TGF-β: from latent to active

The transforming growth factor-betas (TGF-βs) are synthesized as precursor proteins that are modified intracellularly prior to secretion. One of the most relevant intracellular modifications is the cleavage of the C-terminal pro-region from the N-terminal portion of the protein. The C-terminal pro-region is referred to as the latency-associated peptide (LAP) while the N-terminal region is called the mature TGF-β or active TGF-β. However, with some exceptions the LAP noncovalently associates with the mature TGF-β prior to secretion. When the mature TGF-β is associated with the LAP it is called L-TGF-β and cannot interact with its receptor and has no biological effect. The TGF-βs and their receptors are very ubiquitously expressed, suggesting that the regulation of TGF-β activity is likely to be complex and multifactorial. However, one of the most important means of controlling the biological effects of TGF-β is the regulation of converting L-TGF-β to active TGF-β. The current literature supports two major mechanisms of activation of L-TGF-β and suggests that the mechanism of activation of L-TGF-β may be varied and context-dependent. For TGF-β to become biologically active the LAP has to be either released from its associations with L-TGF-β or undergo conformational change such that the LAP is not released from the L-TGF-β complex but exposes the TGF-β receptor binding site. Since TGF-β has been associated with the pathogenesis of numerous diseases, the various mechanisms of activation of L-TGF-β in context offer the possibility of controlling TGF-β activity localized to the organ of involvement and to a more specific disease process.     

Post translational activation of latent transforming growth factor beta (L-TGF-beta): clinical implications

Transforming growth factor-betas (TGF-betas) are multifunctional cytokines that exist in 3 isoforms in mammals. The TGF-betas are ubiquitously expressed and all isoforms are secreted as biologically inactive precursors called latent TGF-beta (L-TGF-beta). L-TGF-betas are generally not effective molecules because they are unable to interact with their receptors. However, the removal of or conformational change of the precursor protein called the latency associated peptide (LAP) results in the generation of biologically active TGF-beta. In vitro active TGF-beta has many biological effects but from a clinical point of view one of the most recognized associations of aberrant TGF-beta production is with diseases characterized by enhanced connective tissue synthesis. Recently a number of observations in the context of fibrotic disorders suggest mechanisms of activation of L-TGF-beta1 in vivo. The recognition of mechanisms that activate L-TGF-beta1 in vivo offers the possibility of interfering with the activation of L-TGF-beta1 for therapeutic purposes.     

The structure of the TGF-beta latency associated peptide region determines the ability of the proprotein convertase furin to cleave TGF-betas

The TGF-beta family members are generated as latent pre-pro-polypeptides. The active mature peptides are cleaved from the latent forms by cellular proteases. TGF-beta 1, for instance, is predominantly processed by a substilisin-like proprotein convertase, furin. TGF-beta 2 has a consensus cleavage site for furin and therefore has been presumed to be cleaved by furin. However, TGF-beta 2 is often secreted as the latent form, which appears to be inconsistent with its postulated sensitivity to furin. We report here that both the regular (short) form of TGF-beta2 and its spliced variant with an additional exon (long form) are insensitive to furin. NIH 3T3 and CHO cells were transfected with expression vectors containing the short or long form of TGF-beta 2 or a chimeric TGF-beta consisting of the TGF-beta1 LAP region, the TGF-beta 2 cleavage site and the TGF-beta 2 mature peptide. The constructs included a c-myc epitope tag in the N-terminal region of the mature peptide. The TGF-betas produced by the transfected cells were analyzed with Western blots and immunocytochemistry. The intracellular proteins harvested from these cells were incubated with furin. Furin only inefficiently cleaved both the long and short forms of TGF-beta 2, but efficiently processed the chimeric TGF-beta. This indicates that the insensitivity of both forms of TGF-beta 2 to furin is a consequence of the tertiary structure of their LAP regions rather than their cleavage site. This differential processing of TGF-beta1 and -beta 2 may be part of the mechanism that generates isoform-specific functions of the TGF-betas.     

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion.

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.     

Molecular interactions that confer latency to transforming growth factor-beta

A major point of regulation of transforming growth factor-beta (TGF-beta) function is through control of activation of the latent TGF-beta complex, which consists of the latency associated peptide (LAP) secreted in non-covalent association with mature TGF-beta. Activation involves proteolysis, dissociation, or altered binding of LAP. However, the mechanism by which LAP confers latency to TGF-beta is poorly understood. Previously, we identified a conserved sequence near the N terminus of LAP as a site of thrombospondin-1 (TSP1) binding to the latent complex. Now we show that expression of the TGF-beta1-latent complex deleted in the TSP1 binding site ((54)LSKL) of LAP (DeltaLSKL LAP) results in secretion of LAP, but not of mature TGF-beta. DeltaLSKL LAP also fails to bind soluble or immobilized TGF-beta1. Consistent with an inability to bind the mature domain, DeltaLSKL LAP is unable to confer latency to TGF-beta, suggesting that the LSKL sequence is important, not only for TSP1 binding and activation, but also for binding to the mature domain. We identified the sequence (94)RKPK in the receptor-binding region of mature TGF-beta1 as the binding site for LAP. Peptides of the RKPK sequence bind LAP and inhibit LAP/TGF-beta association. RKPK peptides also activate latent TGF-beta, presumably by disrupting LAP-mature TGF-beta interactions. These studies provide a molecular basis for both latency and activation by TSP1 through the LSKL sequence of LAP binding to the RKPK sequence of mature TGF-beta.     

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.     

Real-time monitoring of the interactions of transforming growth factor-beta (TGF-beta ) isoforms with latency-associated protein and the ectodomains of the TGF-beta type II and III receptors reveals different kinetic models and stoichiometries of binding

Mature transforming growth factor-beta (TGF-beta) is proteolytically derived from the C terminus of a precursor protein. Latency-associated protein (LAP), the N-terminal remnant of the TGF-beta precursor, is able to bind and neutralize TGF-beta. Mature TGF-beta exerts its activity by binding and complexing members of two subfamilies of receptors, the type I and II receptors. In addition to these signaling receptors, TGF-beta can also interact with an accessory receptor termed the type III receptor. Using a surface plasmon resonance-based biosensor (BIAcore), we determined the mechanisms of interaction of four binding proteins (LAP, the type II and III receptor ectodomains (EDs), and a type II receptor ED/Fc chimera) with three TGF-beta isoforms, and we quantified their related kinetic parameters. Using global fitting based on a numerical integration data analysis method, we demonstrated that LAP and the type II receptor/Fc chimera interacted with the TGF-beta isoforms with a 1:1 stoichiometry. In contrast, the type II ED interactions with TGF-beta were best fit by a kinetic model assuming the presence of two independent binding sites on the ligand molecule. We also showed that the type III ED bound two TGF-beta molecules. Further experiments revealed that LAP was able to block the interactions of TGF-beta with the two EDs, but that the two EDs did not compete or cooperate with each other. Together, these results strongly support the existence of a cell-surface complex consisting of one type III receptor, two TGF-beta molecules, and four type II receptors, prior to the recruitment of the type I receptor for signal transduction. Additionally, our results indicate that the apparent dissociation rate constants are more predictive of the neutralizing potency of these TGF-beta-binding proteins (LAP, the type II and III receptor EDs, and the type II receptor/Fc chimera) than the apparent equilibrium constants.     

Association of the small latent transforming growth factor-beta with an eight cysteine repeat of its binding protein LTBP-1.

Transforming growth factor-betas (TGF-betas) are produced by most cells in large latent complexes of TGF-beta and its propeptide (LAP) associated with a binding protein. The latent TGF-beta binding proteins (LTBPs-1, -2 and -3) mediate the secretion and, subsequently, the association of latent TGF-beta complexes with the extracellular matrix (ECM). The association of beta1-LAP with LTBP-1 was characterized at the molecular level with an expression system in mammalian cells, where TGF-beta1 and various fragments of LTBP-1 were co-expressed and secreted with the aid of a signal peptide synthesized to the LTBP-1 constructs. Immunoblotting of the fusion protein complexes indicated that the third 8-Cys repeat of LTBP-1 bound covalently to the LAP region of TGF-beta1. The cysteine required for the association between LTBP-1 and beta1-LAP was mapped to Cys33 of beta1-LAP. The N-terminal region of LTBP-1 consisting of the first 400 amino acids was found to associate covalently with the ECM. The data indicate that an 8-Cys repeat of LTBP is capable of covalent and specific protein-protein interactions. These interactions are mediated by exchanging cysteine disulfide bonds between the core 8-Cys repeat and an optionally associated protein during the secretion. This is, to our knowledge, the first demonstration of an extracellular protein module that is able to exchange cysteine disulfide bonds with heterologous ligand proteins.     

Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.

Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.     

Immunocytochemical localization of latent transforming growth factor-β1 activation by stimulated macrophages

Transforming growth factor-β1 (TGF-β) is secreted in a latent form consisting of mature TGF-β noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-β from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-β action. We have identified two events associated with latent TGF-β (LTGF-β) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-β concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-γ and lipopolysaccharide reportedly activate LTGF-β via cell membrane–bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-β activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-β epitopes. The induction of TGF-β immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-β activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-β and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-β activation provides an important tool for studies of its regulation. J. Cell. Physiol. 178:275–283, 1999. © 1999 Wiley-Liss, Inc.     

The tryptophan-rich motifs of the thrombospondin type 1 repeats bind VLAL motifs in the latent transforming growth factor-beta complex

Transforming growth factor-beta (TGF-beta) is secreted as a latent complex of the latency-associated peptide (LAP) and the mature domain, which must be activated for TGF-beta to signal. We previously identified thrombospondin 1 (TSP1) as a physiologic activator of TGF-beta in vitro and in vivo. The WSXW sequences in the type 1 repeats of TSP1 interact with the mature domain of TGF-beta, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF-beta latent complex. However, the binding site for the WSXW sequence was not identified. In this report, we show that the WSXW sequences bind the (61)VLAL sequence in mature TGF-beta and also bind (77)VLAL in LAP. A glutathione S-transferase (GST) fusion protein of the second TSP1 type 1 repeat (GST-TSR2) binds immobilized VLAL peptide. VLAL peptides inhibit binding of LAP and mature TGF-beta to soluble GST-TSR2 and immobilized WSXW peptide. VLAL peptide inhibits TSP1-mediated activation of recombinant and endothelial cell-derived latent TGF-beta. Furthermore, TGF-beta or LAP deleted in the VLAL sequence fails to bind immobilized WSXW or soluble GST-TSR2, indicating that binding to both VLAL sequences is important for association of TSP1 and the latent complex. Additionally, TSP1 is unable to activate latent TGF-beta when VLAL is deleted from the mature domain. These data show that the WSXW motif binds VLAL on both LAP and mature TGF-beta, and these interactions are critical for TSP1-mediated activation of the TGF-beta latent complex.     

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.     

Signaling pathways of transforming growth factor beta family members

Transforming growth factor beta (TGF-beta) signaling controls varies of cellular processes, including cell proliferation, differentiation, fibrosis, apoptosis and specification of developmental fate during embryogenesis as well as in mature tissues. The members of TGF-betas family are secreted as inactive (latent) precursors, what prevents uncontrolled activation of the cognate receptors. After activation TGF-beta ligand initiates signaling by binding to and bringing together type I and type II receptor serine/threronine kinases on the cell surface. Recent cellular, biochemical and structural studies have revealed significant insight into the mechanisms of the activation of TGF-beta receptors through ligand binding, the activation of Smad proteins through phosphorylation as well as Smad independent pathways.     

Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology

Regulation of the activation of latent TGF-beta is essential for health as too much or too little TGF-beta activity can have serious, deleterious consequences. The processes that control conversion of the precursor to the biologically active form of TGF-beta in vivo are not well characterized. We have identified a mechanism for the activation of latent TGF-beta that involves binding of the secreted and extracellular matrix protein, thrombospondin-1 (TSP-1), to the latent precursor. Specific sequences in TSP-1 and in the precursor portion (the latency associate peptide-LAP) have been determined to be essential for activation of latent TGF-beta by TSP-1. It is thought that binding of TSP-1 to the latent complex induces a conformational rearrangement of the LAP in such a manner as to prevent the LAP from conferring latency on the mature domain of TGF-beta. A TSP-dependent mechanism of activation may be locally important during wound healing and in post-natal development of epithelial structures. The possible involvement of TSP-1 in TGF-beta activation during several disease processes is also discussed.     

Two distinct domains in pro-region of Nodal-related 3 are essential for BMP inhibition

The transforming growth factor-beta (TGF-beta) superfamily member, Xenopus nodal-related 3 (Xnr3), induces neural tissues through inhibition of bone morphogenetic proteins (BMPs). We recently identified an inhibitory mechanism in which the pro-region of Xenopus tropicalis nodal-related 3 (Xtnr3) physically interacts with BMP ligands. Here, we show that disulfide-linked heterodimerization does not contribute to BMP inhibition by Xtnr3 and that the Xtnr3 mature region, overexpression of which can induce the same phenotype as full-length Xtnr3, does not inhibit BMP signaling. Furthermore, we find that the BMP-inhibitory domains of Xtnr3 are separately located in the N- and C-terminal regions of the pro-region. These results indicate the pro-region of Nodal-related 3 is both necessary and sufficient for its BMP inhibition.     

Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-beta binding proteins and activation of TGF-beta

Transforming growth factors beta (TGF-betas) are multifunctional cytokines that modulate cell growth, differentiation and apoptosis. Numerous effects initiated by TGF-betas in vitro have been described, but the role of TGF-beta targeting and activation under physiological conditions has gained very little attention and understanding. We report here that apoptosis of human umbilical vein endothelial cells (HUVECs) is accompanied by release of truncated large latent TGF-beta complexes from the pericellular matrix followed by activation of TGF-beta. The activation of TGF-beta during apoptosis was accompanied by enhanced secretion of beta1-LAP protein, and apoptotic HUVECs acquired the capacity to induce the release of latent TGF-beta-binding proteins (LTBPs) from extracellular matrices. Activated TGF-beta, in turn, attenuated apoptotic death of HUVECs. Current results indicate that the activation of TGF-beta accompanies the apoptosis of HUVECs, and may play a protective feedback role against apoptotic cell death. The results suggest a role for TGF-beta as a putative extracellular modulator of apoptosis.     

Crystal structure of BMP-9 and functional interactions with pro-region and receptors

Bone morphogenetic proteins (BMPs), a subset of the transforming growth factor (TGF)-beta superfamily, regulate a diverse array of cellular functions during development and in the adult. BMP-9 (also known as growth and differentiation factor (GDF)-2) potently induces osteogenesis and chondrogenesis, has been implicated in the differentiation of cholinergic neurons, and may help regulate glucose metabolism. We have determined the structure of BMP-9 to 2.3 A and examined the differences between our model and existing crystal structures of other BMPs, both in isolation and in complex with their receptors. TGF-beta ligands are translated as precursors, with pro-regions that generally dissociate after cleavage from the ligand, but in some cases (including GDF-8 and TGF-beta1, -2, and -3), the pro-region remains associated after secretion from the cell and inhibits binding of the ligand to its receptor. Although the proregion of BMP-9 remains tightly associated after secretion, we find, in several cell-based assays, that the activities of BMP-9 and BMP-9.pro-region complex were equivalent. Activin receptor-like kinase 1 (ALK-1), an orphan receptor in the TGF-beta family, was also identified as a potential receptor for BMP-9 based on surface plasmon resonance studies (BIAcore) and the ability of soluble ALK-1 to block the activity of BMP-9.pro-region complex in cell-based assays.