The endoplasmic reticulum (ER) orchestrates the secretion of extracellular matrix (ECM) proteins, many of which exceed the size of conventional transport vesicles and therefore require specialized export machinery. TANGO1, encoded by MIA3, organizes ER exit sites for bulky cargo export, yet the molecular basis of cargo recognition and its relationship to the collagen-specific chaperone HSP47 remain unclear. Through quantitative secretome profiling of TANGO1- and HSP47-deficient fibroblasts, structural modeling, and binding analyses, we show that TANGO1 directly and selectively recognizes a defined subset of ECM proteins, including specific collagen and fibrillin isoforms, through a conserved tyrosine residue in its luminal MOTH domain, independently of HSP47. Novel MIA3 variants identified in individuals with previously undescribed skeletal dysplasia disrupt this cargo-binding interface, leading to selective intracellular retention of ECM proteins. These findings identify direct cargo recognition, rather than ER exit-site assembly, as the primary molecular defect underlying MIA3/TANGO1-associated skeletal dysplasia. More broadly, our work establishes TANGO1 as a selective ECM cargo receptor that functions independently of HSP47, providing a new framework for understanding bulky cargo selection at the ER.

Objectives Systemic sclerosis (SSc) is an autoimmune-driven fibrotic disease, characterised by excessive extracellular matrix (ECM) deposition and fibroblast activation. Our study aimed to identify novel fibroblast subpopulations in SSc and determine their functional role. Methods We performed single-cell RNA sequencing on cultured dermal fibroblasts from healthy donors and patients with diffuse cutaneous SSc, verified selected, specific genes at the protein level and used siRNA knockdown experiments to provide evidence for a functional role of these proteins. Results SSc fibroblasts revealed high heterogeneity and in specific activated subsets strong upregulation of CD9 and four and a half LIM domain 1 (FHL1), previously described as a muscle-related protein. Overexpression of FHL1 and CD9 was also detected in fibroblast subsets in patient skin. CD9 was associated with the regulation of FHL1 expression. Downmodulation of FHL1 in primary skin fibroblasts correlated with downregulation of the vestigial-like family member 3 (VGLL3) gene, which is known to be expressed in myofibroblasts in a stiff and fibrotic environment and to upregulate collagen synthesis. Further, VGLL3 was confirmed to be upregulated in SSc donor skin. Conclusions Our study identified novel fibroblast subsets in SSc, characterised by CD9 and/or FHL1 upregulation. The data indicate a functional role of FHL1 in fibroblasts and its involvement in the regulation of ECM production and provide a new mechanistic link to VGLL3 regulation. Our findings suggest new avenues for therapeutic exploration targeting the perpetuating fibroblast activation by the fibrotic environment.

Activation of fibroblasts and formation of myofibroblasts are essential for granulation tissue formation following injury. In fibrotic reactions, excessive deposition of ECM by the activated fibroblasts determines scar formation and functional failure. Although these events critically depend on the activity of a plethora of growth factors and cytokines, TGFβ1 is a unique player controlling the immune response and proliferation of many cell types. Different cell types contribute to its release and activation, which is also regulated by the interaction with the ECM and by mechanical forces. The aim of this study was to elaborate whether fibroblast-derived TGFβ1 plays a critical role during these processes. The data demonstrate a dynamic expression of TGFβ1 during tissue repair. Cell-specific ablation of Tgfb1 in fibroblasts revealed that deletion of TGFβ1 attenuates bleomycin-induced skin fibrosis and perturbs maturation of granulation tissue in skin wounds. Absence of fibroblast-derived TGFβ1 induced vascular alterations (less vascular density and branching, haemorrhage) in early wound healing. This was associated with alterations in the formation of stable ECM structure. This can be explained by paracrine regulation of endothelial cells or pericytes by fibroblast-released TGFβ1 and by impaired expression of pro-angiogenic factors in TGFβ1-deficient fibroblasts. Our findings provide novel mechanistic insights into the central role of fibroblast-derived TGFβ1 for early stages of tissue repair and fibrosis in the skin.

Fibroblast-like synoviocytes (FLS) are key cells promoting cartilage damage and bone loss in rheumatoid arthritis (RA). They are activated to assume an invasive and migratory phenotype. While mechanisms of FLS activation are unknown, evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is increased in RA patients and hTNFtg mice, an RA mouse model. We treated murine chondrocytes with TNFα to produce a damaged, RA-like matrix. Comparison to healthy chondrocyte matrix revealed decreased ECM proteins, e.g. collagens and proteoglycans, increased matrix-degrading proteins and elevated levels of inflammatory cytokines. FLS responded to the damaged chondrocyte matrix with a matrix-remodeling and pro-inflammatory phenotype characterized by a gene signature involved in matrix degradation and increased production of CLL11 and CCL19. Damaged chondrocyte matrix stimulated increased Itga11 expression in FLS, correlating with the increased α11β1 amounts in RA patients. FLS deficient in integrin α11β1 released lower amounts of inflammation-associated cytokines. Our results demonstrate differences in healthy and RA-like chondrocyte ECM and distinctly different responses of wt FLS to damaged versus healthy ECM.

OBJECTIVES:In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) acquire an aggressive, tumour-like phenotype characterised by increased adhesion to extracellular matrix, contributing to joint degradation. The collagen-binding integrin alpha11beta1 is involved in similar processes in cancer-associated fibroblasts, but its role in RA and arthritic mice remains unclear. METHODS:Integrin α11 expression was analysed in synovial tissue and FLS from RA and osteoarthritis patients and human tumour necrosis factor transgenic (hTNFtg) and wild-type mice supported by Accelerating Medicines Partnership Rheumatoid Arthritis and Pathobiology of Early Arthritis Cohort data. A novel 3-dimensional (3D) organoid coculture model and electron microscopy were used to analyse FLS invasion into cartilage explants, Itga11-/- were crossed with hTNFtg mice, and disease severity was evaluated using microcomputed tomography (µCT) and histology. Functional assays using FLS included cell morphology, adhesion, degradation, and matrix metalloproteinase expression and were complemented by osteoclast and coculture studies. RESULTS:In the context of RA, strong α11 expression was detected in the synovium, particularly in sublining clusters of FLS within fibroid-type synovial tissue in vivo and at focal adhesions of arthritic FLS and at invasion sites within the 3D coculture model in vitro. Clinical scores, µCT imaging, and histomorphological analyses revealed significantly reduced cartilage degradation, bone erosions, and FLS attachment to cartilage in Itga11-/-hTNFtg compared to hTNFtg mice. In vitro studies revealed that α11 deficiency led to a decreased receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio along with reduced TNFα-induced proteolytic degradation activity, and signalling pathway activation. CONCLUSIONS:Integrin α11 levels are increased in RA, and its deficiency notably diminishes joint destruction in hTNFtg mice, emphasising its potential as promising therapeutic target.

Supramolecular extracellular matrix (ECM) networks play an essential role in skin architecture and function. Elastin microfibril interface-located proteins (EMILINs) comprise a family of three extracellular glycoproteins that serve as essential structural components of the elastin/fibrillin microfibril network, and exert crucial functions in cellular signaling. Little is known about the structural nature of EMILIN networks in skin. We therefore investigated the spatiotemporal localization of EMILIN-1, -2, -3 in human skin induced by aging, UV-exposure, fibrosis, and connective tissue disorder. Confocal immunofluorescence and immunogold electron microscopy analysis identified all EMILINs as components of elastic fibers and elastin-free oxytalan fibers inserted into the basement membrane (BM). Further, our ultrastructural analysis demonstrates cellular contacts of dermally localized EMILIN-1 positive fibers across the BM with the surface of basal keratinocytes. Analysis of skin biopsies and fibroblast cultures from fibrillin-1 deficient Marfan patients revealed that EMILINs require intact fibrillin-1 as deposition scaffold. In patients with scleroderma and the bleomycin-induced murine fibrosis model EMILIN-2 was upregulated. EMILIN-3 localizes to the tips of candelabra-like oxytalan fibers, and to specialized BMs engulfing hair follicles and sebaceous glands. Our data identify EMILINs as important markers to monitor rearrangements of the dermal ECM architecture induced by aging and pathological conditions.

Transforming growth factor β (TGF-β) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-β remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-β in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-β. The activation of latent TGF-β requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-β, rebalanced TGF-β signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-β in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes. Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring. Here the authors describe membrane permeable peptides that target the interface of TANGO1 and cTAGE5, inhibit secretion of ECM components and could be of therapeutic benefit during wound healing and fibrotic processes.

Excessive production and deposition of extracellular matrix (ECM) constituents by fibroblasts is a hallmark of scleroderma and other fibrotic processes. Collagens represent the major structural component of ECM and controlling its synthesis and secretion provides a means to address human pathologies linked to uncontrolled collagen production. The export of collagen molecules from the ER is mediated by TANGO1 family of proteins. We designed inhibitors of TANGO1 functions and demonstrate their uptake by primary human fibroblasts in a dose-dependent manner. Inhibitor uptake resulted in severely reduced secretion of collagens and other large ECM components as demonstrated by western blotting and detailed proteomic analysis of the secretome. The inhibitors also proved to be highly effective in inhibiting collagen secretion by TGFβ-stimulated fibroblasts. Activated fibroblasts from patients with scleroderma were highly responsive to the inhibitors with a marked reduction in collagen secretion. In addition, granulation tissue formation and ECM deposition subsequent to laser injury in a zebrafish model were significantly reduced by the inhibitorsin vivo. These findings demonstrate that targeting TANGO1 family proteins is a novel approach to modulate matrix deposition in scarring and fibrosis.

Systemic sclerosis (SSc) is characterized by excessive deposition of extracellular matrix (ECM) in the involved organs. Fibroblasts are activated and develop an uncontrolled synthesis of collagen and other ECM constituents. Previous studies have focused on cultured fibroblasts, but the interpretation of the results have been limited due to possible selection of fibroblast populations. To better understand fibroblast heterogeneity in SSc we conducted single cell RNA-sequencing (scRNA-seq) on cultured cells with the aim to 1) identify specific activated populations and 2) to be able to explore potential new therapeutic targets. Our scRNA-seq analysis of human dermal fibroblasts consisted of 4 healthy and 4 SSc donors. We identified numerous up- and downregulated genes in distinct fibroblast populations. A significant upregulation of tetraspanin CD9 was seen in most SSc fibroblast subsets when compared to controls. Flow cytometry confirmed significant CD9 protein upregulation at the cell surface of SSc fibroblasts. CD9 has previously been linked to exosomes in SSc fibroblasts and our further functional analysis revealed, in CD9 knockout (KO) mouse embryonic fibroblasts, a clear reduction of collagen I and other ECM proteins at the protein level, thereby linking CD9 overexpression directly to enhanced collagen synthesis. Furthermore, a strong upregulation of Four and a Half LIM domains 1 (FHL1) was detected in a small cluster of SSc fibroblasts at the RNA and protein level. Although FHL1 has been implicated in myofibroblast differentiation the FHL1 positive cell cluster in our cultures did not correlate with the αSMA expressing subcluster and thus established a novel subpopulation of activated SSc fibroblasts.

Osteoarthritis (OA), a primarily degenerative disorder, and rheumatoid arthritis (RA), a chronic autoimmune inflammatory disease, belong to the most prevalent joint pathologies. Both conditions involve the entire joints with their different tissues and may lead to their complex remodelling and eventually to their destruction. The interaction of articular cartilage with the synovial membrane is a hallmark of both OA and RA and characterized by strong communication between their resident cells such as chondrocytes and fibroblasts as well as invading inflammatory cells such as monocytes with the local extracellular matrix (ECM). Integrins have been assigned a key role in this communication and have, thus, been involved strongly in the pathogenesis of OA and RA. Using OA and RA as examples, this chapter summarizes the role of integrins in both joint pathologies and points to potential therapeutic implications.

ObjectivesActivation of fibroblasts is a hallmark of fibrotic processes. Besides cytokines and growth factors, fibroblasts are regulated by the extracellular matrix environment through receptors such as integrins, which transduce biochemical and mechanical signals enabling cells to mount appropriate responses according to biological demands. The aim of this work was to investigate the in vivo role of collagen–fibroblast interactions for regulating fibroblast functions and fibrosis.MethodsTriple knockout (tKO) mice with a combined ablation of integrins α1β1, α2β1 and α11β1 were created to address the significance of integrin-mediated cell–collagen communication. Properties of primary dermal fibroblasts lacking collagen-binding integrins were delineated in vitro. Response of the tKO mice skin to bleomycin induced fibrotic challenge was assessed.ResultsTriple integrin-deficient mice develop normally, are transiently smaller and reveal mild alterations in mechanoresilience of the skin. Fibroblasts from these mice in culture show defects in cytoskeletal architecture, traction stress generation, matrix production and organisation. Ablation of the three integrins leads to increased levels of discoidin domain receptor 2, an alternative receptor recognising collagens in vivo and in vitro. However, this overexpression fails to compensate adhesion and spreading defects on collagen substrates in vitro. Mice lacking collagen-binding integrins show a severely attenuated fibrotic response with impaired mechanotransduction, reduced collagen production and matrix organisation.ConclusionsThe data provide evidence for a crucial role of collagen-binding integrins in fibroblast force generation and differentiation in vitro and for matrix deposition and tissue remodelling in vivo. Targeting fibroblast–collagen interactions might represent a promising therapeutic approach to regulate connective tissue deposition in fibrotic diseases.

Background In rheumatoid arthritis (RA), fibroblast like synoviocytes (FLS) undergo a “tumor-like” transformation, wherein they develop an aggressive phenotype that is characterized by increased adhesion to components of cartilage extracellular matrix (ECM) and that contributes extensively to joint destruction. The collagen binding integrin α11β1 was previously shown to be involved in similar processes in cancer-associated fibroblasts mediating tumorigenicity and metastasis in certain tumors. Therefore, this study aimed to study the role of integrin α11β1 in RA and to characterize the effects of α11β1 deficiency on the disease course and severity in arthritic hTNFtg mice. Methods The expression levels of integrin α11β1 were analyzed by immunohistochemistry, immunofluorescence, and western blot analysis in synovial samples and FLS of patients with RA and osteoarthritis (OA) as well as in samples from wild type (wt) and arthritic hTNFtg mice. Furthermore, the subcellular expression of integrin α11β1 was investigated in co-culture experiments with cartilage explants and analyzed by transmission electron microscopy. To investigate the effects of integrin α11β1 deficiency, itga11 -/- mice were interbred with hTNFtg mice and disease severity was assessed by clinical scoring of grip strength and paw swelling over the disease course. Hind paws of 12-weeks-old mice of all genotypes were analyzed by µCT imaging followed by stainings of paraffin-embedded tissue sections with Toluidine-blue and tartrate-resistant acid phosphatase (TRAP) to evaluate established parameters of joint destruction such as inflammation area, cartilage destaining, FLS attachment to the cartilage surface, and bone damage. Results Expression levels of integrin α11β1 were clearly elevated in synovial tissues and FLS from RA patients and hTNFtg mice, compared to the controls derived from OA patients and wt mice. Interestingly, this expression was shown to be particularly localized in focal adhesions of the FLS. As revealed by transmission electron microscopy, integrin α11β1 expression was particularly evident in areas of direct cellular contact with the ECM of cartilage. Evaluations of clinical scorings and histomorphological analyses demonstrated that itga11 -/- hTNFtg displayed alleviated clinical symptoms, higher bone volume, less cartilage destruction and reduced FLS attachment to the cartilage in comparison to hTNFtg mice. Conclusions The collagen-binding integrin α11β1 is upregulated in the context of RA and its deficiency in mice with an inflammatory hTNFtg background leads to a significant reduction in the arthritic phenotype which makes integrin α11β1 an interesting target for therapeutical intervention.

BackgroundRheumatoid arthritis (RA) is an autoimmune disorder conducted by fibroblast-like synoviocytes (FLS), which acquire a tumor-like phenotype causing irreversible cartilage and bone damage. FLS attache to the extracellular matrix (ECM) invading the joints, a process mediated by integrins. Integrins are transmembrane proteins regulating several cell functions like cell migration, cell proliferation, tissue invasion and cytokine expression – key mechanisms during the pathogenesis of RA. The collagen-binding Integrin α11β1 (α11) is expressed on FLS mediating their adhesion to the ECM and in this study, we analysed its implication in RA.ObjectivesWe examined the pathogenesis of RA in a murine arthritis model (hTNFtg) lacking α11 to analyze its contribution to joint destructions and the disease course.MethodsExpression levels of α11 were analysed by Western Blot and immunofluorescence staining using FLS and synovial tissue of patients with RA and hTNFtg mice in comparison to their respective controls. Crossbreeding hTNFtg with α11 deficient (itga11-/-) mice enabled us to evaluate arthritis progression using clinical parameters like paw swelling and grip strength. Inflammation area and cartilage damage were quantified by histomorphological techniques such as toluidine blue staining of in paraffin-embedded sections from hind paws. Bone erosion was visualized by µCT imaging and quantification of the bone volume in the tarsal bone area.ResultsHigh expression levels of α11 could be detected in hTNFtg and human RA samples in comparison to their controls. The progression of arthritis in itga11-/-hTNFtg was slower and less severe in comparison to hTNFtg mice, visible in a stronger grip strength and reduced paw swelling at the same timepoint. The histomorphology analysis confirmed these results showing higher cartilage area (3.21% vs 5.22%, p < 0.05), less cartilage destruction (51.73% vs 35.65%, p < 0.05) and reduced inflammation (28.28% vs 12.00%) in itga11-/-hTNFtg compared to the hTNFtg mice. Also, the quantification of the tarsal bone area revealed a higher bone volume (77.94% vs 84.92%, p < 0.01) in itga11-/-hTNFtg.ConclusionThis study showed the important role of α11 in the progression of joint destruction as it is highly expressed in hTNFtg mice and in human synovial tissue of patients with RA. Its absence results in less severe arthritis progression and joint destruction, therefore a possible and interesting target for RA treatment.Disclosure of InterestsNone declared

Systemic sclerosis (SSc) is a chronic autoimmune disease causing vasculopathy and fibrosis in the skin and vital organs. Following an inflammatory reaction, fibroblasts in the connective tissues become activated myofibroblasts and secrete excessive amounts of ECM related proteins, such as collagen, creating fibrosis. Many studies have been carried out in fibroblasts in culture, however, the information obtained remains limited due to high potential heterogeneity of these fibroblasts. We therefore used single cell RNA sequencing to better understand fibroblast heterogeneity in SSc to search for potential new therapeutic targets and new biomarkers. We conducted single cell RNA-sequencing (scRNA-seq) analysis of human dermal fibroblasts from 4 healthy and 4 SSc donors. We identified several up- and downregulated genes in defined cell populations; we also confirmed significant upregulation of tetraspanin CD9 in all SSc fibroblast subsets when compared to controls. CD9 upregulation at the cell surface of SSc fibroblasts was confirmed by flow cytometry. Additionally, we identified strong upregulation of Four and a half LIM domains 1 (FHL1) in a small cluster of SSc fibroblasts at the RNA and protein level. These cells did not express α-SMA and thereby constitute a small subpopulation of activated fibroblasts. CD9 was found in exosomes of SSc fibroblasts and FHL1 has been implicated in myofibroblast differentiation; there is not much known about their role in fibrosis. We are therefore focusing on the direct impact of both CD9 and FHL1 using specific deletion experiments.