Lack of evidence for a role of anthrax toxin receptors as surface receptors for collagen VI and for its cleaved-off C5 domain/endotrophin.

The microfibril-forming collagen VI is proteolytically cleaved and it was proposed that the released C-terminal Kunitz domain (C5) of the α3 chain is an adipokine important for tumor progression and fibrosis. Designated "endotrophin," C5 is a potent biomarker for fibroinflammatory diseases. However, the biochemical mechanisms behind endotrophin activity were not investigated. Earlier, anthrax toxin receptor 1 was found to bind C5, but this potential interaction was not further studied. Given the proposed physiological role of endotrophin, we aimed to determine how the signal is transmitted. Surprisingly, we could not detect any interaction between endotrophin and anthrax toxin receptor 1 or its close relative, anthrax toxin receptor 2. Moreover, we detect no binding of fully assembled collagen VI to either receptor. We also studied the collagen VI receptor NG2 (CSPG4) and confirmed that NG2 binds assembled collagen VI, but not cleaved C5/endotrophin. A cellular receptor for C5/endotrophin, therefore, still remains elusive.

Correlation of metabolic characteristics with maternal, fetal and placental asprosin in human pregnancy.

Objective: Asprosin is a recently discovered hormone associated with obesity and diabetes mellitus. Little is known about asprosin's role during pregnancy, but a contribution of asprosin to pregnancy complications resulting from maternal obesity and gestational diabetes mellitus (GDM) is conceivable. We assessed the potential effects of obesity, GDM and other clinical parameters on maternal and fetal umbilical plasma asprosin concentrations and placental asprosin expression. Design: The Cologne-Placenta Cohort Study comprises 247 female patients, from whom blood and placentas were collected at the University Hospital Cologne. Methods: We studied the maternal and fetal umbilical plasma and placentas of pregnant women with an elective, primary section. Sandwich ELISA measurements of maternal and fetal umbilical plasma and immunohistochemical stainings of placental tissue were performed to determine the asprosin levels. Also, the relation between asprosin levels and clinical blood parameters was studied. Results: There was a strong correlation between the maternal and fetal plasma asprosin levels and both increased with GDM in normal-weight and obese women. Asprosin immunoreactivity was measured in cultivated placental cells and placental tissue. BMI and GDM were not but pre-pregnancy exercise and smoking were correlated with maternal and/or fetal asprosin levels. Placental asprosin levels were associated with maternal but not with fetal plasma asprosin levels and with BMI but not with GDM. Placental asprosin was related to maternal insulin levels and increased upon insulin treatment in GDM patients. Conclusions: Asprosin could potentially act as a biomarker and contribute to the clinical manifestation of pregnancy complications associated with maternal obesity.

Sensitive asprosin detection in clinical samples reveals serum/saliva correlation and indicates cartilage as source for serum asprosin.

The C-terminal pro-fibrillin-1 propeptide asprosin is described as white adipose tissue derived hormone that stimulates rapid hepatic glucose release and activates hunger-promoting hypothalamic neurons. Numerous studies proposed correlations of asprosin levels with clinical parameters. However, the enormous variability of reported serum and plasma asprosin levels illustrates the need for sensitive and reliable detection methods in clinical samples. Here we report on newly developed biochemical methods for asprosin concentration and detection in several body fluids including serum, plasma, saliva, breast milk, and urine. Since we found that glycosylation impacts human asprosin detection we analyzed its glycosylation profile. Employing a new sandwich ELISA revealed that serum and saliva asprosin correlate strongly, depend on biological sex, and feeding status. To investigate the contribution of connective tissue-derived asprosin to serum levels we screened two cohorts with described cartilage turnover. Serum asprosin correlated with COMP, a marker for cartilage degradation upon running exercise and after total hip replacement surgery. This together with our finding that asprosin is produced by primary human chondrocytes and expressed in human cartilage suggests a contribution of cartilage to serum asprosin. Furthermore, we determined asprosin levels in breast milk, and urine, for the first time, and propose saliva asprosin as an accessible clinical marker for future studies.

BMP antagonists in tissue development and disease.

Bone morphogenic proteins (BMPs) are important growth regulators in embryogenesis and postnatal homeostasis. Their tight regulation is crucial for successful embryonic development as well as tissue homeostasis in the adult organism. BMP inhibition by natural extracellular biologic antagonists represents the most intensively studied mechanistic concept of BMP growth factor regulation. It was shown to be critical for numerous developmental programs, including germ layer specification and spatiotemporal gradients required for the establishment of the dorsal-ventral axis and organ formation. The importance of BMP antagonists for extracellular matrix homeostasis is illustrated by the numerous human connective tissue disorders caused by their mutational inactivation. Here, we will focus on the known functional interactions targeting BMP antagonists to the ECM and discuss how these interactions influence BMP antagonist activity. Moreover, we will provide an overview about the current concepts and investigated molecular mechanisms modulating BMP inhibitor function in the context of development and disease.

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.

Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.

C-terminal proteolysis of the collagen VI α3 chain by BMP-1 and proprotein convertase(s) releases endotrophin in fragments of different sizes.

The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.

Myeloid Cell-Restricted STAT3 Signaling Controls a Cell-Autonomous Antifibrotic Repair Program.

Myeloid cells can be beneficial as well as harmful in tissue regenerative responses. The molecular mechanisms by which myeloid cells control this critical decision of the immune system are not well understood. Using two different models of physiological acute or pathological chronic skin damage, in this study we identified myeloid cell-restricted STAT3 signaling as important and an injury context-dependent regulator of skin fibrosis. Targeted disruption of STAT3 signaling in myeloid cells significantly accelerated development of pathological skin fibrosis in a model of chronic bleomycin-induced tissue injury, whereas the impact on wound closure dynamics and quality of healing after acute excision skin injury was minor. Chronic bleomycin-mediated tissue damage in control mice provoked an antifibrotic gene signature in macrophages that was characterized by upregulated expression of IL-10, SOCS3, and decorin. In contrast, in STAT3-deficient macrophages this antifibrotic repair program was abolished whereas TGF-ß1 expression was increased. Notably, TGF-ß1 synthesis in cultured control bone marrow-derived macrophages (BMDMs) was suppressed after IL-10 exposure, and this suppressive effect was alleviated by STAT3 deficiency. Accordingly, coculture of IL-10-stimulated control BMDMs with fibroblasts suppressed expression of the TGF-ß1 downstream target connective tissue growth factor in fibroblasts, whereas this suppressive effect was lost by STAT3 deficiency in BMDMs. Our findings highlight a previously unrecognized protective role of myeloid cell-specific STAT3 signaling in immune cell-mediated skin fibrosis, and its regulatory pathway could be a potential target for therapy.

TGFB1 is secreted through an unconventional pathway dependent on the autophagic machinery and cytoskeletal regulators.

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

The role of chordin fragments generated by partial tolloid cleavage in regulating BMP activity.

Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin-BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner.

AMACO is a component of the basement membrane-associated Fraser complex.

Fraser syndrome (FS) is a phenotypically variable, autosomal recessive disorder characterized by cryptophthalmus, cutaneous syndactyly, and other malformations resulting from mutations in FRAS1, FREM2, and GRIP1. Transient embryonic epidermal blistering causes the characteristic defects of the disorder. Fras1, Frem1, and Frem2 form the extracellular Fraser complex, which is believed to stabilize the basement membrane. However, several cases of FS could not be attributed to mutations in FRAS1, FREM2, or GRIP1, and FS displays high clinical variability, suggesting that there is an additional genetic, possibly modifying contribution to this disorder. An extracellular matrix protein containing VWA-like domains related to those in matrilins and collagens (AMACO), encoded by the VWA2 gene, has a very similar tissue distribution to the Fraser complex proteins in both mouse and zebrafish. Here, we show that AMACO deposition is lost in Fras1-deficient zebrafish and mice and that Fras1 and AMACO interact directly via their chondroitin sulfate proteoglycan (CSPG) and P2 domains. Knockdown of vwa2, which alone causes no phenotype, enhances the phenotype of hypomorphic Fras1 mutant zebrafish. Together, our data suggest that AMACO represents a member of the Fraser complex.

ADAMTSL-6 is a novel extracellular matrix protein that binds to fibrillin-1 and promotes fibrillin-1 fibril formation.

ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs)-like (ADAMTSL) proteins, a subgroup of the ADAMTS superfamily, share several domains with ADAMTS proteinases, including thrombospondin type I repeats, a cysteine-rich domain, and an ADAMTS spacer, but lack a catalytic domain. We identified two new members of ADAMTSL proteins, ADAMTSL-6alpha and -6beta, that differ in their N-terminal amino acid sequences but have common C-terminal regions. When transfected into MG63 osteosarcoma cells, both isoforms were secreted and deposited into pericellular matrices, although ADAMTSL-6alpha, in contrast to -6beta, was barely detectable in the conditioned medium. Immunolabeling at the light and electron microscopic levels showed their close association with fibrillin-1-rich microfibrils in elastic connective tissues. Surface plasmon resonance analyses demonstrated that ADAMTSL-6beta binds to the N-terminal half of fibrillin-1 with a dissociation constant of approximately 80 nm. When MG63 cells were transfected or exogenously supplemented with ADAMTSL-6, fibrillin-1 matrix assembly was promoted in the early but not the late stage of the assembly process. Furthermore, ADAMTSL-6 transgenic mice exhibited excessive fibrillin-1 fibril formation in tissues where ADAMTSL-6 was overexpressed. All together, these results indicated that ADAMTSL-6 is a novel microfibril-associated protein that binds directly to fibrillin-1 and promotes fibrillin-1 matrix assembly.

Latent transforming growth factor beta-binding proteins and fibulins compete for fibrillin-1 and exhibit exquisite specificities in binding sites.

Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFbeta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit "exquisite specificities," a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.

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.

Effects of fibrillin-1 degradation on microfibril ultrastructure.

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.

Identification and characterization of AMACO, a new member of the von Willebrand factor A-like domain protein superfamily with a regulated expression in the kidney.

The genes coding for human and mouse AMACO, an extracellular matrix protein containing VWA-like domains related to those in MAtrilins and COllagens, were detected in databases, the cDNAs were cloned, and the primary structures were deduced from the nucleotide sequences. The genes consist of 14 exons and have a similar exon/intron organization. The protein consists of a signal peptide sequence, an N-terminal VWA domain connected to two additional, tandem VWA domains by a cysteine-rich sequence and an epidermal growth factor (EGF)-like domain. The C terminus is made up of another EGF-like domain followed by a unique sequence present in mouse, but absent in human. The predicted molecular weight of the proteins is 79,485 in human and 83,024 in mouse. Full-length AMACO was expressed in 293-EBNA cells, purified by use of an affinity tag and subjected to biochemical characterization. Both monomers and aggregates of AMACO were recovered, as shown by electron microscopy and SDS-PAGE. AMACO was found in the media of a variety of established cell lines of both fibroblast and epithelial origin. In the matrix formed by 293-EBNA cells overexpressing the protein, AMACO was deposited in patchy structures that were often cell-associated. Affinity-purified antibodies detect expression in cartilage and expression associated with certain basement membranes. In the kidney of adult mice, a second promoter located in intron 4 is active. If the resulting transcript is translated it could not yield a secreted protein because of the lack of a signal peptide sequence. The developmental switch from an AMACO mRNA, expressed by the newborn kidney, to the truncated transcript found in the adult kidney indicates an unusual regulation of AMACO expression.

A ribozyme with michaelase activity: synthesis of the substrate precursors.

The ability to generate RNA molecules that can catalyze complex organic transformations not only facilitates the reconstruction and plausibility of possible prebiotic reaction pathways but is also crucial for elucidating the potential of the application of RNA catalysts in organic syntheses. Iterative RNA selection previously identified a ribozyme that catalyzes the Michael addition of a cysteine thiol to an alpha,beta-unsaturated amide. This reaction is chemically similar to the rate limiting step of the thymidylate synthase reaction, which is the corresponding reaction of a cysteine thiol to the double-bond of the uracil nucleobase. Here we provide a detailed description of the synthesis of the ribozyme substrates and the substrate oligonucleotides used for its characterization and the investigation of the background reaction. We also describe the further characterization of the ribozyme with respect to substrate specificity. We show that the thiol group of the cysteine nucleophile is essential for the reaction to proceed. When substituted for a thiomethyl group, no reaction takes place.