Abstract
Id1 is a member of the inhibitor of differentiation (Id) protein family that regulates a wide range of cell functions. Previous studies have shown that expression of the Id1 gene is down-regulated by TGF-β in epithelial cells, whereas it is up-regulated by BMP in a variety of cell types. During our study of the biological function of TGF-β1, we found that Id1 can be strongly up-regulated by TGF-β1 in the human mammary gland epithelial cell line MCF10A. Quantitative real-time RT-PCR has revealed as high as 7.5-fold induction of Id1 mRNA by TGF-β1 in MCF10A cells after 1 h of TGF-β1 stimulation, and this induction does not require de novo protein synthesis. Using Smad knockdown and knockout approaches, we have identified Smad3 as the responsible R-Smad for mediating transcriptional activation of the Id1 gene. Chromatin immunoprecipitation assay confirms that Smad3 and Smad4 bind to the upstream region of the Id1 gene. Our results demonstrate that Smad3, but not Smad2, mediates TGF-β1-dependent early transcriptional induction of Id1.
Similar content being viewed by others
Introduction
The TGF-β superfamily signaling is mediated by cell surface type I and type II receptors (i.e. TβRI and TβRII in the case of TGF-β ligands), both of which are serine/threonine kinases, and intracellular Smad proteins 1, 2. Upon ligand binding to the cell surface TβRII, TβRII phosphorylates the downstream kinase TβRI. The activated TβRI then phosphorylates the two TGF-β-specific R-Smad proteins, i.e. Smad2 and Smad3, at their C-terminal SXS motifs. Phospho-Smad2/3 form a complex with the common mediator Smad4, and the heteromeric Smad complex then accumulates in the nucleus, where it binds to a Smad-binding element (SBE) AGAC and cooperates with a large number of transcription co-regulators at the promoters of diverse arrays of target genes 3. On many TGF-β target gene promoters, Smads cooperate with a context-dependent sequence-specific DNA-binding transcription activator to activate the gene promoter. For instance, Smads physically interact and functionally synergize with Sp1 to up-regulate the p15 transcription in response to TGF-β 4. Smads also mediate active repression of certain genes such as the Id1 gene in cooperation with ATF3 5.
Id1 is a member of the helix-loop-helix (HLH) protein family that comprises four members (Id1 through 4) 6. They mostly act as negative transcriptional regulators in many biological processes such as cell differentiation, cell senescence, neurogenesis, apoptosis and angiogenesis 6, 7, 8, 9, 10, 11, 12, 13. As all of them lack a DNA-binding domain, they heterodimerize with and inhibit the function of basic HLH (bHLH) transcription factors 6, 14, for examples, myogenic regulatory factors, such as MyoD, myogenin, Myf5 and MRF4/Myf6 15, 16, or class A of E proteins, such as E12, E47, E2-2 and ITF-2 15, 17, 18, 19. Inhibitor of differentiation (Id) proteins also interact with non-HLH proteins such as Ets2 20 and MIDA1 21 to regulate their activities. It is reported that Id1 protein is required for BCR/ABL-mediated leukemogenesis 22. A series of downstream target genes of Id1 have been identified by hybridization and microarray analysis 10, 23, 24. Id1 can localize to centrosomes and induce abnormal centrosome numbers in human primary cells and tumor cell lines 25. Expression of Id1 is regulated by a variety of ligands, including BMPs of the TGF-β superfamily 26.
During the course of our study on TGF-β-dependent gene regulation, we noticed that Id1 gene could be transiently, yet significantly, induced by TGF-β. Although both positive and negative transcriptional regulation of Id1 by TGF-β1 has been reported 5, 27, it is generally believed that Id1 gene is repressed for TGF-β to achieve its cytostatic and stress responses in epithelial cells. Furthermore, Id1 induction in certain cell types may be mediated through the ALK1-activated Smad1 pathway, but not through the TβRI-activated Smad2/3 pathway 28. Therefore, the molecular mechanism underlying transcriptional activation of Id1 by TGF-β1 still remains elusive. In this study, we have demonstrated that TGF-β1 transiently and profoundly induces transcription of Id1 in human epithelial cells and mouse embryonic fibroblasts (MEFs). The Id1 induction is then switched to repression after continuous TGF-β stimulation. For the first time, we have determined that Smad3, but not Smad2, mediates TGF-β1-dependent transcriptional induction of the Id1 gene in human epithelial cells and MEFs. The Id1 induction is directly associated with the binding of Smad3 to the upstream region of the Id1 promoter and TGF-β-induced acetylation of histones H3 and H4.
Results
Id1 is induced by TGF-β1 in various human epithelial cell lines
Although previous studies have reported both repression and induction of the Id1 gene by TGF-β 5, 27, it is a dominant view that TGF-β primarily represses Id1 transcription 5. Most of these studies focus on the regulation of the Id1 gene with prolonged TGF-β1 treatment (e.g. >3 h TGF-β1 treatment). It remains unclear whether the induction or repression is cell type-specific or can take place in the same cell. In this study, we re-examined the regulation of Id1 expression by TGF-β1, particularly by short TGF-β1 exposure. The steady-state level of endogenous Id1 protein was analyzed in three human cell lines: MCF10A, HaCaT and HeLa cells. These cells were treated with TGF-β1 for various time periods from 15 min up to 4 h, and the level of Id1 protein was measured by anti-Id1 western blotting analysis. In order to eliminate the effect of residual TGF-β1 in serum-containing medium, cells were serum starved overnight, and the TGF-β1 treatment was carried out in serum-free medium. As shown in Figure 1, Id1 protein could be induced transiently by TGF-β1 in all the three human cell lines. Id1 protein induction was observed at 15 min of TGF-β1 treatment, and Id1 protein level reached the peak at 1 h after TGF-β1 addition, with an exception that Id1 protein was more gradually induced and reached its peak at 1.5 h in MCF10A cells. AS the positive controls, we observed a rapid induction in phosphorylation of Smad2 and Smad3 with 15 min of TGF-β1 treatment. After reaching its peak, Id1 protein level gradually decreased even with the presence of TGF-β1. In accordance with previous reports 5, 27, the Id1 protein level at 4 h after TGF-β1 treatment was below the basal level, indicating that the Id1 gene was repressed or Id1 protein was rapidly degraded. Thus, Id1 protein is transiently induced by TGF-β1 in various human cell lines.
Id1 is an immediate early response gene of TGF-β1
To determine whether the early increase in the Id1 protein level is attributed to the increased Id1 transcription, real-time RT-PCR was carried out to quantify the Id1 mRNA level in MCF10A cells. Total RNAs were prepared from MCF10A cells that were treated with TGF-β1 for various time periods up to 12 h. As shown in Figure 2A and 2B, the peak of Id1 mRNA induction was observed at 1 h after TGF-β1 stimulation regardless of the growth conditions. At the peak time, TGF-β1 induced a 2.2- and 7.5-fold increase of Id1 mRNA when MCF10A cells were cultured in low-serum (Figure 2A) and serum-free media (Figure 2B), respectively. This result suggests that the increased level of Id1 protein in Figure 1 results from the increased level of Id1 mRNA induced by TGF-β1. As observed with Id1 protein level, Id1 mRNA level decreased gradually after 1 h of TGF-β1 treatment, and the level of Id1 mRNA returned to the basal level after 4 h of TGF-β1 treatment. Later time points showed that prolonged TGF-β1 treatment caused the decline of Id1 mRNA to fall below the basal level, indicating a repression of the Id1 transcription. Therefore, TGF-β1 has a dual effect on Id1 transcription. Whilst cells exposed to TGF-β1 for short time (< 1 h) exhibit an acute response to induce Id1 mRNA, chronic TGF-β1 treatment (> 4 h) has a repressive effect on Id1 transcription.
The observation that Id1 mRNA was induced by TGF-β1 within 1 h implies that Id1 induction by TGF-β1 is an immediate early response, independent of new protein synthesis. To confirm this, we treated MCF10A cells with the protein synthesis inhibitor cycloheximide (CHX) for 30 min prior to and during TGF-β1 stimulation. RT-PCR analysis showed that CHX treatment did not affect the early induction of Id1 transcription by TGF-β1 (Figure 2C). Interestingly, under such conditions TGF-β1-induced Id1 mRNA continues to rise with continuous TGF-β1 stimulation (up to 4 h, Figure 2C). These results suggest that TGF-β1-mediated early induction of Id1 does not require new protein synthesis, whereas TGF-β1-mediated late repression of Id1 transcription is dependent on de novo protein synthesis. The observation that Id1 repression requires new protein synthesis is consistent with a previous report that TGF-β-induced ATF3 mediates Id1 repression 5.
TGF-β1-dependent Id1 induction does not require Smad2
Smad2 and Smad3 are key intracellular signal transducers for TGF-β1 responses. Here we took a loss-of-function approach to determine whether TGF-β1-mediated Id1 induction is a Smad-dependent response. As a first step, we established cell lines derived from MCF10A that stably express Smad2 shRNA (shSmad2). The level of Smad3 was unchanged when 80% knockdown of endogenous Smad2 was achieved (Figure 3A). Control and Smad2 shRNA cells were then treated with TGF-β1 for different time periods, and Id1 protein level was determined by western blotting analysis (Figure 3A). Compared to control cells, knockdown of Smad2 had no effect on the induction profile of Id1 protein by TGF-β1 (Figure 3A).
To rule out the possibility that a low level of Smad2 in Smad2 shRNA cells suffices to mediate TGF-β1-induced Id1 expression, we analyzed the Id1 induction in Smad2-null MEFs (Smad2KO). Smad2-null MEFs were treated with TGF-β1 for 1 h, and Id1 protein level was determined by western blotting analysis. We found that Smad2-null MEFs responded well to TGF-β1 to induce Smad3 phosphorylation and Id1 expression (Figure 3C, lane 4). In addition, we included BMP2 as a control because it is a well-known inducer of Id1 expression 29, 30. As expected, BMP2 normally induces Smad1 phosphorylation and does not require Smad2 for Id1 induction (Figure 3C, lane 2). These data suggest that Smad2 is dispensable for Id1 induction by TGF-β1 or BMP. In the same experiment, we also treated Smad2KO MEFs with the proteasome inhibitor MG132 on top of BMP2 and TGF-β1 (Figure 3C, lanes 3 and 5) or alone (Figure 3C, lane 6), and MG132 did not significantly increase the Id1 protein level when Id1 was strongly induced by either BMP2 or TGF-β1 under the Smad2KO background.
Smad3 is indispensable for TGF-β1-triggered Id1 induction
Smad2 and Smad3, two close homologous that share 92% identities, are both activated by the same TGF-β type I receptor to mediate specific and sometimes distinct TGF-β1 responses. To compare the functions of Smad2 and Smad3 in mediating TGF-β1-mediated Id1 induction, we also generated MCF10A stable cells (shSmad3) that express Smad3 shRNA to knock down the expression of Smad3 protein. As shown in Figure 4A, depletion of Smad3 expression was evident, and it had no effect on Smad2 or β-actin expression. In these shSmad3 stable cells, Id1 induction by TGF-β1 was abolished. Thus, even though Smad2 protein was normally phosphorylated in response to TGF-β1 in the shSmad3 cells, Smad2 apparently failed to compensate for the loss of Smad3 function. This result suggests that Smad3 is a rate-limiting and perhaps indispensable factor for Id1 induction by TGF-β1 in MCF10A cells.
To confirm the critical role of Smad3 in TGF-β1-mediated induction of Id1, we also performed the experiment with Smad3-null MEFs. In Smad3-null MEFs, the ability of TGF-β1 to induce Id1 expression was abolished (Figure 4B, lane 4). To prove that the effect of Smad3 deletion is specific for TGF-β1 responses and thus the integrity of BMP signaling is not affected, we examined Id1 induction by BMP2. It is clear that Smad3-null MEFs still retained the response to BMP2 (Figure 4B, lane 2). The presence of the proteasome inhibitor MG132 in Smad3-null MEFs leads to a further increased accumulation of Id1 protein in the presence of BMP2 (Figure 4B, lane 3), suggesting that a quick turn-over mechanism by the 26S proteasome exists for Id1 protein in Smad3-null MEFs.
Smad3 and Smad4 bind to the mouse Id1 promoter
We next attempted to analyze the mouse Id1 gene promoter and investigate its chromatin alteration in response to TGF-β. The BMP-responsive element of the Id1 promoter has been localized to a small fragment containing a cluster of SBE and binding sites of other transcription factors 29, 30. To determine the TGF-β1 responsive element in the Id1 promoter, we analyzed the ∼2-kb sequence upstream of the transcriptional initiation site of the Id1 gene. Within the proximal 2-kb region (nt −2 000 to −1), twenty five potential SBE, GTCT or AGAC have been found. Three of these regions (−1 850 to −1 467, −1 265 to −926, and −255 to +47) contain 8, 6 and 2 potential SBE motifs, respectively (Figure 5A).
Since Smad3 is essential for mediating TGF-β-induced Id1 expression, we sought to determine if Smad3 and Smad4 (as an obligatory cofactor of Smad3) bind to the native Id1 promoter in vivo, by chromatin immunoprecipitation (ChIP) assays with anti-Smad3 and anti-Smad4 antibodies. Since we have already excluded the involvement of Smad2 for Id1 activation by TGF-β1, we chose Smad2-null MEFs for ChIP assays to avoid residual antibody cross-reactivity between Smad2 and Smad3. Four pairs of primers were then selected to amplify the indicated specific regions in the mouse Id1 promoter (Figure 5A), with the downstream sequence nt +757 to +1 114 as a negative control. As shown in Figure 5B, Smad3 bound to only one region of the mouse Id1 promoter (−1 265 to −926) after 1 h of TGF-β1 treatment. In the absence of TGF-β stimulation, anti-Smad3 antibody could not enrich any region of the mouse Id1 promoter. Smad4 was capable of binding to all three upstream promoter regions in the presence, but not absence, of TGF-β (Figure 5C). As a negative control, neither Smad3 nor Smad4 immunoprecipitation could enrich the 3′ region (+757/+1 114) (Figure 5B and 5C). Our results suggest that Smad3 and Smad4 only bind to the upstream region of the mouse Id1 promoter after TGF-β stimulation, consistent with the indispensable role of Smad3 protein in the Id1 induction by TGF-β.
TGF-β1 induces histone acetylation at the mouse Id1 promoter
It is well documented that acetylation of lysine residues in the N-terminal tails of histones H3 and H4 correlates positively with gene transcription 31, 32. Upon TGF-β stimulation, activated Smads can recruit transcription coactivators such as histone acetyltransferase CBP/p300 to the promoter 33, 34, 35. To analyze the acetylation status of histones H3 and H4 during the activation of Id1 gene by TGF-β1, we carried out ChIP assays with anti-acetyl histone H3K14 and anti-acetyl histone H4 antibodies.
We assessed the presence of acetylated H3 and H4 in the same Smad-binding regions by ChIP assays. As shown in Figure 5D and 5E, TGF-β1 stimulation (1 and 3 h) induced a significant increase in acetylation of both histone H3 and histone H4 associated with all the three upstream regions of the Id1 gene promoter. However, the region of nt −1 850 to −1 467 appeared to have higher levels of acetylated H3/H4 than the other two regions. The levels of H3/H4 acetylation gradually decreased after TGF-β1 treatment for a long time and reached the basal level at 6 h, which correlates well with the repressed state of the Id1 promoter. These results suggest that histone H3 and H4 acetylation is associated with the transactivated Id1 promoter in the early response phase to TGF-β1.
Discussion
This study provides a mechanistic view on how the Id1 gene is regulated by TGF-β. We found that Smad3, but not Smad2, is required for the immediate early induction of the Id1 gene in both human and mouse cells. Smad3, together with Smad4, binds to the Id1 promoter. The ability of TGF-β1 to induce acetylation of H3 and H4 in the same promoter region suggests that the Smad3/4 activator complex likely recruits coactivators such as p300/CBP to facilitate transcriptional induction of the Id1 gene. However, long-term TGF-β1 stimulation (> 4 h) resulted in transcriptional repression of the Id1 gene, which depends on new protein synthesis, as previously reported 5. It has been shown that ATF3 interacts with Smad3 and binds to the CRE/ATF binding sequence in this region to repress the expression of Id1 after 2 h of TGF-β1 stimulation 5. It has also been reported that Smad3 can interact with HDAC4 and HDAC5 to repress the expression of Runx2 gene 36. It is conceivable that ATF3, whose expression is induced by TGF-β1, recruits HDACs to the Smad complex, thereby converting it from an activator to a repressor complex. We found that the timing of transcriptional repression is well correlated with decreases in the levels of acetylated H3 and H4 (Figure 5D and 5E). Therefore, regulation of the Id1 gene by TGF-β1 is a complicated event, shifting from an early induction phase to a late repression phase.
The biphasic regulation of Id1 implicates a complex role of Id1 in modulating TGF-β1 responses. Although the exact role of Id1's early induction in TGF-β responses awaits further investigation, certain possible roles of Id1 in regulating TGF-β signaling can be envisioned. As Id1 is an inhibitor of differentiation via its association with bHLH transcription factors 6, 14, the early induction of Id1 may confer TGF-β the ability to inhibit differentiation by controlling the activities of bHLH proteins. For instance, considering that TGF-β is a potent inhibitor of myogenic differentiation 37, it is possible that TGF-β-induced Id1 early expression contributes to early inhibition or delay of the myogenic function of transcription factors such as MyoD during skeletal muscle differentiation. Similarly, a previous report clearly shows that Id1 induction regulates smooth muscle cell differentiation in response to TGF-β, during which USF, a bHLH factor, is involved in E box-dependent expression of smooth muscle genes 27. Therefore, it is tempting to speculate that in response to TGF-β, Id1 induction contributes to inhibition of bHLH transcription factors that are involved in the differentiation of various cell lineages.
Repression of Id1 in the late phase of gene expression in response to TGF-β is also critical to maintain proper cellular responses to TGF-β. As there is good evidence demonstrating that Id1 promotes cell proliferation likely through inhibition of cyclin-dependent kinase inhibitors (CKIs) 6, 10, induction of Id1 would be contradictory to the ability of TGF-β to arrest cells in the G1 phase of cell cycle. Thus, it may be necessary to remove the excess amount of Id1 to ensure the antiproliferative response to TGF-β in epithelial cells such as HaCaT keratinocytes and MCF10A mammary epithelial cells. During the immediate early phase of response to TGF-β (before 4 h), CKIs such as p21 and p15 are transcriptionally induced along with Id1 and its repressor ATF3. Four hours after TGF-β stimulation, cell cycle inhibitors continue to rise while Id1 becomes transcriptionally repressed by ATF3. Loss of Id1 expression also coincides with TGF-β-dependent repression of c-Myc, a factor that positively controls Id1 expression 38. It has been reported that down-regulation of Id1 expression is associated with TGF-β-induced cell cycle arrest in prostate epithelial cells 39, 40. The negative role of Id1 in TGF-β-induced antiproliferative response is further supported by previous studies showing that forced overexpression of Ids in human keratinocytes and endothelial cells delays senescence, probably through inactivation of CKIs 41, 42, 43. Together with these studies, our findings provide valuable information on the regulation of the Id1 gene and its potential functions in influencing TGF-β responses during cell proliferation and differentiation.
Materials and Methods
Cell cultures, TGF-β1 treatment, antibodies and western blotting
Human mammary epithelial MCF10A cells were maintained in D-MEM/F-12 medium complemented with 5% horse serum, 10 μg/ml insulin (Sigma), 0.5 μg/ml hydrocortisone (Sigma), 0.02 μg/ml epidermal growth factor (Sigma) and 1% of penicillin and streptomycin (Invitrogen). HeLa cells, Smad2−/− and Smad3−/− MEFs were cultured in D-MEM medium supplemented with 15% of fetal bovine serum (FBS) and 1% of penicillin and streptomycin. HaCaT cells were maintained in MEM medium + 1% of non-essential amino acids (NEAA, Invitrogen) + 10% of FBS and 1% of penicillin and streptomycin.
For Id1 induction, MCF10A cells were washed once with and maintained in serum-free medium for 12 h before TGF-β1 treatment (5 ng/ml). For cycloheximide (CHX, Sigma) treatment, it was added to the medium at 100 μM for 30 min prior to TGF-β1 stimulation. Endogenous Id1 was detected using a polyclonal anti-Id1 antibody (C2, Santa Cruz Biotechnology). Endogenous Smad2, Smad3 and phosphorylated Smad2 were detected using polyclonal antibodies from Zymed. Anti-phosphorylated Smad3 was a kind gift of Dr Ed Leof (Mayo Clinic). Anti-β-actin, a monoclonal antibody, was obtained from Sigma. Western analysis was detected by Western Lightning Kit (Perkin-Elmer Life Science, Inc.) or SuperSignal West Dura and Femto (Pierce) with Kodak Image Station 440.
RT-PCR
Total RNAs were prepared from TGF-β1-treated or untreated MCF10A cells with Trizol reagent (Invitrogen). The first-strand cDNAs were synthesized with 2.5 μg of total RNA. All real-time RT-PCR quantifications were carried out with the 7300 real-time PCR system and TaqMan reagents with triplicate (Applied BioSystem), and regular RT-PCRs were performed in a PCR machine (PTC-200 Peltier Thermal Cycler of DNA Engine).
Retroviral infection
Retroviral vector pSRG expressing shRNAs against human Smad2 and Smad3 have been previously described 44. pSRG-shSmad2 or pSRG-shSmad3 DNA was co-transfected with the plasmid pCL into 293T cell using CaCl2 method to produce retrovirus particles. MCF10A cells were infected overnight by freshly prepared retroviruses and selected puromycin (2.0 μg/ml, Sigma). All puromycin-resistant colonies were pooled together for further studies.
Cell transfection and luciferase assays
Mouse Id1-luciferase constructs were kind gifts of Dr Benezra 45. HepG2 cells were co-transfected with 0.5 μg of mouse Id1-luciferase reporter plasmid and 0.05 μg of SV40 β-galactosidase plasmid by FuGene 6 in 12-well plates according to the manufacturer's instructions (Roche). At 48 h after transfection, cells were treated with TGF-β1 (5 ng/ml) overnight and harvested with reporter lysis buffer (Promega). All assays were repeated at least twice with duplicates in the Microplate Luminometer LB96V (EG&G BERTHOLD).
ChIP assays
ChIP assays were carried out with chromatins prepared as reported previously 46 from Smad2−/− MEFs with or without TGF-β1 treatment. Briefly, 2 μg of anti-Smad4 (B8, Santa Cruz Biotechnology) or anti-Smad2/3 (E20, Santa Cruz Biotechnology), and 50 U of chromatin at A260 were used for each ChIP assay. ChIP products were amplified by primers in the mouse Id1 upstream sequences as follows:
−1 850F: 5′-GTG CCC TAG ATA TAC CAA TAC TT-3′
−1 467R: 5′-TTC TGG ACT CCG AAA CTG ACT CA-3′
−1 265F: 5′-GGA GGT AAG TTG ACC CTT GGT CA-3′
−926R: 5′-TTG AAG GCC TCC GAG CAA GCT CT-3′
−255F: 5′-ACC GCA AAA TTA GCT TAG TCT CT-3′
+47R: 5′-AAC AGA GTG TGG GAA GAG AAC AA-3′
+757F: 5′-GAC GGT ACC AGT GGG TAG AGG GTT TGA T-3′
+1 114R: 5′-GGA AGA TCT CAT AGA ACT ATT GTA AAA CAA TAT-3′
PCR products were separated by a 2% agarose gel.
References
ten Dijke P, Heldin CH, eds. Smad Signal Transduction. Dordrecht: Springer, 2006.
Derynck R, Miyazono K . The TGF-β Family. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2008.
Feng X-H, Derynck R . Specificity and versatility in TGF-β signaling through Smads. Annu Rev Cell Dev Biol 2005; 21:659–693.
Feng X-H, Lin X, Derynck R . Smad2, Smad3 and Smad4 cooperate with Sp1 to induce p15Ink4B transcription in response to TGF-β. EMBO J 2000; 19:5178–5193.
Kang Y, Chen CR, Massagué J . A self-enabling TGFβ response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells. Mol Cell 2003; 11:915–926.
Perk J, Iavarone A, Benezra R . Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 2005; 5:603–614.
Andres-Barquin PJ, Hernandez MC, Israel MA . Id genes in nervous system development. Histol Histopathol 2000; 15:603–618.
Benezra R, Rafii S, Lyden D . The Id proteins and angiogenesis. Oncogene 2001; 20:8334–8341.
Yokota Y, Mori S . Role of Id family proteins in growth control. J Cell Physiol 2002; 190:21–28.
Ruzinova MB, Benezra R . Id proteins in development, cell cycle and cancer. Trends Cell Biol 2003; 13:410–418.
de Candia P, Benera R, Solit DB . A role for Id proteins in mammary gland physiology and tumorigenesis. Adv Cancer Res 2004; 92:81–94.
Sugai M, Gonda H, Nambu Y, Yokota Y, Shimizu A . Role of Id proteins in B lymphocyte activation: new insights from knockout mouse studies. J Mol Med 2004; 82:592–599.
Wong YC, Wang X, Ling MT . Id-1 expression and cell survival. Apoptosis 2004; 9:279–289.
Sun XH, Copeland NG, Jenkins NA, Baltimore D . Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins. Mol Cell Biol 1991; 11:5603–5611.
Langlands K, Yin X, Anand G, Prochownik EV . Differential interactions of Id proteins with basic-helix-loop-helix transcription factors. J Biol Chem 1997; 272:19785–19793.
Vinals F, Ventura F . Myogenin protein stability is decreased by BMP-2 through a mechanism implicating Id1. J Biol Chem 2004; 279:45766–45772.
Einarson MB, Chao MV . Regulation of Id1 and its association with basic helix-loop-helix proteins during nerve growth factor-induced differentiation of PC12 cells. Mol Cell Biol 1995; 15:4175–4183.
Parrinello S, Lin CQ, Murata K, et al. Id-1, ITF-2, and Id-2 comprise a network of helix-loop-helix proteins that regulate mammary epithelial cell proliferation, differentiation, and apoptosis. J Biol Chem 2001; 276:39213–39219.
Zheng W, Wang H, Xue L, Zhang Z, Tong T . Regulation of cellular senescence and p16INK4A expression by Id1 and E47 proteins in human diploid fibroblast. J Biol Chem 2004; 279:31524–31532.
Ohtani N, Zebedee Z, Huot TJ, et al. Opposing effects of Ets and Id proteins on p16INK4A expression during cellular senescence. Nature 2001; 409:1067–1070.
Inoue T, Shoji W, Obinata M . MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein. Biochem Biophys Res Commun 1999; 266:147–151.
Nieborowska-Skorska M, Hoser G, Rink L, et al. Id1 transcription inhibitor-matrix metalloproteinase 9 axis enhances invasiveness of the breakpoint cluster region/Abelson tyrosine kinase-transformed leukemia cells. Cancer Res 2006; 66:4108–4116.
Singh J, Itahana Y, Parrinello S, Murata K, Desprez PY . Molecular cloning and characterization of a zinc finger protein involved in Id-1-stimulated mammary epithelial cell growth. J Biol Chem 2001; 276:11852–11858.
Volpert OV, Pili R, Sikder HA, et al. Id1 regulates angiogenesis through transcriptional repression of thrombospondin-1. Cancer Cell 2002; 2:473–483.
Hasskarl J, Duensing S, Manuel E, Munger, K . The helix-loop-helix protein ID1 localizes to centrosomes and rapidly induces abnormal centrosome numbers. Oncogene 2004; 23:1930–1938.
Miyazono K, Miyazawa K . Id: a target of BMP signaling. Sci STKE 2002; 151:PE40.
Chambers RC, Leoni P, Kaminski N, Laurent GJ, Heller RA . Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching. Am J Pathol 2003; 162:533–546.
Wiercinska E, Wickert L, Denecke B, et al. Id1 is a critical mediator in TGF-β-induced transdifferentiation of rat hepatic stellate cells. Hepatology 2006; 43:1032–1041.
Lopez-Rovira T, Chalaux E, Massagué J, Rosa JL, Ventura F . Direct binding of Smad1 and Smad4 to two distinct motifs mediates bone morphogenetic protein-specific transcriptional activation of Id1 gene. J Biol Chem 2002; 277:3176–3185.
Korchynskyi O, ten Dijke P . Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem 2002; 277:4883–4891.
Agalioti T, Chen G, Thanos D . Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002; 111:381–392.
Kurdistani SK, Tavazoie S, Grunstein M . Mapping global histone acetylation patterns to gene expression. Cell 2004; 117:721–733.
Feng X-H, Zhang Y, Wu R-Y, Derynck R . The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smad3 in TGF-β-induced transcriptional activation. Genes Dev 1998; 12:2153–2163.
Janknecht R, Wells NJ, Hunter T . TGF-beta-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev 1998; 12:2114–2119.
Shen X, Hu PP, Liberati NT, Datto MB, Frederick JP, Wang XF TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein. Mol Biol Cell 1998; 9:3309–3319.
Kang JS, Alliston T, Delston R, Derynck R . Repression of Runx2 function by TGF-β through recruitment of class II histone deacetylases by Smad3. EMBO J 2005; 24:2543–2555.
Derynck R, Piek E, Schneider RA, Choy L, Alliston T . TGF-β family signaling in mesenchymal differentiation. In: Derynck R, Miyazono K, eds. The TGF-β Family. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2008:613–666.
Swarbrick A, Akerfeldt MC, Lee CS, et al. Regulation of cyclin expression and cell cycle progression in breast epithelial cells by the helix-loop-helix protein Id1. Oncogene 2005; 24:381–389.
Di K, Ling MT, Tsao SW, Wong YC, Wang X . Id-1 modulates senescence and TGF-β1 sensitivity in prostate epithelial cells. Biol Cell 2006; 98:523–533.
Ling MT, Wang X, Tsao SW, Wong YC . Down-regulation of Id-1 expression is associated with TGF-β1-induced growth arrest in prostate epithelial cells. Biochim Biophys Acta 2002; 1570:145–152.
Alani RM, Young AZ, Shifflett CB . Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a. Proc Natl Acad Sci USA 2001; 98:7812–7816.
Nickoloff BJ, Chaturvedi V, Bacon P, Qin JZ, Denning MF, Diaz Mo . Id-1 delays senescence but does not immortalize keratinocytes. J Biol Chem 2000; 275:27501–27504.
Tang J, Gordon GM, Nickoloff BJ, Foreman KE . The helix-loop-helix protein Id-1 delays onset of replicative senescence in human endothelial cells. Lab Invest 2002; 82:1073–1079.
Lin X, Duan X, Liang YY, et al. PPM1A functions as a Smad phosphatase to terminate TGF-β signaling. Cell 2006; 125:915–928.
Tournay O, Benezra R . Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1. Mol Cell Biol 1996; 16:2418–2430.
Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M . Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 2000; 103:843–852.
Acknowledgements
We thank Robert Benezra (Sloan-Kettering Institute, NY, USA) for Id1-luc reporter constructs and Ed Leof (Mayo Clinic, MN, USA) for anti-phospho-Smad3 antibody. We are grateful to Xin-Hua Feng (Baylor College of Medicine, TX, USA)for critically reading the manuscript and stimulating discussions. This research was supported by NIH grant R01DK073932, USA and partly by the Baylor Breast Center SPORE career development award, USA.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Liang, YY., Brunicardi, F. & Lin, X. Smad3 mediates immediate early induction of Id1 by TGF-β. Cell Res 19, 140–148 (2009). https://doi.org/10.1038/cr.2008.321
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/cr.2008.321
Keywords
This article is cited by
-
Deep learning for de-convolution of Smad2 versus Smad3 binding sites
BMC Genomics (2022)
-
Id proteins: emerging roles in CNS disease and targets for modifying neural stemcell behavior
Cell and Tissue Research (2022)
-
Cancer microenvironment and genomics: evolution in process
Clinical & Experimental Metastasis (2022)
-
Interplay between transforming growth factor-β and Nur77 in dual regulations of inhibitor of differentiation 1 for colonic tumorigenesis
Nature Communications (2021)
-
CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer
Oncogene (2021)