A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-beta 1 in platelets is composed of three components, i.e. the mature TGF-beta 1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-beta 1 precursor (TGF-beta 1-latency associated peptide) and the latent TGF-beta 1 binding protein (LTBP). The TGF-beta 1-latency associated peptide is sufficient for the latency of TGF-beta 1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-beta 1 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-beta 1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-beta 1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-beta 1 was studied by pulse-chase analysis. LTBP became covalently associated with the TGF-beta 1 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-beta 1 complex was observed as early as 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-beta 1 precursor was seen. In contrast, the TGF-beta 1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-beta 1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-beta 1.     

Dual role for the latent transforming growth factor-beta binding protein in storage of latent TGF-beta in the extracellular matrix and as a structural matrix protein

The role of the latent TGF-beta binding protein (LTBP) is unclear. In cultures of fetal rat calvarial cells, which form mineralized bonelike nodules, both LTBP and the TGF-beta 1 precursor localized to large fibrillar structures in the extracellular matrix. The appearance of these fibrillar structures preceded the appearance of type I collagen fibers. Plasmin treatment abolished the fibrillar staining pattern for LTBP and released a complex containing both LTBP and TGF-beta. Antibodies and antisense oligonucleotides against LTBP inhibited the formation of mineralized bonelike nodules in long-term fetal rat calvarial cultures. Immunohistochemistry of fetal and adult rat bone confirmed a fibrillar staining pattern for LTBP in vivo. These findings, together with the known homology of LTBP to the fibrillin family of proteins, suggest a novel function for LTBP, in addition to its role in matrix storage of latent TGF-beta, as a structural matrix protein that may play a role in bone formation.     

GDF11 forms a bone morphogenetic protein 1-activated latent complex that can modulate nerve growth factor-induced differentiation of PC12 cells

All transforming growth factor beta (TGF-beta) superfamily members are synthesized as precursors with prodomain sequences that are proteolytically removed by subtilisin-like proprotein convertases (SPCs). For most superfamily members, this is believed sufficient for activation. Exceptions are TGF-betas 1 to 3 and growth differentiation factor 8 (GDF8), also known as myostatin, which form noncovalent, latent complexes with their SPC-cleaved prodomains. Sequence similarities between TGF-betas 1 to 3, myostatin, and superfamily member GDF11, also known as bone morphogenetic protein 11 (BMP11), prompted us to examine whether GDF11 might be capable of forming a latent complex with its cleaved prodomain. Here we demonstrate that GDF11 forms a noncovalent latent complex with its SPC-cleaved prodomain and that this latent complex is activated via cleavage at a single specific site by members of the developmentally important BMP1/Tolloid family of metalloproteinases. Evidence is provided for a molecular model whereby formation and activation of this complex may play a general role in modulating neural differentiation. In particular, mutant GDF11 prodomains impervious to cleavage by BMP1/Tolloid proteinases are shown to be potent stimulators of neurodifferentiation, with potential for therapeutic applications.     

Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system

Transforming growth factor-beta1 (TGF-beta1) is secreted by most cells as a high molecular weight latent complex, which consists of latent TGF-beta1 disulfide bonded to latent TGF-beta1-binding protein (LTBP). Current recombinant expression systems yield less than 1-2 mg of the mature TGF-beta1 per liter of cell culture medium. In an effort to produce large quantities of the recombinant cytokine for structural studies, we have constructed a mammalian expression system based on a modified pcDNA3.1(+) vector with a glutamine synthetase gene inserted for gene amplification. The leader peptide of TGF-beta1 was replaced with that of rat serum albumin, and an eight-histidine tag was inserted immediately after the leader sequence to facilitate protein purification. In addition, Cys 33 of TGF-beta1, which forms a disulfide bond with LTBP, was replaced by a serine residue. The resulting expression construct produced a stable clone expressing 30 mg of mature TGF-beta1 per liter of spent medium. Purified TGF-beta1 bound with high affinity to its type II receptor with a solution dissociation constant of approximately 70 nM, and was fully active in both a Mv1Lu cell growth inhibition assay and in a PAI-1 luciferase reporter assay. Owing to similarities in the synthesis, secretion, and structure of TGF-beta family members, this recombinant expression system may also be applied to the overexpression of other TGF-beta isomers and even to members of the TGF-beta superfamily to facilitate their preparation.     

Latent TGF-beta binding protein-3 (LTBP-3) requires binding to TGF-beta for secretion

Latent transforming growth factor-beta (TGF-beta) binding protein (LTBP)-1, which is easily secreted, has been shown to enhance the secretion of TGF-beta. Here we show that another member of the LTBP family, LTBP-3, is not secreted by several cell types, but secretion occurs after coexpression with TGF-beta. The secretion of LTBP-3 requires complexing of LTBP-3 with Cys33 of the TGF-beta propeptide.     

Retention of the transforming growth factor-beta 1 precursor in the Golgi complex in a latent endoglycosidase H-sensitive form.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as a latent high molecular weight complex in a human erythroleukemia cell line, HEL, treated with phorbol 12-myristate 13-acetate. The complex is comprised of three components: mature TGF-beta 1, the TGF-beta 1 latency-associated peptide (beta 1-LAP), and the latent TGF-beta 1-binding protein (LTBP). LTBP plays an important role in the assembly and secretion of the latent TGF-beta 1 complex; if the TGF-beta 1 precursor fails to bind to LTBP, much of it remains inside the cells and may contain anomalous disulfide bond(s) between beta 1-LAP and the mature TGF-beta 1 molecule (Miyazono, K., Olofsson, A., Colosetti, P., and Heldin, C.-H. (1991) EMBO J. 10, 1091-1101). In the present work, we have investigated the subcellular localization and properties of the TGF-beta 1 precursor retained intracellularly. When the HEL cells were metabolically labeled and chased for up to 72 h, a considerable part of the TGF-beta 1 precursor was still observed intracellularly in an unprocessed form. The secreted form of the TGF-beta 1 precursor was resistant to endoglycosidase H, whereas the intracellular form of the TGF-beta 1 precursor was sensitive to endoglycosidase H, regardless of the presence or absence of swainsonine, an inhibitor of mannosidase II. Indirect immunofluorescence microscopy revealed that the TGF-beta 1 precursor co-localized with mannosidase II, a marker for the Golgi complex, but not with protein disulfide isomerase, a marker for the endoplasmic reticulum. The intracellular TGF-beta 1 precursor was prepared from phorbol 12-myristate 13-acetate-treated HEL cells and tested for TGF-beta 1 bioactivity. Half-maximal inhibition of the DNA synthesis in mink lung epithelial cells, Mv1Lu, was observed at 80 pM of the acid-treated TGF-beta 1 precursor, whereas nontreated material showed minimal growth inhibitory activity. Taken together, these results indicate that the TGF-beta 1 precursor is retained inside the cells in the Golgi complex, mainly in a latent, immature form.     

Vitamin D3 metabolites regulate LTBP1 and latent TGF-beta1 expression and latent TGF-beta1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.     

Targeting of bone morphogenetic protein growth factor complexes to fibrillin

Both latent transforming growth factor-beta (TGF-beta)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-beta family of growth factors. Interactions between latent TGF-beta-binding protein-1 and TGF-beta and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-beta family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.     

Increased abdominal obesity,insulin and glucose levels in nondiabetic subjects with a T29C polymorphism of the transforming growth factor-beta1 gene

BACKGROUND: In humans, a T-->C transition at nucleotide 29 in the region encoding the signal peptide sequence of the transforming growth factor (TGF)-beta(1), which results in a Leu-->Pro substitution at codon 10, has been associated with myocardial infarction. AIMS/METHODS: In the present study, we genotyped 284 unrelated, nondiabetic Swedish men born in 1944 to assess the impact of the Leu10Pro variant on obesity, including abdominal obesity, and estimates of insulin, glucose and lipid metabolism as well as blood pressure. RESULTS: The frequency of the Pro10 variant was 38.9% (95% CI 32.2-46.0%), and the distribution of genotypes was in Hardy-Weinberg equilibrium. Data analysis showed that heterozygotes had significantly higher body mass index compared to homozygous carriers to the Leu10 variant. In addition, homozygous carriers of the Leu10 variant had significantly lower abdominal sagittal diameter than both Leu10Pro and Pro10Pro carriers. We also found that heterozygotes had significantly higher fasting insulin values as well as higher HOMA insulin-resistance index in comparison to homozygous carriers of the Leu10. Fasting glucose levels were significantly higher in subjects with the Pro10Pro variant compared to subjects with either the Leu10Leu or Leu10Pro variant. CONCLUSION: These findings suggest that the Pro10 allele in the TGF-beta(1) gene pathway might contribute to prevalent diseases such as obesity and type 2 diabetes mellitus.     

TGF-beta1 codon 25 gene polymorphism is associated with cirrhosis in patients with hereditary hemochromatosis

Hereditary hemochromatosis (HHC) is an autosomal recessive disorder of iron metabolism with variable penetrance. Only a minority of C282Y homozygotes develop clinical overt disease and cirrhosis. The phenotypic heterogeneity of HHC may be due to host genetic factors influencing fibrogenesis such as cytokine gene polymorphisms. In this respect, we investigated the impact of functional genetic polymorphisms of TGF-beta1 (codon 10 Leu/Pro, codon 25 Arg/Pro), TNF-alpha (-308 G/A, -238 G/A) and angiotensinogen (-6 G/A) on the development of cirrhosis in HHC. One hundred and forty-nine (111 male, mean age: 51.0+/-12.9) C282Y homozygotes who underwent liver biopsy were studied. Genotyping was performed by RFLP analysis. TGF-beta1 codon 25 genotypes Arg/Pro and Pro/Pro were more common in patients with cirrhosis than in those without (23.6% vs. 7.4%, p = 0.005). In contrast, the distribution of TGF-beta1 codon 10, TNF-alpha and angiotensinogen genotypes was not different. Logistic regression analysis identified male sex, age, serum ferritin and TGF-beta1 codon 25 Arg/Pro and Pro/Pro as independent predictors for the presence of cirrhosis. The adjusted odds ratio for TGF-beta1 codon 25 Arg/Pro and Pro/Pro was 2.8 (95% CI 1.4-5.7, p = 0.004). In conclusion, C282Y homozygotes carrying TGF-beta1 genotypes Arg/Pro and Pro/Pro are more likely to develop cirrhosis than those with genotype Arg/Arg.     

Transforming growth factor-beta 1, -beta 2, and -beta 3 secreted by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.

Transforming growth factor-beta 1 (TGF-beta 1) has been found to occur as latent high molecular weight complexes, with or without an associated component denoted latent TGF-beta 1-binding protein (LTBP). We show here that a human glioblastoma cell line (U-1240 MG) secretes all isoforms of TGF-beta s found in mammalian cells (TGF-beta 1, -beta 2, and -beta 3). Approximately 26% of the secreted TGF-beta is in an active form. Latent TGF-beta s were partially purified from medium conditioned by the U-1240 MG cell line using anion exchange chromatography. Analysis of the different fractions by immunoblotting using antisera against precursor parts of the different TGF-beta isoforms, and against LTBP, revealed that not only TGF-beta 1 but also other isoforms of TGF-beta may occur in high molecular weight forms containing LTBP. In addition, each one of the TGF-beta isoforms occurred in smaller forms not containing LTBP. Interestingly, each of the TGF-beta isoforms was also seen in complexes of about 210 kDa containing associated component(s) distinct from LTBP. These results indicate that each of the different isoforms of TGF-beta is synthesized and secreted by this glioblastoma cell line in several different high molecular weight latent forms; the biological importance of the various latent TGF-beta complexes is discussed.     

Proteolysis of latent transforming growth factor-beta (TGF-beta )-binding protein-1 by osteoclasts. A cellular mechanism for release of TGF-beta from bone matrix

The binding of growth factors to the extracellular matrix (ECM) may be a key pathway for regulation of their activity. We have shown that a major mechanism for storage of transforming growth factor-beta (TGF-beta) in bone ECM is via its association with latent TGF-beta-binding protein-1 (LTBP1). Although proteolytic cleavage of LTBP1 has been reported, it remains unclear whether this represents a physiological mechanism for release of matrix-bound TGF-beta. Here we examined the role of LTBP1 in cell-mediated release of TGF-beta from bone ECM. We first characterized the soluble and ECM-bound forms of latent TGF-beta produced by primary osteoblasts. Next, we examined release of ECM-bound TGF-beta by bone resorbing cells. Isolated avian osteoclasts and rabbit bone marrow-derived osteoclasts released bone matrix-bound TGF-beta via LTBP1 cleavage. 1,25-Dihydroxyvitamin D3 enhanced LTBP1 cleavage, resulting in release of 90% of the ECM-bound LTBP1. In contrast, osteoblasts failed to cleave LTBP1 or release TGF-beta from bone ECM. Cleavage of LTBP1 by avian osteoclasts was inhibited by serine protease and metalloproteinase (MMP) inhibitors. Studies using purified proteases showed that plasmin, elastase, MMP2, and MMP9 were able to cleave LTBP1 to produce 125-165-kDa fragments. These studies identify LTBP1 as a novel substrate for MMPs and provide the first demonstration that LTBP1 proteolysis may be a physiological mechanism for release of TGF-beta from ECM-bound stores, potentially the first step in the pathway by which matrix-bound TGF-beta is rendered active.     

A vitamin D analog regulates mesangial cell smooth muscle phenotypes in a transforming growth factor-beta type II receptor-mediated manner.

Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-beta (TGF-beta) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D(3) (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)) in a dose-dependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle alpha actin mRNA, but 1, 25(OH)(2)D(3)-treated MC-E cells did not. The addition of a TGF-beta(1)-neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels. However, after exposure to 1, 25(OH)(2)D(3) or OCT, there was no significant difference in the secretion of active TGF-beta. We next investigated whether TGF-beta type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.     

Growth on type I collagen promotes expression of the osteoblastic phenotype in human osteosarcoma MG-63 cells.

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.     

Latent transforming growth factor-beta 1 associates to fibroblast extracellular matrix via latent TGF-beta binding protein

The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse- chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.     

Identification of a functional binding site for activin on the type I receptor ALK4

Activins, like other members of the transforming growth factor-beta (TGF-beta) superfamily, initiate signaling by assembling a complex of two types of transmembrane serine/threonine receptor kinases classified as type II (ActRII or ActRIIB) and type I (ALK4). A kinase-deleted version of ALK4 can form an inactive complex with activin and ActRII/IIB and thereby acts in a dominant negative manner to block activin signaling. Using the complex structure of bone morphogenetic protein-2 bound to its type I receptor (ALK3) as a guide, we introduced extracellular domain mutations in the context of the truncated ALK4 (ALK4-trunc) construct and assessed the ability of the mutants to inhibit activin function. We have identified five hydrophobic amino acid residues on the ALK4 extracellular domain (Leu40, Ile70, Val73, Leu75, and Pro77) that, when mutated to alanine, have substantial effects on ALK4-trunc dominant negative activity. In addition, eleven mutants partially affected activin binding to ALK4. Together, these residues likely constitute the binding surface for activin on ALK4. Cross-linking studies measuring binding of 125I-activin-A to the ALK4-trunc mutants in the presence of ActRII implicated the same residues. Our results indicate that there is only a partial overlap of the binding sites on ALK4 and ALK3 for activin-A and bone morphogenetic protein-2, respectively. In addition three of the residues required for activin binding to ALK4 are conserved on the type I TGF-beta receptor ALK5, suggesting the corresponding region on ALK5 may be important for TGF-beta binding.     

Role of the latent TGF-beta binding protein in the activation of latent TGF-beta by co-cultures of endothelial and smooth muscle cells

Transforming growth factor beta (TGF-beta) is released from cells in a latent form consisting of the mature growth factor associated with an aminoterminal propeptide and latent TGF-beta binding protein (LTBP). The endogenous activation of latent TGF-beta has been described in co- cultures of endothelial and smooth muscle cells. However, the mechanism of this activation remains unknown. Antibodies to native platelet LTBP and to a peptide fragment of LTBP inhibit in a dose-dependent manner the activation of latent TGF-beta normally observed when endothelial cells are cocultured with smooth muscle cells. Inhibition of latent TGF- beta activation was also observed when cells were co-cultured in the presence of an excess of free LTBP. These data represent the first demonstration of a function for the LTBP in the extracellular regulation of TGF-beta activity and indicate that LTBP participates in the activation of latent TGF-beta, perhaps by concentrating the latent growth factor on the cell surface where activation occurs.     

The role and regulation of GDF11 in Smad2 activation during tailbud formation in the Xenopus embryo

A key role for phosphorylation of Smad2 by TGFβ superfamily ligands in the axial patterning of early embryos is well established. The regulation and role of Smad2 signaling in post-neurula embryonic patterning, however, is less well understood. While a variety of TGFβ superfamily ligands are implicated in various stages of anterior–posterior patterning, the ligand GDF11 has been shown to have a particular role in post-gastrula patterning in the mouse. Mouse GDF11 is specifically localized to the developing tail and is essential for normal posterior axial patterning. Mature GDF11 ligand is inhibited by its own prodomain, and extracellular proteolysis of this prodomain is thought to be necessary for GDF11 activity. The contribution of this proteolytic regulatory mechanism to Smad activation during embryogenesis in vivo, and to the development of posterior pattern, has not been characterized. We investigate here the role of Xenopus GDF11 in the activation of Smad2 during the development of tailbud-stage embryos, and the role of this activation in larval development. We also demonstrate that the activity of BMP-1/Tolloid-like proteases is necessary for the normal GDF11-dependent activation of Smad2 phosphorylation during post-gastrula development. These data demonstrate that GDF11 has a central role in the activation of Smad2 phosphorylation in tailbud stage Xenopus embryos, and provide the first evidence that BMP-1/Tolloid-mediated prodomain cleavage is important for activation of GDF11 in vivo.     

A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2

Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5^W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.