Activation Of Latent TGF-beta Research Articles

TGF‐beta Regulation by the Matricellular Protein Thrombospondin 1

Thrombospondin 1 (TSP1) is a matricellular protein that plays multiple roles in disease. Activation of the TGF-beta latent complex is a major regulatory node for controlling TGF-beta in homeostasis and disease. TSP1 can regulate the conversion of latent transforming growth factor-beta (TGF-beta) to its biologically active form in certain fibrotic diseases and cancers. We have developed peptide and small molecule antagonists that block TSP1 binding to the latent complex and inhibit TSP1-dependent activation of latent TGF-beta. These antagonists reduce TGF-beta activity and extracellular matrix production and fibrosis in multiple rodent models, particularly in fibrotic complications of diabetes. TGF-beta also plays a role in the osteolytic bone destruction characteristic of the plasma cell cancer, multiple myeloma. In both immune competent syngeneic mouse and human xenograft models, we showed that treatment with the TSP1-TGF-beta antagonist reduced disease burden, TGF-beta activity, and prevented bone destruction via a decrease in osteoclasts and an increase in osteoblasts. Current studies are examining the role of the TSP1-TGF-beta pathway in immune dysregulation in multiple myeloma.

Transcriptional activation of urokinase by the Krüppel-like factor Zf9/COPEB activates latent TGF-β1 in vascular endothelial cells

Understanding the regulation of genes controlling fibrinolysis and matrix homeostasis is essential for elucidating the basis of tissue repair. A recently described novel Krüppel-like factor, Zf9, is up-regulated in acute liver injury in activated hepatic stellate cells. Because Zf9 can be induced widely, its activity was examined in vascular endothelium, a key cell in vascular injury. Zf9 is induced as an immediate-early response gene in bovine aortic endothelial cells (BAECs) following treatment with serum or phorbol ester. Zf9 transcriptionally activates urokinase plasminogen activator (uPA). Recombinant Zf9-GST binds to wild-type but not mutated 'GC-box' motifs within the human uPA promoter (-63 to -32), with greatest affinity to the middle of 3 contiguous GC boxes. Transient transfection of Zf9 drives transactivation of a full-length uPA promoter- and GC box-construct, but not a uPA promoter-construct devoid of GC boxes. Transactivation of uPA by Zf9 is also supported in Drosophila S2 cells. Most importantly, transiently transfected Zf9 up-regulates endogenous uPA messenger RNA and activity in BAECs, resulting in increased bioactive transforming growth factor-beta (TGF-beta) via enhancement of proteolytic activation of the latent molecule. Furthermore, concomitant expression of Zf9 and uPA proteins was observed in arterial endothelial cells after balloon injury in rats, suggesting a potential role of Zf9 in uPA expression not only in vitro but also in vivo. These findings suggest a role of Zf9 in the injury response by enhancing uPA synthesis and subsequent activation of latent TGF-beta. (Blood. 2000;95:1309-1316)

Arthrofibrotic remodeling after knee replacement surgery, a stress-related disease? - Clinic, biochemical model, therapy, research -

Aims and Objectives:The predicted incidence of arthrofibrosis after knee replacement surgery is 5 - 10%. Arthrofibrosis is defined as painful impairment of joint flexibility occurring postoperatively. To counteract symptoms and to accomplish quality standards (E/F 0-0-90) several therapeutic strategies as for instance physiotherapy or mobilization under anaesthesia are applied. Although it is known that mechanical and emotional stress induce myofibroblast differentiation, extracellular matrix (ECM) synthesis and xylosyltransferase (XT) activity, biochemical processes of fibrotic diseases receive little attention in orthopaedics. Until today, neither an adequate disease model nor a diagnostic marker for arthrofibrosis has been described.Materials and Methods:Surgical procedures (e.g. endoprosthesis, cruciate ligament replacement) initiate physiological wound healing and the release of inflammatory mediators from platelets, damaged tissue and other cells of the immune system. Secreted cytokines such as transforming growth factor (TGF-beta 1) or platelet derived growth factor (PDGF) promote myofibroblast proliferation and differentiation. Mechanical strain (stretching exercises with pain inhibition) leads to activation of latent TGF-beta 1, which triggers scarring via ECM/XT synthesis and prevents myofibroblast apoptosis. Our recent study carried out a decreased platelet count in arthrofibrosis patients compared to healthy controls. Thus, an elevated platelet consumption might take place. Analogous to TGF-beta 1, PDGF also inhibits myofibroblast apoptosis and expression of matrix metalloproteinase expression, which degrade ECM. Emotional stress might not only matter in other fibrotic diseases e.g. tako-tsubo cardiomyopathy (TTC) but also in arthrofibrosis. Sympathicotonic destabilization (due to pain, frustration or social burden) results in increased catecholamine synthesis and sympathetic activity. These effects are recognizable by vegetative symptoms (e.g. insomnia, sweating, muscle tension). Catecholamines directly activate fibroblasts and increase ECM synthesis by binding to Beta 2-adrenergic receptors. In TTC, a decrease in catecholamines and profibrotic cytokines restores organ functionality.Results:Causal therapy Several therapeutic approaches might exert a positive influence on degradation of fibrotic tissue: less of postisometric stretching, avoidance of emotional workload, positive expectations due to medical consultation about reversibility of fibrotic mechanisms, appropriate medication as well as osteopathy.Conclusion:Research Operating surgeons are able to detect an unfavorable development of arthrofibrosis relatively early. Nevertheless, they are afraid to name the possible complication. Therefore, quantification of a reliable biomarker would bring great advantages for diagnostics of pre- and after-treatment. Our current studies examine whether increased XT activity might display a biomarker in synovial fluid.

The Thrombospondin1-TGF-β Pathway and Glaucoma.

Glaucoma is characterized by abnormal remodeling of the extracellular matrix (ECM) in the trabecular meshwork and in the connective tissue beams of the lamina cribrosa (LC) at the optic nerve head (ONH), which is associated with axonal damage. Mechanical strain can stimulate ECM remodeling and increased expression of matricellular proteins. Thrombospondins 1 and 2 are induced by cyclic mechanical strain in the eye in both the trabecular meshwork and in the LC region of the ONH. TGF-betas 1 and 2 are increased in glaucoma and play a role in the pathologic remodeling of the ECM in the eye in glaucoma. In this study, we address the role of thrombospondin1 as a regulator of latent TGF-beta activation and discuss the potential therapeutic use of antagonists of the thrombospondin1-TGF-beta pathway for treatment of glaucoma.

Xiamenmycin Attenuates Hypertrophic Scars by Suppressing Local Inflammation and the Effects of Mechanical Stress

Hypertrophic scarring is a common disease affecting millions of people around the world, but there are currently no satisfactory drugs to treat the disease. Exaggerated inflammation and mechanical stress have been shown to be two main mechanisms of excessive fibrotic diseases. Here we found that a benzopyran natural product, xiamenmycin, could significantly attenuate hypertrophic scar formation in a mechanical stretch-induced mouse model. The compound suppressed local inflammation by reducing CD4+ lymphocyte and monocyte/macrophage retention in fibrotic foci and blocked fibroblast adhesion with monocytes. Both in vivo and in vitro studies found that the compound inhibited the mechanical stress-induced profibrotic effects by suppressing proliferation, activation, fibroblast contraction, and inactivating FAK, p38, and Rho guanosine triphosphatase signaling. Taken together, the compound could simultaneously suppress both the inflammatory and mechanical stress responses, which are the two pivotal pathological processes in hypertrophic scar formation, thus suggesting that xiamenmycin can serve as a potential agent for treating hypertrophic scar formation and other excessive fibrotic diseases.

Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin alpha(v)beta(5)-dependent latent transforming growth factor (TGF)-beta1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin alpha(v)beta(5), both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin alpha(v)beta(5) interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin alpha(v)beta(5)-dependent latent TGF-beta1 activation and TGF-beta1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-alpha(v)beta(5) interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-beta1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin alpha(v)beta(5) interactions inhibit contraction-induced latent TGF-beta1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-beta1 with integrin alpha(v)beta(5). Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.

Hypoxia stimulates hepatocyte epithelial to mesenchymal transition by hypoxia-inducible factor and transforming growth factor-β-dependent mechanisms

During development of liver fibrosis, an important source of myofibroblasts is hepatocytes, which differentiate into myofibroblasts by epithelial to mesenchymal transition (EMT). In epithelial tumours and kidney fibrosis, hypoxia, through activation of hypoxia-inducible factors (HIFs), is an important stimulus of EMT. Our recent studies demonstrated that HIF-1alpha is important for the development of liver fibrosis. Accordingly, the hypothesis was tested that hypoxia stimulates hepatocyte EMT by a HIF-dependent mechanism. Primary mouse hepatocytes were exposed to room air or 1% oxygen and EMT evaluated. In addition, bile duct ligations (BDLs) were performed in control and HIF-1alpha-deficient mice and EMT quantified. Exposure of hepatocytes to 1% oxygen increased expression of alpha-smooth muscle actin, vimentin, Snail and fibroblast-specific protein-1 (FSP-1). Levels of E-cadherin and zona occludens-1 were decreased. Upregulation of FSP-1 and Snail by hypoxia was completely prevented in HIF-1beta-deficient hepatocytes and by pretreatment with SB431542, a transforming growth factor-beta (TGF-beta) receptor inhibitor. HIFs promoted TGF-beta-dependent EMT by stimulating activation of latent TGF-beta1. To determine whether HIF-1alpha contributes to EMT in the liver during the development of fibrosis, control and HIF-1alpha-deficient mice were subjected to BDL. FSP-1 was increased to a greater extent in the livers of control mice when compared with HIF-1alpha-deficient mice. Results from these studies demonstrate that hypoxia stimulates hepatocyte EMT by a HIF and TGF-beta-dependent mechanism. Furthermore, these studies suggest that HIF-1alpha is important for EMT in the liver during the development of fibrosis.

Transforming growth factor β as a therapeutic target in systemic sclerosis

Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine with vital homeostatic functions. Aberrant TGF-beta expression is implicated in the pathogenesis of fibrosis in systemic sclerosis (SSc); thus, TGF-beta represents a molecular therapeutic target in this disease. Anti-TGF-beta monoclonal antibody has been evaluated in a small trial of early SSc, with disappointing results. Antibodies against the alphavbeta6 integrin that prevent latent TGF-beta activation, however, have shown promise in preclinical studies. Small-molecule inhibitors of TGF-beta-receptor activity are effective in animal models of fibrosis. Imatinib mesylate and related tyrosine kinase inhibitors also block TGF-beta pathways and abrogate fibrotic responses. The blocking of TGF-beta activity might lead to spontaneous immune activation, epithelial hyperplasia and impaired wound healing. Loss of immune tolerance is a potential concern in an autoimmune disease such as SSc. Novel insights from microarray-based gene expression analyses and studies of genetic polymorphisms in TGF-beta signaling could aid in identifying patients who are most likely to respond to anti-TGF-beta treatment. This intervention promises to have a major impact on the treatment of SSc. Concerns regarding efficacy and safety and whether biomarkers can indicate these features, questions regarding appropriate dosing and timing of therapy, and identification of potential responders are critical challenges ahead.

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.

Influence of ginsenoside Rg1, a panaxatriol saponin from Panax notoginseng, on renal fibrosis in rats with unilateral ureteral obstruction

Total saponins of Panax notoginseng (PNS) have been shown to ameliorate renal interstitial fibrosis. Ginsenoside Rg1, a panaxatriol saponin, is one of the major active molecules from PNS. The present study was undertaken to investigate the effect of ginsenoside Rg1 on renal fibrosis in rats with unilateral ureteral obstruction (UUO). The rats were randomly divided into 3 groups: sham-operation (n=15), UUO (n=15) and UUO with ginsenoside Rg1 treatment (n=15, 50 mg per kg body weight, intraperitoneally (i.p.) injected). The rats were sacrificed on Days 7 and 14 after the surgery. Histological examination demonstrated that ginsenoside Rg1 significantly inhibited interstitial fibrosis including tubular injury as well as collagen deposition. alpha-smooth muscle actin (alpha-SMA) and E-cadherin are two markers of tubular epithelial-myofibroblast transition (TEMT). Interestingly, ginsenoside Rg1 notably decreased alpha-SMA expression and simultaneously enhanced E-cadherin expression. The messenger RNA (mRNA) of transforming growth factor-beta1 (TGF-beta1), a key mediator to regulate TEMT, in the obstructed kidney increased dramatically, but was found to decrease significantly after administration of ginsenoside Rg1. Further study showed that ginsenoside Rg1 considerably decreased the levels of both active TGF-beta1 and phosphorylated Smad2 (pSmad2). Moreover, ginsenoside Rg1 substantially suppressed the expression of thrombospondin-1 (TSP-1), a cytokine which can promote the transcription of TGF-beta1 mRNA and the activation of latent TGF-beta1. These results suggest that ginsenoside Rg1 inhibits renal interstitial fibrosis in rats with UUO. The mechanism might be partly related to the blocking of TEMT via suppressing the expression of TSP-1.

Antitransforming growth factor-β therapy in fibrosis: recent progress and implications for systemic sclerosis

Transforming growth factor-beta (TGF-beta) is required for tissue homeostasis but is also implicated in disease processes including fibrosis, and thus represents a molecular target for therapy. Multiple strategies for inhibiting excessive TGF-beta function exist. The three principal platforms are RNA-based technologies, monoclonal antibodies and small molecules. Monoclonal antibodies targeting TGF-beta have been used in a small clinical trial, with disappointing results to date. Antibodies to the alphavbeta6 integrin prevent local activation of latent TGF-beta and show promise in preclinical studies. Over a dozen small molecules inhibit the kinase activity of TGF-beta receptors. Several commonly used drugs appear to have unanticipated anti-TGF-beta activity and may therefore have a role in antifibrotic therapy. Because TGF-beta has important physiological functions, inhibiting its activity might potentially lead to aberrant immune activation, epithelial hyperplasia and impaired wound healing; spontaneous autoimmunity in particular is a concern in an autoimmune disease such as systemic sclerosis. Novel insights from DNA microarray analysis and genetic polymorphisms in TGF-beta signaling will aid in defining patient populations most likely to respond to anti-TGF-beta treatment. Anti-TGF-beta therapies promise to have a major impact in systemic sclerosis. Significant concerns regarding efficacy and safety need to be addresed. The identification of optimal candidates for therapy, and of biomarkers of safety and efficacy, are critical challenges ahead.

Integrin αvβ6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies

The integrin alphavbeta6 promotes proliferation of specialized epithelia and acts as a receptor for the activation of latent TGFbeta1. We studied alphavbeta6 expression in experimental and human liver fibrosis and the potential of its pharmacological inhibition for treatment of hepatic fibrosis. alphavbeta6 expression was studied by quantitative PCR and immunohistochemistry in rats with cirrhosis due to bile duct ligation (BDL), administration of thioacetamide (TAA), in Mdr2(Abcb4)(-/-) mice with spontaneous biliary fibrosis, and in livers of patients with chronic hepatitis C (n=79) and end-stage liver disease due to various etiologies (n=18). The effect of a selective alphavbeta6 inhibitor was evaluated in Mdr2(Abcb4)(-/-) mice with ongoing fibrogenesis. Integrin beta6 mRNA increased with fibrosis stage in hepatitis C and was upregulated between 25- and 100-fold in TAA- and BDL-induced cirrhosis, in Mdr2(Abcb4)(-/-) mice and in human end-stage liver disease. alphavbeta6 protein was absent in normal livers and expressed de novo on (activated) bile duct epithelia and transitional hepatocytes. A single dose of the alphavbeta6 inhibitor injected into Mdr2(Abcb4)(-/-) mice significantly induced profibrolytic matrix metalloproteinases (MMP)-8 and -9 after 3 h, with a corresponding increase in extracellular matrix-degrading activities. In parallel profibrogenic transcripts (procollagen alpha1(I), TGFbeta2, and MMP-2) showed a trend of downregulation. (1) Integrin alphavbeta6 is induced de novo in rodent and human liver fibrosis, where it is expressed on activated bile duct epithelia and (transitional) hepatocytes during fibrosis progression. (2) In vivo a single dose of a small molecule alphavbeta6 inhibitor induced antifibrogenic and profibrolytic genes and activities, suggesting alphavbeta6 is a unique target for treatment of liver fibrosis.

A Thrombospondin-1 Antagonist of Transforming Growth Factor-β Activation Blocks Cardiomyopathy in Rats with Diabetes and Elevated Angiotensin II

In diabetes and hypertension, the induction of increased transforming growth factor-beta (TGF-beta) activity due to glucose and angiotensin II is a significant factor in the development of fibrosis and organ failure. We showed previously that glucose and angiotensin II induce the latent TGF-beta activator thrombospondin-1 (TSP1). Because activation of latent TGF-beta is a major means of regulating TGF-beta, we addressed the role of TSP1-mediated TGF-beta activation in the development of diabetic cardiomyopathy exacerbated by abdominal aortic coarctation in a rat model of type 1 diabetes using a peptide antagonist of TSP1-dependent TGF-beta activation. This surgical manipulation elevates initial blood pressure and angiotensin II. The hearts of these rats had increased TSP1, collagen, and TGF-beta activity, and cardiac function was diminished. A peptide antagonist of TSP1-dependent TGF-beta activation prevented progression of cardiac fibrosis and improved cardiac function by reducing TGF-beta activity. These data suggest that TSP1 is a significant mediator of fibrotic complications of diabetes associated with stimulation of the renin-angiotensin system, and further studies to assess the blockade of TSP1-dependent TGF-beta activation as a potential antifibrotic therapeutic strategy are warranted.

Integrin β6 Mediates Phospholipid and Collectin Homeostasis by Activation of Latent TGF-β1

Surfactant lines the alveolar surface and prevents alveolar collapse. Derangements of surfactant cause respiratory failure and interstitial lung diseases. The collectins, surfactant proteins A and D, are also important in innate host defense. However, surfactant regulation in the postnatal lung is poorly understood. We found that the epithelial integrin, alphavbeta6, regulates surfactant homeostasis in vivo by activating latent transforming growth factor (TGF)-beta. Adult mice lacking the beta-subunit of alphavbeta6 (Itgb6-/-) developed increased bronchoalveolar lavage phospholipids and surfactant proteins A and D, and demonstrated abnormal-appearing alveolar macrophages, reminiscent of the human disease pulmonary alveolar proteinosis. Using lung-specific expression of constitutively active TGF-beta1 in Itgb6-/- mice, we found that TGF-beta1 was sufficient to normalize these abnormalities. Tgfbeta1-deficient mice also demonstrated increased phospholipids and surfactant proteins A and D, but mice lacking the key TGF-beta signaling molecule, SMAD3, did not. Therefore, integrin-mediated activation of latent TGF-beta1 regulates surfactant constituents independent of intracellular SMAD3. In vivo increases in surfactant protein A and D were not associated with increases in mRNA for these proteins in alveolar tissue from Itgb6-/- mice. On the other hand, isolated alveolar macrophages from Itgb6-/- mice were defective in processing phospholipids in vitro, suggesting that reduced surfactant clearance contributes to altered surfactant homeostasis in these mice in vivo. These findings show that alphavbeta6 and TGF-beta1 regulate homeostasis of phospholipids and collectins in adult mouse lungs and may have implications for anti-fibrotic therapeutics that inhibit active TGF-beta in the lung.

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter: Effect on Latent Transforming Growth Factor-β Binding Protein-1L Expression Level and Possible Prognostic Significance in Ovarian Cancer

Latent transforming growth factor (TGF)binding proteins (LTBPs) play important roles in the secretion and activation of TGF. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, 202G/C and 20A/C. While the alleles with 202C and 20C were initially reported, our data demonstrated that 202G and 20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with 202G and 20A. Interestingly , ovarian cancer patients (n 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P 0.02). These findings suggest that the single nucleotide polymorphisms 202G/C and 20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients , probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer. (J Mol Diagn 2006, 8:342–350; DOI: 10.2353/jmoldx.2006.050133) Transforming growth factor (TGF)is a potent growth inhibitor for most cell types, including epithelial cells. The secretion of TGFby tumor cells may contribute to tumor suppression by autocrine growth inhibition, but on the other hand, it may also promote tumor progression by stimulating tumor invasion and angiogenesis, and inhibiting immune response. TGFis synthesized as latent high-molecular weight complexes, composed of TGF, the NH2-terminal part of the TGFprecursor, and the latent TGFbinding protein (LTBP). LTBP is a glycoprotein with a molecular weight of more than 190 kd; it possesses 16 to 18 epidermal growth factor (EGF)-like domains and several repeats of a unique motif containing eight cysteine residues. Four isoforms of LTBP have been found in mammalian species (LTBP-1 to LTBP4). LTBP-1 binds to TGF1 through one of the eight cysteine motifs and facilitates assembly and secretion and activation of TGF1. Immunoelectronmicroscopic observations indicate that LTBP-1 is one of the extracellular microfibrillar components; it targets TGF1 to extracellular structures and participates in the activation of latent TGF1, perhaps by concentrating latent TGF1 on the cell surface where activation occurs. LTBP-1S (short) and LTBP-1L (long) are derived from independent promoters and alternative splicing between codons 145 and 146 of LTBP-1S. LTBP-1L has a N-terminal extension of 346 amino acids that is not found in the LTBP-1S. The N-terminal extension contains an EGF-like domain that facilitates matrix incorporation, and LTBP-1L has been confirmed to associate more efficiently than LTBP-1S with the extracellular matrix.

Sublytic complement C5b-9 complexes induce thrombospondin-1 production in rat glomerular mesangial cells via PI3-k/Akt: association with activation of latent transforming growth factor-beta1.

Mesangial cell proliferation is a common cellular response to a variety of different types of glomerular injury. Complement C5b-9 is a prime candidate to mediate mesangial cell proliferation, especially sublytic C5b-9, which can induce the production of multiple inflammatory factors and cytokines. Transforming growth factor (TGF)-beta1 plays a major role in the accumulation of extracellular matrix (ECM), while thrombospondin (TSP)-1 has been identified as an activator of latent TGF-beta1 in an in vitro system. Using rat glomerular mesangial cells (GMCs) as a model system, we assessed the effect of sublytic C5b-9 on the expression of TSP-1 and TGF-beta1 and explored the relevant pathway of signal transduction. First, we ensured the concentrations of anti-Thy1 antibody and complement, which were regarded as a sublytic C5b-9 dose, and examined whether the sublytic C5b-9 induced expression of TSP-1 in rat GMCs which, in turn, activated latent TGF-beta1 by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, we investigated the role of the PI3-k/Akt pathway in sublytic C5b-9-induced TSP-1 production in rat GMCs by Western blot analysis. The addition of sublytic C5b-9 (5% anti-Thy1 antibody and 4% normal serum) to rat GMCs induced activation of latent TGF-beta1 via TSP-1. The addition of sublytic C5b-9 apparently increased the protein of Akt phosphorylation, whereas PI3-k inhibitor LY294002 could clearly reduce the increase of TSP-1 induced by sublytic C5b-9. These results indicate that TSP-1 is an activator of latent TGF-beta1 in sublytic C5b-9-induced rat GMCs; furthermore, the PI3-k/Akt signal transduction pathway may play a key role in sublytic C5b-9-induced TSP-1 production.

Function-blocking antithrombospondin-1 monoclonal antibodies.

Thrombospondin-1 (TSP-1) has been implicated in many different processes based in part on inhibitory activities of anti-TSP-1 monoclonal antibodies (mAbs). To map epitopes of 13 anti-TSP-1 mAbs to individual modules or groups of modules spanning TSP-1 and the closely related TSP-2 homolog. The mapping has led to assignment or reassignment of the epitopes of four mAbs, refinement of the epitopes of six mAbs, and confirmation of the epitopes of the remaining three mAbs. ESTs10, P12, and MA-II map to the N-terminal domain; 5G11, TSP127.6, and ESTs12 to the third properdin module; C6.7, HB8432, and P10 to epidermal growth factor (EGF)-like modules 1 and/or 2; and A6.1, mAb133, MA-I, and D4.6 to the calcium-binding wire module. A6.1, which recognizes a region of the wire that is identical in mouse and human TSP-1, reacts with TSP-1 from both species, and also reacts weakly with human TSP-2. Two other mouse antihuman TSP-1 mAbs, A4.1 and D4.6, also react with mouse TSP-1. Consideration of previous literature and mapping of epitopes of inhibitory mAbs suggest that biological activities are present throughout TSP-1, including the EGF-like modules that have not been implicated in the past. Because the epitopes for 10 of the antibodies likely are within 18 nm of one another in calcium-replete TSP-1, some of the inhibitory effects may result from steric hindrance. Such seems to be the case for mAb133, which binds the calcium-binding wire but is still able to interfere with the activation of latent TGF-beta by the properdin modules.

Fibronectin is required for integrin o:vβ6‐mediated activation of latent TGF‐β complexes containing LTBP‐1

Transforming growth factor-betas (TGF-beta) are secreted as latent complexes consisting of the TGF-beta dimer, the TGF-beta propeptide dimer, and the latent TGF-beta binding protein (LTBP). Although the bonds between TGF-beta and its propeptide are cleaved intracellulary, the propeptide associates with TGF-beta by electrostatic interactions, thereby conferring latency to the complex. We reported that a specific sequence of LTBP-1 is required for latent TGF-beta activation by the integrin alphavbeta6. Here we describe a 24 amino acid sequence from the hinge domain required for activation. The LTBP-1 polypeptide rL1N, which includes the hinge, associates with fibronectin in binding assays. We present evidence that fibronectin null cells minimally activate latent TGF-beta and poorly incorporate the active hinge sequence into their matrix. In addition, cells missing the fibronectin receptor alpha5beta1 exhibit defective activation of latent TGF-beta by alphavbeta6 and decreased matrix incorporation. The results indicate specificity for integrin-mediated latent TGF-beta activation that include unique sequences in LTBP-1 and an appropriate matrix molecule.

Lung Alveolar Septation Defects in Ltbp-3-Null Mice

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) modulate the secretion and activation of latent TGF-beta. To explore LTBP function in vivo, we created an Ltbp-3(-/-) mouse that has developmental emphysema with decreased septation in terminal alveoli. Differences in distal airspace enlargement were obvious at day 6 after birth. Secondary septation was inhibited, so by days 21 to 28 the mean linear intercept was approximately twofold greater in mutant versus control lungs. There were no differences in lung collagen and elastin, visualized by immunohistochemistry, or in myofibroblast numbers, determined by alpha-smooth muscle actin-positive cells, between mutant or wild-type lungs as the animals aged, other than differences associated with altered lung structure in mutant animals. However, from day 10 there was twice the number of alveolar type II cells in mutant alveoli compared to controls. At days 6 and 10, a transient enhancement in cell proliferation in the mutant lungs was observed by both 5-bromo-2'-deoxy-uridine and proliferating cell nuclear antigen labeling, accompanied by enhanced numbers of terminal dUTP nick-end labeling-positive cells at days 4, 6, and 10. Finally, there was a transient decrease in TGF-beta signaling at days 4 to 6 in Ltbp-3(-/-) lungs. These results indicate that in the absence of Ltbp-3, a temporary decrease in TGF-beta signaling in the lungs at days 4 to 6 alters cell proliferation, correlating with inhibition of septation and developmental emphysema.

Constitutive Thrombospondin-1 Overexpression Contributes to Autocrine Transforming Growth Factor-β Signaling in Cultured Scleroderma Fibroblasts

The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.