Smad-mediated Transcriptional Activation Research Articles

Vertebrate-conserved Schnurri zinc fingers restrain Drosophila vein patterning.

The Drosophila Smad-interacting co-factor, Schnurri (Shn) confers transcriptional repression in response to Decapentaplegic (Dpp) signaling. Shn zinc fingers 6-8 mediate this Smad interaction but are lacking in vertebrate Shn homologs. In contrast, the vertebrate-conserved zinc finger 1,2 and 4,5 pairs have been reported to engage in Smad-mediated transcriptional activation in fly and vertebrate systems, and to contribute to Dpp-dependent tissue repair in the fly retina. We report that mutation of zinc coordination residues within vertebrate-conserved Shn zinc finger pairs 1,2 and 4,5 results in ectopic venation that is sensitive to Dpp signaling.

Abstract 3802: Effect of CS-7017, a selective PPARδ agonist, on the TGFβ/Smad-mediated enhanced cell motility of EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cells

Abstract Background: CS-7017, a novel selective peroxisome proliferator-activated receptor gamma (PPARδ) agonist of thiazolidinedione class, has been reported to induce morphological change and cellular differentiation as well as its antiproliferative effects. A phase II clinical trial of CS-7017 in combination with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib is currently ongoing in advanced non-small cell lung cancer (NSCLC) patients. Though EGFR-TKIs are effective for NSCLCs with EGFR-activating mutations, the majority of responders eventually develop acquired resistance. We have previously reported that EGFR-TKI-resistant NSCLC cells acquire enhanced cell motility via activation of TGFβ pathway. In this study, we aim to investigate the effects of CS-7017 in EGFR-TKI-resistant NSCLC cells with a focus on cell motility. Materials and Methods: Erlotinib-resistant PC-9ER and gefitinib-resistant PC-9/ZD cells, established from PC-9 NSCLC cells with EGFR-activating mutation, were used for this study and constitutive PI3K/Akt activation and the EGFR T790M mutation have been previously shown to account for the acquired resistance, respectively. Growth-inhibitory effect of CS-7017 was evaluated by MTT assay. Wound closure assay and transwell assay were performed to evaluate the cell motility. The levels of TGFβ1 and 2 secretion were quantified by ELISA. Cellular mRNA levels of TGFB1 and TGFB2 were measured by qRT-PCR. Smad- or PPARδ-mediated transcriptional activity was analyzed by luciferase assay. Effects of CS-7017 on the molecules relevant to EGFR, TGFβ and PPARδ pathways were observed by immunoblot analysis. Results: Both PC-9ER and PC-9/ZD resistant cells showed the enhanced cell motility resulting from TGFβ2-induced activation of TGFβ pathway. CS-7017 showed significant inhibitory effect on the cell motility of the resistant cells but showed no growth-inhibitory effect. Moreover, combination treatment of CS-7017 and erlotinib resulted in better inhibition of the cell motility of PC-9ER cells. CS-7017 treatment was found to suppress the TGFβ2 secretion significantly resulting from downregulation of TGFB2 at transcriptional level and attenuated Smad2 phosphorylation was observed in the resistant cells. Smad-mediated transcriptional activity was also suppressed by CS-7017 treatment. CS-7017 treatment induced PPARδ-mediated transcriptional activity in PC-9 and PC-9/ZD cells but not in PC-9ER cells, suggesting that the inhibitory effect of CS-7017 on the cell motility of PC-9ER cells may not be due to direct activation of PPARδ signaling by CS-7017. Conclusions: These results imply that CS-7017 may serve as a potential therapeutic agent to prevent metastasis in NSCLC patients who develop acquired resistance to EGFR-TKIs regardless of the types of resistance mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3802. doi:1538-7445.AM2012-3802

Abstract A224: CS-7017, a selective PPARγ agonist, attenuates the TGF-β/Smad-mediated enhanced cell motility of EGFR-tyrosine kinase inhibitor-resistant non-small cell lung cancer cells.

Abstract Background: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been shown to possess antitumor property in preclinical models of human cancers including non-small cell lung cancer (NSCLC). CS-7017, a novel selective PPARγ agonist of thiazolidinedione class, has been reported to induce morphological change and cellular differentiation as well as its antiproliferative effects. A phase II clinical trial of CS-7017 in combination with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib is currently ongoing in advanced NSCLC patients. Though EGFR-TKIs are effective for NSCLCs with EGFR-activating mutations, the majority of responders eventually develop acquired resistance. We have previously reported that EGFR-TKI-resistant NSCLC cells acquire enhanced cell motility via activation of TGF-β/Smad pathway. In this study, we aim to investigate the effects of CS-7017 in EGFR-TKI-resistant NSCLC cells with a focus on cell motility. Materials and Methods: Erlotinib-resistant PC-9/ER and gefitinib-resistant PC-9/ZD cells, established from PC-9 NSCLC cells with EGFR-activating mutation, were used for this study and constitutive PI3K/Akt activation and secondary T790M mutation have been previously shown to account for the acquired resistance, respectively. Growth-inhibitory effect of CS-7017 was evaluated by MTT assay. Wound closure assay and transwell assay were performed to evaluate the cell motility. Secretion level of TGF-β ligands was quantified by ELISA. Transcriptional activity of Smad and PPARγ was measured by luciferase assay. Effects of CS-7017 on the molecules relevant to EGFR, TGF-β and PPARγ pathways were observed by immunoblot analysis. Results: Both PC-9/ER and PC-9/ZD resistant cells showed enhanced cell motility compared with PC-9 parental cells. Significantly higher amount of secreted TGF-β2 was detected in both resistant cells than in PC-9 cells and induction of phosphorylated Smad2 was observed in the resistant cell lines. Activation of TGF-β/Smad pathway by the increased TGF-β2 secretion was suggested to confer the enhanced cell motility of the resistant cells. CS-7017 showed significant inhibitory effect on the cell motility of the resistant cells but showed no growth-inhibitory effect. Moreover, combined treatment with CS-7017 and erlotinib resulted in better inhibition of the cell motility of PC-9/ER cells. CS-7017 treatment was found to suppress the TGF-β2 secretion significantly and attenuated Smad2 phosphorylation was observed in the resistant cells. Smad-mediated transcriptional activity was also suppressed by CS-7017 treatment. CS-7017 treatment induced PPARγ-mediated transcriptional activity in PC-9 cells but not in PC-9/ER cells, suggesting that the inhibitory effect of CS-7017 on the cell motility of PC-9/ER cells may not be due to direct activation of PPARγ signaling by CS-7017. Conclusions: These results imply that CS-7017 may serve as a potential therapeutic agent to prevent metastasis in NSCLC patients who develop acquired resistance to EGFR-TKIs regardless of the types of resistance mechanisms. The mechanism of TGF-β2 downregulation by CS-7017 needs to be further elucidated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A224.

Transforming Growth Factor β-Induced Reactivation of Epstein-Barr Virus Involves Multiple Smad-Binding Elements Cooperatively Activating Expression of the Latent-Lytic SwitchBZLF1Gene

Transforming growth factor β (TGF-β) physiologically induces Epstein-Barr virus (EBV) lytic infection by activating the expression of EBV's latent-lytic switch BZLF1 gene. Liang et al. (J. Biol. Chem. 277:23345-23357, 2002) previously identified a Smad-binding element (SBE) within the BZLF1 promoter, Zp; however, it accounts for only 20 to 30% of TGF-β-mediated activation of transcription from Zp. Here, we identified additional factors responsible for the rest of this activation. The incubation of EBV-positive MutuI cells with a TGF-β neutralizing antibody or inhibitors of the TGF-β type I receptor (TβRI) or Smad3 eliminated the TGF-β-induced reactivation of EBV. The coexpression of Smad2, Smad3, and Smad4 together with a constitutively active form of TβRI induced 15- to 25-fold transcription from Zp in gastric carcinoma AGS cells. By electrophoretic mobility shift assays, we identified four additional Smad-binding elements, named SBE2 to SBE5. Substitution mutations in individual SBEs reduced Smad-mediated activation of Zp by 20 to 60%; together, these mutations essentially eliminated it. Chromatin immunoprecipitation assays confirmed that Smad4 newly bound the Zp region of the EBV genome following the incubation of MutuI cells with TGF-β. SBE2 overlaps the ZEB-binding ZV silencing element of Zp. Depending upon posttranslational modifications, Smad4 either competed with ZEB1 for binding or formed a complex with ZEB1 on the Zp ZV element in a cell-free assay system. In transiently transfected cells, exogenously expressed ZEB1 inhibited Smad-mediated transcriptional activation from Zp. We conclude that TGF-β induces EBV lytic reactivation via the canonical Smad pathway by activating BZLF1 gene expression through multiple SBEs acting in concert.

Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS.

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-β (TGF-β) signaling. However, the identities of the Smad partners mediating TGF-β signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-β increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-β-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-β receptor type I (TβR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

Group 13 HOX proteins interact with the MH2 domain of R-Smads and modulate Smad transcriptional activation functions independent of HOX DNA-binding capability

Interactions with co-factors provide a means by which HOX proteins exert specificity. To identify candidate protein interactors of HOXA13, we created and screened an E11.5–E12.5, distal limb bud yeast two-hybrid prey library. Among the interactors, we isolated the BMP-signaling effector Smad5, which interacted with the paralogous HOXD13 but not with HOXA11 or HOXA9, revealing unique interaction capabilities of the AbdB-like HOX proteins. Using deletion mutants, we determined that the MH2 domain of Smad5 is necessary for HOXA13 interaction. This is the first report demonstrating an interaction between HOX proteins and the MH2 domain of Smad proteins. HOXA13 and HOXD13 also bind to other BMP and TGF-β/Activin-regulated Smad proteins including Smad1 and Smad2, but not Smad4. Furthermore, HOXD13 could be co-immunoprecipitated with Smad1 from cells. Expression of HOXA13, HOXD13 or a HOXD13 homeodomain mutant (HOXD13IQN>AAA) antagonized TGF-β-stimulated transcriptional activation of the pAdtrack-3TP-Lux reporter vector in Mv1Lu cells as well as the Smad3/Smad4-activated pTRS6-E1b promoter in Hep3B cells. Finally, using mammalian one-hybrid assay, we show that transcriptional activation by a GAL4/Smad3-C-terminus fusion protein is specifically inhibited by HOXA13. Our results identify a new co-factor for HOX group 13 proteins and suggest that HOX proteins may modulate Smad-mediated transcriptional activity through protein–protein interactions without the requirement for HOX monomeric DNA-binding capability.

Transforming Growth Factor-β (TGF-β) Type I Receptor/ALK5-dependent Activation of the GADD45β Gene Mediates the Induction of Biglycan Expression by TGF-β

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.

The Kaposi's sarcoma-associated herpesvirus K-bZIP protein represses transforming growth factor beta signaling through interaction with CREB-binding protein.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is involved in the pathogenesis of KS, primary effusion lymphoma, and multicentric Castleman's disease. K-bZIP, the protein encoded by the open reading frame K8 of KSHV, is a member of the basic region-leucine zipper family of transcription factors. We studied the mechanisms that underlie KSHV-induced oncogenesis by investigating whether K-bZIP perturbs signaling through transforming growth factor beta (TGF-beta), which inhibits proliferation of a wide range of cell types. K-bZIP repressed TGF-beta-induced, Smad-mediated transcriptional activity and antagonized the growth-inhibitory effects of TGF-beta. Since both K-bZIP and Smad are known to interact with CREB-binding protein (CBP), the effect of CBP on inhibition of Smad-mediated transcriptional activation by K-bZIP was examined. K-bZIP mutants, which lacked the CBP-binding site, could not repress TGF-beta-induced or Smad3-mediated transcriptional activity. Overexpression of CBP restored K-bZIP-induced inhibition of Smad3-mediated transcriptional activity. Competitive interaction studies showed that K-bZIP inhibited the interaction of Smad3 with CBP. These results suggest that K-bZIP, through its binding to CBP, disrupts TGF-beta signaling by interfering with the recruitment of CBP into transcription initiation complexes on TGF-beta-responsive elements. We propose a possibility that K-bZIP may contribute to oncogenesis through its ability to promote cell survival by repressing TGF-beta signaling.

RUNX3 inhibits cell proliferation and induces apoptosis by reinstating transforming growth factor beta responsiveness in esophageal adenocarcinoma cells

BackgroundSEG-1, a Barrett's-derived esophageal adenocarcinoma cell line, is not responsive to transforming growth factor beta (TGF-β) growth effects. We hypothesize that SEG-1 cells lack the tumor-suppressor gene Runt domain transcription factor 3 (RUNX3) and that its reinstatement can restore the antiproliferative and apoptotic effects of TGF-β. MethodsRUNX3 expression was assessed by immunoblotting. SEG-1 cells were transfected with RUNX3 and treated with TGF-β. The effects of RUNX3 transfection on cell proliferation and apoptosis were determined. Smad-mediated TGF-β transcriptional activity was evaluated with the use of dual-luciferase assay. ResultsSEG-1 cells are not responsive to TGF-β. SEG-1 cells lack RUNX3 protein expression, while RUNX3 is highly expressed in normal human gastric and esophageal epithelium. Although the Smad-2 signaling is activated by TGF-β, SEG-1 cells lack Smad-mediated TGF-β transcriptional activity. In cells transfected with RUNX3, TGF-β acquired an antiproliferative effect and induced apoptosis (P=.001). RUNX3 transfection, in the absence of TGF-β, had no effect on proliferation and apoptosis of SEG-1 cells. RUNX3 expression dramatically increases SMAD-mediated TGF-β–induced transcriptional activity when compared with controls (P=.0001). ConclusionsRUNX3 is not expressed in SEG-1 cells, while it is present in normal esophageal mucosa. RUNX3 is essential for the antiproliferative and apoptotic effects of TGF-β in SEG-1 cells and for the Smad-mediated transcriptional activity of TGF-β.

C-Ski inhibits the TGF-beta signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements.

c-Ski inhibits transforming growth factor-beta (TGF-beta) signaling through interaction with Smad proteins. c-Ski represses Smad-mediated transcriptional activation, probably through its action as a transcriptional co-repressor. c-Ski also inhibits TGF-beta-induced downregulation of genes such as c-myc. However, mechanisms for transcriptional regulation of target genes by c-Ski have not been fully determined. In this study, we examined how c-Ski inhibits both TGF-beta-induced transcriptional activation and repression. DNA-affinity precipitation analysis revealed that c-Ski enhances the binding of Smad2 and 4, and to a lesser extent Smad3, to both CAGA and TGF-beta1 inhibitory element probes. A c-Ski mutant, which is unable to interact with Smad4, failed to enhance the binding of Smad complex on these probes and to inhibit the Smad-responsive promoter. These results suggest that stabilization of inactive Smad complexes on DNA is a critical event in c-Ski-mediated inhibition of TGF-beta signaling.

Transforming Growth Factor-β1 Regulation of Collagenase-3 Expression in Osteoblastic Cells by Cross-talk between the Smad and MAPK Signaling Pathways and Their Components, Smad2 and Runx2

Transforming growth factor-beta (TGF-beta) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblasts and seems to be involved in osteoclastic bone resorption. Here, we show that TGF-beta 1 stimulates collagenase-3 expression in the rat osteoblastic cell line UMR 106-01 and requires de novo protein synthesis. Dominant-negative Smad2/3 constructs indicated that Smad signaling is essential for TGF-beta 1-stimulated collagenase-3 promoter activity. Inhibitors of the ERK1/2 and p38 MAPK pathways, but not the JNK pathway, reduced TGF-beta 1-stimulated collagenase-3 expression, indicating that the p38 MAPK and ERK1/2 pathways are also required for TGF-beta 1-stimulated collagenase-3 expression in UMR 106-01 cells. These inhibitors did not prevent nuclear localization of Smad proteins, but they inhibited Smad-mediated transcriptional activation. We have shown for the first time that Runx2 (a bone transcription factor and a potential substrate for the MAPK pathway) is phosphorylated in response to TGF-beta 1 treatment in osteoblastic cells. Cotransfection of Smad2 and Runx2 constructs had a cooperative effect on TGF-beta 1-stimulated collagenase-3 promoter activity in these cells. We further identified ligand-independent physical interaction between Smad2 and Runx2. Taken together, our results provide an important role for cross-talk between the Smad and MAPK pathways and their components in expression of collagenase-3 following TGF-beta 1 treatment in UMR 106-01 cells.

Activation of TGF-beta-Smad signaling pathway following polyamine depletion in intestinal epithelial cells.

Smad proteins are transcription activators that are critical for transmitting transforming growth factor-beta (TGF-beta) superfamily signals from the cell surface receptors to the nucleus. Our previous studies have shown that cellular polyamines are essential for normal intestinal mucosal growth and that a decreased level of polyamines inhibits intestinal epithelial cell proliferation, at least partially, by increasing expression of TGF-beta/TGF-beta receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-beta/TGF-beta receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) increased basal levels of Smad3 and Smad4 proteins, induced their nuclear translocation, and stimulated Smad sequence-specific DNA-binding activity. Polyamine depletion-induced Smads were also associated with a significant increase in transcription activation as measured by luciferase reporter gene activity of Smad-dependent promoters. Inhibition of Smads by a dominant-negative mutant Smad4 in the DFMO-treated cells prevented the increased Smad transcription activation. Polyamine-deficient cells highly expressed TGF-beta and were growth-arrested at the G1 phase. Inhibition of TGF-beta by treatment with either immunoneutralizing anti-TGF-beta antibody or TGF-beta antisense oligodeoxyribonucleotides not only blocked the induction of Smad activity but also decreased the Smad-mediated transcriptional activation in polyamine-depleted cells. These findings suggest that Smads are involved in the downstream cellular processes mediated by cellular polyamines and that increased TGF-beta/TGF-beta receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad target gene expression.

Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins.

Balancing signals derived from the TGFbeta family is crucial for regulating cell proliferation and differentiation, and in establishing the embryonic axis during development. TGFbeta/BMP signaling leads to the activation and nuclear translocation of Smad proteins, which activate transcription of specific target genes by recruiting P/CAF and p300. The two members of the ZEB family of zinc finger factors (ZEB-1/deltaEF1 and ZEB-2/SIP1) regulate TGFbeta/BMP signaling in opposite ways: ZEB-1/deltaEF1 synergizes with Smad-mediated transcriptional activation, while ZEB-2/SIP1 represses it. Here we report that these antagonistic effects by the ZEB proteins arise from the differential recruitment of transcriptional coactivators (p300 and P/CAF) and corepressors (CtBP) to the Smads. Thus, while ZEB-1/deltaEF1 binds to p300 and promotes the formation of a p300-Smad transcriptional complex, ZEB-2/SIP1 acts as a repressor by recruiting CtBP. This model of regulation by ZEB proteins also functions in vivo, where they have opposing effects on the regulation of TGFbeta family-dependent genes during Xenopus development.

Regulation of TG-interacting factor by transforming growth factor-beta.

TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix (Santa Clara, CA, U.S.A.) oligonucleotide microarray analysis, we found that TGIF mRNA level was elevated by transforming-growth-factor-beta (TGF-beta) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicated that TGF-beta1 and activin were able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4 kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter was stimulated by TGF-beta1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-beta responsive region of the promoter. In addition, expression of TGIF was able to cause a dose-dependent inhibition of TGF-beta and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-beta and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-beta/activin action.

Negative Regulation of BMP/Smad Signaling by Tob in Osteoblasts

Bone morphogenetic protein (BMP) controls osteoblast proliferation and differentiation through Smad proteins. Here we show that Tob, a member of the emerging family of antiproliferative proteins, is a negative regulator of BMP/Smad signaling in osteoblasts. Mice carrying a targeted deletion of the tob gene have a greater bone mass resulting from increased numbers of osteoblasts. Orthotopic bone formation in response to BMP2 is elevated in tob-deficient mice. Overproduction of Tob represses BMP2-induced, Smad-mediated transcriptional activation. Finally, Tob associates with receptor-regulated Smads (Smad1, 5, and 8) and colocalizes with these Smads in the nuclear bodies upon BMP2 stimulation. The results indicate that Tob negatively regulates osteoblast proliferation and differentiation by suppressing the activity of the receptor-regulated Smad proteins.

Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-beta involves functional cooperation with p300/CBP transcriptional coactivators.

Transforming growth factor-beta (TGF-beta) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-beta- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected fibroblasts, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-beta to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-beta. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-beta stimulation of collagen gene expression in fibroblasts. Oncogene (2000) 19, 3546 - 3555

Physical and Functional Interaction of SMADs and p300/CBP

SMADs are transforming growth factor beta (TGF-beta) receptor substrates and mediators of TGF-beta transcriptional responses. Here we provide evidence that the coactivators p300 and CBP interact with Smads 1 through 4. The biological relevance of this interaction is shown in vivo by overexpression of the adenovirus E1A protein and mutant forms of E1A that lack p300-binding sites. Wild-type E1A, but not the mutants, inhibits SMAD-dependent transcriptional responses to TGF-beta. E1A also inhibits the intrinsic transactivating function of the Smad4 MH2 domain. In addition, overexpression of p300 enhances SMAD-dependent transactivation. Our results suggest a role for p300/CBP in SMAD-mediated transcriptional activation and provide an explanation for the observed ability of E1A to interfere with TGF-beta action.