Making sense of latent TGFß activation

Justin P. Annes*, John S. Munger and Daniel B Rifkin

Departments of Cell Biology and Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA

* Author for correspondence (e-mail: annesj01{at}med.nyu.edu)


    Summary
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
TGFß is secreted as part of a latent complex that is targeted to the extracellular matrix. A variety of molecules, `TGFß activators,' release TGFß from its latent state. The unusual temporal discontinuity of TGFß synthesis and action and the panoply of TGFß effects contribute to the interest in TGF-ß. However, the logical connections between TGFß synthesis, storage and action are obscure. We consider the latent TGFß complex as an extracellular sensor in which the TGFß propeptide functions as the detector, latent-TGFß-binding protein (LTBP) functions as the localizer, and TGF-ß functions as the effector. Such a view provides a logical continuity for various aspects of TGFß biology and allows us to appreciate TGFß biology from a new perspective.

Key words: Transforming growth factor-ß, Activation, Sensor


    Introduction
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
The transforming growth factors ß (TGFß) are multipotent cytokines that are important modulators of cell growth, inflammation, matrix synthesis and apoptosis (Taipale et al., 1998Go). Defects in TGFß function are associated with a number of pathological states, including tumor cell growth, fibrosis and autoimmune disease (Blobe et al., 2000Go). The TGFß signal transduction pathway is a topic of intense investigation, and much progress has been achieved in characterizing the proteins involved. The extracellular concentration of TGFß activity is primarily regulated by the conversion of latent TGFß to active TGFß; tissues contain significant quantities of latent TGFß and activation of only a small fraction of this latent TGFß generates maximal cellular responses. Yet, despite this fact, many researchers overlook or misunderstand latent TGFß activation. This may be because TGFß biology is unusual: (1) the TGFß propeptide remains tightly bound to the cytokine after the bonds between the propeptide and mature TGFß are cleaved; (2) the interaction between TGFß and its propeptide renders the growth factor latent; (3) the TGFßs are secreted as a complex in which a second gene product is covalently bound to the TGFß propeptide; and (4) upon secretion, the TGFß large latent complex (LLC) may be covalently linked to the extracellular matrix (ECM) (Fig. 1). Moreover, the multiple activators of the latent TGFß complex comprise a seemingly unrelated group of molecules, and the three TGFß isoforms — TGFß1, TGFß2 and TGFß3 — have similar properties in vitro, but distinct effects in vivo. Here, we present a model in which latent TGFß is considered to be a molecular sensor that responds to specific signals by releasing TGFß. These signals are often perturbations of the ECM that are associated with phenomena such as angiogenesis, wound repair, inflammation and, perhaps, cell growth. Changes in the cell's environment are relayed to the sensor by a number of different molecules, including proteases, integrins and thrombospondin (TSP). We propose that consideration of latent TGFß in this manner unifies the processes of TGFß secretion, sequestration and activation and clarifies features of TGFß biology.



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Fig. 1. The TGFß large latent complex (LLC). The LLC comprises TGFß (black), LAP (red) and LTBP. TGFß and LAP are proteolytically separated at the site indicated by the arrowhead. After processing, TGFß remains noncovalently associated with LAP. LAP and LTBP are joined by disulfide bonds (light blue lines). The LLC is covalently linked to the extracellular matrix (ECM) through an isopeptide bond (green) between the N-terminus of LTBP (somewhere between EGF2 and the hinge domain) and a currently unidentified matrix protein. The hinge domain (arrow) of LTBP is a protease-sensitive region that allows LLC to be proteolytically released from the ECM.

 


    The components and assembly of the sensor
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
Before presenting the model, we must first describe the synthesis of TGFß and its latent complex. The three TGFßs are all synthesized as homodimeric proproteins (proTGFß) that have a mass of 75 kDa. The dimeric propeptides, also known as the latency-associated proteins (LAPs){dagger}, are cleaved from the mature TGFß 24-kDa dimer in the trans Golgi by furin-type enzymes. Early in the assembly of the TGFß LLC, disulfide linkages are formed between cysteine residues of LAP and specific cysteine residues in the latent-TGFß-binding protein (LTBP) (Fig. 2, step 1) (Saharinen et al., 1996Go; Gleizes et al., 1996Go; Miyazono et al., 1991Go). LTBP is a member of the LTBP/fibrillin protein family, which comprises fibrillin-1, fibrillin-2 and fibrillin-3, and LTBP-1, LTBP-2, LTBP-3, and LTBP-4 (Ramirez and Pereira, 1999Go). These proteins contain multiple epidermal-growth-factor-like repeats as well as unique domains containing eight cysteine residues (8-cys domains) (Fig. 1) (Kanzaki et al., 1990Go; Tsuji et al., 1990Go; Sinha et al., 1998Go). LTBP-1, LTBP-3 and LTBP-4 form a subset within the family based on their ability to bind LAP. Only the third of the four 8-cys domains within each of the LAP-binding LTBPs can disulfide bond to LAP (Saharinen and Keski-Oja, 2000Go); the other 8-cys domains may localize LTBPs to the ECM (Unsold et al., 2001Go). As part of the LLC, TGFß cannot interact with its receptors, because the TGFß1, 2 and 3 prodomains (LAPs) function as inhibitors owing to their noncovalent, high-affinity association with TGFß (Lawrence et al., 1984Go; Dubois et al., 1995Go). We use the term `TGFß activation' to refer to the liberation of TGFß from the latent complex. LTBP and its bound latent TGFß are found primarily as components of the matrix. Indeed, the N-terminal region of LTBP-1 is covalently cross-linked to ECM proteins by transglutaminase (tTGase) (Fig. 1; Fig. 2, step 3) (Nunes et al., 1997Go). However, an LTBP binding partner in the ECM has not been unambiguously identified. (Although our discussion is based primarily upon LTBP-1, the similar sequences and domain structures of the LTBPs suggest that most of our statements are generally applicable.) LTBP-1 exists in a range of sizes (125-210 kDa) owing to the use of two independent promoters as well as differences in splicing and glycosylation (Koski et al., 1999Go). Most forms of LTBP-1 have two protease-sensitive regions; proteolysis at the more N-terminal site can release a truncated form of LTBP-1 (or LLC) from the ECM (Fig. 2, step 4) (Taipale et al., 1994Go). The functions of LTBP-1 may vary depending on its size. For example, LTBP-1 that contains an N-terminal extension (LTBP-1L) generated by use of the upstream promoter associates more readily with the ECM than does LTBP-1 (LTBP-1S) formed by use of the downstream promoter (Olofsson et al., 1995Go).



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Fig. 2. The latent TGFß sensor model. The figure depicts the sequential events in the bioavailability of TGFß, from synthesis to signaling consequences, according to consideration of the TGFß LLC as a sensor. Sensor assembly (1) occurs cotranslationally when the localizer (LTBP; L) is covalently linked to pro-TGFß (D-E). As shown, the next step (step 2) is the proteolytic cleavage of the bonds between the detector (LAP; D) and the effector (TGFß; E). This step turns the sensor `on' or, in other words, makes the sensor competent (note that the timing of this step is variable and may occur after secretion). Once secreted, the sensor is stored in the ECM (step 3). Subsequently, the complex may be solubilized from the matrix (step 4) by cleavage of LTBP in the hinge region. This soluble form of TGFß is still latent and may be activated (step 5). Under other conditions, activation of the matrix-bound sensor occurs (step 5'). Binding of liberated TGFß (E) to its receptors (step 6) with subsequent signal transduction has multiple results (green arrows; A), including induction of TGFß expression (B), enhanced expression of transcripts encoding TGFß activators (C), and increased synthesis of ECM components (D).

 

In our model the three components of the LLC —TGFß, LAP and LTBP- constitute a sensor (Fig. 1). This sensor consists of an effector (TGFß), a localizer (LTBP) and a detector (LAP). We consider TGFß to be the effector because it is the output of the sensor, LTBP to be the localizer because it interacts with the ECM, and LAP to be a detector because any activation mechanism must act on LAP, since LAP is sufficient to inhibit TGFß bioactivity (Gentry and Nash, 1990Go). The characterization of the mechanisms controlling the liberation of TGFß from the latent complex is central to the consideration of TGFß action because the release of TGFß determines the free TGFß levels. Several mechanisms for the activation of latent TGFß complexes are known (Munger et al., 1997Go; Koli et al., 2001Go), and a diverse group of activators, including proteases, TSP-1, the integrin {alpha}vß6, reactive oxygen species (ROS) and low pH, can activate TGFß. However, the biological advantage of releasing TGFß as a latent complex and the relationships between the various activators are obscure. By considering the LLC as a sensor, we think that the role of the latent complex and its activators is clarified.


    The latent TGFß complex as a sensor
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
What general properties do sensors have and how do these properties relate to latent TGFß? Consider, as an example, a smoke detector. Before it is used, it must be assembled correctly, placed in an appropriate location and put into a competent state (turned on). The sensor can then change in response to a stimulus (smoke) above a certain threshold, and this change relays information about the environment in the form of an effector (an alarm). Modification of the assembly or the location of the device can alter its effectiveness to respond to smoke.

These features of a smoke detector have analogies in the structure/function of the LLC. The latent TGFß complex is a sensor that responds to extracellular perturbations and couples these events with the activation of latent TGFß. As in the case of a smoke detector, the LLC must be appropriately assembled to function properly. The latent TGFß complex is formed intracellularly and proTGFß that fails to complex with LTBP is inefficiently secreted (Miyazono et al., 1991Go). Furthermore, failure to localize appropriately the latent TGFß complex in the extracellular milieu alters the effectiveness of activation of latent TGFß. Evidence to support this supposition derives from the ability of both inhibiters of tTGase (Kojima and Rifkin, 1993Go) and antibodies raised against LTBP-1 to block the activation of latent TGFß (Flaumenhaft et al., 1993Go; Dallas et al., 1995Go; Nakajima et al., 1997Go; Gualandris et al., 2000Go). In addition, mice that are null for LTBP-3 or LTBP-4 demonstrate phenotypes consistent with altered TGFß signaling (Dabovic et al., 2002Go; Sterner-Kock et al., 2002Go). Specific LTBP isoforms may differentially localize the latent complex, and different LTBP isoforms may preferentially associate with specific TGFß isoforms. In fact, the third 8-cys domain of LTBP-4 is reported to bind only to TGFß1 (Saharinen and Keski-Oja, 2000Go).

As with many sensing devices, the TGFß complex must be made competent to signal (i.e. turned on). Competence requires proteolytic separation of LAP from TGFß (i.e. processing of proLLC into LLC; Fig. 2, step 2). ProLLC cannot be activated by any known mechanism, including heat (85°C for 10 min) or pH (1.5). Although proteolytic cleavage of proTGFß may occur in the Golgi, this is not always the case. For example, multiple glioblastoma cell lines primarily secrete unprocessed proTGFß as part of proLLC (Leitlein et al., 2001Go). To be a substrate for TGFß activation, this proTGFß must be processed at the furin protease site by a plasma-membrane-bound furin or another extracellular protease, such as plasmin [(Lyons et al., 1988Go) our own observation]. Indeed, the addition of furin inhibitors to glioma cultures blocks proTGFß processing. Once pro-TGFß is processed, the complex is `on' (competent), and it can be activated. In our model, we distinguish between the processing of proTGFß (turning the sensor on or making it competent) and activating TGFß. Thus, processing of proTGFß is a regulated step affecting TGFß bioavailability. Furthermore, it is interesting to speculate that proTGFß performs a distinct signaling function from TGFß (perhaps through integrin ligation) similar to the separate signaling capacities of proNGF and NGF (Lee et al., 2001Go).

We propose that the sensing function of the latent TGFß complex resides mainly within LAP. This conclusion is supported by several facts: (1) the known TGFß activators (e.g. plasmin, TSP-1 and {alpha}vß6 integrin) interact directly with LAP (Lyons et al., 1988Go; Ribeiro et al., 1999Go; Munger et al., 1999Go); (2) the physical conditions that release active TGFß (e.g. heat and pH extremes) denature LAP but not TGFß (Lawrence et al., 1985Go); and (3) LAP adopts different conformations in unbound and TGFß1-bound states (McMahon et al., 1996Go). Moreover, the relative lack of amino acid sequence conservation among LAP isoforms compared with TGFß isoforms may provide a mechanism for diversification of TGFß activation. For example, latent TGFß1 and TGFß3 can be activated by {alpha}vß6, whereas TGFß2 cannot (Annes et al., 2002Go; Munger et al., 1999Go). This is due to the presence of the integrin-binding sequence RGD in TGFß1 and three LAPs but not TGFß2 LAP. Sequence analysis reveals only 34-38% amino acid sequence identity among LAP isoforms (LAPß1, ß2, ß3) compared with 75% identity among TGFß isoforms (TGFß1, 2, 3). However, there is considerable conservation of LAP isoform sequences across species (Table 1). The amino acid sequence identity shared by human TGFß1 LAP and chicken TGFß1 LAP is 90% (Table 1). We suggest that the relative lack of conservation between LAP isoforms allows LAPs to act as isoform-specific detectors. The divergence between LAP amino acid sequences may explain, in part, the isoform-specific functions of TGFß in vivo, despite the overlapping expression patterns of the isoforms in vivo and their virtually identical functions in vitro. For example, TGFß1 and TGFß2 mRNAs are the predominant isoforms observed in the mouse heart during endocardial cushion and valvular genesis (Akhurst et al., 1990Go; Millan et al., 1991Go), and both recombinant TGFß1 and TGFß2 function in in vitro assays of endocardial cell transformation (Nakajima et al., 1997Go). However, TGFß2-/- but not TGFß1-/- mice have defects in endocardial and valvular genesis (Sanford et al., 1997Go). Structural differences in LAP may provide a mechanistic basis for activation of TGFß2 and not TGFß1 in this setting.


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Table 1. The amino acid identities among the LAP isoforms of humans, mice and chickens

 

The paradigm of latent TGFß as a sensor also suggests that the response threshold of the latent TGFß complex might be modulated. Although no examples have been reported, the existence of molecules that either bind LAP and prevent an activator from binding or, conversely, alter the conformation of LAP to facilitate recognition by an activating molecule is a likely possibility.


    TGFß activation, or tripping the TGFß sensor
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
A variety of molecules, from protons to proteases, have been described as latent TGFß activators (Fig. 2, steps 5,5'). A commonality among these activators is that they are all indicative of ECM perturbations. Indeed, given the profound effects of TGFß on matrix homeostasis, the primary change that the TGFß sensor detects may be alterations in the matrix. In this section we discuss some of the known TGFß activators.

Proteolytic activation of latent TGFß
A number of proteases including plasmin, MMP-2 and MMP-9 have been identified in vitro as latent TGFß activators (Sato and Rifkin, 1989Go; Yu and Stamenkovic, 2000Go). Plasmin and MMP2/9 belong to the serine protease and metalloprotease families, respectively. These protease families, along with the adamalysin-related membrane proteinases, are the primary enzymes involved in ECM degradation (Werb, 1997Go). The ability of these enzymes to activate the latent TGFß complex couples matrix turnover with the production of a molecule, TGFß, that has a primary role in maintaining matrix integrity and stability (Ignotz and Massague, 1986Go; Verrecchia et al., 2001Go). There are three ways in which proteases might facilitate the activation of latent TGFß. First, the protease-sensitive hinge region in LTBP is a potential target for the liberation of a still-latent remnant of the LLC, which would have to be further processed for activation (Taipale et al., 1994Go). Second, as discussed above, proteases can act in the extracellular environment to convert proLLC to LLC and thereby render the latent complex activation competent. Third, proteolytic cleavage of LAP, resulting in destabilization of LAP-TGFß interactions, might release active TGFß from its latent complex (Lyons et al., 1988Go). Degradation of LAP is an attractive mechanism for sensor activation because heightened levels of proteases are associated with several processes that involve increased TGFß activation. However, thus far, mice that have null mutations in the genes that encode the known activating proteases do not demonstrate any phenotype consistent with TGFß deficiency. This may reflect redundancy among the activating enzymes or the fact that these mice have not been studied in the correct context.

Activation by thrombospondin-1
The matricellular protein TSP-1 activates latent TGFß (Schultz-Cherry and Murphy-Ullrich, 1993Go). The mechanism involves a direct interaction between TSP-1 and LAP (reviewed by Murphy-Ullrich and Poczatek, 2000Go). A short amino acid sequence (RFK) located between the first and second type 1 properdin-like repeats is believed to be responsible for latent TGFß activation. Surprisingly, a tetrapeptide (KRFK) also functions as a TGFß activator in vitro and in vivo (Crawford et al., 1998Go). This peptide probably acts by disrupting the non-covalent interactions between LAP and TGFß. Interestingly, TSP-1 null mice demonstrate a partial phenotypic overlap with TGFß1-null animals, thereby supporting the contention that TSP-1 is an in vivo activator of latent TGFß (Crawford et al., 1998Go). TSP-1 facilitates wound repair in several ways: modulation of cell adhesion, promotion of angiogenesis, and reconstruction of the matrix (Frazier, 1991Go). The correlation between wounding and enhanced TSP-1 expression suggests that TSP-1 is an appropriate molecule for activation of the latent complex, since TGFß plays a prominent role in wound healing (Border and Ruoslahti, 1992Go). TSP-1 is also expressed throughout development in a number of tissues, where it may function as a TGFß activator (Iruela-Arispe et al., 1993Go; Majack et al., 1987Go).

Activation by integrins
Integrins are dimeric cell surface receptors composed of {alpha} and ß subunits (reviewed by van der Flier and Sonnenberg, 2001Go). The first integrin to be identified as a TGFß activator was {alpha}vß6 (Munger et al., 1999Go). The mechanism of activation depends upon a direct interaction between {alpha}vß6 and the RGD amino acid sequence present in LAP ß1 and LAP ß3 (Fig. 1). The expression of {alpha}vß6 is restricted to epithelia, and in most epithelia the integrin is normally expressed at low levels (Breuss et al., 1993Go). In response to wounding or inflammation, the expression of {alpha}vß6 increases (Breuss et al., 1995Go; Miller et al., 2001Go). Therefore, epithelial cell upregulation of {alpha}vß6 and subsequent TGFß activation is a situation in which the cellular response to a process (inflammation) produces a potent suppressor of that process. Consistent with both the ability of ß6 integrin to activate latent TGFß and the pro-fibrotic effects of TGFß (Border and Ruoslahti, 1992Go) is the observation that wild-type mice develop pulmonary inflammation followed by fibrosis in response to the inflammatory and profibrotic drug bleomycin, but integrin ß6-/- mice have only a minor fibrotic response (Munger et al., 1999Go). In addition, global analysis of gene expression in the lungs of integrin ß6-/- mice treated with bleomycin compared with similarly treated wild-type mice demonstrates a pronounced failure to induce expression of TGFß-regulated genes in the mutant mice. These results indicate that fibrosis is the result of excess TGFß produced by heightened expression of {alpha}vß6 in response to the inflammatory stimulus. Since TGFß dramatically increases the generation of {alpha}vß6 by primary airway epithelial cells in vitro (Wang et al., 1996Go), it is likely that bleomycin triggers a feed-forward mechanism for coordinately up-regulating integrin expression and TGFß generation. We suggest that fibrosis is the result of a failure to interrupt this feed-forward loop that is perpetuated by persistent ECM perturbation after wounding or inflammation.

Recently, Mu et al., reported that the integrin {alpha}vß8 can activate latent TGFß1 (Mu et al., 2002Go). It is interesting that activation by {alpha}vß8 requires protease (MT1-MMP) activity in addition to the integrin. Although the exact roles of MT1-MMP and {alpha}vß8 in this activation mechanism remain to be elucidated, the authors suggest that the integrin concentrates latent TGFß on the cell surface, where it is subsequently activated by MT1-MMP. A cooperative interaction between different classes of latent TGFß activator has been suggested previously (Yehualaeshet et al., 1999Go): the cell-surface-associated proteins (CD36 and TSP-1) concentrate latent TGFß on the membrane where it is subsequently activated by plasmin.

Activation by reactive oxygen species (ROS)
Barcellos-Hoff and her co-workers showed that when ROS are produced in vitro (either by ionizing radiation or a metal-catalyzed ascorbate system) or in vivo after irradiation, latent TGFß1 is activated (Barcellos-Hoff et al., 1994Go; Barcellos-Hoff and Dix, 1996Go). This is probably a result of scissions and side group modifications caused by hydroxyl radicals that disable LAP. The response of the TGFß sensor to certain types of oxidative stress may reflect a need to produce TGFß during processes such as inflammation and apoptosis that can cause ECM damage through the production of ROS.

Activation by pH
Latent TGFß present in conditioned medium is activated by mild acid treatment (pH 4.5) (Lyons et al., 1988Go), which probably denatures LAP, thereby disturbing the interaction between LAP and TGFß. In vivo, a similar pH is generated by osteoclasts during bone resorption when an integrin-dependent sealing zone is generated between the bone and the cell (Teitelbaum, 2000Go). Since the bone matrix deposited by osteoblasts is rich in latent TGFß, the acidic environment created by osteoclasts in vitro might result in latent TGFß activation (Oreffo et al., 1989Go; Oursler, 1994Go).


    TGFß biology and the role of the sensor
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
The evidence that TGFß is released in a latent form and must be activated is derived primarily from in vitro studies. There is little in vivo evidence demonstrating a requirement for latent TGFß activation for several reasons, including the fact that measurement of changes in active TGFß levels in tissues or animals is extremely difficult. In this section we discuss the in vivo evidence supporting the importance of extracellular TGFß activation by examining the phenotypes of animals or people in whom specific steps in the post-translational assembly and/or processing of latent TGFß are defective (Fig. 2). By incorporating the sensor model into our analysis, we have arrived at new interpretations of these phenotypically complex situations.

The effect of improper LLC assembly is illustrated in the phenotypes of mice that have null mutations in the LTBP-3 or LTBP-4 genes. LTBP-3-/- mice display bone phenotypes including osteoarthritis and osteopetrosis (Dabovic et al., 2002Go), which also occur in mice that have defective TGFß signaling pathways resulting from either mutations in Smad3 (osteoarthritis) (Yang et al., 2001Go) or the expression of a dominant negative type II TGFß receptor in osteoblasts (osteopetrosis) (Filvaroff et al., 1999Go). LTBP-4-/- mice develop pulmonary emphysema, cardiac myopathy and colorectal cancer (Sterner-Kock et al., 2002Go). It is interesting that the defects in LTBP-4-/- animals are consistent with both increased and decreased TGFß activity: (1) emphysema has been associated with both increased and decreased TGFß activity (Kaartinen et al., 1995Go; Zhou et al., 1996Go); (2) cardiac myopathy is associated with increased TGFß activity (Schultz Jel et al., 2002Go); and (3) colorectal cancer is associated with a lack of TGFß activity (reviewed by Gold, 1999Go). Thus, the phenotypes displayed by the LTBP-mutant mice are not necessarily described by a simple deficit in TGFß.

Does consideration of TGFß biology in terms of the sensor model clarify aspects of this situation? In the absence of a specific LTBP, TGFß may be (a) inefficiently secreted and unable to localize to the ECM or (b) secreted in a complex with a different LTBP, presuming the cell expresses more than one LTBP isoform. According to the sensor model, these scenarios have varying effects on TGFß activity. Whereas decreasing TGFß secretion results in less TGFß activity, eliminating or changing the isoform of LTBP is predicted to modulate the localization and/or activation pattern of the complex in a context-dependent manner. Therefore, it is not accurate to say that there is more or less TGFß in these LTBP-null mice; rather, the distribution and timing of TGFß activities may be modified. For instance, LTBP-3-null mice have increased bone density, which is similar to transgenic mice expressing a dominant negative type II TGFß receptor under control of the osteocalcin promoter, but TGFß1-null mice become osteoporotic rather than osteopetrotic as they age (Geiser et al., 1998Go). It is likely that the LTBP-3-/- phenotype emphasizes the effect of altered local distribution of a TGFß in a cell or tissue type, whereas the TGFß1-null phenotype illustrates the result of a global loss of the cytokine.

A localization defect can occur not only when there is a defect in LLC assembly but also if there is an alteration in ECM binding. This might occur if the binding partner for LTBP is missing or defective or if tTGase, which cross-links LLC to the matrix, is absent. However, mice with a null mutation in the TGase2 gene do not display a phenotype consistent with a global deficit in TGFß (Nanda et al., 2001Go; De Laurenzi and Melino, 2001Go). This may indicate the existence of redundant TGases. We suggest that closer examination will reveal TGFß-related changes in those tissues or cells that depend exclusively upon TGase2 for fixing of the sensor into the ECM.

An example of a human pathology related to altered TGFß latency is Camurati-Engelmann disease (CED). This autosomal dominant disease results from mutations in the TGFß1 LAP sequence and is characterized by hyperostosis and sclerosis of the base of the skull and long bones, respectively (Janssens et al., 2000Go; Kinoshita et al., 2000Go; Nishimura et al., 2002Go). Most of the mutations in CED occur at or close to the cysteine residues involved in the interchain bonds of the LAP dimer. Earlier work with mutated TGFß cDNAs indicated that proper disulfide bond formation is required to produce latent TGFß, because mutation of C223 and C225 yields constitutively active TGFß (Brunner et al., 1989Go). Studies with fibroblasts from three patients with CED mutations at or close to C225 indicate that the mutant cells produce substantially more active TGFß1 than do wild-type cells (Saito et al., 2001Go). Why the CED cells generate enhanced levels of active TGFß is not clear, since disulfide bonds between the appropriate cysteine residues do form; however, the answer to this question may be clarified by consideration of the available data on CED in terms of the sensor model of latent TGFß.

There are curious differences between the TGFß produced by wild-type and CED fibroblasts. First, CED and normal cells produce similar amounts of total TGFß1 as judged by TGFß1 LAP immunoblotting; however, after acid activation of the latent TGFß, medium conditioned by CED cells contains five times the amount of active TGFß1 compared with medium conditioned by normal cells (Saito et al., 2001Go). Thus, there is a discrepancy between the amounts of immunoreactive and biologically active TGFß1 produced by the two cell types. Second, there is a difference in the degree of proteolytic processing of proTGFß1 by CED fibroblasts compared with wild-type cells (Saito et al., 2001Go). Whereas wild-type cells produce substantial amounts of unprocessed proTGFß1, CED cells process all of the proTGFß1 to LAP and TGFß1. According to the sensor model, all of the latent complex produced by CED, but not wild-type, cells is in an activation-competent state (i.e. the CED LLC is `on' because it has been proteolytically cleaved) (Fig. 2, step 2). This is in contrast to the primarily proTGFß1 produced by wild-type cells. This form of TGFß is considered to be `off' and cannot be activated by any known mechanism. Our definition of `on' or competent latent TGFß clarifies why there is significantly more TGFß activity in CED, compared with wild-type, conditioned medium following acid activation, despite the fact that the cells secrete equal amounts of the TGFß propeptide. Apparently, the CED mutation alters the susceptibility of LAP to proteolysis by furin and or other processing proteases. It is interesting to speculate that this same conformational change might make LAP more sensitive to activating proteolytic events. Therefore, we suggest that the latent TGFß complex of CED individuals is assembled and localized normally but is hyper-responsive.

Two reports indicate that altered expression of molecules that activate the latent complex result in pathologies. The first report describes lung fibrosis after bleomycin treatment (Munger et al., 1999Go). In this example, fibrosis is impaired in mice missing ß6 integrin, an activator important for generating TGFß during inflammatory states (Munger et al., 1999Go). A second example is the developmental pulmonary emphysema observed in fibrillin-1-hypomorphic mice (E. R. Neptune, P. A. Frischmeyer, D. A. Arking et al., personal communication). These animals have a defect in the terminal septation of the alveoli that correlates with excess of both TGFß and TGFß signaling. It is likely that the defect in terminal alveolar septation in these mice is due to excess TGFß, because higher levels of TGFß activity were detected in the lungs of mutant animals, and the administration of TGFß-neutralizing antibodies reverses the pathology. The lack of fibrillin might result in defective localization of LLC and subsequent TGFß activation, because the LLC normally localizes with fibrillin-1 (Taipale et al., 1996Go). Thus, the abnormal distribution of LLC results in inappropriate activation. An additional explanation as to why fibrillin-1-/- mice have altered TGFß levels is revealed through consideration of latent TGFß as a sensor. We propose that the altered ECM of the mutant mice cues cells to remodel the matrix and that this remodeling is associated with the inappropriate and persistent expression of a TGFß activator.


    Conclusion
 Top
 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 
We have conceptualized latent TGFß as an ECM-localized sensor in order to unify our current understanding of TGFß biology. In our model, the sensor comprises a localizer (LTBP), a detector (LAP) and an effector (TGFß). Failure to localize latent TGFß appropriately results in altered TGFß activity. The role of the latent TGFß complex in coordinating ECM perturbation with ECM reorganization is emphasized if one considers latent TGFß as a sensor that responds to ECM damage or other extracellular perturbations. The storage of latent TGFß in the ECM provides a mechanism for spatially and temporally linking perturbation with restructuring. The sensor model provides a framework for understanding the complex and varied nature of TGFß activity: the primary role of TGFß is to `report' an alteration of the extracellular milieu and initiate a response.

The sensor model clearly separates two aspects of TGFß biology that are often misunderstood: the processing of the proTGFß (turning the sensor `on') and the liberation of TGFß from the latent complex. Visualizing the latent TGFß complex as a sensor has offered insight into the somewhat confusing results reported for Camurati-Engelmann syndrome. Moreover, the consideration of active TGFß formation in terms of a matrix-localized sensor makes it easier to imagine the existence of accessory molecules that interact with the sensor and either potentiate or dampen activation as well as the context-specific use of or localization by specific LTBP forms. In addition, a commonality of TGFß activators is made apparent by representing TGFß activation as a process involving sensor detection: all identified TGFß activators are associated with ECM perturbation. Finally, the latent TGFß sensor could allow the activities of the three nearly identical TGFß cytokines to be distinguished, in part, through a diversity in LAP sequences that permits differential response to individual activators. By viewing latent TGFß as a matrix-localized sensor, we can understand TGFß assembly, latency, activation and activity as coordinated events rather than as disparate aspects of TGFß biology.


    Acknowledgments
 
The authors thank A. Roberts, R. Derynk and B. Dabovic for critical readings of the manuscript. This work was supported by NIH grants HL 63786 (J.S.M.), CA34282, CA78422 and DE13742 (D.B.R.), and T32 GM07308 (J.P.A.) and Sonneborn Fund (J.S.M.).


    Footnotes
 
{dagger} We refer to the N-terminal sequence of the TGFß proprotein (proTGFß) as either the TGFß propeptide or LAP. We also distinguish between two forms of LLC; LLC consists of LTBP, TGFß and LAP, whereas complexes that contain LTBP plus proTGFß are called proLLC. Back


    References
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 Summary
 Introduction
 The components and assembly...
 The latent TGFß...
 TGFß activation, or...
 TGFß biology and the...
 Conclusion
 References
 

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