Latent Transforming Growth Factor-β Binding Protein Domains Involved in Activation and Transglutaminase-dependent Cross-Linking of Latent Transforming Growth Factor-β

Transforming growth factor-β (TGF-β) is secreted by many cell types as part of a large latent complex composed of three subunits: TGF-β, the TGF-β propeptide, and the latent TGF-β binding protein (LTBP). To interact with its cell surface receptors, TGF-β must be released from the latent complex by disrupting noncovalent interactions between mature TGF-β and its propeptide. Previously, we identified LTBP-1 and transglutaminase, a cross-linking enzyme, as reactants involved in the formation of TGF-β. In this study, we demonstrate that LTBP-1 and large latent complex are substrates for transglutaminase. Furthermore, we show that the covalent association between LTBP-1 and the extracellular matrix is transglutaminase dependent, as little LTBP-1 is recovered from matrix digests prepared from cultures treated with transglutaminase inhibitors. Three polyclonal antisera to glutathione S–transferase fusion proteins containing amino, middle, or carboxyl regions of LTBP-1S were used to identify domains of LTBP-1 involved in crosslinking and formation of TGF-β by transglutaminase. Antibodies to the amino and carboxyl regions of LTBP-1S abrogate TGF-β generation by vascular cell cocultures or macrophages. However, only antibodies to the amino-terminal region of LTBP-1 block transglutaminase-dependent cross-linking of large latent complex or LTBP-1. To further identify transglutaminase-reactive domains within the amino-terminal region of LTBP-1S, mutants of LTBP-1S with deletions of either the amino-terminal 293 (ΔN293) or 441 (ΔN441) amino acids were expressed transiently in CHO cells. Analysis of the LTBP-1S content in matrices of transfected CHO cultures revealed that ΔN293 LTBP-1S was matrix associated via a transglutaminasedependent reaction, whereas ΔN441 LTBP-1S was not. This suggests that residues 294–441 are critical to the transglutaminase reactivity of LTBP-1S.     

LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) belongs to the fibrillin-LTBP superfamily of extracellular matrix proteins. LTBPs and fibrillins are involved in the sequestration and storage of latent growth factors, particularly transforming growth factor β (TGF-β), in tissues. Unlike other LTBPs, LTBP-2 does not covalently bind TGF-β, and its molecular functions remain unclear. We are screening LTBP-2 for binding to other growth factors and have found very strong saturable binding to fibroblast growth factor-2 (FGF-2) (Kd = 1.1 nM). Using a series of recombinant LTBP-2 fragments a single binding site for FGF-2 was identified in a central region of LTBP-2 consisting of six tandem epidermal growth factor-like (EGF-like) motifs (EGFs 9–14). This region was also shown to contain a heparin/heparan sulphate-binding site. FGF-2 stimulation of fibroblast proliferation was completely negated by the addition of 5-fold molar excess of LTBP-2 to the assay. Confocal microscopy showed strong co-localisation of LTBP-2 and FGF-2 in fibrotic keloid tissue suggesting that the two proteins may interact in vivo. Overall the study indicates that LTBP-2 is a potent inhibitor of FGF-2 that may influence FGF-2 bioactivity during wound repair particularly in fibrotic tissues.     

The Effect of Antifibrotic Drugs in Rat Precision-Cut Fibrotic Liver Slices

Two important signaling pathways in liver fibrosis are the PDGF- and TGFβ pathway and compounds inhibiting these pathways are currently developed as antifibrotic drugs. Testing antifibrotic drugs requires large numbers of animal experiments with high discomfort. Therefore, a method to study these drugs ex vivo was developed using precision-cut liver slices from fibrotic rat livers (fPCLS), representing an ex vivo model with a multicellular fibrotic environment. We characterized the fibrotic process in fPCLS from rat livers after 3 weeks of bile duct ligation (BDL) during incubation and tested compounds predominantly inhibiting the TGFβ pathway (perindopril, valproic acid, rosmarinic acid, tetrandrine and pirfenidone) and PDGF pathway (imatinib, sorafenib and sunitinib). Gene expression of heat shock protein 47 (Hsp47), α smooth muscle actin (αSma) and pro-collagen 1A1 (Pcol1A1) and protein expression of collagens were determined. During 48 hours of incubation, the fibrosis process continued in control fPCLS as judged by the increased gene expression of the three fibrosis markers, and the protein expression of collagen 1, mature fibrillar collagen and total collagen. Most PDGF-inhibitors and TGFβ-inhibitors significantly inhibited the increase in gene expression of Hsp47, αSma and Pcol1A1. Protein expression of collagen 1 was significantly reduced by all PDGF-inhibitors and TGFβ-inhibitors, while total collagen was decreased by rosmarinic acid and tetrandrine only. However, fibrillar collagen expression was not changed by any of the drugs. In conclusion, rat fPCLS can be used as a functional ex vivo model of established liver fibrosis to test antifibrotic compounds inhibiting the PDGF- and TGFβ signalling pathway.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

Matrix metalloproteinase 2-integrin alpha(v)beta3 binding is required for mesenchymal cell invasive activity but not epithelial locomotion: a computational time-lapse study

Cellular invasive behavior through three-dimensional collagen gels was analyzed using computational time-lapse imaging. A subpopulation of endocardial cells, derived from explanted quail cardiac cushions, undergoes an epithelial-to-mesenchymal transition and invades the substance of the collagen gels when placed in culture. In contrast, other endocardial cells remain epithelial and move over the gel surface. Here, we show that integrin αvβ3 and matrix metalloproteinase (MMP)2 are present and active in cushion mesenchymal tissue. More importantly, functional assays show that mesenchymal invasive behavior is dependent on MMP2 activity and integrin αvβ3 binding. Inhibitors of MMP enzymatic activity and molecules that prevent integrin αvβ3 binding to MMP2, via its hemopexin domain, result in significantly reduced cellular protrusive activity and invasive behavior. Computational analyses show diminished intensity and persistence time of motility in treated invasive mesenchymal cells, but no reduction in motility of the epithelial-like cells moving over the gel surface. Thus, quantitative time-lapse data show that mesenchymal cell invasive behavior, but not epithelial cell locomotion over the gel surface, is partially regulated by the MMP2–integrin interactions.     

miR-218 regulates focal adhesion kinase–dependent TGFβ signaling in fibroblasts

Scarring, which occurs in essentially all adult tissue, is characterized by the excessive production and remodeling of extracellular matrix by α-smooth muscle actin (SMA)–expressing myofibroblasts located within connective tissue. Excessive scarring can cause organ failure and death. Oral gingivae do not scar. Compared to dermal fibroblasts, gingival fibroblasts are less responsive to transforming growth factor β (TGFβ) due to the reduced expression, due to the reduced expression and activity of focal adhesion kinase (FAK) by this cell type. Here we show that, compared with dermal fibroblasts, gingival fibroblasts show reduced expression of miR-218. Introduction of pre–miR-218 into gingival fibroblasts elevates FAK expression and, via a FAK/src-dependent mechanism, results in the ability of TGFβ to induce α-SMA. The deubiquitinase cezanne is a direct target of miR-218 and has increased expression in gingival fibroblasts compared with dermal fibroblasts. Knockdown of cezanne in gingival fibroblasts increases FAK expression and causes TGFβ to induce α-smooth muscle actin (α-SMA). These results suggest that miR-218 regulates the ability of TGFβ to induce myofibroblast differentiation in fibroblasts via cezanne/FAK.     

5Z-7-Oxozeanol Inhibits the Effects of TGFβ1 on Human Gingival Fibroblasts

Transforming growth factor (TGF)β acts on fibroblasts to promote the production and remodeling of extracellular matrix (ECM). In adult humans, excessive action of TGFβ is associated with fibrotic disease and fibroproliferative conditions, including gingival hyperplasia. Understanding how the TGFβ1 signals in fibroblasts is therefore likely to result in valuable insights into the fundamental mechanisms underlying fibroproliferative disorders. Previously, we used the TAK1 inhibitor (5Z)-7-Oxozeaenol to show that, in dermal fibroblasts, the non-canonical TAK1 pathway mediates the ability of TGFβ1 to induce genes promoting tissue remodeling and repair. However, the extent to which TAK1 mediates fibroproliferative responses in fibroblasts in response to TGFβ1 remains unclear. Herein, we show that, in gingival fibroblasts, (5Z)-7-Oxozeaenol blocks the ability of TGFβ1 to induce expression of the pro-fibrotic mediator CCN2 (connective tissue growth factor, CTGF) and type I collagen protein. Moreover, genome-wide expression profiling revealed that, in gingival fibroblasts, (5Z)-7-Oxozeaenol reduces the ability of TGFβ1 to induce mRNA expression of essentially all TGFβ1-responsive genes (139/147), including those involved with a hyperproliferative response. Results from microarray analysis were confirmed using real time polymerase chain reaction analysis and a functional cell proliferation assay. Our results are consistent with the hypothesis that TAK1 inhibitors might be useful in treating fibroproliferative disorders, including that in the oral cavity.     

Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFβ.     

Reprogramming during epithelial to mesenchymal transition under the control of TGFβ

Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor β (TGFβ), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFβ signaling is considered to be abnormal in the context of cancer development; however, TGFβ acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFβ, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.     

Role of the prostaglandin E2/E-prostanoid 2 receptor signalling pathway in TGFβ-induced mice mesangial cell damage

The prostaglandin E2 receptor, EP2 (E-prostanoid 2), plays an important role in mice glomerular MCs (mesangial cells) damage induced by TGFβ1 (transforming growth factor-β1); however, the molecular mechanisms for this remain unknown. The present study examined the role of the EP2 signalling pathway in TGFβ1-induced MCs proliferation, ECM (extracellular matrix) accumulation and expression of PGES (prostaglandin E₂ synthase). We generated primary mice MCs. Results showed MCs proliferation promoted by TGFβ1 were increased; however, the production of cAMP and PGE₂ (prostaglandin E₂) was decreased. EP2 deficiency in these MCs augmented FN (fibronectin), Col I (collagen type I), COX2 (cyclooxygenase-2), mPGES-1 (membrane-associated prostaglandin E1), CTGF (connective tissue growth factor) and CyclinD1 expression stimulated by TGFβ1. Silencing of EP2 also strengthened TGFβ1-induced p38MAPK (mitogen-activated protein kinase), ERK1/2 (extracellular-signal-regulated kinase 1/2) and CREB1 (cAMP responsive element-binding protein 1) phosphorylation. In contrast, Adenovirus-mediated EP2 overexpression reversed the effects of EP2-siRNA (small interfering RNA). Collectively, the investigation indicates that EP2 may block p38MAPK, ERK1/2 and CREB1 phosphorylation via activation of cAMP production and stimulation of PGE₂ through EP2 receptors which prevent TGFβ1-induced MCs damage. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the damage induced by TGFβ1.     

Genotype-Specific Interaction of Latent TGFβ Binding Protein 4 with TGFβ

Latent TGFβ binding proteins are extracellular matrix proteins that bind latent TGFβ to form the large latent complex. Nonsynonymous polymorphisms in LTBP4, a member of the latent TGFβ binding protein gene family, have been linked to several human diseases, underscoring the importance of TGFβ regulation for a range of phenotypes. Because of strong linkage disequilibrium across the LTBP4 gene, humans have two main LTBP4 alleles that differ at four amino acid positions, referred to as IAAM and VTTT for the encoded residues. VTTT is considered the “risk” allele and associates with increased intracellular TGFβ signaling and more deleterious phenotypes in muscular dystrophy and other diseases. We now evaluated LTBP4 nsSNPs in dilated cardiomyopathy, a distinct disorder associated with TGFβ signaling. We stratified based on self-identified ethnicity and found that the LTBP4 VTTT allele is associated with increased risk of dilated cardiomyopathy in European Americans extending the diseases that associate with LTBP4 genotype. However, the association of LTBP4 SNPs with dilated cardiomyopathy was not observed in African Americans. To elucidate the mechanism by which LTBP4 genotype exerts this differential effect, TGFβ’s association with LTBP4 protein was examined. LTBP4 protein with the IAAM residues bound more latent TGFβ compared to the LTBP4 VTTT protein. Together these data provide support that LTBP4 genotype exerts its effect through differential avidity for TGFβ accounting for the differences in TGFβ signaling attributed to these two alleles.     

MT1-MMP releases latent TGF-beta1 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.     

Metabolic reprogramming of glycolysis and glutamine metabolism are key events in myofibroblast transition in systemic sclerosis pathogenesis

Systemic Sclerosis (SSc) is a rare fibrotic autoimmune disorder for which no curative treatments currently exist. Metabolic remodelling has recently been implicated in other autoimmune diseases; however, its potential role in SSc has received little attention. Here, we aimed to determine whether changes to glycolysis and glutaminolysis are important features of skin fibrosis. TGF‐β1, the quintessential pro‐fibrotic stimulus, was used to activate fibrotic pathways in NHDFs in vitro. Dermal fibroblasts derived from lesions of SSc patients were also used for in vitro experiments. Parameters of glycolytic function were assessed using by measuring extracellular acidification in response to glycolytic activators/inhibitors, whilst markers of fibrosis were measured by Western blotting following the use of the glycolysis inhibitors 2‐dg and 3PO and the glutaminolysis inhibitor G968. Succinate was also measured after TGF‐β1 stimulation. Itaconate was added to SSc fibroblasts and collagen examined. TGF‐β1 up‐regulates glycolysis in dermal fibroblasts, and inhibition of glycolysis attenuates its pro‐fibrotic effects. Furthermore, inhibition of glutamine metabolism also reverses TGF‐β1‐induced fibrosis, whilst glutaminase expression is up‐regulated in dermal fibroblasts derived from SSc patient skin lesions, suggesting that enhanced glutamine metabolism is another aspect of the pro‐fibrotic metabolic phenotype in skin fibrosis. TGF‐β1 was also able to enhance succinate production, with increased succinate shown to be associated with increased collagen expression. Incubation of SSc cells with itaconate, an important metabolite, reduced collagen expression. TGF‐β1 activation of glycolysis is a key feature of the fibrotic phenotype induced by TGF‐B1 in skin cells, whilst increased glutaminolysis is also evident in SSc fibroblasts.     

The latent transforming growth factor-beta-binding protein-1 promotes in vitro differentiation of embryonic stem cells into endothelium

The latent transforming growth factor-beta-binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-beta-masking protein, LTBP-1 is involved both in the sequestration of latent TGF-beta in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-beta or the latency associated peptide, which neutralize TGF-beta. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-beta 1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-beta-dependent mechanism.     

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.     

How Soluble GARP Enhances TGFβ Activation

GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to α_vβ₆ or α_vβ₈ integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo.