A FoxO–Smad synexpression group in human keratinocytes

Roger Gomis

Claudio Alarcón

Wei He

Qiongqing Wang

Joan Seoane

Alex Lash

Joan Massagué

*Cancer Biology and Genetics Program, Howard Hughes Medical Institute, and

^{Bioinformatics Core Facility, Memorial Sloan–Kettering Cancer Center, New York, NY 10021}

^{To whom correspondence should be addressed. E-mail:}gro.ccksm.iks@eugassam-j

Contributed by Joan Massagué, June 27, 2006

^{Present address: Oncology Programme, Institute for Research in Biomedicine, 08028 Barcelona, Spain.}

^{Present address: Medical Oncology Research Program, Institut Català per la Recerca i Estudis Avançats, Vall d’Hebron University Hospital Research Institute, 08028 Barcelona, Spain.}

Author contributions: R.R.G. and J.M. designed research; R.R.G., C.A., W.H., and Q.W. performed research; R.R.G., J.S., and A.L. contributed new reagents/analytic tools; R.R.G., C.A., W.H., Q.W., and J.M. analyzed data; and R.R.G. and J.M. wrote the paper.

Abstract

Transforming growth factor β (TGF-β) signals through activation of Smad transcription factors. Activated Smad proteins associate with different DNA-binding cofactors for the recognition and regulation of specific target genes. Members of the forkhead box O family (FoxO1, FoxO3, and FoxO4) play such a role in the induction of the cyclin-dependent kinase inhibitors p15Ink4b and p21Cip1. To delineate the organization of the TGF-β response in human keratinocytes, we defined the set of genes whose activation by TGF-β requires both FoxO and Smad functions. FoxO factors are shown to be essential for 11 of the 115 immediate gene activation responses to TGF-β in these cells. FoxO1, FoxO3, and FoxO4 act redundantly as mediators of these effects. Smad4, which functions as a partner of receptor-phosphorylated Smad2/3, is required for all of these responses. These results define a FoxO–Smad synexpression group or group of genes that are jointly induced by a common mechanism in response to TGF-β. In addition to p15INK4b and p21CIP1, these genes include mediators of stress responses (GADD45A, GADD45B, and IER1) and adaptive cell signaling responses (CTGF, JAG1, LEMD3, SGK, CDC42EP3, and OVOL1). Bioinformatic analysis of the promoter region of these genes reveals diverse configurations of Smad and FoxO binding elements, implying differences in the regulatory properties of this group of genes. Indeed, a subset of FoxO/Smad-dependent TGF-β gene responses additionally require the transcription factor CCAAT/enhancer-binding protein β. The composition of the FoxO–Smad synexpression group suggests that stress reactions and adaptive functions accompany the cytostatic response of keratinocytes to TGF-β.

Keywords: Forkhead, TGF-β, transcription

Abstract

Transforming growth factor β (TGF-β) is a member of a large family of secreted growth factors of central importance in metazoan development and homeostasis (1 –5). TGF-β signaling induces a large set of gene responses that control cell behavior and fate. These responses are susceptible to disruption in inherited and somatic disorders including cancer (1, 3, 4). Delineating the organization of TGF-β transcriptional programs is therefore important for understanding the basis for the multifunctional action of TGF-β.

TGF-β activates a membrane receptor serine/threonine kinase complex that phosphorylates the transcription factors Smad2 and Smad3 (6). Thus activated, Smad2/3 accumulate in the nucleus and bind Smad4, which is essential for many, but not all, Smad-dependent responses (7 –9). Smad proteins bind DNA, preferentially at the sequence AGAC, denoted the Smad-binding element (SBE) (6, 10). Alone, the affinity of Smad proteins for the SBE is insufficient to support binding to endogenous promoters in vivo except in genes with multiple SBE clusters. In most cases, activated Smad complexes must associate with other DNA-binding proteins and cooperatively bind compound elements in gene regulatory regions (11). Members of diverse families of DNA-binding proteins fulfill this role as Smad partners. Based on this model, one can envision repertoires of Smad transcriptional complexes regulating distinct subset of genes in a cell type-specific manner. The specificity of TGF-β action would thus depend on the Smad cofactors and chromatin status provided by the developmental state and environmental context of the target cell.

This model predicts that the transcriptional response to TGF-β in a given cell type could be parsed into groups of genes that are controlled by specific Smad–cofactor combinations. Each group of genes would be regulated in a unified manner. Groups of genes that are synchronously regulated by a common signal, referred to as “synexpression groups,” have been described during embryo development (12). Synexpression groups may support coordinated events for the completion of a developmental step. By their nature, Smad–cofactor combinations could provide a mechanistic basis for the coordinated regulation of selected gene sets and, therefore, a mechanism-based definition of synexpression groups. Evidence for this role has been provided in the action of the TGF-β family member BMP4 (bone morphogenetic protein 4) in Xenopus development (13).

In the present studies we tested these predictions by focusing on FoxO factors (FoxO1, FoxO3, and FoxO4) as Smad partners. FoxO factors are critical players in growth inhibitory responses to stress in human cells and the control of starvation responses and longevity in lower organisms (14, 15). We recently found that FoxO factors act as Smad partners in the induction of p21CIP1 and p15INK4b as part of the cytostatic response of epithelial cells (16, 17). By means of FoxO and Smad4 genetic depletion and gene expression profiling in human keratinocytes, we have now defined a set of genes that are transcriptionally induced by TGF-β through the FoxO–Smad combination. This FoxO–Smad synexpression group includes cytostatic, stress, and adaptive activities, providing insights into the organization of the overall TGF-β response in this cell type.

Click here to view.

Acknowledgments

We thank D. Accili (Columbia University, New York, NY) and R. Bernards for reagents; D. A. Thomas, H.-V. Le, D. Padua, and the members of J.M.’s laboratory for helpful discussion; E. Kim and the High Throughput Screening Core Facility at the Memorial Sloan–Kettering Cancer Center; and A. Viale and the Genomic Core Facility at the Memorial Sloan–Kettering Cancer Center. R.R.G. is a recipient of a postdoctoral fellowship from the Ministerio de Educación y Cultura of Spain. W.H. and Q.W. are postdoctoral fellows and J.M. is an Investigator of the Howard Hughes Medical Institute.

Acknowledgments

Abbreviations

shRNA	short hairpin RNA
FHBE	forkhead-binding element
SBE	Smad-binding element
C/EBP	CCAAT/enhancer-binding protein
ChIP	chromatin immunoprecipitation
qRT-PCR	quantitative real-time PCR
CDK	cyclin-dependent kinase.

Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

Footnotes

References

1. Derynck R., Akhurst RJ., Balmain A. Nat. Genet. 2001;29:117–129.[PubMed][Google Scholar]
2. Gorelik L., Flavell RA. Nat. Rev. Immunol. 2002;2:46–53.[PubMed][Google Scholar]
3. Massagué J., Blain S. W., Lo R. S. Cell. 2000;103:295–309.[PubMed]
4. Roberts A. B., Wakefield L. M. Proc. Natl. Acad. Sci. USA. 2003;100:8621–8623.
5. Schier A. F., Shen M. M. Nature. 2000;403:385–389.[PubMed]
6. Shi Y., Massagué J. Cell. 2003;113:685–700.[PubMed]
7. Chu G. C., Dunn N. R., Anderson D. C., Oxburgh L., Robertson E. J. Development (Cambridge, U.K.) 2004;131:3501–3512.[PubMed]
8. He W., Dorn D. C., Erdjument-Bromage H., Tempst P., Moore M. A., Massagué J. Cell. 2006;125:929–941.[PubMed]
9. Sirard C., de la Pompa J. L., Elia A., Itie A., Mirtsos C., Cheung A., Hahn S., Wakeham A., Schwartz L., Kern S. E., et al. Genes Dev. 1998;12:107–119.
10. Gao S., Steffen J., Laughon A. J. Biol. Chem. 2005;280:36158–36164.[PubMed]
11. Massagué J., Seoane J., Wotton D. Genes Dev. 2005;19:2783–2810.[PubMed]
12. Niehrs C., Pollet N. Nature. 1999;402:483–487.[PubMed]
13. Karaulanov E., Knochel W., Niehrs C. EMBO J. 2004;23:844–856.
14. Greer EL., Brunet A. Oncogene. 2005;24:7410–7425.[PubMed][Google Scholar]
15. Tran H., Brunet A., Grenier J. M., Datta S. R., Fornace A. J., Jr., DiStefano P. S., Chiang L. W., Greenberg M. E. Science. 2002;296:530–534.[PubMed]
16. Seoane J., Le H. V., Shen L., Anderson S. A., Massagué J. Cell. 2004;117:211–223.[PubMed]
17. Gomis RR., Alarcón C., Nadal C., VanPoznak C., Massagué J. Cancer Cell. 2006 in press. [[PubMed][Google Scholar]
18. Kang Y., Chen CR., Massagué J. Mol. Cell. 2003;11:915–926.[PubMed][Google Scholar]
19. Siegel PM., Massagué J. Nat. Rev. Cancer. 2003;3:807–821.[PubMed][Google Scholar]
20. Moussad E. E., Brigstock D. R. Mol. Genet Metab. 2000;71:276–292.[PubMed]
21. Artavanis-Tsakonas S., Rand M. D., Lake R. J. Science. 1999;284:770–776.[PubMed]
22. Takekawa M., Tatebayashi K., Itoh F., Adachi M., Imai K., Saito H. EMBO J. 2002;21:6473–6482.
23. Yoo J., Ghiassi M., Jirmanova L., Balliet A. G., Hoffman B., Fornace A. J., Jr., Liebermann D. A., Bottinger E. P., Roberts A. B. J. Biol. Chem. 2003;278:43001–43007.[PubMed]
24. Garcia J., Ye Y., Arranz V., Letourneux C., Pezeron G., Porteu F. EMBO J. 2002;21:5151–5163.
25. Hellemans J., Preobrazhenska O., Willaert A., Debeer P., Verdonk P. C., Costa T., Janssens K., Menten B., Van Roy N., Vermeulen S. J., et al. Nat. Genet. 2004;36:1213–1218.[PubMed]
26. Pan D., Estevez-Salmeron L. D., Stroschein S. L., Zhu X., He J., Zhou S., Luo K. J. Biol. Chem. 2005;280:15992–16001.[PubMed]
27. Brunet A., Park J., Tran H., Hu L. S., Hemmings B. A., Greenberg M. E. Mol. Cell. Biol. 2001;21:952–965.
28. Czech MP. Proc. Natl. Acad. Sci. USA. 2003;100:11198–11200.[Google Scholar]
29. Joberty G., Perlungher R. R., Sheffield P. J., Kinoshita M., Noda M., Haystead T., Macara I. G. Nat. Cell Biol. 2001;3:861–866.[PubMed]
30. Li B., Nair M., Mackay D. R., Bilanchone V., Hu M., Fallahi M., Song H., Dai Q., Cohen P. E., Dai X. Development (Cambridge, U.K.) 2005;132:1463–1473.
31. Wrana JL., Attisano L., Wieser R., Ventura F., Massagué J. Nature. 1994;370:341–347.[PubMed][Google Scholar]
32. Levy L., Hill CS. Mol. Cell. Biol. 2005;25:8108–8125.[Google Scholar]
33. McKnight SL. Cell. 2001;107:259–261.[PubMed][Google Scholar]
34. Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Nucleic Acids Res. 1997;25:3389–3402.
35. Baldessari D., Shin Y., Krebs O., Konig R., Koide T., Vinayagam A., Fenger U., Mochii M., Terasaka C., Kitayama A., et al Mech. Dev. 2005;122:441–475.[PubMed][Google Scholar]
36. Beer MA., Tavazoie S. Cell. 2004;117:185–198.[PubMed][Google Scholar]
37. Xie X., Lu J., Kulbokas E. J., Golub T. R., Mootha V., Linblad-Toh K., Lander E. S., Kellis M. Nature. 2005;434:338–345.
38. Vivanco I., Sawyers CL. Nat. Rev. Cancer. 2002;2:489–501.[PubMed][Google Scholar]
39. Staller P., Peukert K., Kiermaier A., Seoane J., Lukas J., Karsunky H., Moroy T., Bartek J., Massagué J., Hanel F., Eilers M. Nat. Cell Biol. 2001;3:392–399.[PubMed]
40. Carcamo J., Zentella A., Massagué J. Mol. Cell. Biol. 1995;15:1573–1581.
41. Kang Y., He W., Tulley S., Gupta G. P., Serganova I., Chen C. R., Manova-Todorova K., Blasberg R., Gerald W. L., Massagué J. Proc. Natl. Acad. Sci. USA. 2005;102:13909–13914.
42. Shang Y., Hu X., DiRenzo J., Lazar MA., Brown M. Cell. 2000;103:843–852.[PubMed][Google Scholar]
43. Kretzschmar M., Doody J., Timokhina I., Massagué J. Genes Dev. 1999;13:804–816.