BIOGRAPHY: Dr. Roberts obtained her Ph.D. degree from the University of Wisconsin for study of the metabolism of retinoic acid. Following post-doctoral research at Harvard University Medical School and teaching in the Chemistry Department at Indiana University, she joined the Laboratory of Chemopreventionin 1976 and became Chief in 1995. She serves on numerous editorial boards, is a past President of the Wound Healing Society, and has recently been elected to the Senior Biomedical Research Staff.

RESEARCH INTERESTS: The objective of my research group is to gain insight into mechanisms of Transforming Growth Factor-ßeta (TGF-ß) action by study of its signal transduction pathways and the roles that altered signaling might play in pathogenetic effects of TGF-ß, including especially wound healing, fibrosis, and carcinogenesis.

Signal transduction: Important new insights have been gained into the signal transduction pathways down stream of the receptor serine-threonine kinases of ligands belonging to the TGF-ß superfamily. A novel family of proteins called SMADs (Sma and Mad-related protein) has been identified which mediate signals directly from the receptor serine-threonine kinases. A subset of these proteins, the receptor-activated SMADs, act as latent transcriptional activators in that they are phosphorylated directly by the type I receptor kinase and then, in heteromeric complex with a common mediator, Smad4, translocate to the nucleus where they participate in DNA-bound complexes to activate transcription of target genes. In general, Smads 2 and 3 mediate signals from TGF-ß and activin receptors, and Smads 1, 5, and 8 from the BMP receptors.

Study of Smad-Interacting Proteins

We are studying several Smad-interacting proteins, both nuclear and cytoplasmic, for their roles in SMAD-mediated signal transduction. These include:

• TRAP1 - a cytoplasmic protein first described by Charng et al., JBC 273, 9365, 1998 which we now show to bind to TGF-ß and activin receptors and to serve as a putative chaperone for Smad4, possibly facilitating the presentation of Smad4 to receptor-activated Smad proteins. TRAP1 associates with the receptor complex in the quiescent state and, upon activation of signaling, dissociates from the receptor complex and binds transiently with Smad4, ultimately again dissociating from Smad4 in favor of the active Smad4/Smad2 complex. Future studies are focused on identification and characterization of homologs of TRAP1 and of other cellular proteins that interact with TRAP1.

• Sorting nexin 6 (SNX6) - a cytoplasmic receptor-interacting protein. We have shown for the first time that various members of the SNX family of proteins bind to receptor serine-threonine kinases as well as to receptor tyrosine kinases and suggest that they might play a role in receptor trafficking.

• Smad nuclear interacting protein 1 (SNIP1) - a nuclear protein which inhibits Smad-dependent transcriptional activation of target genes by binding to Smad4 and competing with Smad4 for binding to the C/H1 locus of the transcriptional coactivators CBP/p300. Recent data show that SNIP1 also interferes with transcriptional activation by p65RelA of the Nf-kB family of transcription factors which also binds the C/H1 domain. Regulated expression of SNIP1 in embryogenesis suggests that it may represent a mechanism for selective restriction of the activity of certain transcription factors while being permissive for other transcription factors which interact through other domains of CBP/p300.

We were one of the first laboratories to clone Smad1, which mediates signals from members of the BMP subfamily of proteins and, in certain instances,from TGF-ß. By homologous recombination, we have now created mice null for the Smad1 gene. These mice die at about embryonic day 9.5, and demonstrate that Smad1 is necessary for allantois formation in the mouse. Based on this early embryonic lethality, we have now characterized the genomic structure of Smad1 and created a conditional knockout of Smad1 which can be crossed with mice expressing Cre recombinase from tissue-specific promoters to generate mice in which Smad1 is deleted in a tissue-specific manner. Study of these mice is presently ongoing.

A. Creation of the Smad1 targeting vector. B. Tissues stained: a. Intestinal villi b. Bone marrow c. Mammary gland d. Endocrine pancreas e. IgG control Antibody: rabbit anti-Smad1 IgG (Zymed)

Study of Smad3 null mice

In collaboration with Dr. Chuxia Deng, NIDDK, we generated and characterized Smad3 null mice (Yang et al., EMBO J 18:1280-1291 1999). These mice die between 1 and 8 months due to a primary defect in immune function. To begin to understand the effects of loss of Smad3 on specific TGF-ß signaling pathways in vivo, we have used a defined system in which effects of TGF-ß on specific participating cell types have been thoroughly described. Study of healing of cutaneous wounds in these mice have provided new insights into the roles of TGF-ß in the healing process and specifically into the roles of Smad3 in mediating TGF-ß or activin signals in keratinocytes, macrophages, and fibroblasts. Based on the finding that Smad3 null macrophages are unable to chemotax toward TGF-ß and fail to autoinduce TGF-ß, we predicted that Smad3 null mice would be resistant to fibrosis. Studies are now on going using models of radiation-induced fibrosis in the skin (in collaboration with Angelo Russo and James Mitchell, NCI) as well as TGF-ß-induced fibrosis of the lung (in collaboration with Jack Gauldie, MacMasters University). Preliminary results suggest that Smad3 null mice are resistant to fibrosis and that Smad3 plays a critical role in the pathogenetic effects of TGF-ß. Future approaches will involve the use of cDNA microarray methodology to identify specific targets of TGF-ß or activin signaling in cells derived from these Smad3 null mice.

Sections of skin of either wildtype or Smad3 null mice at 6 weeks post-irradiation (30Gy). Note the hyperplastic epidermis and dense dermis in the wildtype mice compared to Smad3 null mice.

The role of Smad signaling in wound healing and carcinogenesis

On-going studies are aimed at identifying the roles of specific Smad proteins in the mechanistically related processes of wound healing and carcinogenesis. Tumors have been described as wounds which do not heal. The comparatively small percentage of tumors in which either TGF-ß receptors or Smad4 has been totally lost as with biallelic loss or mutational inactivation, suggest that there is selective advantage in tumorigenesis to loss of the tumor suppressor activities of TGF but to retention of aspects of its signaling which promote expression of the transformed phenotype. We are using both overexpression of Smad proteins and cells null for particular Smad proteins to attempt to discriminate between endpoints of Smad2 and Smad3 signaling pathways downstream of the TGF-ß receptors. We are testing the hypothesis, based on data obtained from wound healing studies and microarray analysis, that Smad3 and also mitogen-activated protein kinase (MAPK) pathways will play a central roles in both wound healing and tumorigenesis.

Most tumor cells retain some responsivity to TGF-ß even though growth- inhibitory pathways have been lost. These autocrine pathways likely contribute to the oncogenic behavior of the tumor cell, as do paracrine effects of TGF-ß- on stromal elements.

Representative Recent Publications:

1. Yang, X., Letterio, J.J., Chen, L., Lechleider, R.J., Hayman, R., Gu, H., Roberts, A.B., Deng, C. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T-cell responsiveness to TGF-ß. EMBO J. 18:1280-1291, 1999.
2. Ashcroft, G.S., Yang, X., Glick, A., Weinstein, M., Letterio, J.J., Mizel, D.E., Anzano, M., Greenwell-Wild, T., Wahl, S.M., Deng, C., Roberts,A.B. Mice lacking SMAD 3 show accelerated wound healing and an impaired local inflammatory response.Nature Cell Biol. 1:260-266, 1999.
3. de Caestecker, M.P., Yahata, T., Wang, D., Parks, W.T., Huang, S., Hill,C.S., Shioda, T., Roberts, A.B., Lechleider, R.J.: The Smad4 activation domain (SAD) is a proline-rich, p300-dependent transcriptional activation domain. J. Biol. Chem. 275:2115-2122, 2000
4. Koch, R.M., Roche, N.S., Parks, W.T., Ashcroft, G.S., Letterio, J.J., and Roberts, A.B.: Incisional Wound Healing in TGF-ß1 Null Mice, Wound Rep. Reg. 8:179-191, 2000.
5. Kim, R.H., Wang, D., Martin, J., Huff, C., de Caestecker, M.P., Parks, W.T., Meng, X., Lechleider, R.J., Wang, T., and Roberts, A.B. A Novel Smad Nuclear Interacting Protein (SNIP1) Suppresses p300-dependent TGF-ß Signal-transduction. Genes and Devel. 14: 1605-1616, 2000.
6. de Caestecker, M.P., Piek, E., and Roberts, A.B. TGF-ß signaling and Cancer. J. Natl. Can. Inst. 92: 1388-1402, 2000.
7. Larisch, S., Yi, Y., Lotan, R., Kerner, H., Eimerl, S., Parks, W.T., Yossi, G., Reffey, S.B., de Caestecker, M.P., Danielpour, D., Book-Melamed , N., Timberg, R., Duckett, C., Lechleider, R.J., Steller, H., Orly, J., Kim, S.J., and Roberts, A.B. ARTS, a novel mitochondrial septin-like protein mediates apoptosis dependent on its P-loop motif. Nature Cell Biol. 2:915-921, 2000.
8. Huang, S., Flanders, K.C., and Roberts, A.B. Characterization of the mouse Smad1 gene and its expression pattern in adult mouse tissues, Gene, 258: 43-43, 2000.
9. Flanders, K.C., Kim, E.S., and Roberts, A.B. Immunohistochemical expression of Smads 1-6 in the 15-day gestation mouse embryo: signaling by BMPs and TGF-ßs. Devel. Dynamics, 220:141-154, 2001
10. Wurthner, J.U., Frank, D.B., Felici, A., Green M., Cao, Z., McNally, J., Schneider, M.D., Lechleider, R.J., Roberts, A.B. TGF-ß receptor-associated protein1 (TRAP1) is a Smad4 chaperone. J. Biol. Chem. 276:19495-19502, 2001.
11. Piek, E., Ju, W., Heyer, J., Escalante-Alcalde, D., Stewart, C.L., Weinstein, M., Deng, C., Kucherlapati, K., Böttinger, E.P., Roberts, A.B. Functional characterization of TGF-ß signaling in mouse embryonic fibroblasts lacking expression of Smad2 or Smad3. J. Biol. Chem. 276:19945-19953, 2001.
12. Parks, W.T., Frank, D.B., Huff, C., Renfrew-Haft, C., McNally, J.G., Reddi, A., Taylor, S.I., Roberts, A.B., Wang, T., Lechleider, R.L. Sorting Nexin 6, a novel SNX, interacts with the TGF-ß Family of serine-threonine kinase receptors. J. Biol. Chem. 276:19332-19339, 2001.
13. Reffey, S.B., Wurthner, J.U., Roberts, A.B., Duckett, C.S. X-linked inhibitor of apoptosis protein functions as a cofactor in transforming growth factor-ß signaling. J. Biol. Chem. 276:26542-26549, 2001.
14. Kim, R.H., Flanders, K.C., Birkey-Reffey, S., Anderson, L.A., Perkins, N.D., Duckett, C.S., Roberts, A.B. SNIP1 inhibits NF-kB signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators. J. Biol. Chem. 276:46297-46304, 2001.
15. Lechleider, R.J., Ryan, J.L., Garrett, L., Eng, C., Deng, C., Wynshaw-Boris, T., Roberts, A.B. Targeted mutagenesis of Smad1 reveals an essential role in chorioallantoic fusion. Devel. Biol. 240:157-167, 2001.
16. Huang, S., Tang, B., Usoskin, D., Lechleider, R.J., Jamin, S.P., Li, C., Anzano, M.A., Ebendal, T., Deng, C., Roberts, A.B., Conditional Knockout of the Smad1 gene, Genesis 32:76-79, 2002.
17. Flanders, K.C., Sullivan, C.D., Fujii, M., Sowers, A., Anzano, M., Arabshahi, A., Major, C., Deng, C., Russo, A., Mitchell, J.B., Roberts, A.B. Mice lacking Smad3 are protected against cutaneous injury induced by ionizing radiation. Am. J. Pathol.160:1057-68, 2002.

Reviews:

1. Piek, E. Roberts, A.B. Suppressor and oncogenic roles of TGF-ß? and its signaling pathways in tumorigenesis. Adv. Cancer Res., 83:1-54, 2001.
2. Wakefield, L.M., Roberts, A.B. TGF-ß Signaling: Positive and Negative Effects on Tumorigenesis. Curr. Opin. Genet. Dev. 12:22-29, 2002.