Latent Complex Research Articles

Extracellular regulation of growth factor signaling by fibrillin microfibrils

Fibrillins regulate large latent complexes of TGFβ through interactions with Latent TGFβ Binding Proteins (LTBPs). These interactions are mediated by a single region in fibrillin‐1 that contains the first hybrid domain. In Fbn1 mutant mice, TGFβ signaling is activated, demonstrating that fibrillin microfibrils regulate TGFβ signaling in vivo. Fibrillins also interact directly with the prodomains of Bone Morphogenetic Proteins (BMPs) and target and sequester BMP‐4, BMP‐5, and BMP‐7 into the extracellular matrix. In the skeletal muscle of Fbn2 null mice, BMP signaling is activated, and skeletal muscle is poorly differentiated. These studies indicate that fibrillin microfibrils control BMP signaling in vivo. In order to investigate the molecular mechanisms underlying the regulation of growth factor signaling in more detail, we have generated two additional Fbn1 mouse models. Investigations of mice in which fibrillin‐1 is truncated and eGFP‐tagged will be discussed. This "gain of function" mouse model will be compared with Fbn1 null mice. In addition, the effect of deleting the first hybrid 1 domain in fibrillin‐1 on TGFβ signaling will be reported.

Active TGF‐β1 correlates with myofibroblasts and malignancy in the colorectal adenoma‐carcinoma sequence

Transforming growth factor-beta1 (TGF-beta1), a cytokine involved in various stages of cancer, is produced as a latent complex and requires processing to become active. We have determined total and active TGF-beta1 levels in homogenates of colorectal neoplasia. In contrast to total TGF-b levels, showing a stepwise increase in the mucosa-adenoma-carcinoma sequence, active TGF-beta1 levels are increased only in carcinomas but not in premalignant adenomas. Furthermore, solely active TGF-beta1 levels are associated with the stage of the carcinomas and worse patient prognosis. Active TGF-beta1 levels correlated significantly with plasminogen activator inhibitor (PAI)-1, alpha-smooth muscle actin (SMA) and several matrix-remodeling proteinases. Interestingly, SMA levels are also significantly increased in colorectal carcinomas but not in adenomas, suggesting that despite the enhanced total TGF-beta1 levels, myofibroblast accumulation is not (yet) occurring in these premalignant neoplasias. The correlation between active TGF-beta1 and SMA expression in tumors indicates that tumor-promoting myofibroblasts might arise as a result of increased TGF-beta1 activation. These data underline the significance of the interaction between malignant cells and (myo)-fibroblasts in the tumor microenvironment, modulating the biologic behavior of colorectal cancer.

P38 MAPK Is an Early Determinant of Promiscuous Smad2/3 Signaling in the Aortas of Fibrillin-1 (Fbn1)-null Mice

Excessive transforming growth factor-beta (TGF-beta) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-beta complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-beta-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome.

Preparation from bovine colostrum of a fraction rich in latent transforming growth factor-β2 and cleared of most allergenic proteins by one-step Cibacron Blue chromatography

A strategy was developed for the preparation of a latent transforming growth factor-β2 (latent TGF-β2) rich fraction from bovine colostrum. Different conventional chromatography techniques were tested to investigate some physicochemical properties of latent TGF-β2 complexes to optimize their recovery. Three latent TGF-β2 forms were detected by gel filtration (600, 150–250 and 65–150 kDa). Cation exchange chromatography showed that the p I of latent TGF-β2 was above pH 7; hydrophobic interaction chromatography showed that the hydrophobicity of latent TGF-β2 was much higher than that of other whey proteins. Affinity chromatography on Cibacron Blue 3G was used to separate latent TGF-β2 and immunoglobulin G from the most immunogenic proteins, α-lactalbumin, β-lactoglobulin and albumin. Optimum conditions were determined: preparation of a fraction 26 times richer in TGF-β2 than in serocolostrum was obtained with a good yield (up to 70%) of TGF-β2, 80% of which was in the latent form.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

Perturbation of transforming growth factor (TGF)-beta1 association with latent TGF-beta binding protein yields inflammation and tumors.

Transforming growth factor-beta (TGF-beta) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-beta is often released as part of an inactive tripartite complex consisting of TGF-beta, the TGF-beta propeptide, and a molecule of latent TGF-beta binding protein (LTBP). The interaction of TGF-beta and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-beta from this state is crucial for signaling. To examine the contribution of LTBP to TGF-beta function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1(C33S/C33S) mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-beta1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1(-/-) animals. Tgfb1(C33S/C33S) mice exhibited decreased levels of active TGF-beta1, decreased TGF-beta signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-beta complex is important for proper TGF-beta1 function and that Tgfb1(C33S/C33S) mice are hypomorphs for active TGF-beta1. Moreover, although mechanisms exist to activate latent TGF-beta1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.

Lysyl Oxidase Binds Transforming Growth Factor-; and Regulates Its Signaling via Amine Oxidase Activity

Lysyl oxidase (LOX), an amine oxidase critical for the initiation of collagen and elastin cross-linking, has recently been shown to regulate cellular activities possibly by modulating the functions of growth factors. In this study, we investigated the interaction between LOX and transforming growth factor-beta1 (TGF-beta1), a potent growth factor abundant in bone, the effect of LOX on TGF-beta1 signaling, and its potential mechanism. The specific binding between mature LOX and mature TGF-beta1 was demonstrated by immunoprecipitation and glutathione S-transferase pulldown assay in vitro. Both proteins were colocalized in the extracellular matrix in an osteoblastic cell culture system, and the binding complex was identified in the mineral-associated fraction of bone matrix. Furthermore, LOX suppressed TGF-beta1-induced Smad3 phosphorylation likely through its amine oxidase activity. The data indicate that LOX binds to mature TGF-beta1 and enzymatically regulates its signaling in bone and thus may play an important role in bone maintenance and remodeling.

REBUS‐PLS: A response‐based procedure for detecting unit segments in PLS path modelling

AbstractStructural equation models (SEMs) make it possible to estimate the causal relationships, defined according to a theoretical model, linking two or more latent complex concepts, each measured through a number of observable indicators, usually called manifest variables. Traditionally, the component‐based estimation of SEMs by means of partial least squares (PLS path modelling, PLS‐PM) assumes homogeneity over the observed set of units: all units are supposed to be well represented by a unique model estimated on the overall data set. In many cases, however, it is reasonable to expect classes made of units showing heterogeneous behaviours to exist.Two different kinds of heterogeneity could be affecting the data: observed and unobserved heterogeneity. The first refers to the case of a priori existing classes, whereas in unobserved heterogeneity no information is available either on the number of classes or on their composition.If a group structure for the statistical units is given, the aim of the analysis is to search for any differences in the behaviours of the a priori given classes. In PLS‐PM this would mean studying the effect of directly observed moderating variables, i.e. estimating as many (local) models as there are classes.Unobserved heterogeneity, instead, implies identifying classes of units (a priori unknown) having similar behaviours. Such heterogeneity is captured by an unobserved (latent) discrete moderating variable defining both the number of classes and the class membership.A new method for unobserved heterogeneity detection in PLS‐PM is proposed in this paper: response‐based procedure for detecting unit segments in PLS‐PM (REBUS‐PLS). REBUS‐PLS, according to PLS‐PM features, does not require distributional hypotheses and may lead to local models that are different in terms of both structural and measurement models. An application of REBUS‐PLS on real data will be shown. Copyright © 2008 John Wiley & Sons, Ltd.

Spreading tolerance by converting

Unlike some religious leaders, suppressive T cells need more than just charisma to spread messages of tolerance: the cells rely on a cell surface cytokine complex to convert others, according to Andersson et al. (page 1975). Figure 1 Naive-turned-suppressor cells dampen colitis (left). Regulatory T (T reg) cells suppress other T cells in part via the cytokine TGF-β, which can convert naive CD4+ T cells into suppressor cells. Andersson et al. now find that naive CD4+ T cells turn into suppressor cells only when they bump into T reg cells that have an inactive TGF-β complex on their surface. Proteolysis of the inactive complex, which liberates soluble protein, was required for conversion. Inhibiting the protease or swamping the cells with inactive complex prevented cleavage and thus conversion. The converted cells could suppress gut inflammation when transferred into mice suffering from colitis. Other immune cells also secrete soluble TGF-β, but they do not seem to force conversion—at least in this system. Previous experiments showed that naive T cells cultured with T reg cell–depleted spleen cells, which secrete TGFβ, fail to convert. Non-T reg cells don't express the inactive surface complex, so perhaps its cleavage is needed to trigger other conversion-supporting signals. The latent TGF-β complex was present only on T reg cells that had been stimulated via their T cell receptors. This specificity might allow T reg cells to limit conversion by delivering the TGF-β signal only to T cells bound to the same antigen-presenting cell and thus on the verge of being activated. “We wouldn't want T reg cells to run amok and convert all naive T cells,” says lead author Ethan Shevach. How often T reg cells rely on this mechanism and its impact on the induction of tolerance in vivo are unknown.

Latent TGF-β binding proteins (LTBPs)-1 and -3 coordinate proliferation and osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) possess the capability to differentiate into bone forming cells, osteoblasts, and thus represent a new therapeutic tool in regenerative medicine. Transforming growth factor (TGF)-β is abundantly present in bone tissue where it regulates osteoblast and osteoclast functions in a complex manner. Latent TGF-β binding protein (LTBP)-1 mediates the extracellular matrix (ECM) targeting and accumulation of most TGF-β in the bone. We describe here an important regulatory role for LTBP-3 in TGF-β activation and autocrine growth control in MSCs. LTBP-3 knockdown via siRNA mediated silencing resulted in reduced cell proliferation and reduced osteogenic differentiation. When MSCs were induced to undergo differentiation, LTBP-3 levels became downregulated in parallel with reduced TGF-β activation. These changes coincided with the matrix maturation phase of osteogenic differentiation. The mechanism of LTBP-3 is most likely via TGF-β activation in the early proliferative phase of the differentiation process. Later, when TGF-β activity would inhibit further maturation and mineralization, LTBP-3 expression becomes downregulated and LTBP-1 containing large latent TGF-β1 complexes accumulate into the ECM. These complexes represent readily available targets for osteoclast mediated release and activation of TGF-β in bone tissue. Our results provide evidence that LTBP isoforms can differentially regulate TGF-β activation and ECM accumulation during osteogenic differentiation.

Structure/function analysis of LTBP-1

The latent TGF-β binding proteins are multidomain extracellular matrix glycoproteins, comprising repeating units of EGF-like, calcium-binding EGF-like, eight-cysteine, four-cysteine and RGD motifs (Sinha et al. 1998). Four LTBP isoforms have been isolated, sharing sequence identity and domain homology with the fibrillin family of extracellular matrix proteins. LTBP-1 was first characterized as a 125-kD component of the large latent TGF-β complex secreted from platelets (Wakefield et al. 1988; Kanzaki et al. 1990) in which LTBP-1 is linked by a disulphide bond to the TGF-β propeptide, which remains noncovalently associated with TGF-β until activated (Saharinen et al. 1996; Gleizes et al. 1996). Four alternatively spliced variants of LTBP-1 have been identified and have been designated LTBP-1S, LTBP-1L and LTBP-1Δ53. The LTBP-1S cDNA encodes 1394 amino acids (Kanzaki et al. 1990). LTBP-1L encodes 1740 amino acids, arising as a result of a splicing event at codons 145–146 of LTBP-1S and incorporating an amino terminal extension of 346 highly basic amino acids (Olofsson et al. 1995). The LTBP-1Δ53 isoforms lack 53 amino acids from the first eight-cysteine motif (Okluet al. 1998) and a ‘hinge’ region (Gong et al. 1998). We are especially interested in the differential expression and deposition of the LTBP-1S and 1L isoforms in the extracellular matrix, their molecular composition, and the matrix molecules that they might target and bind to.

MT1-MMP releases latent TGF-β1 from endothelial cell extracellular matrix via proteolytic processing of LTBP-1

Targeting of transforming growth factor beta (TGF-β) to the extracellular matrix (ECM) by latent TGF-β binding proteins (LTBPs) regulates the availability of TGF-β for interactions with endothelial cells during their quiescence and activation. However, the mechanisms which release TGF-β complexes from the ECM need elucidation. We find here that morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) resulted in membrane-type 1 matrix metalloproteinase (MT1-MMP) -mediated solubilization of latent TGF-β complexes from the ECM by proteolytic processing of LTBP-1. These processes required the activities of PKC and ERK1/2 signaling pathways and were coupled with markedly increased MT1-MMP expression. The functional role of MT1-MMP in LTBP-1 release was demonstrated by gene silencing using lentiviral short-hairpin RNA as well as by the inhibition with tissue inhibitors of metalloproteinases, TIMP-2 and TIMP-3. Negligible effects of TIMP-1 and uPA/plasmin system inhibitors indicated that secreted MMPs or uPA/plasmin system did not contribute to the release of LTBP-1. Current results identify MT1-MMP-mediated proteolytic processing of ECM-bound LTBP-1 as a mechanism to release latent TGF-β from the subendothelial matrix.

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.

NF-κB p52, RelB and c-Rel are transported into the nucleus via a subset of importin α molecules

In resting cells NF-κB transcription factors are retained in the cytoplasm as latent inactive complexes, until they are activated and rapidly transported into the nucleus. We show that all NF-κB proteins are imported into the nucleus via a subset of importin α isoforms. Our data indicate that the NF-κB components of the classical and alternative pathways have somewhat different specifities to importin α molecules. Based on the results from binding experiments of in vitro-translated and Sendai virus infection-induced or TNF-α-stimulated endogenous NF-κB proteins, it can be predicted that the specifity of NF-κB proteins to importin α molecules is different and changes upon the composition of the imported dimer. p52 protein binds directly to importin α3, α4, α5 and α6 and c-Rel binds to importin α5, α6 and α7 via a previously described monopartite nuclear localization signals (NLSs). Here we show that RelB, instead, has a bipartite arginine/lysine-rich NLS that mediates the binding of RelB to importin α5 and α6 and subsequent nuclear translocation of the protein. Moreover, we show that the nuclear import of p52/RelB heterodimers is mediated exclusively by the NLS of RelB. In addition, we found that the NLS of p52 mediates the nuclear import of p52/p65 heterodimers.

Induction of an Epithelial Integrin αvβ6 in Human Cytomegalovirus-Infected Endothelial Cells Leads to Activation of Transforming Growth Factor-β1 and Increased Collagen Production

Human cytomegalovirus (CMV) infection is a major cause of morbidity in immunosuppressed individuals, and congenital CMV infection is a leading cause of birth defects in newborns. Infection with pathogenic viral strains alters cell-cell and cell-matrix interactions, affecting extracellular matrix remodeling and endothelial cell migration. The multifunctional cytokine transforming growth factor (TGF)-beta1 regulates cell proliferation, differentiation, and extracellular matrix remodeling. Secreted as a latent protein complex, TGF-beta1 requires activation before binding to receptors that phosphorylate intracellular effectors. TGF-beta1 is activated by integrin alphavbeta6, which is strongly induced in the epithelium by injury and inflammation but has not previously been found in endothelial cells. Here, we report that CMV infection induces integrin alphavbeta6 expression in endothelial cells, leading to activation of TGF-beta1, signaling through its receptor ALK5, and phosphorylation of its intracellular effector Smad3. Infection of endothelial cells was also found to stimulate collagen synthesis through a mechanism dependent on both TGF-beta1 and integrin alphavbeta6. Immunohistochemical analysis showed integrin alphavbeta6 up-regulation in capillaries proximal to foci of CMV infection in lungs, salivary glands, uterine decidua, and injured chorionic villi of the placenta, demonstrating both its induction in endothelium and up-regulation in epithelium in vivo. Our results suggest that activation of TGF-beta1 by integrin alphavbeta6 contributes to pathological changes and may impair endothelial cell functions in tissues that are chronically infected with CMV.

Tryptase activates TGFβ in human airway smooth muscle cells via direct proteolysis

Transforming growth factor β (TGFβ) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFβ bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFβ from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139–2141]. The aim of this study was to determine if tryptase could cause TGFβ activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFβ activation without affecting levels of total TGFβ. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH2. Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFβ via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.

Integrins and the activation of latent transforming growth factor β1 – An intimate relationship

Integrins are crucial for the ability of cells to sense mechanical perturbations and to transmit intracellular stress to their environment. We here review the more recently discovered role of integrins in activating the pleiotrophic cytokine transforming growth factor beta 1 (TGF- β1). TGF- β1 controls tissue homeostasis in embryonic and normal adult tissues and contributes to the development of fibrosis, cancer, autoimmune and vascular diseases when being mis-regulated. In most of these conditions, active TGF- β1 is generated by dissociation from a large latent protein complex that sequesters latent TGF- β1 in the extracellular matrix (ECM). Two main models are proposed how integrins contribute to latent TGF- β1 activation: (1) In a protease-dependent mechanism, integrins αv β8 and αv β3 are suggested to simultaneously bind the latent TGF- β1 complex and proteinases. This close vicinity at the cell surface improves enzymatic cleavage of the latent complex to release active TGF- β1. (2) Integrins αv β3, αv β5, αv β6, and αv β8 appear to change the conformation of the latent TGF- β1 complex by transmitting cell traction forces. This action requires association of the latent complex with a mechanically resistant ECM and is independent from proteolysis. Understanding that different integrins use different mechanisms to activate latent TGF- β1 opens new possibilities to develop cell-specific therapeutic strategies for TGF- β1-induced pathologies.

Activation of Transforming Growth Factor β(TGFβ)by interaction of Matrix Metalloproteinase 13 (MMP‐13) with the large latent complex

Dysregulated TGFβ activation has been indicated in the pathology of osteoarthritis. We have reported that hypertrophic chondrocytes produce large quantities of activated TGFβ and a large latent complex of TGFβ that contains MMP‐13 in non‐covalent association. Bioinformatics revealed that the hemopexin domains of MMP‐13 can non‐covalently interact with the calcium‐EGF‐like domains of Latent TGF Binding Protein (LTBP1), the extracellular matrix anchor protein for the TGFβ large latent complex. The catalytic domain of MMP‐13 is in alignment with the protease‐sensitive hinge region of LTBP1, the site of release of this molecule from the extracellular matrix, suggesting a novel mechanism of TGFβ large latent complex release. To test this proposed interaction, we designed peptides of the MMP‐13 hemopexin domain that are most likely to interact with LTBP1. Tissue extract of avian hypertrophic cartilage bound five times more MMP‐13 peptide than extracts from resting zone cartilage. In addition, rat tibial growth plate cartilage immunoprecipitated with antibody to LTBP1 demonstrated four times more binding affinity for MMP‐13 peptide than the scrambled peptide control. These data support the specific interaction between MMP‐13 and LTBP1 predicted in our bioinformatics model and our proposed model of the mechanism of release of the TGFβ large latent complex by MMP‐13. Sponsored in part by a grant from the CCDA.

Genetic Analysis of the Role of Proteolysis in the Activation of Latent Myostatin

Myostatin is a secreted protein that normally acts to limit skeletal muscle growth. As a result, there is considerable interest in developing agents capable of blocking myostatin activity, as such agents could have widespread applications for the treatment of muscle degenerative and wasting conditions. Myostatin normally exists in an inactive state in which the mature C-terminal portion of the molecule is bound non-covalently to its N-terminal propeptide. We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases. Here, I show that mice engineered to carry a germline point mutation rendering the propeptide protease-resistant exhibit increases in muscle mass approaching those seen in mice completely lacking myostatin. Mice homozygous for the point mutation have increased muscling even though their circulating levels of myostatin protein are dramatically increased, consistent with an inability of myostatin to be activated from its latent state. Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members. These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.