COPB2 haploinsufficiency causes a coatopathy with osteoporosis and developmental delay

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures and developmental delay of variable severity. Because the role of COPB2 in bone has not been characterized, we studied the effect of COPB2 deficiency on skeletal development in mice and zebrafish. Copb2+/− mice showed low bone mass and decreased bone strength. In zebrafish, larvae carrying a copb2 heterozygous frameshift variant showed delayed mineralization. copb2-null embryos showed endoplasmic reticulum (ER) and Golgi disorganization, and embryonic lethality. COPB2 siRNA-treated fibroblasts showed delayed collagen trafficking with retention of type I collagen in the ER and Golgi, and altered distribution of Golgi markers. Our data suggest that COPB2 haploinsufficiency leads to disruption of intracellular collagen trafficking and osteoporosis, which may improve with ascorbic acid supplementation. This work highlights the role of COPI complex as a critical regulator of bone mass and identifies a new form of coatopathy due to COPB2 deficiency.

AB0078 ANTI-FIBROTIC EFFECT OF DIFFERENT FUCOIDANS IN OSTEOATHRITIC FIBROBLAST-LIKE-SYNOVIOCYTES

Background:Synovial fibrosis is a pathological process observed in several musculoskeletal disorders that contributes to articular pain and stiffness. It is characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation, being TGF-β main inductor of these processes. Fucoidans are sulfated polysaccharide obtained mainly from various species of brown algae and brown seaweed such as Fucus vesiculosus, Undaria pinnatifida, or Macrocystis pyrifera. Recent studies show that fucoidans are promising candidates to address the symptoms of OA. Although a wide spectrum of biological activities has been registered in these polysaccharides, their properties vary between fucoidans from different species.Objectives:Our aim was to evaluate and to compare the anti-fibrotic effects of different fucoidans on fibroblast-like synoviocytes (FLS).Methods:FLS were isolated from OA patients. Primary cultured cells were treated with fucoidans from Fucus vesiculosus (FF), Undaria pinnatifida (FU) and Macrocystis pyrifera (FM) at 5, 30. To activate pro-fibrotic pathways, cells were stimulated with TGF-β and cell viability and proliferation were analyzed by MTT and BrdU assay, respectively. Then gene expression of extracellular matrix proteins, collagen I and III and fibronectin, as well as plod2b, gene coding for alterative splice-variant of lysyl hydroxylases LDH2b, were evaluated by RT-qPCR. The presence of fibrotic maker, alpha smooth muscle actin (α-sma), was analyzed by immunocytochemistry and intracellular collagen levels were assessed by picrosirius red staining. Wound-clousure and transwell cell invasion assay were also performed to evaluate the capacity of cell motility and invasiveness.Figure 1.Measurement of expression of pro-fibrotic markersResults:TGF-β induced a clear pro-fibrotic response (Figure 1). Cell proliferation induced by TGF-β was attenuated in the presence of all tested fucoidans. These polysaccharides also reduced the gene expression of TGF-β-elicited collagen I and III (p< 0.05), although FF failed to downregulate fibronectin levels and none of them showed modulation of plod2b expression. Results were confirmed at protein level. FF30 and FM5 reduced intracellular collagen accumulation induced by TGF-β (p< 0.05). Likewise, the expression of a-sma in TGFb-activated FLS was significantly diminished in the presence of all fucoidans (p< 0.05). By scratch wound assay, we observed that TGF-b induced cell mobility to close the scratch, which was inhibited by all fucoidans. Similarly, FU and FM were also able to attenuated cell invasion after TGF stimulation. Additionally, we also detected that fucoidans showed anti-inflammatory effects in FLS as they reduced the NO production and IL-6 expression induced by IL-1.Conclusion:Our results indicate a protective effect of fucoidans against activated pro-fibrotic pathways in fibroblast-like synoviocytes. However, we detected that these beneficial activities vary between fucoidans from different species and further studies are warranted.Disclosure of Interests:Carlos Vaamonde García: None declared, Herminia Domínguez: None declared, Francisco J. Blanco Grant/research support from: Sanofi-Aventis, Lilly, Bristol MS, Amgen, Pfizer, Abbvie, TRB Chemedica International, Glaxo SmithKline, Archigen Biotech Limited, Novartis, Nichi-iko pharmaceutical Co, Genentech, Jannsen Research & Development, UCB Biopharma, Centrexion Theurapeutics, Celgene, Roche, Regeneron Pharmaceuticals Inc, Biohope, Corbus Pharmaceutical, Tedec Meiji Pharma, Kiniksa Pharmaceuticals, Ltd, Gilead Sciences Inc, Consultant of: Lilly, Bristol MS, Pfizer, Rosa Meijide Failde: None declared

Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Broadly conserved roles of TMEM131 family proteins in intracellular collagen assembly and secretory cargo trafficking

Collagen is the most abundant protein in animals. Its dysregulation contributes to aging and many human disorders, including pathological tissue fibrosis in major organs. How premature collagen proteins in the endoplasmic reticulum (ER) assemble and route for secretion remains molecularly undefined. From an RNA interference screen, we identified an uncharacterized Caenorhabditis elegans gene tmem-131, deficiency of which impairs collagen production and activates ER stress response. We find that amino termini of human TMEM131 contain bacterial PapD chaperone–like domains, which recruit premature collagen monomers for proper assembly and secretion. Carboxy termini of TMEM131 interact with TRAPPC8, a component of the TRAPP tethering complex, to drive collagen cargo trafficking from ER to the Golgi. We provide evidence that previously undescribed roles of TMEM131 in collagen recruitment and secretion are evolutionarily conserved in C. elegans, Drosophila, and humans.

Collagen assembly and turnover imaged with a CRISPR-Cas9 engineered Dendra2 tag

Electron microscopy has been the “gold standard” for studying collagen networks but dynamic information on how cells synthesise the networks has been lacking. Live imaging methods have been unable to distinguish newly-synthesised fibrils from pre-existing fibrils and intracellular collagen. Here, we tagged endogenous collagen-I using CRISPR-Cas9 with photoswitchable Dendra2 and demonstrate live cells synthesising, migrating on, and interacting with, collagen fibrils. This strategy is applicable for other long half-life proteins.

The Preliminary Study of Effects of Tolfenamic Acid on Cell Proliferation, Cell Apoptosis, and Intracellular Collagen Deposition in Keloid FibroblastsIn Vitro

Keloid scarring is a fibroproliferative disorder due to the accumulation of collagen type I. Tolfenamic acid (TA), a nonsteroidal anti-inflammatory drug, has been found to potentially affect the synthesis of collagen in rats. In this preliminary study, we aimed to test the effects of TA on cell proliferation, cell apoptosis, and the deposition of intracellular collagen in keloid fibroblasts. Normal fibroblasts (NFs) and keloid fibroblasts (KFs) were obtained from human dermis tissue. Within the dose range 10−3–10−6 M and exposure times 24 h, 48 h, and 72 h, we found that 0.55 × 10−3 M TA at 48 h exposure exhibited significantly decreased cell proliferation in both NFs and KFs. Under these experimental conditions, we demonstrated that (1) TA treatment induced a remarkable apoptotic rate in KFs compared to NFs; (2) TA treatment reduced collagen production in KFs versus NFs; (3) TA treatment decreased collagen type I expression in KFs comparing to that of NFs. In summary, our data suggest that TA decreases cell proliferation, induces cell apoptosis, and inhibits collagen accumulation in KFs.

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis

Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production.