Secreted ADAMTS-like proteins as regulators of connective tissue function.

The extracellular matrix (ECM) determines functional properties of connective tissues through structural components, such as collagens, elastic fibers, or proteoglycans. The ECM also instructs cell behavior through regulatory proteins, including proteases, growth factors, and matricellular proteins, which can be soluble or tethered to ECM scaffolds. The secreted a disintegrin and metalloproteinase with thrombospondin type 1 repeats/motifs-like (ADAMTSL) proteins constitute a family of regulatory ECM proteins that are related to ADAMTS proteases but lack their protease domains. In mammals, the ADAMTSL protein family comprises seven members, ADAMTSL1-6 and papilin. ADAMTSL orthologs are also present in the worm, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster. Like other matricellular proteins, ADAMTSL expression is characterized by tight spatiotemporal regulation during embryonic development and early postnatal growth and by cell type- and tissue-specific functional pleiotropy. Although largely quiescent during adult tissue homeostasis, reexpression of ADAMTSL proteins is frequently observed in the context of physiological and pathological tissue remodeling and during regeneration and repair after injury. The diverse functions of ADAMTSL proteins are further evident from disorders caused by mutations in individual ADAMTSL proteins, which can affect multiple organ systems. In addition, genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) in ADAMTSL genes to complex traits, such as lung function, asthma, height, body mass, fibrosis, or schizophrenia. In this review, we summarize the current knowledge about individual members of the ADAMTSL protein family and highlight recent mechanistic studies that began to elucidate their diverse functions.

Alternative splicing of the metalloprotease ADAMTS17 spacer regulates secretion and modulates autoproteolytic activity.

ADAMTS proteases mediate biosynthesis and breakdown of secreted extracellular matrix (ECM) molecules in numerous physiological and disease processes. In addition to their catalytic domains, ADAMTS proteases contain ancillary domains, which mediate substrate recognition and ECM binding and confer distinctive properties and roles to individual ADAMTS proteases. Although alternative splicing can greatly expand the structural and functional diversity of ADAMTS proteases, it has been infrequently reported and functional consequences have been rarely investigated. Here, we characterize the structural and functional impact of alternative splicing of ADAMTS17, mutations in which cause Weill-Marchesani syndrome 4. Two novel ADAMTS17 splice variants, ADAMTS17A and ADAMTS17B, were investigated by structural modeling, mass spectrometry, and biochemical approaches. Our results identify a novel disulfide-bridged insertion in the ADAMTS17A spacer that originates from inclusion of a novel exon. This insertion results in differential autoproteolysis of ADAMTS17, and thus, predicts altered proteolytic activity against other substrates. The second variant, ADAMTS17B, results from an in-frame exon deletion and prevents ADAMTS17B secretion. Thus, alternative splicing of the ADAMTS spacer significantly regulates the physiologically relevant proteolytic activity of ADAMTS17, either by altering proteolytic specificity (ADAMTS17A) or by altering cellular localization (ADAMTS17B).

The quest for substrates and binding partners: A critical barrier for understanding the role of ADAMTS proteases in musculoskeletal development and disease.

Secreted ADAMTS metalloproteases are involved in the sculpting, remodeling, and erosion of connective tissues throughout the body, including in the musculoskeletal system. ADAMTS proteases contribute to musculoskeletal development, pathological tissue destruction, and are mutated in congenital musculoskeletal disorders. Examples include versican cleavage by ADAMTS9 which is required for interdigital web regression during limb development, ADAMTS5-mediated aggrecan degradation in osteoarthritis resulting in joint erosion, and mutations in ADAMTS10 or ADAMTS17 that cause Weill-Marchesani syndrome, a short stature syndrome with bone, joint, muscle, cardiac, and eye involvement. Since the function of ADAMTS proteases and proteases in general is primarily defined by the molecular consequences of proteolysis of their respective substrates, it is paramount to identify all physiological substrates for each individual ADAMTS protease. Here, we review the current knowledge of ADAMTS proteases and their involvement in musculoskeletal development and disease, focusing on some of their known physiological substrates and the consequences of substrate cleavage. We further emphasize the critical need for the identification and validation of novel ADAMTS substrates and binding partners by describing the principles of mass spectrometry-based approaches and by emphasizing strategies that need to be considered for validating the physiological relevance for ADAMTS-mediated proteolysis of novel putative substrates.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

Early fibrillin-1 assembly monitored through a modifiable recombinant cell approach.

Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length fibrillin-1 assembly system that allows for rapid analysis of critical roles in fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.

Biomaterial-Based Regenerative Strategies for Volumetric Muscle Loss: Challenges and Solutions.

Significance: Volumetric muscle loss (VML) is caused by the loss of significant amounts of skeletal muscle tissue. VML cannot be repaired by intrinsic regenerative processes, resulting in permanent loss of muscle function and disability. Current rehabilitative-focused treatment strategies lack efficacy and do not restore muscle function, indicating the need for the development of effective regenerative strategies. Recent Advances: Recent developments implicate biomaterial-based approaches for promoting muscle repair and functional restoration post-VML. Specifically, bioscaffolds transplanted in the injury site have been utilized to mimic endogenous cues of the ablated tissue to promote myogenic pathways, increase neo-myofiber synthesis, and ultimately restore contractile function to the injured unit. Critical Issues: Despite the development and preclinical testing of various biomaterial-based regenerative strategies, effective therapies for patients are not available. The unique challenges posed for biomaterial-based treatments of VML injuries, including its scalability and clinical applicability beyond small-animal models, impede progress. Furthermore, production of tissue-engineered constructs is technically demanding, with reproducibility issues at scale and complexities in achieving vascularization and innervation of large constructs. Future Directions: Biomaterial-based regenerative strategies designed to comprehensively address the pathophysiology of VML are needed. Considerations for clinical translation, including scalability and regulatory compliance, should also be considered when developing such strategies. In addition, an integrated approach that combines regenerative and rehabilitative strategies is essential for ensuring functional improvement.

The biology of the extracellular matrix: novel insights.

PURPOSE OF REVIEW: Extracellular matrix (ECM) has both structural and regulatory roles. This update reviews the representative recent developments in diverse aspects of ECM biology relevant to inflammation, tissue destruction, fibrosis, and regeneration. RECENT FINDINGS: Biological regulation by ECM is emerging as a major research area, driven by several new directions. Sensing of mechanical cues provided by ECM was found to be crucial in regulating cell differentiation. Transforming growth factor-ß (TGF-ß) is a pivotal agent in fibrosis and inflammation. A combination of structural biology and cell biology provided novel insights on the mechanisms of its activation by cellular traction and ECM. Improved understanding of how fibrillin microfibrils and associated proteins regulated TGF-ß sequestration and activation was achieved by analysis of inherited connective tissue disorders having TGF-ß dysregulation as an underlying pathologic mechanism. Insights on microRNA-mediated ECM regulation suggest a key role for miR-29, for which potential therapeutic roles are emerging. Advances in understanding the ECM turnover by proteinases provided novel insights on cell regulation and identified useful disease biomarkers. SUMMARY: As a crucial modulator of cell behavior, ECM has exceptionally strong relevance and translational implications for human disease, opening novel opportunities for mechanistic understanding of disease pathogenesis as well as treatment.

En route towards a personalized medicine approach: Innovative therapeutic modalities for connective tissue disorders.

Connective tissue disorders can be caused by pathogenic variants (mutations) in genes encoding extracellular matrix (ECM) proteins. Such disorders typically manifest during development or postnatal growth and result in significant morbidity and mortality. The development of curative treatments for connective tissue disorders is hampered in part by the inability of many mature connective tissues to efficiently regenerate. To be most effective, therapeutic strategies designed to preserve or restore tissue function will likely need to be initiated during phases of significant endogenous connective tissue remodeling and organ sculpting postnatally and directly target the underlying ECM protein mutations. With recent advances in whole exome sequencing, in-vitro and in-vivo disease modeling, and the development of mutation-specific molecular therapeutic modalities, it is now feasible to directly correct disease-causing mutations underlying connective tissue disorders and ameliorate their pathogenic consequences. These technological advances may lead to potentially curative personalized medicine approaches for connective tissue disorders that have previously been considered incurable. In this review, we highlight innovative therapeutic modalities including gene replacement, exon skipping, DNA/mRNA editing, and pharmacological approaches that were used to preserve or restore tissue function in the context of connective tissue disorders. Inherent to a successful application of these approaches is the need to deepen the understanding of mechanisms that regulate ECM formation and homeostasis, and to decipher how individual mutations in ECM proteins compromise ECM and connective tissue development and function.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling.

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFß signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFß and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function.

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.

Genetic and functional linkage between ADAMTS superfamily proteins and fibrillin-1: a novel mechanism influencing microfibril assembly and function.

Tissue microfibrils contain fibrillin-1 as a major constituent. Microfibrils regulate bioavailability of TGFß superfamily growth factors and are structurally crucial in the ocular zonule. FBN1 mutations typically cause the Marfan syndrome, an autosomal dominant disorder manifesting with skeletal overgrowth, aortic aneurysm, and lens dislocation (ectopia lentis). Infrequently, FBN1 mutations cause dominantly inherited Weill-Marchesani syndrome (WMS), isolated ectopia lentis (IEL), or the fibrotic condition, geleophysic dysplasia (GD). Intriguingly, mutations in ADAMTS [a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] family members phenocopy these disorders, leading to recessive WMS (ADAMTS10), WMS-like syndrome (ADAMTS17), IEL (ADAMTSL4 and ADAMTS17) and GD (ADAMTSL2). An ADAMTSL2 founder mutation causes Musladin-Lueke syndrome, a fibrotic disorder in beagle dogs. The overlapping disease spectra resulting from fibrillin-1 and ADAMTS mutations, interaction of ADAMTS10 and ADAMTSL2 with fibrillin-1, and evidence that these ADAMTS proteins accelerate microfibril biogenesis, constitutes a consilience suggesting that some ADAMTS proteins evolved to provide a novel mechanism regulating microfibril formation and consequently cell behavior.

The matrix in focus: new directions in extracellular matrix research from the 2021 ASMB hybrid meeting.

The extracellular matrix (ECM) is a complex assembly of macromolecules that provides both architectural support and molecular signals to cells and modulate their behaviors. Originally considered a passive mechanical structure, decades of research have since demonstrated how the ECM dynamically regulates a diverse set of cellular processes in development, homeostasis, and disease progression. In September 2021, the American Society for Matrix Biology (ASMB) organized a hybrid scientific meeting, integrating in-person and virtual formats, to discuss the latest developments in ECM research. Here, we highlight exciting scientific advances that emerged from the meeting including (1) the use of model systems for fundamental and translation ECM research, (2) ECM-targeting approaches as therapeutic modalities, (3) cell-ECM interactions, and (4) the ECM as a critical component of tissue engineering strategies. In addition, we discuss how the ASMB incorporated mentoring, career development, and diversity, equity, and inclusion initiatives in both virtual and in-person events. Finally, we reflect on the hybrid scientific conference format and how it will help the ASMB accomplish its mission moving forward.

Homocysteine modifies structural and functional properties of fibronectin and interferes with the fibronectin-fibrillin-1 interaction.

Homocystinuria is a genetic disorder resulting in elevated levels of homocysteine in plasma and tissues. Some of the skeletal and ocular symptoms such as long bone overgrowth, scoliosis, and ectopia lentis overlap with symptoms seen in Marfan syndrome. Marfan syndrome is caused by mutations in the extracellular matrix protein fibrillin-1. We previously showed that fibrillin-1 is a target for homocysteine and that the deposition of homocysteinylated fibrillin-1 in the extracellular matrix is compromised. Since the assembly of fibrillin-1 is critically dependent on fibronectin, we analyzed the consequences of fibronectin homocysteinylation and its interaction with fibrillin-1. Cellular fibronectin and proteolytic fragments were homocysteinylated and tested in various interaction assays with recombinant fibrillin-1 and heparin. Fibronectin homocysteinylation consistently compromised the fibronectin-fibrillin-1 interaction, while the interaction with heparin was not affected. Fibronectin homocysteinylation, but not cysteinylation, reduced the fibronectin dimers to monomers as shown by Western blotting. ELISA analyses of homocysteinylated fibronectin with three monoclonal antibodies demonstrated structural changes in the disulfide-containing FNI domains FNI(2), FNI(4), and FNI(9). Using fluorescently labeled fibronectin, we studied the consequence of fibronectin homocysteinylation on assembly in cell culture. Modified fibronectin showed deficiencies in denovo matrix incorporation and initial assembly. In conclusion, we define here characteristic structural changes of fibronectin upon homocysteinylation that translate into functional deficiencies in the fibronectin-fibrillin-1 interaction and in fibronectin assembly. Since fibronectin is a major organizer of various extracellular protein networks, these structural and functional alterations may contribute to the pathogenesis of homocystinuria and Marfan syndrome.

Functional consequences of homocysteinylation of the elastic fiber proteins fibrillin-1 and tropoelastin.

Homocystinuria caused by cystathionine-beta-synthase deficiency represents a severe form of homocysteinemias, which generally result in various degrees of elevated plasma homocysteine levels. Marfan syndrome is caused by mutations in fibrillin-1, which is one of the major constituents of connective tissue microfibrils. Despite the fundamentally different origins, both diseases share common clinical symptoms in the connective tissue such as long bone overgrowth, scoliosis, and ectopia lentis, whereas they differ in others. Fibrillin-1 contains approximately 13% cysteine residues and can be modified by homocysteine. We report here that homocysteinylation affects functional properties of fibrillin-1 and tropoelastin. We used recombinant fragments spanning the entire fibrillin-1 molecule to demonstrate that homocysteinylation, but not cysteinylation leads to abnormal self-interaction, which was attributed to a reduced amount of multimerization of the fibrillin-1 C terminus. The deposition of the fibrillin-1 network by human dermal fibroblasts was greatly reduced by homocysteine, but not by cysteine. Furthermore, homocysteinylation, but not cysteinylation of elastin-like polypeptides resulted in modified coacervation properties. In summary, the results provide new insights into pathogenetic mechanisms potentially involved in cystathionine-beta-synthase-deficient homocystinuria.

Biogenesis of extracellular microfibrils: Multimerization of the fibrillin-1 C terminus into bead-like structures enables self-assembly.

Microfibrils are essential elements in elastic and nonelastic tissues contributing to homeostasis and growth factor regulation. Fibrillins form the core of these multicomponent assemblies. Various human genetic disorders, the fibrillinopathies, arise from mutations in fibrillins and are frequently associated with aberrant microfibril assembly. These disorders include Marfan syndrome, Weill-Marchesani syndrome, Beals syndrome, and others. Although homotypic and heterotypic fibrillin self-interactions are considered to provide critical initial steps, the detailed mechanisms for microfibril assembly are unknown. We show here that the C-terminal recombinant half of fibrillin-1 assembles into disulfide-bonded multimeric globular structures with peripheral arms and a dense core. These globules are similar to the beaded structures observed in microfibrils isolated from tissues. Only these C-terminal fibrillin-1 multimers interacted strongly with the fibrillin-1 N terminus, whereas the monomers showed very little self-interaction activity. The multimers strongly inhibited microfibril formation in cell culture, providing evidence that these recombinant assemblies can also interact with endogenous fibrillin-1. The C-terminal self-interaction site was fine-mapped to the last three calcium-binding EGF domains in fibrillin-1. These results suggest a new mechanism for microfibril formation where fibrillin-1 first oligomerizes via its C terminus before the partially or fully assembled bead-like structures can further interact with other beads via the fibrillin-1 N termini.

Acromelic dysplasias: how rare musculoskeletal disorders reveal biological functions of extracellular matrix proteins.

Acromelic dysplasias are a group of rare musculoskeletal disorders that collectively present with short stature, pseudomuscular build, stiff joints, and tight skin. Acromelic dysplasias are caused by mutations in genes (FBN1, ADAMTSL2, ADAMTS10, ADAMTS17, LTBP2, and LTBP3) that encode secreted extracellular matrix proteins, and in SMAD4, an intracellular coregulator of transforming growth factor-ß (TGF-ß) signaling. The shared musculoskeletal presentations in acromelic dysplasias suggest that these proteins cooperate in a biological pathway, but also fulfill distinct roles in specific tissues that are affected in individual disorders of the acromelic dysplasia group. In addition, most of the affected proteins directly interact with fibrillin microfibrils in the extracellular matrix and have been linked to the regulation of TGF-ß signaling. Together with recently developed knockout mouse models targeting the affected genes, novel insights into molecular mechanisms of how these proteins regulate musculoskeletal development and homeostasis have emerged. Here, we summarize the current knowledge highlighting pathogenic mechanisms of the different disorders that compose acromelic dysplasias and provide an overview of the emerging biological roles of the individual proteins that are compromised. Finally, we develop a conceptual model of how these proteins may interact and form an "acromelic dysplasia complex" on fibrillin microfibrils in connective tissues of the musculoskeletal system.

Regulation of ADAMTS Proteases.

A disintegrin and metalloprotease with thrombospondin type I motifs (ADAMTS) proteases are secreted metalloproteinases that play key roles in the formation, homeostasis and remodeling of the extracellular matrix (ECM). The substrate spectrum of ADAMTS proteases can range from individual ECM proteins to entire families of ECM proteins, such as the hyalectans. ADAMTS-mediated substrate cleavage is required for the formation, remodeling and physiological adaptation of the ECM to the needs of individual tissues and organ systems. However, ADAMTS proteases can also be involved in the destruction of tissues, resulting in pathologies such as arthritis. Specifically, ADAMTS4 and ADAMTS5 contribute to irreparable cartilage erosion by degrading aggrecan, which is a major constituent of cartilage. Arthritic joint damage is a major contributor to musculoskeletal morbidity and the most frequent clinical indication for total joint arthroplasty. Due to the high sequence homology of ADAMTS proteases in their catalytically active site, it remains a formidable challenge to design ADAMTS isotype-specific inhibitors that selectively inhibit ADAMTS proteases responsible for tissue destruction without affecting the beneficial functions of other ADAMTS proteases. In vivo, proteolytic activity of ADAMTS proteases is regulated on the transcriptional and posttranslational level. Here, we review the current knowledge of mechanisms that regulate ADAMTS protease activity in tissues including factors that induce ADAMTS gene expression, consequences of posttranslational modifications such as furin processing, the role of endogenous inhibitors and pharmacological approaches to limit ADAMTS protease activity in tissues, which almost exclusively focus on inhibiting the aggrecanase activity of ADAMTS4 and ADAMTS5.

The extracellular matrix glycoprotein ADAMTSL2 is increased in heart failure and inhibits TGFß signalling in cardiac fibroblasts.

Fibrosis accompanies most heart diseases and is associated with adverse patient outcomes. Transforming growth factor (TGF)ß drives extracellular matrix remodelling and fibrosis in the failing heart. Some members of the ADAMTSL (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motifs-like) family of secreted glycoproteins bind to matrix microfibrils, and although their function in the heart remains largely unknown, they are suggested to regulate TGFß activity. The aims of this study were to determine ADAMTSL2 levels in failing hearts, and to elucidate the role of ADAMTSL2 in fibrosis using cultured human cardiac fibroblasts (CFBs). Cardiac ADAMTSL2 mRNA was robustly increased in human and experimental heart failure, and mainly expressed by fibroblasts. Over-expression and treatment with extracellular ADAMTSL2 in human CFBs led to reduced TGFß production and signalling. Increased ADAMTSL2 attenuated myofibroblast differentiation, with reduced expression of the signature molecules α-smooth muscle actin and osteopontin. Finally, ADAMTSL2 mitigated the pro-fibrotic CFB phenotypes, proliferation, migration and contractility. In conclusion, the extracellular matrix-localized glycoprotein ADAMTSL2 was upregulated in fibrotic and failing hearts of patients and mice. We identified ADAMTSL2 as a negative regulator of TGFß in human cardiac fibroblasts, inhibiting myofibroblast differentiation and pro-fibrotic properties.

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-beta.

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.