Transforming growth factor-beta inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors.
Transforming growth factor-beta (TGF-beta) represses surfactant protein B (Sp-B) gene transcription through a mechanism that remains unknown. A homeodomain and a forkhead transcription factor, NKX2.1 and HNF-3, respectively, are known activators of Sp-B transcription. Because SMADs are the effectors of TGF-beta-induced gene activation, we examined the possibility that gene repression by TGF-beta may also occur through interactions of SMADs with NKX2.1 and HNF-3. We found that lung epithelial carcinoma H441 cells contain SMAD2/3 and -4, which localize to the nucleus in response to TGF-beta treatment. The activity of a transfected Sp-B promoter/reporter construct was reduced in a dose-dependent manner by TGF-beta. Cotransfection with a mutant, constitutively activated form of the Tgf-beta type I receptor repressed Sp-B promoter activity in the absence of TGF-beta ligand. Dominant negative mutants of Smads blocked the repressor activity of TGF-beta. SMAD3, but not SMAD2, mediated the repressor activity of TGF-beta on the Sp-B promoter. Mutations within a 70-base pair domain that includes binding sites for NKX2.1, hepatocyte nuclear factor 3 (HNF-3), or cAMP response element-binding protein (CREB) eliminated SMAD3-dependent repression of Sp-B transcription. Electrophoretic mobility shift analysis showed no evidence for direct binding of SMAD3 to the Sp-B promoter, and a DNA binding mutant of SMAD3 also repressed Sp-B, suggesting that direct DNA binding of SMAD3 may not be required. Using a mammalian two hybrid assay, we found physical and functional interactions between SMAD3 and both NKX2.1 and HNF-3. Also, a glutathione S-transferase-fused SMAD3 directly binds to in vitro synthesized NKX2.1 or HNF-3, demonstrating protein-protein interactions between SMAD3 and the two transcriptional factors. The DNA binding of NKX2.1 to Sp-B promoter was reduced in response to TGF-beta treatment, although expression of Nkx2.1 was not affected. We conclude that SMAD3 interactions with the positive regulators NKX2.1 and HNF-3 underlie the molecular basis for TGF-beta-induced repression of Sp-B gene transcription.
Li
Changgong
C
Department of Pediatrics, Women's and Children's Hospital, University of Southern California School of Medicine, Los Angeles, California 90033, USA.
Zhu
Nian-Ling
NL
Tan
Rosemarie C
RC
Ballard
Philip L
PL
Derynck
Rik
R
Minoo
Parviz
P
D030201:Activin Receptors, Type I / Q000378:metabolism; D002277:Carcinoma; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D037701:Pulmonary Surfactant-Associated Protein B / Q000235:genetics* / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D051900:Smad3 Protein; D000074482:Thyroid Nuclear Factor 1; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D037701:Pulmonary Surfactant-Associated Protein B; D018125:Receptors, Transforming Growth Factor beta; D012097:Repressor Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D000074482:Thyroid Nuclear Factor 1; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I