1 Department of Developmental Biology, Harvard School of Dental Medicine,
Boston, MA 02115, USA
2 Cutaneous Biology Research Center, Massachusetts General Hospital/Harvard
Medical School, Charlestown, MA 02129, USA
* Author for correspondence (e-mail: mwhitman{at}hms.harvard.edu)
SUMMARY
Ligands belonging to the transforming growth factor (TGF) ß superfamily have emerged as major regulators of a wide variety of developmental events, ranging from the earliest steps in germ layer patterning of the pre-gastrula embryo to tissue healing, regeneration and homeostasis in the adult. Recently, Caroline Hill and Bob Lechleider organized the third in a bi-annual series of FASEB meetings on TGFß signaling and development at Snowmass (CO, USA). This meeting highlighted the ongoing interplay between advances in our understanding of the molecular biology of TGFß family signaling and in investigations into its roles in specific developmental events.
Introduction
Since their identification in 1995, the Smad proteins have emerged as the
major transducers of the TGFß signaling pathway, which regulates
transcription during embryogenesis and adulthood
(Fig. 1) (Massague and Wotton, 2000).
Although important responses to TGFß signaling can occur both without
Smads (Derynck and Zhang,
2003
) and without transcriptional regulation
(Ozdamar et al., 2005
), the
predominant focus of this meeting was on mechanisms that regulate Smad
activity and on how these mechanisms confer specific transcriptional
responses. This emphasis arose largely from the sheer number and variety of
candidate regulators and targets of Smad function that have been identified
over the past few years (Fig.
1, Table 1)
(Derynck and Zhang, 2003
;
ten Dijke and Hill, 2004
). A
second focus of the meeting concerned how the TGFß ligand superfamily are
extracellulary regulated (Fig.
2). It has been well recognized for many years that a complex set
of extracellular antagonists and co-factors modulate TGFß ligand
activity. How these regulators fit together to generate spatially and
temporally complex patterns of highly specific gene activation remains a
crucial area of investigation. A number of talks reported on novel mechanisms
of ligand regulation, some of which were placed in specific developmental
contexts.
|
Aristidis Moustakas (Ludwig Institute, Uppsala, Sweden) focused on responses that either coordinate or distinguish TGFß effects on epithelial-to-mesenchymal transitions (EMTs) and on inhibition of cell proliferation. He described the functional characterization of genes identified in a broadly targeted microarray screen that compared genes that are induced by TGFß in NMuMg mammary epithelial cells (in which TGFß concomitantly induces cytostasis and EMT) with those that are induced by Bmp7 (which induces neither response in these cells). Members of the Id family of basic helix-loop-helix (bHLH) factors, particularly Id2, emerged from this study as being crucial targets for the downregulation of gene expression by TGFß during the regulation of both cytostasis and EMT. Two additional gene targets of TGFß signaling were identified: a high-mobility group protein, which acts as a potential upstream regulator of EMT; and a homeodomain protein, which regulates the epithelial cytostatic response.
Kunxin Luo (University of California, Berkeley, CA, USA) approached the
issue of TGFß-regulated transcription and EMT versus cytostasis from a
different angle by examining the role of the co-repressors Sno and Ski in
these processes. Sno and Ski have previously been shown by the Luo laboratory
and others to act as co-repressors that interact with Smads to suppress
TGFß-stimulated transcription (Luo,
2004). Although the functions of Sno and Ski have generally been
viewed as being overlapping, Luo used RNAi analysis to show that their
individual knock down produces distinctive effects on cell motility, EMT and
growth arrest, suggesting that the relative endogenous levels of Sno and Ski
may lead to distinctive responses to TGFß signals.
The differential interaction of Smad complexes with co-repressors and
co-activators is emerging as a major issue in mechanisms that generate
specific cell-type responses to TGFß signals. Rik Derynck (University of
California, San Francisco, CA, USA) touched on this issue for Smad3/Runx2
complexes, which interact with specific histone deacetylases (HDACs) to shut
off bone-differentiation genes in mesenchymal stem cells. By contrast, Runx1
and Runx3 appear to form transcriptional activation complexes with Smads in
other cell types (Ito and Miyazono,
2003).
|
Distinct and overlapping Smad functions
Although functional distinctions between different Smads have been
established in assays in vitro, the significance of these distinctions has
been awaiting in vivo tests. Smad2 and Smad3 are regulated by similar upstream
pathways (Fig. 1) but differ
biochemically in that Smad3 can bind directly to DNA, whereas Smad2 cannot,
owing to an insert in the otherwise conserved DNA-binding region
(Dennler et al., 1999). This
insert is excised in an alternative splice variant of Smad2 (Smad2
exon3), enabling it to bind DNA, but the significance of this splice variant
in vivo is not known. Inactivation of the Smad3 gene in mice does not
result in disruption of early embryogenesis, indicating that Smad2 can
effectively compensate for Smad3 at these stages
(Datto et al., 1999
;
Yang et al., 1999
). Liz
Robertson (Oxford University, Oxford, UK) presented new evidence indicating
that Smad2
exon3 can functionally substitute for both Smad3 and Smad2
when it is expressed in the mouse epiblast. Her genetic interaction data
suggest that Smad3 can partially compensate for some Smad2 functions. Thus,
the DNA-binding activities of Smad2
exon3 and Smad3 appear to be of
crucial importance in the early mouse embryo. By contrast, Lalage Wakefield
(National Cancer Institute, Bethesda, MD, USA) reported significant
differences in how mammary epithelial cells that lack either Smad2 or Smad3
genes or proteins (?) respond to TGFß. Thus, functional divergence
between Smad2 and Smad3 seems to be a cell-context-dependent phenomenon, and
defining the basis for this divergence will be an important area for further
investigation. Findings from both the Robertson and Wakefield laboratories
indicate that the level of Smads (as controlled by gene dose) is an important
component of phenotypic outcome, consistent with work from many other
laboratories demonstrating that the ubiquitin ligase-targeted degradation of
Smads is an important mechanism of Smad regulation
(Datto and Wang, 2005
).
TGFß signaling and other signaling pathways: integration and relays
A common theme at the meeting among studies in different developmental
systems was the integration of TGFß signaling with other extracellular
signals. Xiao-Fan Wang (Duke University, Durham, NC, USA) discussed mechanisms
that integrate cooperative transcriptional regulation by Wnt and TGFß in
mesenchymal progenitor cells. Peter ten Dijke (Leiden University Medical
Center, Leiden, The Netherlands) reported that Wnt signaling is required for
BMP-induced osteoblast differentiation. This cooperative interaction may be
important in vivo because a Wnt antagonist is required for bone homeostasis in
vivo. Several talks described the induction of a second signaling cascade by a
TGFß signal. For example, Laurel Raftery (Massachusetts General Hospital,
Boston, MA, USA) described that stimulation of Notch signaling by BMP
signaling is important in Drosophila follicle cell patterning. The
activation of FGF signaling was reported to be important for the
TGFß-induced proliferation of craniofacial bone development by Yang Chai
(University of Southern California, Los Angeles, CA, USA). However, this
TGFß-FGF relay mechanism appears to be specific for the induction of bone
formation from neural crest mesenchyme, as opposed to the induction of smooth
muscle differentiation. Bob Lechleider (Georgetown University Medical Center,
Washington, DC, USA) reported that it is downregulation of FGF signaling that
is important for TGFß-induced smooth muscle differentiation. The close
intertwining of TGFß signaling with other pathways appears to be an
important component of cell fate determination by TGFß family
members.
Extracellular regulation of TGFß ligands
The extracellular regulation of TGFß ligand activity is an expanding
area of investigation, and has been intensely investigated during
Drosophila dorsoventral patterning. In Drosophila blastoderm
embryos, extracellular BMP-binding proteins (Sog and Tsg) and the
metalloprotease Tolloid are required to localize BMP activity to a narrow
spatial domain at the dorsal midline of the late blastoderm embryo (reviewed
by Ashe, 2005;
Raftery and Sutherland, 2003
).
Mike O'Connor (University of Minnesota, Minneapolis, MN, USA) presented his
group's studies of the related extracellular DPP/BMP-binding proteins,
Crossveinless2 (Cv2), Crossveinless (Cv) and Tolloid-related (Tlr), which are
required to localize BMP activity to the narrow line of primordial wing cells
in Drosophila where the posterior crossvein will form
(Ralston and Blair, 2005
;
Serpe et al., 2005
;
Shimmi et al., 2005
).
Differences in kinetics between the Sog/Tsg/Tolloid system and the Cv2/Cv/Tlr
system correlate with the differences in temporal constraints for patterning
in these two tissues. O'Connor described a bind and release mechanism for
localizing active BMP ligands that almost certainly also occurs in vertebrate
tissues because all three classes of extracellular regulators are also found
in vertebrates: the Sog/CV/Chordin type of BMP-binding proteins;
Tolloid/Tlr/BMP1 extracellular metalloproteases; and Tsg-like proteins
(reviewed by Dale, 2000
).
Metalloproteases related to Tolloid are proving to be a versatile group of
BMP regulatory proteins. Tolloid and its Xenopus homolog Xolloid have
previously been shown to have similar abilities to cleave the BMP antagonists
Sog and Chordin (reviewed by Mullins,
1998). Each is now reported to release active TGFß family
ligands by targeting a different class of latent complexes. Some TGFß
ligands form latent complexes when the initially synthesized propeptide is
cleaved during secretion; the C-terminal ligand domain remains associated with
the N-terminal pro-domain in a latent complex (reviewed by
Massague, 1998
). Malcolm
Whitman (Harvard Dental School, Boston, MA, USA) reported that
Xenopus GDF11, a TGFß ligand, is secreted as a latent complex of
mature ligand and pro-domain. This complex is cleaved, so that GDF11 is
activated by Xolloid in the developing tail, where these factors are
co-expressed. Mihaela Serpe (University of Minnesota, Minneapolis, MN, USA)
reported that Drosophila Tolloid and Tolloid-related both can
activate latent ligands in a cell culture system. Murine Bmp1, another member
of this family, can also activate latent ligands
(Ge et al., 2005
). This class
of metalloproteases has broad biological activities, as Bmp1 cleaves a number
of extracellular matrix proteins in biologically important reactions
(Gonzalez et al., 2005
).
The ability of pro-domains to remain associated with the ligand in a
secreted latent complex was first identified for TGFß1, TGFß2 and
TGFß3 (reviewed by Massague,
1998). However, not all ligands in this family form such
complexes; for example, there have been no reports of such complexes for Bmp2
and Bmp4. Intriguingly, Bmp9 appears to retain full biological activity, while
it is associated with its prodomain (Senyon Choe, Salk Institute, San Diego,
CA, USA). Perhaps the formation of pro-domain latent complexes is a
characteristic of broadly expressed or circulating ligands, whereas ligands
with more limited expression patterns might be regulated by other
mechanisms.
|
The functional pairing of TGFß superfamily receptors to ligands, like functional divergence among the Smads, has been established in vitro but has not been fully investigated in vivo. TBRII and Alk5 have been identified as type II and type I receptors, respectively, for TGFß. Yang Chai (University of Southern California, Los Angeles, CA, USA) and Vesa Kaartinen (Saban Research Institute, Children's Hospital, Los Angeles, CA, USA) presented data on the loss of TßRII and Alk5, respectively, in neural crest derivatives in mice. In each case, dramatic craniofacial malformations result, consistent with the action of these receptors in a common neural crest pathway. Intriguingly, however, loss of Alk5 results in additional defects not seen with loss of the gene encoding TßRII, raising the possibility that the Alk5 Type I receptor may act in conjunction with Type II receptors other than TßRII in the developing neural crest.
TGFß signaling and vascular development
Studies of TGFß signaling in mouse angiogenesis have been informative
both for uncovering novel mechanisms of TGFß signal transduction and for
elucidating the etiology of vascular defects in human hereditary hemorrhagic
telangiectasia (HHT) (reviewed by Marchuk
et al., 2003). HHT1 is associated with mutations in eng,
the gene for endoglin. HHT2 is associated with mutations in ACVRL1,
the gene for Alk1, a type I receptor that can bind TGFß. Both syndromes
are marked by the formation of direct arterial-venous malformations in some
tissues, in which arteries and veins are directly connected with no
intervening capillary network. These syndromes suggest that a sufficient level
of TGFß signal transduction is necessary to maintain capillary networks
in certain tissues. Consistent with this hypothesis, TGFß has
dose-dependent effects on endothelial cells in vitro. Doug Marchuk (Duke
University, Durham, NC, USA) presented the human genetic perspective on
TGFß signaling. In addition to the two autosomal dominant HHT syndromes
that have been described in humans, Marchuk reported that individuals with
juvenile polyposis, which is associated with mutations in MADH4
(Smad4), also exhibit HHT-like lesions
(Gallione et al., 2004
). It
seems likely that these human syndromes identify rate-limiting steps for
TGFß regulation of capillary remodeling.
There was lively discussion during the vasculogenesis session, as
investigators sought to resolve the complex phenotypes observed in different
studies of receptor mutants in murine endothelial cells. A substantial effort
has been directed towards developing mouse genetic models of HHT, and towards
understanding the mechanisms of TGFß signaling in angiogenesis. In vitro
studies of angiogenesis divide the process into two phases, both of which are
stimulated by TGFß1 (reviewed by
Lebrin et al., 2005;
Marchuk et al., 2003
).
Vascular remodeling begins with endothelial cell migration, proliferation and
lumen formation, which are aspects of the activation phase of angiogenesis.
Remodeling is completed with the cessation of endothelial cell proliferation
and migration, the production of new basement membrane, and the recruitment
and differentiation of smooth muscle cells, i.e. the maturation, or
resolution, phase of angiogenesis. Mice homozygous null for either
Eng or Alk1 (Acvrl1 - Mouse Genome Informatics)
have defects in the embryonic vasculature; initial vasculature formation
occurs, but subsequent remodeling is defective
(Li et al., 1999
;
Oh et al., 2000
). The effects
of these mutations on angiogenesis in vivo and in embryonic endothelial cells
are under intensive investigation.
|
Endoglin was identified soon after betaglycan as a potential endothelial
component of TGFß receptor complexes; initial data suggested that it
antagonizes TGFß signaling (Barbara et
al., 1999). ten Dijke reported that endoglin can promote signaling
through Alk1 and antagonize signaling through Alk5; Jeff Wrana (Samuel
Lunenfeld Institute, Toronto, Canada) touched on results that indicate that
endoglin antagonizes signaling by regulating Alk5 levels
(Lebrin et al., 2004
;
Pece-Barbara et al., 2005
).
The molecular mechanisms by which endoglin can bias receptor choice by
TGFß ligands are still unclear. Understanding the mechanisms for receptor
choice and the specific cell types that are most sensitive to each signal
transduction pathway remain crucial issues for this field. These studies of
TGFß signaling in mouse angiogenesis, like studies of BMP signaling in
Drosophila patterning, underscore the importance of developmental
genetics for uncovering the delicate balance of mechanisms that mediate
dose-dependent responses to TGFß family ligands in normal
development.
Conclusions
The TGFß field has made dramatic progress in the identification of
components of the pathways that regulate TGFß signaling both
extracellularly and intracellularly. The importance of in vivo developmental
analyses to test these molecular mechanisms is highlighted by the evolving
story on TGFß1 signaling in angiogenesis. Similar studies of other in
vivo mechanisms will be fertile ground for presentations in 2 years at the
next meeting.
ACKNOWLEDGMENTS
The authors thank Bob Lechleider and Caroline Hill for organizing a superb meeting, and Doug Marchuk and Mike O'Connor for providing thoughtful comments on the manuscript. They also acknowledge the many excellent talks and posters that could not be discussed here for reasons of space. M.W. is supported by a grant from the NICHD and L.R. is supported by a grant from NIHGM.
REFERENCES
Ashe, H. L. (2005). BMP signalling: synergy and feedback create a step gradient. Curr. Biol. 15,R375 -R377.[CrossRef][Medline]
Babitt, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J.,
Campagna, J. A., Schneyer, A. L., Woolf, C. J. and Lin, H. Y.
(2005). Repulsive guidance molecule (RGMa), a DRAGON homologue,
is a bone morphogenetic protein co-receptor. J. Biol.
Chem. 280,29820
-29827.
Barbara, N. P., Wrana, J. L. and Letarte, M.
(1999). Endoglin is an accessory protein that interacts with the
signaling receptor complex of multiple members of the transforming growth
factor-beta superfamily. J. Biol. Chem.
274,584
-594.
Byfield, S. D. and Roberts, A. B. (2004). Lateral signaling enhances TGF-beta response complexity. Trends Cell. Biol. 14,107 -111.[CrossRef][Medline]
Chen, X., Rubock, M. J. and Whitman, M. (1996). A transcriptional partner for MAD proteins in TGF-beta signalling. Nature 383,691 -696.[CrossRef][Medline]
Dale, L. (2000). Pattern formation: a new twist to BMP signalling. Curr. Biol. 10,R671 -R673.[CrossRef][Medline]
Datto, M. and Wang, X. F. (2005). Ubiquitin-mediated degradation a mechanism for fine-tuning TGF-beta signaling. Cell 121,2 -4.[CrossRef][Medline]
Datto, M. B., Frederick, J. P., Pan, L., Borton, A. J., Zhuang,
Y. and Wang, X. F. (1999). Targeted disruption of Smad3
reveals an essential role in transforming growth factor beta-mediated signal
transduction. Mol. Cell. Biol.
19,2495
-2504.
Dennler, S., Huet, S. and Gauthier, J. M. (1999). A short amino-acid sequence in MH1 domain is responsible for functional differences between Smad2 and Smad3. Oncogene 18,1643 -1648.[CrossRef][Medline]
Derynck, R. and Zhang, Y. E. (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425,577 -584.[CrossRef][Medline]
Gallione, C. J., Repetto, G. M., Legius, E., Rustgi, A. K., Schelley, S. L., Tejpar, S., Mitchell, G., Drouin, E., Westermann, C. J. and Marchuk, D. A. (2004). A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet 363,852 -889.[CrossRef][Medline]
Ge, G., Hopkins, D. R., Ho, W. B. and Greenspan, D. S.
(2005). GDF11 forms a bone morphogenetic protein 1-activated
latent complex that can modulate nerve growth factor-induced differentiation
of PC12 cells. Mol. Cell. Biol.
25,5846
-5858.
Gonzalez, E. M., Reed, C. C., Bix, G., Fu, J., Zhang, Y.,
Gopalakrishnan, B., Greenspan, D. S. and Iozzo, R. V. (2005).
BMP-1/Tolloid-like metalloproteases process endorepellin, the angiostatic
C-terminal fragment of perlecan. J. Biol. Chem.
280,7080
-7087.
Goumans, M. J., Valdimarsdottir, G., Itoh, S., Rosendahl, A.,
Sideras, P. and ten Dijke, P. (2002). Balancing the
activation state of the endothelium via two distinct TGF-beta type I
receptors. EMBO J. 21,1743
-1753.
Ito, Y. and Miyazono, K. (2003). RUNX transcription factors as key targets of TGF-beta superfamily signaling. Curr. Opin. Genet. Dev. 13, 43-47.[CrossRef][Medline]
Lamouille, S., Mallet, C., Feige, J. J. and Bailly, S.
(2002). Activin receptor-like kinase 1 is implicated in the
maturation phase of angiogenesis. Blood
100,4495
-4501.
Lebrin, F., Goumans, M. J., Jonker, L., Carvalho, R. L.,
Valdimarsdottir, G., Thorikay, M., Mummery, C., Arthur, H. M. and ten Dijke,
P. (2004). Endoglin promotes endothelial cell proliferation
and TGF-beta/ALK1 signal transduction. EMBO J.
23,4018
-4028.
Lebrin, F., Deckers, M., Bertolino, P. and Ten Dijke, P. (2005). TGF-beta receptor function in the endothelium. Cardiovasc. Res. 65,599 -608.[CrossRef][Medline]
Lewis, K. A., Gray, P. C., Blount, A. L., MacConell, L. A., Wiater, E., Bilezikjian, L. M. and Vale, W. (2000). Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature 404,411 -414.[CrossRef][Medline]
Li, D. Y., Sorensen, L. K., Brooke, B. S., Urness, L. D., Davis,
E. C., Taylor, D. G., Boak, B. B. and Wendel, D. P. (1999).
Defective angiogenesis in mice lacking endoglin.
Science 284,1534
-1537.
Luo, K. (2004). Ski and SnoN: negative regulators of TGF-beta signaling. Curr. Opin. Genet. Dev. 14,65 -70.[CrossRef][Medline]
Marchuk, D. A., Srinivasan, S., Squire, T. L. and Zawistowski,
J. S. (2003). Vascular morphogenesis: tales of two syndromes.
Hum. Mol. Genet. 12,R97
-R112.
Massague, J. (1998). TGF-beta signal transduction. Annu. Rev. Biochem. 67,753 -791.[CrossRef][Medline]
Massague, J. and Wotton, D. (2000).
Transcriptional control by the TGF-beta/Smad signaling system. EMBO
J. 19,1745
-1754.
Mullins, M. C. (1998). Holy tolloido: tolloid cleaves SOG/Chordin to free DPP/BMPs. Trends Genet. 14,127 -129.[CrossRef][Medline]
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe,
P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S. et al.
(2000). Activin receptor-like kinase 1 modulates transforming
growth factor-beta 1 signaling in the regulation of angiogenesis.
Proc. Natl. Acad. Sci. USA
97,2626
-2631.
Ozdamar, B., Bose, R., Barrios-Rodiles, M., Wang, H. R., Zhang,
Y. and Wrana, J. L. (2005). Regulation of the polarity
protein Par6 by TGFbeta receptors controls epithelial cell plasticity.
Science 307,1603
-1609.
Pece-Barbara, N., Vera, S., Kathirkamathamby, K., Liebner, S.,
Di Guglielmo, G. M., Dejana, E., Wrana, J. L. and Letarte, M.
(2005). Endoglin null endothelial cells proliferate faster, and
more responsive to TGFbeta 1 with higher affinity receptors and an activated
ALK1 pathway. J. Biol. Chem.
280,27800
-27808
Raftery, L. A. and Sutherland, D. J. (2003). Gradients and thresholds: BMP response gradients unveiled in Drosophila embryos. Trends Genet. 19,701 -708.[CrossRef][Medline]
Ralston, A. and Blair, S. S. (2005). Long-range Dpp signaling is regulated to restrict BMP signaling to a crossvein competent zone. Dev. Biol. 280,187 -200.[CrossRef][Medline]
Schier, A. F. (2003). Nodal signaling in vertebrate development. Annu. Rev. Cell Dev. Biol. 19,589 -621.[CrossRef][Medline]
Seki, T., Yun, J. and Oh, S. P. (2003).
Arterial endothelium-specific activin receptor-like kinase 1 expression
suggests its role in arterialization and vascular remodeling. Circ.
Res. 93,682
-689.
Seoane, J., Le, H. V., Shen, L., Anderson, S. A. and Massague, J. (2004). Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117,211 -223.[CrossRef][Medline]
Serpe, M., Ralston, A., Blair, S. S. and O'Connor, M. B.
(2005). Matching catalytic activity to developmental function:
tolloid-related processes Sog in order to help specify the posterior crossvein
in the Drosophila wing. Development
132,2645
-2656.
Shimmi, O., Ralston, A., Blair, S. S. and O'Connor, M. B. (2005). The crossveinless gene encodes a new member of the Twisted gastrulation family of BMP-binding proteins which, with Short gastrulation, promotes BMP signaling in the crossveins of the Drosophila wing. Dev. Biol. 282,70 -83.[CrossRef][Medline]
ten Dijke, P. and Hill, C. S. (2004). New insights into TGF-beta-Smad signalling. Trends Biochem. Sci. 29,265 -273.[CrossRef][Medline]
Yan, Y. T., Liu, J. J., Luo, Y. E. C., Haltiwanger, R. S.,
Abate-Shen, C. and Shen, M. M. (2002). Dual roles of Cripto
as a ligand and coreceptor in the nodal signaling pathway. Mol.
Cell. Biol. 22,4439
-4449.
Yang, X., Letterio, J. J., Lechleider, R. J., Chen, L., Hayman,
R., Gu, H., Roberts, A. B. and Deng, C. (1999). Targeted
disruption of SMAD3 results in impaired mucosal immunity and diminished T cell
responsiveness to TGF-beta. EMBO J.
18,1280
-1291.