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(Circulation Research. 1996;78:173-179.)
© 1996 American Heart Association, Inc.

Articles

Transforming Growth Factor-ß Signal Transduction

Thomas Brand, Michael D. Schneider

From the Abteilung für Zell- und Molekularbiologie (T.B.), Institut für Biochemie und Biotechnologie, Technische Universität Braunschweig (Germany), and the Molecular Cardiology Unit (M.D.S.), Departments of Medicine, Cell Biology, and Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, Tex.

Correspondence to Dr Michael D. Schneider, Molecular Cardiology Unit, Baylor College of Medicine, One Baylor Plaza, Room 506 C, Houston, TX 77030.

Key Words: growth factors • receptors • signal transduction

	Introduction

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
The TGF-ß superfamily of cytokines comprises an array of more than two dozen secreted peptides that have been implicated as multifunctional regulators of cell growth, differentiation, and function.^{1 2} Members of this family are pivotal for normal development in a variety of species, providing diffusible signals that influence pattern formation, body axes, cell fate, and other aspects of morphogenesis. In the embryonic heart, developmental functions for TGF-ß inferred from the genes' program of expression and from model systems for cardiac organogenesis include the specification of cardiogenic precursor cells in lateral mesoderm as well as epithelial-mesenchymal transitions involved in valve formation.² In postnatal myocardium, TGF-ß controls the mixed histocompatibility genes, whose deregulation may explain the myocarditis found in mice lacking TGF-ß₁. In isolated cardiac muscle cells, TGF-ß counters the suppression of myocyte contraction by interleukin-1ß, at least in part through a block to induction of nitric oxide synthase. Conversely, TGF-ß evokes a "fetal" program of myocardial gene expression, which, together with the upregulation of TGF-ß by mechanical load and other trophic signals, suggests the operation of an autocrine or paracrine loop in certain forms of cardiac hypertrophy.²

The TGF-ß superfamily consists of at least 25 different peptides, classified into three subgroups based on sequence similarities: (1) TGF-ßs themselves, of which three isoforms are found in mammals, (2) activins, and (3) a complex third subfamily of proteins (BMPs, nodal, Xenopus Vg-1, Drosophila dpp, and screw) with prominent effects on mesoderm induction and formation of axial structures.^{1 2} All known members of the superfamily are produced as large precursor molecules that are cleaved at a conserved RXXR motif into the mature C-terminal dimer and the N-terminal proregion; binding of the ligand by the N-terminal remnant results in a biologically inactive latent complex. This requirement for activation of latent TGF-ß is highlighted by the selective deficiency of active TGF-ß, versus total TGF-ß, in experimental and clinical atherosclerosis. Transgenic mice overexpressing apolipoprotein (a) show reduced plasminogen activation, low levels of plasmin in the vessel wall, and, consequently, low abundance of active TGF-ß; active TGF-ß likewise is decreased in advanced atherosclerosis in humans.^{3 4} Because TGF-ß commonly acts to arrest the cell cycle, it was postulated that proliferation in the vessel wall is contingent not on hyperlipidemia per se but on the resulting depressed activity of this potential anti-mitogen.^{3 4} Ventricular myocytes likewise are targets for growth inhibition by TGF-ß.²

We recently reviewed in detail the available biological data concerning functional activities of TGF-ß in myocardium.² In the present article, we emphasize progress toward deciphering the exact mechanisms of TGF-ß receptor activation, determining the "downstream" cytoplasmic and nuclear targets of the TGF-ß receptor, and the engineering of signal-deficient TGF-ß receptors as dominant inhibitors of the TGF-ß cascade. The panoply of actions shown for TGF-ß has led to long-standing quandaries, some of which can be answered with greater assurance than others. Can a single receptor or receptor complex be reconciled with all activities of TGF-ß? Alternatively, do distinguishable receptors mediate discrete actions of a given family member? Does signal diversity arise from combinatorial interactions among TGF-ß receptor proteins? What cellular proteins physically associate with TGF-ß receptors, and which signaling properties of the receptor do they confer?

	A Novel Superfamily of STK Receptors

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
Chemical cross-linking with radioiodinated TGF-ß reveals three predominant receptors—type I (TßRI; M_r, 50 kD), type II (TßRII; M_r, 80 kD), and type III (betaglycan; M_r, 280 kD)—in cardiac myocytes and most mammalian cells. Betaglycan and TßRII were isolated by expression cloning; TßRI, in turn, was cloned by exploiting its similarity to TßRII.^{1 2} Betaglycan is a membrane-anchored 853-amino-acid proteoglycan, which binds all three TGF-ß isoforms with high affinity (K_d, 10^-10 mol/L). The short cytoplasmic domain of betaglycan is rich in serine and threonine but has no apparent signaling function. Hence, cells lacking betaglycan can respond to TGF-ß, although betaglycan serves an auxiliary role by presenting TGF-ß to TßRII, a component of the signaling receptor complex. TßRII binds TGF-ß with higher affinity when ligand is associated with betaglycan and has inherently low affinity for TGF-ß₂. Thus, cells lacking betaglycan, such as endothelial cells, respond poorly, if at all, to TGF-ß₂. Soluble forms of betaglycan are released from the cell surface by endogenous proteases and can act as a TGF-ß inhibitor by sequestering ligand.

Both TßRI and TßRII belong to a recently discovered family of membrane-spanning STKs, including receptors for all three classes of TGF-ß–like cytokines (Figure).^{1 2} Type II receptors are more divergent structurally than type I receptors, sharing 30% to 40% sequence homology in the kinase domain, versus 60% to 90% for type I receptor kinase domains. TßRII is a 567-amino-acid protein characterized by a single transmembrane domain and by an intracellular conserved STK domain, the distinguishing feature of this receptor superfamily. The kinase domain is interrupted by two kinase inserts, between subdomains VIa and VIb (insert I) and between subdomains X and XI (insert II); the C terminal to the kinase domain is a Ser/Thr-rich tail. The ligand-binding domain contains six to nine variable cysteines and an invariant cysteine box (CCX_4-5CN) close to the transmembrane domain in all STK receptors. Positioning of cysteine residues determines ligand binding and is conserved in receptors with the same ligand specificity.

View larger version (20K): [in this window] [in a new window]   Figure 1. TGF-ß signaling via type I and type II STK receptors acting in concert. A, The type II receptor binds ligand and renders the type I receptor competent to bind, forming a heteromeric complex with TGF-ß. For clarity, only one type I and one type II subunit are shown. Phosphorylation of the type II receptor is independent of ligand binding and type I receptor. Phosphorylation of type I receptor at the GS box is dependent on complex formation and the type II receptor kinase. The ligand-dependent trans phosphorylation of TßRI is sufficient to trigger most, if not all, downstream events. B, Type I and type II receptors that are deficient for signaling are dominant inhibitors of TGF-ß–dependent responses. The illustrated classes of TGF-ß receptor mutations, from left to right, are as follows: the kinase-defective truncation of TßRII, inactivating point mutations of the TßRII kinase domain or kinase insert II, the kinase-defective truncation of TßRI, inactivating point mutations of the TßRI kinase, inactivating point mutations of the TßRI GS box, and constitutively activating mutations of the GS box. The constitutively active TßRI functions autonomously in the absence of both ligand and TßRII.

Overall, TßRI is quite similar in structure to TßRII, with three distinguishing features. The extracellular ligand-binding domains of all type I receptors contain seven cysteine residues at nearly invariant positions plus the cysteine box. At the carboxy terminus, only a very short nonkinase sequence is present, lacking Ser/Thr residues. Most important, the kinase domain is immediately preceded by a type I receptor–specific domain that is rich in serine (SGSGSGLP, the GS box).

	Type I and Type II Receptors Form Heteromeric Protein Kinase Complexes

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
When the TGF-ß receptor complexes visualized by affinity labeling are immunoprecipitated, using antibodies selective for TßRII versus TßRI, both receptor types can be coprecipitated from cardiac myocytes, as in other cell types.⁵ TßRI is essentially unable to bind ligand in the absence of TßRII, as shown using mutant mink lung epithelial cells that lack TßRII; such cells fail to exhibit cross-linking of TGF-ß to either TßRI or TßRII, despite the presence of wild-type TßRI.⁶ Similarly, COS cells transfected with TßRI display ligand-binding activity only after cotransfection of TßRII. In contrast to type I receptors for TGF-ß and activin, type I receptors for BMP and dpp did not require cotransfection with a corresponding type II receptor to produce high-affinity binding in COS cells⁷ ; however, an endogenous type II receptor, not detected by cross-linking, is a formal possibility.

The requirement for TGF-ß in complex formation is not absolute. Heterodimers can be formed in the absence of TGF-ß using transiently transfected COS cells that express especially high numbers of receptors as a result of vector amplification, using baculoviral expression in insect cells, and using the two recombinant receptors in vitro.⁸ Thus, heterodimer formation can occur in the absence of ligand if both receptors are expressed at supraphysiological concentrations, whereas TGF-ß is needed for assembly of the heteromeric complex at physiological concentrations of TßRI and TßRII. Although the exact stoichiometry of the heteromeric complex is uncertain, two-dimensional gel electrophoresis of high molecular weight TßRI/TßRII complexes provides evidence for an oligomeric complex composed of at least two subunits of each receptor type per TGF-ß dimer.⁹

The two cytoplasmic kinase domains have weak but measurable affinity for each other in the yeast two-hybrid system, whereas the individual kinase domains do not homodimerize.⁸ Interaction cloning, using the TßRI kinase domain as "bait," has identified a type II BMP receptor that is highly expressed in the heart.¹⁰ Whereas the cytoplasmic domains of the type II BMP receptor and TßRI can associate in yeast, the full-length proteins in COS cells do not. A crucial function of the ligand, therefore, is to ensure specificity by determining complex formation.

	Dominant-Inhibitory TGF-ß Receptors Demonstrate the Interdependence of TßRI and TßRII

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
What is the operational relation between TßRII and TßRI? Earlier analysis of mutant cell lines resistant to growth inhibition by TGF-ß established two principles: (1) TßRI cannot bind TGF-ß in the absence of TßRII. (2) TßRII cannot signal in the absence of TßRI.⁶ If type II receptors were needed solely to confer ligand binding to TßRI, then a truncation of TßRII containing the extracellular and transmembrane regions, omitting the kinase, would be expected to rescue cells lacking endogenous TßRII. Conversely, if signal generation requires the function of both protein kinases in tandem, then a kinase-defective truncation of TßRII should not restore signaling to cells deficient for wild-type protein. Moreover, by analogy to the perhaps simpler case of receptor tyrosine kinases, such as the receptors for platelet-derived and fibroblast growth factors, which undergo reciprocal autophosphorylation in trans, deletion of either STK domain might be predicted to result in a dominant inhibitor of the TGF-ß cascade, a dominant-negative receptor.

Indeed, a truncation of TßRII lacking the kinase domain produces a dominant-negative phenotype when transfected into cardiac myocytes¹¹ (Figure). All three isoforms of TGF-ß upregulate the skeletal -actin promoter in ventricular muscle cells, and the truncated TßRII effectively blocks gene induction by all three peptides. Conversely, the truncated receptor also interferes with downregulation of the -myosin heavy chain promoter by TGF-ß.¹¹ Thus, by itself, this kinase-defective mutation of TßRII is sufficient to block both positive and negative control of gene expression by TGF-ß and markedly impairs signaling by all known mammalian isoforms of TGF-ß. Specificity of the dominant-negative phenotype was demonstrated using a corresponding truncation of the type II activin receptor, which had no effect, and by restoring TGF-ß–dependent transcription with increasing amounts of wild-type TßRII. Similarly, transfection of mink lung epithelial cells with kinase-deficient truncations of TßRII suppressed the induction of TGF-ß–dependent genes^{11 12} and blocked the antiproliferative effects as well.¹²

Similar findings resulted from the replacement of charged amino acids with alanine at conserved or invariant residues of subdomains II and VI-B (K277, R378, D379, K381, D397, and R528), associated in other protein kinases with ATP binding, phosphoamino acid recognition, or the catalytic loop for substrate binding and phosphate transfer.¹¹ All mutations that failed to rescue a TßRII-deficient cell line suppressed TGF-ß–dependent transcription in both cardiac myocytes and wild-type Mv1Lu cells. Loss of signaling capacity and a dominant-negative phenotype in the wild-type background also resulted from substitution of charged amino acids in kinase insert II (R497 and H507), a region whose deletion had been shown to impair autophosphorylation of TßRII, and from replacement of several charged residues specific to STK receptors (E272, H362, D397, D446, and R528), whose impact on phosphorylation has not yet been shown.¹¹ A corresponding kinase-defective truncation of TßRI and the mutation of the invariant lysine in subdomain II of TßRI each produce signal-deficient receptors that act as dominant inhibitors of TGF-ß–dependent transcription in a wild-type background¹¹ (M.-J. Charng and M.D. Schneider, unpublished data). Thus, the loss of function in either TßRI or TßRII is sufficient for a dominant-negative protein that interferes with the TGF-ß transcriptional cascade (Figure). Such results highlight the interdependence of these two STK receptors. A contrasting two-pathway model suggests that TßRII is responsible for growth inhibition by TGF-ß, whereas TßRI mediates the induction of Jun B and certain other genes.¹³

Loss-of-function mutations, resulting in dominant-negative proteins, have also been defined for other type II and type I receptors. In Xenopus, a truncated type II receptor for activin prevented the induction of dorsal mesoderm.¹⁴ In this system, BMP-4 elicits ventral mesoderm; a similar truncation of a type I BMP receptor blocked the induction of ventral mesoderm by BMP-4 and converted ventral mesoderm to dorsal mesoderm.¹⁵ That the kinase activity of both BMP receptors is needed for signal transduction has recently been confirmed.¹⁰ A two-receptor model also is supported by the genetics of dpp receptor mutations in Drosophila. Inactivating mutations of the gene saxophone or thick veins, which encode type I receptors, cause defects in dorsoventral patterning defects equivalent to a partial or complete loss of dpp, respectively.⁷ Mutations in punt, encoding a type II dpp receptor, similarly ventralize the embryo and interfere with the dpp-dependent expression of genes, including labial and dpp itself.¹⁶

Intriguing chimeric receptors have been constructed by fusing the TßRII ligand-binding domain to the TßRI kinase and vice versa.¹⁷ The first of these can restore ligand binding to cells lacking functional TßRII and presents TGF-ß appropriately to TßRI; conversely, the latter is competent to bind TGF-ß, contingent on the presence of the TßRII extracellular domain. Neither chimera is sufficient for TGF-ß signaling, on its own. Moreover, neither can collaborate functionally with wild-type TßRI or TßRII. By contrast, cotransfection of the two chimerae can rescue TGF-ß–dependent signaling.¹⁷ Thus, only heteromeric complexes that contain both a TßRII and TßRI kinase domain were effective.

The contrasting two-pathway model has drawn support from the selective loss of growth inhibition, sparing other responses, in certain cells that express TßRII at seemingly negligible levels or at an unexplained diminished size.^{5 13} As with the preferential inhibition of growth control after stable transfection by a dominant-negative TßRII,¹³ such results would also support an alternative interpretation (ie, when levels of TßRII decrease below an operational threshold, growth control is selectively impaired despite enough TßRII for gene induction). As shown below, gain-of-function mutations based on the biochemical interplay between TßRII and TßRI have furnished a means to distinguish between these possibilities.

	TßRI Is Phosphorylated by TßRII and Mediates Signal Transduction

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
Phosphorylation of TßRII is augmented only marginally, upon TGF-ß binding.⁶ Kinase-inactive mutants lack certain phosphopeptides, but the overall phosphorylation pattern suggests that TßRII is phosphorylated at multiple sites by cellular kinases and at few sites by autophosphorylation. One major phosphorylated peptide is derived from the Ser/Thr-rich C-terminal tail; however, a distally truncated receptor lacking these residues has normal signaling activity in TßRII-deficient cells and cannot elicit a dominant-negative phenotype in a wild-type background.^{11 12} Thus, the phosphorylation state of TßRII has not yet been shown to be important for signaling.

In contrast, phosphorylation of TßRI was increased 50-fold in Mv1Lu cells by TGF-ß binding and is a critical step in receptor activation.⁶ By cotransfection with defective variants of TßRII versus TßRI, ligand-dependent phosphorylation of TßRI was shown to require the kinase activity of TßRII⁶ and recognition of TßRI as substrate.¹⁸ Tryptic peptide digests of phosphorylated TßRI contained a single phosphorylated peptide, mapped to the previously mentioned GS box, with phosphorylation of both serine and threonine residues. Mutation of the seven phosphorylatable amino acids in this domain abolished both gene induction and antiproliferative signaling by TGF-ß.⁶ The GS domain has a central cluster of two threonine and three serine residues (T185TSGSGSG) and two more distal threonines. Mutation of single Ser/Thr residues in the central cluster had no effect on signaling activity, whereas double and triple point mutations crippled the receptor with increasing efficiency, and mutations at four or all five sites rendered it inactive.¹⁹ Unlike each residue of the central cluster itself, mutation of Thr 200 to valine was sufficient to inactivate receptor function and to confer a potent dominant-negative phenotype¹⁹ (M.-J. Charng, J. Hawker, T. Brand, M.D. Schneider, unpublished data).

Although altering Thr 204 to valine only partially inactivates the type I receptor, replacement with aspartic acid, an acidic amino acid used as a surrogate for negatively charged phosphate groups, caused a gain-of-function mutation that was highly active even in the absence of ligand and TßRII¹⁹ (Figure). Notably, the T204D mutation constitutively generates both transcriptional and antiproliferative signals, providing conclusive support for the directional interdependence of TßRII and its substrate, TßRI. Although Thr 204 is replaced by asparagine in the majority of type I receptors, substitution with aspartic acid caused constitutive signaling even for a chimera of TßRI containing the GS domain of an activin type I receptor, with glutamine at this position. In principle, therefore, gain-of-function mutations can now be engineered for other type I receptors, including `orphan` receptors, to define their biological activities as well as their biochemical partners.

	Cytoplasmic Intermediaries

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
One approach to identify postreceptor components of the TGF-ß signaling cascade has been cloning by the yeast `two-hybrid` system, on the basis of protein-protein association. A predominant protein from neonatal rat heart that bound the TßRI kinase is the immunophilin FK506 binding protein-12 (FKBP-12),²⁰ a cellular target of the immunosuppressant FK506 and a modulator of the ryanodine receptor. The complex of FKBP-12 with FK506 interferes with activity of the phosphatase calcineurin and thereby blocks T-cell activation. Under the conditions of the two-hybrid system, FKBP-12 bound to all known type I receptor kinase domains with comparable affinity. Association with either a kinase-deficient point mutation of TßRI or internal deletions of the activin type I receptor kinase was markedly weakened, and type II receptor kinase domains did not interact.²⁰ FK506 competed with TßRI for FKBP-12 binding, and mutants of FKBP-12 deficient in binding FK506 could not interact with type I receptors. Such results have suggested that type I receptors share binding sites on FKBP-12 with FK506 and might be the native ligand for the immunophilin. However, it remains unproved whether FKBP-12 becomes phosphorylated by TßRI or participates in any manner in TGF-ß signaling.

A second approach relies on candidates, such as the guanine nucleotide–binding protein Ras, drawn from more established signaling pathways. Classically, mitogens operating through receptor tyrosine kinases and various other agonists increase GTP occupancy on Ras; GTP-bound Ras, in turn, binds and activates downstream effectors, including the Raf protein kinase and phosphatidylinositol 3-kinase. In the Xenopus assays discussed earlier, dominant-negative mutations of Ras (locked in the GDP-bound state) block the ability of activin, BMP-4, and TGF-ß to provoke mesoderm formation.²¹ The simplest and most gratifying interpretation of such findings would be that TGF-ß family members signal through Ras. At least three counterarguments, however, suggest caution and alternative conclusions. First, mesoderm formation by activin requires fibroblast growth factor²² ; the Ras effect can be reconciled with inhibition of signaling by the fibroblast growth factor receptor. Second, the induction of mesoderm by activated Ras can be mimicked by supplying the rate-limiting factor for protein translation, and the dominant-negative Ras effect has been reinterpreted as Ras-dependent translation of certain growth factor mRNAs.²³ Both gain-of-function and loss-of-function mutations in Ras may manipulate autocrine pathways. Third, in cardiac myocytes, dominant-negative N17 Ras inhibited TGF-ß–induced and basal transcription of a skeletal -actin reporter gene equally.²⁴ Indeed, a generalized requirement for Ras activity was found for efficient expression of all reporter genes tested including neutral core promoters.²⁴ When a recombinant adenovirus was used for highly efficient delivery of N17 Ras, the dominant-negative protein was shown to inhibit global runoff transcription in isolated cardiac nuclei (M. Abdellatif and M.D. Schneider, unpublished data).

	Cell-Cycle Regulators and Transcription Factors

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
More is known of the nuclear targets for TGF-ß cascades in both growth control and gene transcription. Proliferation of eukaryotic cells is regulated by a delicate balance of positive and negative cell-cycle regulators—cyclins, Cdks, Cdk inhibitors, and cyclin-activating kinase.²⁵ Acting in the G₁ phase, D-type cyclins assemble with Cdk4 and Cdk6; cyclin E combines with Cdk2. A key substrate for G₁ Cdks is Rb, a tumor suppressor "pocket protein." Pocket proteins complex with E2F transcription factors that, in turn, regulate the expression of DNA polymerase-, dihydrofolate reductase, and cdc2, which are required for DNA synthesis. Mitogenic growth factors activate Cdks, which increase Rb phosphorylation, resulting in release of E2F, sequential binding of E2F to differing pocket proteins, and ultimately cell-cycle progression. Operation of a pocket protein pathway has been substantiated in cardiac myocytes by using adenoviral E1A proteins that displace E2F from the pocket²⁶ or by forced expression of E2F itself (L. Kirshenbaum and M.D. Schneider, unpublished data).

TGF-ß acts as an anti-mitogen for many cell types, including cardiac myocytes.² Studies of how TGF-ß exerts these effects have disclosed multiple targets, which cooperate to arrest proliferative growth. The activity or abundance of several key Cdk inhibitors (levels of p15^INK4b and p21^Cip1 and the activity of p27^Kip1) is specifically increased in TGF-ß–treated cells.²⁷ Conversely, TGF-ß suppresses the synthesis of the late G₁- and S-phase cyclins E and A along with Cdk2 and Cdk4, perhaps as secondary adaptations to the growth-arrested state.²⁷ Since deregulated expression of E2F can override growth suppression by TGF-ß, control of Rb phosphorylation by G₁ cyclins is pivotal to the antiproliferative pathway through which TGF-ß signals.²⁸ Currently, however, the precise events coupling receptor activation to increased expression of Cdk inhibitors and reduced cyclin/Cdk expression remain undefined. These mechanisms hold particular interest, given the reciprocal relation between active TGF-ß and atherosclerosis in both experimental animals and humans.^{3 4}

The ability of TGF-ß to activate coordinate changes in gene expression that correspond well to those triggered by mechanical load, taken with the induction of TGF-ß during hypertrophy, has provided an impetus to defining the mechanisms for transcriptional control by TGF-ß in myocardium.²⁹ The first 200 nucleotides of the skeletal -actin promoter proved sufficient not only to confer tissue specificity in neonatal ventricular myocytes but also to regulate the gene by TGF-ß and fibroblast growth factors. Basal transcription of the promoter in cardiac myocytes required the MADS box protein SRF, Sp1, and TEF-1 in concert, reminiscent of the need for SRF, Sp1, and helix-loop-helix factors for activity of the cardiac -actin promoter. SRF and TEF-1 binding sites in particular were necessary for efficient induction by TGF-ß and were each, independently, sufficient to confer TGF-ß responsiveness to a neutral promoter.²⁹ Thus, SRF and TEF-1 each serve as TGF-ß response factors, distinct from proteins previously implicated in transcriptional activation by TGF-ß. That this mechanism might be generalized to other hypertrophic signals is shown by the shared requirement for SRF and TEF-1, along with Sp1, in ₁-adrenergic activation of the skeletal -actin gene.³⁰

	A TGF-ß Family Member Controlling Heart Formation in Drosophila

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
Remarkable information in the signaling machinery for a TGF-ß–related factor, dpp, has come from genetic studies in Drosophila. The gene dpp acts as a morphogen that establishes the dorsoventral body axis.³¹ Later in development, dpp is also involved in gut development, axis specification of individual imaginal disks, and the induction of the cardiac equivalent in Drosophila, the dorsal vessel. Two genes have been isolated that modulate dpp activity, acting upstream of the receptor. A novel member of the TGF-ß family, screw is postulated to form heterodimers with dpp; tolloid encodes a metalloprotease that may process dpp propeptide into the active ligand.³² Three dpp receptor genes, cited earlier, have been identified: sax and tkv, which code for type I receptors, and punt, encoding a type II receptor. By screening for Drosophila mutants with phenotypes similar to dpp, tkv, or punt, the schnurri gene, which is essential for dpp signaling in endoderm and visceral mesoderm and codes for a putative zinc finger transcription factor resembling major histocompatibility complex–binding proteins, was isolated.³²

In dorsal ectoderm, dpp acts as an inductive signal that activates and maintains the expression of tinman, a homeobox gene controlling both visceral and cardiac myocyte differentiation.³¹ Loss of tinman in dpp null mutants causes a failure to form cardiac mesoderm; conversely, ectopic expression of dpp causes ventral expansion of tinman expression but no increase in cardiac progenitors, suggesting that other dorsal-restricted factors also are required.³¹ Vertebrate homologues of dpp (BMP-2 and -4) are 90% homologous in the processed peptide domain and can functionally substitute for dpp in Drosophila or vice versa.¹ Though BMP-4 itself is expressed in early mouse myocardium and induces cardiac muscle formation in embryonic stem cells,³³ it is unknown to what extent the dpp cascade parallels or predicts the roles performed by BMPs in vertebrate embryogenesis. However, activin and a chick homologue of nodal now have been shown to participate in a molecular pathway that determines left-right asymmetry of the chick embryo, including the sidedness of cardiac development.³⁴

	Conclusions

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
Recent progress in identifying the molecular basis for TGF-ß receptor activation and downstream effectors of TGF-ß signaling cascades has begun to yield at least a preliminary framework for signal transduction by this growth factor superfamily. Maternal effects illustrated by the transplacental transfer of TGF-ß³⁵ and a marked potential for functional redundancy among the dozens of ligands and receptors are likely to confound interpretation of simple loss-of-function mutations in this system. Dominant-negative mutations and constitutively activated receptors can complement alternative approaches to provide a more detailed understanding of the developmental and physiological functions of TGF-ß in the cardiovascular system and in vertebrates more generally.

	Selected Abbreviations and Acronyms

 

BMP = bone morphogenetic protein Cdk = cyclin-dependent protein kinase dpp = decapentaplegic protein FKBP-12 = FK506 binding protein-12 Rb = retinoblastoma gene product SRF = serum response factor STK = serine/threonine kinase TßRI = type I radioiodinated TGF-ß receptor TßRII = type II radioiodinated TGF-ß receptor TEF-1 = transcription enhancer factor-1 TGF = transforming growth factor

	Acknowledgments

 
This study was supported in part by National Institutes of Health grants (R01 HL-47567, R01 52555, P01 HL-49953, P50 HL-42267, and T32 HL-07706) to Dr Schneider. Dr Brand was a fellow of the Deutsche Forschungsgemeinschaft. The authors thank M.-J. Charng for detailed comments on the manuscript. The authors also acknowledge profound contributions by many investigators whose work could not be explicitly cited here.

Received August 30, 1995; accepted October 5, 1995.

	References

Top Introduction A Novel Superfamily of... Type I and Type... Dominant-Inhibitory TGF-ß... TßRI Is Phosphorylated by... Cytoplasmic Intermediaries Cell-Cycle Regulators and... A TGF-ß Family Member... Conclusions References

 
1. Kingsley DM. The TGF-ß superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 1994;8:133-146.

2. Brand T, Schneider MD. The TGF-ß superfamily in myocardium: ligands, receptors, transduction, and function. J Mol Cell Cardiol. 1995;27:5-18.

3. Grainger DJ, Kemp PR, Liu AC, Lawn RM, Metcalfe JC. Activation of transforming growth factor-ß is inhibited in transgenic apolipoprotein(a) mice. Nature. 1994;370:460-462.

4. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-ß is severely depressed in advanced atherosclerosis. Nat Med. 1995;1:74-79.

5. Brand T, Schneider MD. Inactive type II and type I receptors for TGF ß are dominant inhibitors of TGF ß-dependent transcription. J Biol Chem. 1995;270:8274-8284.

6. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J. Mechanism of activation of the TGF-ß receptor. Nature. 1994;370:341-347.

7. Penton A, Chen YJ, Staehling-Hampton K, Wrana JL, Attisano L, Szidonya J, Cassill JA, Massagué J, Hoffmann FM. Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor. Cell. 1994;78:239-250.

8. Ventura F, Doody J, Liu F, Wrana JL, Massague J. Reconstitution and transphosphorylation of TGF-ß receptor complexes. EMBO J. 1994;13:5581-5589.

9. Yamashita H, ten Dijke P, Franzen P, Miyazono K, Heldin CH. Formation of hetero-oligomeric complexes of type I and type II receptors for transforming growth factor-ß. J Biol Chem. 1994;269:20172-20178.

10. Liu F, Ventura F, Doody J, Massague J. Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Mol Cell Biol. 1995;15:3479-3486.

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