normal Human Mammary Epithelial Cells—TGFβ induces a G1 cell cycle arrest and epithelial to mesenchymal transition, but not apoptosis, in primary normal human mammary epithelial cells grown in culture (15Hosobuchi M. Stampfer M.R. In Vitro Cell Dev. Biol. 1989; 25: 705-713Crossref PubMed Scopus (62) Google Scholar) (data not shown). To understand the mechanisms behind these TGFβ-induced phenotypes, we partially defined the TGFβ transcriptome in normal human mammary epithelial cells (HMEC). Specifically, we used cDNA microarray expression analysis to determine the relative expression of 7000 genes at 2 and 12 h after TGFβ treatment in HMEC. Data analysis revealed 54 up-regulated and 10 down-regulated TGFβ-regulated genes. Genes included in this list had a -fold change of greater than 1.3 or less 0.7 at both time points and a p value of less than 0.05 at both time points (Supplemental Table I and methods therein). Next, we identified genes within this data set that were in common to TGFβ-regulated genes identified through transcriptional profiling in other TGFβ-responsive cell systems. We surmised that because genes in this subgroup were regulated by TGFβ irrespective of cell origin or transformation status, they would be of central importance to the TGFβ cytostatic program. Plasminogen activator inhibitor-1 (PAI1) is a well established TGFβ-induced gene and was induced 8-fold 2 h after TGFβ treatment in HMEC (Supplemental Table I). Consequently, PAI1 served as an important positive control in the microarray, in Northern blots, and in subsequent experiments (Fig. 1A). A second common TGFβ target gene identified in our expression analysis was gadd45β/hMyD118. In addition to our studies in primary normal HMEC, previous findings indicate that gadd45β is a TGFβ-induced gene in transformed cell lines derived from myeloid, breast, skin, breast, pancreas, and bone (4Zhang W. Bae I. Krishnaraju K. Azam N. Fan W. Smith K. Hoffman B. Liebermann D.A. Oncogene. 1999; 18: 4899-4907Crossref PubMed Scopus (131) Google Scholar, 5Takekawa M. Tatebayashi K. Itoh F. Adachi M. Imai K. Saito H. EMBO J. 2002; 21: 6473-6482Crossref PubMed Scopus (151) Google Scholar, 11Yoo J. Ghiassi M. Jirmanova L. Balliet A.G. Hoffman B. Fornace Jr., A.J. Liebermann D.A. Bottinger E.P. Roberts A.B. J. Biol. Chem. 2003; 278: 43001-43007Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Zawel L. Yu J. Torrance C.J. Markowitz S. Kinzler K.W. Vogelstein B. Zhou S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2848-2853Crossref PubMed Scopus (80) Google Scholar). Because of its frequent presence in the TGFβ transcriptional response and because of its previously described role in growth arrest, differentiation, and apoptosis, we chose to characterize the upstream signal transduction pathway necessary for gadd45β transcriptional induction and, second, to examine the role of GADD45β in the TGFβ response. We first determined the scope of gadd45β transcriptional activation. Specifically, we monitored its induction by TGFβ in several cell lines and by other members of the TGFβ superfamily in HMEC. To determine whether other TGFβ-responsive cell lines responded similarly to HMEC with respect to gadd45β transcription, several cell lines were treated with TGFβ or vehicle for 1 h. The gadd45β and PAI1 transcripts were induced by TGFβ in the following cell lines: HMEC, HaCaT, Mv1Lu, PANC-1, primary breast organoid outgrowths, and to a lesser extent in HepG2 and HeLa cells (data not shown) (Fig. 1A). The Madin Darby canine kidney and 293 cell lines did not respond to TGFβ stimulation by inducing either gadd45β or PAI1. TGFβ treatment of 10T1/2 murine fibroblasts caused a moderate increase in PAI1 transcription but did not affect gadd45β mRNA levels. We also asked whether other members of the TGFβ superfamily of growth factors could regulate gadd45β transcription. Fig. 1C illustrates that both TGFβ and BMP2 induced gadd45β transcription. However, the kinetics of gadd45β induction as well as the strength of induction differed between the two ligands. Finally, of the three genes that comprise the gadd45 family, only gadd45β was found to be TGFβ-inducible in HMEC; gadd45α was not affected by TGFβ treatment, and Gadd545γ was not detected (Fig. 1B). Gadd153/Chop10, a GADD family member by virtue of its induction by cellular stress, was transiently repressed by TGFβ. To distinguish whether TGFβ treatment resulted in increased gadd45β transcription or increased gadd45β mRNA stability, we measured the gadd45β mRNA half-life before and after TGFβ treatment. HMEC were treated with TGFβ for 1 h before the addition of the transcription inhibitor actinomycin D for various periods of time. Quantitative analysis of the Northern blot revealed that TGFβ failed to stabilize the gadd45β mRNA (Fig. 2, A and B). The accumulation of gadd45β mRNA within2hofTGFβ treatment suggested that it is an immediate early TGFβ-induced target gene. To test this idea, we pretreated HMEC with the protein translation inhibitor cyclohexamide 15 min before a 3-h combined TGFβ/cyclohexamide treatment. We found that the levels of gadd45β increased in a TGFβ-dependent manner irrespective of cyclohexamide pretreatment, indicating that new protein synthesis is not required for TGFβ induction of gadd45β (Fig. 2C). These data indicate that gadd45β is a direct TGFβ transcriptional target. gadd45β Is Partly Dependent upon SMAD3 and Independent of SMAD2 in Its Regulation by TGFβ —We first sought to determine whether specific inhibition of SMAD2, SMAD3, and SMAD4 abrogated gadd45β responsiveness to TGFβ. To approach this, we employed siRNA-mediated silencing of the SMAD2, SMAD3, and SMAD4 proteins. Because we were unable to achieve silencing greater than 60% of wild-type levels in HMEC, we chose to use Mv1Lu cells for our siRNA studies. Transfection of Mv1Lu cells with siRNAs specific to SMAD2 or SMAD3 reduced the respective protein expression to nearly undetectable levels (Fig. 3A). Loss of SMAD2 caused a 70% decrease in the induction of PAI1 by TGFβ. In contrast, siRNA silencing of SMAD2 had no significant effect on gadd45β induction following TGFβ treatment (Fig. 3A). SMAD3-deficient cells, however, responded to TGFβ stimulation with reduced levels of induction for both gadd45β and PAI1. Although the decrease in PAI1 induction by TGFβ observed in the SMAD2 and the SMAD3 single-knockout cells was enhanced in the double-knockout cells, the SMAD2/SMAD3 double-knockout cells behaved similarly to SMAD3-deficient cells with respect to gadd45β induction (Fig. 3A). Dose-response curves with the Smad3 siRNA (IC50 ∼1 nm) further demonstrated that TGFβ activates PAI1 and gadd45β through a mechanism that is partly dependent upon SMAD3 (Fig. 3B). SMAD4 Silencing Prevents gadd45β and PAI1 Induction by TGFβ —Of the many proteins involved in mediating the different facets of TGFβ signal transduction, SMAD4 is considered central to many of the responses. Two different siRNAs were designed against mink smad4, and the efficacy of their silencing was tested in Mv1Lu cells by Western blot (Fig. 4A). Consistent with the central role of SMAD4 in TGFβ signaling, siRNA silencing of SMAD4 resulted in a dramatic loss of gadd45β transcriptional induction following TGFβ treatment (Fig. 4B). As a confirmation of specificity, a human-specific SMAD4 siRNA, which contains mismatches at two positions relative to the mink sequence, did not affect SMAD4 protein expression or TGFβ-regulated transcription of gadd45β or PAI1. siSmad4-A and siSmad4-B both robustly silenced SMAD4 protein expression and did not interfere with SMAD3 protein expression (Fig. 4A). Examination of the gadd45β and PAI1 transcript" @default.
• W2070750236 created "2016-06-24" @default.
• W2070750236 creator A5007350449 @default.
• W2070750236 creator A5052754584 @default.
• W2070750236 date "2004-02-01" @default.
• W2070750236 modified "2023-10-09" @default.
• W2070750236 title "Identification of a β 3′ Enhancer That Mediates SMAD3- and SMAD4-dependent Transcriptional Induction by Transforming Growth Factor β" @default.
• W2070750236 cites W1512214997 @default.
• W2070750236 cites W1563090311 @default.
• W2070750236 cites W1636022999 @default.
• W2070750236 cites W1966615054 @default.
• W2070750236 cites W1972922971 @default.
• W2070750236 cites W1992105713 @default.
• W2070750236 cites W1998230419 @default.
• W2070750236 cites W2009228099 @default.
• W2070750236 cites W2014716127 @default.
• W2070750236 cites W2015604710 @default.
• W2070750236 cites W2015609160 @default.
• W2070750236 cites W2040630551 @default.
• W2070750236 cites W2040805319 @default.
• W2070750236 cites W2048355846 @default.
• W2070750236 cites W2052647271 @default.
• W2070750236 cites W2058759929 @default.
• W2070750236 cites W2061089834 @default.
• W2070750236 cites W2065445811 @default.
• W2070750236 cites W2065951163 @default.
• W2070750236 cites W2080239627 @default.
• W2070750236 cites W2083555035 @default.
• W2070750236 cites W2085871516 @default.
• W2070750236 cites W2086554912 @default.
• W2070750236 cites W2087988643 @default.
• W2070750236 cites W2099368049 @default.
• W2070750236 cites W2099476552 @default.
• W2070750236 cites W2110291136 @default.
• W2070750236 cites W2128212057 @default.
• W2070750236 cites W2154568166 @default.
• W2070750236 cites W2158169867 @default.
• W2070750236 cites W2164615751 @default.
• W2070750236 cites W2313812356 @default.
• W2070750236 cites W425633012 @default.
• W2070750236 doi "https://doi.org/10.1074/jbc.m311517200" @default.
• W2070750236 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14630914" @default.
• W2070750236 hasPublicationYear "2004" @default.
• W2070750236 type Work @default.
• W2070750236 sameAs 2070750236 @default.
• W2070750236 citedByCount "42" @default.
• W2070750236 countsByYear W20707502362012 @default.
• W2070750236 countsByYear W20707502362013 @default.
• W2070750236 countsByYear W20707502362015 @default.
• W2070750236 countsByYear W20707502362016 @default.
• W2070750236 countsByYear W20707502362018 @default.
• W2070750236 countsByYear W20707502362019 @default.
• W2070750236 countsByYear W20707502362022 @default.
• W2070750236 countsByYear W20707502362023 @default.
• W2070750236 crossrefType "journal-article" @default.
• W2070750236 hasAuthorship W2070750236A5007350449 @default.
• W2070750236 hasAuthorship W2070750236A5052754584 @default.
• W2070750236 hasBestOaLocation W20707502361 @default.
• W2070750236 hasConcept C104317684 @default.
• W2070750236 hasConcept C111936080 @default.
• W2070750236 hasConcept C116834253 @default.
• W2070750236 hasConcept C118131993 @default.
• W2070750236 hasConcept C185592680 @default.
• W2070750236 hasConcept C54355233 @default.
• W2070750236 hasConcept C59822182 @default.
• W2070750236 hasConcept C70721500 @default.
• W2070750236 hasConcept C86339819 @default.
• W2070750236 hasConcept C86803240 @default.
• W2070750236 hasConcept C95444343 @default.
• W2070750236 hasConceptScore W2070750236C104317684 @default.
• W2070750236 hasConceptScore W2070750236C111936080 @default.
• W2070750236 hasConceptScore W2070750236C116834253 @default.
• W2070750236 hasConceptScore W2070750236C118131993 @default.
• W2070750236 hasConceptScore W2070750236C185592680 @default.
• W2070750236 hasConceptScore W2070750236C54355233 @default.
• W2070750236 hasConceptScore W2070750236C59822182 @default.
• W2070750236 hasConceptScore W2070750236C70721500 @default.
• W2070750236 hasConceptScore W2070750236C86339819 @default.
• W2070750236 hasConceptScore W2070750236C86803240 @default.
• W2070750236 hasConceptScore W2070750236C95444343 @default.
• W2070750236 hasIssue "7" @default.
• W2070750236 hasLocation W20707502361 @default.
• W2070750236 hasOpenAccess W2070750236 @default.
• W2070750236 hasPrimaryLocation W20707502361 @default.
• W2070750236 hasRelatedWork W2061914098 @default.
• W2070750236 hasRelatedWork W2113330652 @default.
• W2070750236 hasRelatedWork W2118646732 @default.
• W2070750236 hasRelatedWork W2152841149 @default.
• W2070750236 hasRelatedWork W2586783543 @default.
• W2070750236 hasRelatedWork W2745970610 @default.
• W2070750236 hasRelatedWork W2973036402 @default.
• W2070750236 hasRelatedWork W4283022034 @default.
• W2070750236 hasRelatedWork W4303699336 @default.
• W2070750236 hasRelatedWork W4309683845 @default.
• W2070750236 hasVolume "279" @default.
• W2070750236 isParatext "false" @default.
• W2070750236 isRetracted "false" @default.
• W2070750236 magId "2070750236" @default.

• next