SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation.

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D-pellet culture led to significant increases in the expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific -10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N-acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co-IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B-SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC-chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Fibrillin microfibrils and elastic fibre proteins: Functional interactions and extracellular regulation of growth factors.

Fibrillin microfibrils are extensible polymers that endow connective tissues with long-range elasticity and have widespread distributions in both elastic and non-elastic tissues. They act as a template for elastin deposition during elastic fibre formation and are essential for maintaining the integrity of tissues such as blood vessels, lung, skin and ocular ligaments. A reduction in fibrillin is seen in tissues in vascular ageing, chronic obstructive pulmonary disease, skin ageing and UV induced skin damage, and age-related vision deterioration. Most mutations in fibrillin cause Marfan syndrome, a genetic disease characterised by overgrowth of the long bones and other skeletal abnormalities with cardiovascular and eye defects. However, mutations in fibrillin and fibrillin-binding proteins can also cause short-stature pathologies. All of these diseases have been linked to dysregulated growth factor signalling which forms a major functional role for fibrillin.

ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-ß superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill-Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

A high-content platform to characterise human induced pluripotent stem cell lines.

Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components. Here we report the establishment of a high-content platform for phenotypic analysis of human iPSC lines. In the described assay, cells are dissociated and seeded as single cells onto 96-well plates coated with fibronectin at three different concentrations. This method allows assessment of cell number, proliferation, morphology and intercellular adhesion. Altogether, our strategy delivers robust quantification of phenotypic diversity within complex cell populations facilitating future identification of the genetic, biological and technical determinants of variance. Approaches such as the one described can be used to benchmark iPSCs from multiple donors and create novel platforms that can readily be tailored for disease modelling and drug discovery.

Epithelial-mesenchymal status influences how cells deposit fibrillin microfibrils.

Here, we show that epithelial-mesenchymal status influences how cells deposit extracellular matrix. Retinal pigmented epithelial (RPE) cells that expressed high levels of E-cadherin and had cell-cell junctions rich in zona occludens (ZO)-1, ß-catenin and heparan sulfate, required syndecan-4 but not fibronectin or protein kinase C α (PKCα) to assemble extracellular matrix (fibrillin microfibrils and perlecan). In contrast, RPE cells that strongly expressed mesenchymal smooth muscle α-actin but little ZO-1 or E-cadherin, required fibronectin (like fibroblasts) and PKCα, but not syndecan-4. Integrins α5ß1 and/or α8ß1 and actomyosin tension were common requirements for microfibril deposition, as was heparan sulfate biosynthesis. TGFß, which stimulates epithelial-mesenchymal transition, altered gene expression and overcame the dependency on syndecan-4 for microfibril deposition in epithelial RPE cells, whereas blocking cadherin interactions disrupted microfibril deposition. Renal podocytes had a transitional phenotype with pericellular ß-catenin but little ZO-1; they required syndecan-4 and fibronectin for efficient microfibril deposition. Thus, epithelial-mesenchymal status modulates microfibril deposition.

Leri's pleonosteosis, a congenital rheumatic disease, results from microduplication at 8q22.1 encompassing GDF6 and SDC2 and provides insight into systemic sclerosis pathogenesis.

OBJECTIVES: Leri's pleonosteosis (LP) is an autosomal dominant rheumatic condition characterised by flexion contractures of the interphalangeal joints, limited motion of multiple joints, and short broad metacarpals, metatarsals and phalanges. Scleroderma-like skin thickening can be seen in some individuals with LP. We undertook a study to characterise the phenotype of LP and identify its genetic basis. METHODS AND RESULTS: Whole-genome single-nucleotide polymorphism genotyping in two families with LP defined microduplications of chromosome 8q22.1 as the cause of this condition. Expression analysis of dermal fibroblasts from affected individuals showed overexpression of two genes, GDF6 and SDC2, within the duplicated region, leading to dysregulation of genes that encode proteins of the extracellular matrix and downstream players in the transforming growth factor (TGF)-ß pathway. Western blot analysis revealed markedly decreased inhibitory SMAD6 levels in patients with LP. Furthermore, in a cohort of 330 systemic sclerosis cases, we show that the minor allele of a missense SDC2 variant, p.Ser71Thr, could confer protection against disease (p<1×10(-5)). CONCLUSIONS: Our work identifies the genetic cause of LP in these two families, demonstrates the phenotypic range of the condition, implicates dysregulation of extracellular matrix homoeostasis genes in its pathogenesis, and highlights the link between TGF-ß/SMAD signalling, growth/differentiation factor 6 and syndecan-2. We propose that LP is an additional member of the growing 'TGF-ß-pathies' group of musculoskeletal disorders, which includes Myhre syndrome, acromicric dysplasia, geleophysic dysplasias, Weill-Marchesani syndromes and stiff skin syndrome. Identification of a systemic sclerosis-protective SDC2 variant lays the foundation for exploration of the role of syndecan-2 in systemic sclerosis in the future.

Elastic fibres in health and disease.

Elastic fibres are insoluble components of the extracellular matrix of dynamic connective tissues such as skin, arteries, lungs and ligaments. They are laid down during development, and comprise a cross-linked elastin core within a template of fibrillin-based microfibrils. Their function is to endow tissues with the property of elastic recoil, and they also regulate the bioavailability of transforming growth factor ß. Severe heritable elastic fibre diseases are caused by mutations in elastic fibre components; for example, mutations in elastin cause supravalvular aortic stenosis and autosomal dominant cutis laxa, mutations in fibrillin-1 cause Marfan syndrome and Weill-Marchesani syndrome, and mutations in fibulins-4 and -5 cause autosomal recessive cutis laxa. Acquired elastic fibre defects include dermal elastosis, whereas inflammatory damage to fibres contributes to pathologies such as pulmonary emphysema and vascular disease. This review outlines the latest understanding of the composition and assembly of elastic fibres, and describes elastic fibre diseases and current therapeutic approaches.

Fibrillin-1 regulates the bioavailability of TGFbeta1.

We have discovered that fibrillin-1, which forms extracellular microfibrils, can regulate the bioavailability of transforming growth factor (TGF) beta1, a powerful cytokine that modulates cell survival and phenotype. Altered TGFbeta signaling is a major contributor to the pathology of Marfan syndrome (MFS) and related diseases. In the presence of cell layer extracellular matrix, a fibrillin-1 sequence encoded by exons 44-49 releases endogenous TGFbeta1, thereby stimulating TGFbeta receptor-mediated Smad2 signaling. This altered TGFbeta1 bioavailability does not require intact cells, proteolysis, or the altered expression of TGFbeta1 or its receptors. Mass spectrometry revealed that a fibrillin-1 fragment containing the TGFbeta1-releasing sequence specifically associates with full-length fibrillin-1 in cell layers. Solid-phase and BIAcore binding studies showed that this fragment interacts strongly and specifically with N-terminal fibrillin-1, thereby inhibiting the association of C-terminal latent TGFbeta-binding protein 1 (a component of the large latent complex [LLC]) with N-terminal fibrillin-1. By releasing LLC from microfibrils, the fibrillin-1 sequence encoded by exons 44-49 can contribute to MFS and related diseases.

ADAMTS6 cleaves the large latent TGFß complex and increases the mechanotension of cells to activate TGFß.

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFß. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFß homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFß-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFß homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFß activation. We found that their expression increases TGFß activation in a dose dependent manner, following stimulation with mature TGFß1. For ADAMTS6, the catalytically active protease is required for effective TGFß activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFß complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFß is perturbed ADAMTS6 can influence TGFß activation via two mechanisms. It directly cleaves the latent TGFß complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFß from large latent complexes which may contribute to disease pathogenesis.

Latent TGFß complexes are transglutaminase cross-linked to fibrillin to facilitate TGFß activation.

TGFß superfamily members are potent growth factors in the extracellular matrix with essential roles in all aspects of cellular behaviour. Latent TGFß binding proteins (LTBPs) are co-expressed with TGFß, essential for correct folding and secretion of the growth factor, to form large latent complexes. These large latent complexes bind extracellular proteins such as fibrillin for sequestration of TGFß in the matrix, essential for normal tissue function, and dysregulated TGFß signalling is a hallmark of many fibrillinopathies. Transglutaminase-2 (TG2) cross-linking of LTBPs is known to play a role in TGFß activation but the underlying molecular mechanisms are not resolved. Here we show that fibrillin is a matrix substrate for TG2 and that TG2 cross-linked complexes can be formed between fibrillin and LTBP-1 and -3, and their latent TGFß complexes. The structure of the fibrillin-LTBP1 complex shows that the two elongated proteins interact in a perpendicular arrangement which would allow them to form distal interactions between the matrix and the cell surface. Formation of the cross-link with fibrillin does not change the interaction between latent TGFß and integrin αVß6 but does increase TGFß activation in cell-based assays. The activating effect may be due to direction of the latent complexes to the cell surface by fibrillin, as competition with heparan sulphate can ameliorate the activating effect. Together, these data support that TGFß activation can be enhanced by covalent tethering of LTBPs to the matrix via fibrillin.

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development.

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet few mechanisms ensuring ECM proteostasis during this period are known. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding the major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of Adamts6 and Adamts10 mutant mouse embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics discovery of ADAMTS6-cleaved sites, identifies a proteostatic mechanism contributing to postnatal fibrillin-2 reduction and fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development defined by a drastic reduction of aggrecan and cartilage link protein, impaired BMP signaling in cartilage, and increased GDF5 sequestration in fibrillin-2-rich tissue. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of the defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

Defining elastic fiber interactions by molecular fishing: an affinity purification and mass spectrometry approach.

Deciphering interacting networks of the extracellular matrix is a major challenge. We describe an affinity purification and mass spectrometry strategy that has provided new insights into the molecular interactions of elastic fibers, essential extracellular assemblies that provide elastic recoil in dynamic tissues. Using cell culture models, we defined primary and secondary elastic fiber interaction networks by identifying molecular interactions with the elastic fiber molecules fibrillin-1, MAGP-1, fibulin-5, and lysyl oxidase. The sensitivity and validity of our method was confirmed by identification of known interactions with the bait proteins. Our study revealed novel extracellular protein interactions with elastic fiber molecules and delineated secondary interacting networks with fibronectin and heparan sulfate-associated molecules. This strategy is a novel approach to define the macromolecular interactions that sustain complex extracellular matrix assemblies and to gain insights into how they are integrated into their surrounding matrix.

Tropoelastin and Elastin Assembly.

Elastic fibers are an important component of the extracellular matrix, providing stretch, resilience, and cell interactivity to a broad range of elastic tissues. Elastin makes up the majority of elastic fibers and is formed by the hierarchical assembly of its monomer, tropoelastin. Our understanding of key aspects of the assembly process have been unclear due to the intrinsic properties of elastin and tropoelastin that render them difficult to study. This review focuses on recent developments that have shaped our current knowledge of elastin assembly through understanding the relationship between tropoelastin's structure and function.

Autosomal Recessive Cutis Laxa 1C Mutations Disrupt the Structure and Interactions of Latent TGFß Binding Protein-4.

Latent TGFß binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFß and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues.

Regulation of TGFß Signalling by TRPV4 in Chondrocytes.

The growth factor TGFß and the mechanosensitive calcium-permeable cation channel TRPV4 are both important for the development and maintenance of many tissues. Although TRPV4 and TGFß both affect core cellular functions, how their signals are integrated is unknown. Here we show that pharmacological activation of TRPV4 significantly increased the canonical response to TGFß stimulation in chondrocytes. Critically, this increase was only observed when TRPV4 was activated after, but not before TGFß stimulation. The increase was prevented by pharmacological TRPV4 inhibition or knockdown and is calcium/CamKII dependent. RNA-seq analysis after TRPV4 activation showed enrichment for the TGFß signalling pathway and identified JUN and SP1 as key transcription factors involved in this response. TRPV4 modulation of TGFß signalling represents an important pathway linking mechanical signalling to tissue development and homeostasis.

Molecular Cloning, Lentiviral Transduction, and Expression of Recombinant ADAMTSL2 and ADAMTSL4.

Lentiviral systems have proven advantageous in the delivery and long-term integration of gene sequences into the genome of several cell types in vitro, in vivo, as well as in clinical trials. Here we detail the protocols involved in the molecular cloning of ADAMTSL2 and ADAMTSL4 into the human immunodeficiency virus (HIV)-derived pCDH lentiviral system. We also describe the lentiviral transduction of ADAMTSL2 and ADAMTSL4 into mammalian HEK293-EBNA cells to create stable cell lines, as well as their recombinant expression.

Optogenetic Control of the BMP Signaling Pathway.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

Development of a microtiter plate-based glycosaminoglycan array for the investigation of glycosaminoglycan-protein interactions.

The interactions of glycosaminoglycans (GAGs) with proteins underlie a wide range of important biological processes. However, the study of such binding reactions has been hampered by the lack of a simple frontline analysis technique. Previously, we have reported that cold plasma polymerization can be used to coat microtiter plate surfaces with allyl amine to which GAGs (e.g., heparin) can be noncovalently immobilized retaining their ability to interact with proteins. Here, we have assessed the capabilities of surface coats derived from different ratios of allyl amine and octadiene (100:0 to 0:100) to support the binding of diverse GAGs (e.g., chondroitin-4-sulfate, dermatan sulfate, heparin preparations, and hyaluronan) in a functionally active state. The Link module from TSG-6 was used as a probe to determine the level of functional binding because of its broad (and unique) specificity for both sulfated and nonsulfated GAGs. All of the GAGs tested could bind this domain following their immobilization, although there were clear differences in their protein-binding activities depending on the surface chemistry to which they were adsorbed. On the basis of these experiments, 100% allyl amine was chosen for the generation of a microtiter plate-based "sugar array"; X-ray photoelectron spectroscopy revealed that similar relative amounts of chondroitin-4-sulfate, dermatan sulfate, and heparin (including two selectively de-sulfated derivatives) were immobilized onto this surface. Analysis of four unrelated proteins (i.e., TSG-6, complement factor H, fibrillin-1, and versican) illustrated the utility of this array to determine the GAG-binding profile and specificity for a particular target protein.

Effect of Topography and Physical Stimulus on hMSC Phenotype Using a 3D In Vitro Model.

This communication reports the first comparative study addressing the effects of both structural architecture and mechanical loading on human mesenchymal stem cells (hMSC) positioned at the interface of a 3D in vitro model composed of a nanofibre/hydrogel laminate composite. hMSC phenotype was affected by both stimuli over a seven-day period. Cells were orientated parallel to the underlying fibre direction irrespective of environment (electrospun 2D fibre sheet or laminate 2D sheet with collagen gel layer). Application of cyclical tensile force (5% strain, 1 Hz, 1 h per day) encouraged hMSCs to remain at the fibre/gel interface, whereas cells cultured in static conditions migrated from the interface to the upper hydrogel layer. Depending on the stimulus applied, hMSCs presented an up-regulation in gene expression, indicative of several cell lineages, with those cultured at the interface and physically stimulated expressing markers indicative of angiogenesis, osteogenesis, and tenogenesis. This study highlights the importance of developing biomaterial scaffolds with environmental cues to specifically drive cells towards the tissue intended for bioengineering.

The role of fibrillin and microfibril binding proteins in elastin and elastic fibre assembly.

Fibrillin is a large evolutionarily ancient extracellular glycoprotein that assembles to form beaded microfibrils which are essential components of most extracellular matrices. Fibrillin microfibrils have specific biomechanical properties to endow animal tissues with limited elasticity, a fundamental feature of the durable function of large blood vessels, skin and lungs. They also form a template for elastin deposition and provide a platform for microfibril-elastin binding proteins to interact in elastic fibre assembly. In addition to their structural role, fibrillin microfibrils mediate cell signalling via integrin and syndecan receptors, and microfibrils sequester transforming growth factor (TGF)ß family growth factors within the matrix to provide a tissue store which is critical for homeostasis and remodelling.