Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis.

Osteoarthritis (OA) is the most common joint disease. Currently there are no effective methods that simultaneously prevent joint degeneration and reduce pain1. Although limited evidence suggests the existence of voltage-gated sodium channels (VGSCs) in chondrocytes2, their expression and function in chondrocytes and in OA remain essentially unknown. Here we identify Nav1.7 as an OA-associated VGSC and demonstrate that human OA chondrocytes express functional Nav1.7 channels, with a density of 0.1 to 0.15 channels per µm2 and 350 to 525 channels per cell. Serial genetic ablation of Nav1.7 in multiple mouse models demonstrates that Nav1.7 expressed in dorsal root ganglia neurons is involved in pain, whereas Nav1.7 in chondrocytes regulates OA progression. Pharmacological blockade of Nav1.7 with selective or clinically used pan-Nav channel blockers significantly ameliorates the progression of structural joint damage, and reduces OA pain behaviour. Mechanistically, Nav1.7 blockers regulate intracellular Ca2+ signalling and the chondrocyte secretome, which in turn affects chondrocyte biology and OA progression. Identification of Nav1.7 as a novel chondrocyte-expressed, OA-associated channel uncovers a dual target for the development of disease-modifying and non-opioid pain relief treatment for OA.

PGRN deficiency exacerbates, whereas a brain penetrant PGRN derivative protects, GBA1 mutation-associated pathologies and diseases.

Mutations in GBA1, encoding glucocerebrosidase (GCase), cause Gaucher disease (GD) and are also genetic risks in developing Parkinson's disease (PD). Currently, the approved therapies are only effective for directly treating visceral symptoms, but not for primary neuronopathic involvement in GD (nGD). Progranulin (PGRN), encoded by GRN, is a novel modifier of GCase, but the impact of PGRN in GBA1 mutation-associated pathologies in vivo remains unknown. Herein, Grn-/- mice crossed into Gba9v/9v mice, a Gba1 mutant line homozygous for the Gba1 D409V mutation, generating Grn-/-Gba9v/9v (PG9V) mice. PG9V mice exhibited neurobehavioral deficits, early onset, and more severe GD phenotypes compared to Grn-/- and Gba9v/9v mice. Moreover, PG9V mice also displayed PD-like phenotype. Mechanistic analysis revealed that PGRN deficiency caused severe neuroinflammation with microgliosis and astrogliosis, along with impaired autophagy associated with the Gba1 mutation. A PGRN-derived peptide, termed ND7, ameliorated the disease phenotype in GD patient fibroblasts ex vivo. Unexpectedly, ND7 penetrated the blood-brain barrier (BBB) and effectively ameliorated the nGD manifestations and PD pathology in Gba9v/null and PG9V mice. Collectively, this study not only provides the first line of in vivo but also ex vivo evidence demonstrating the crucial role of PGRN in GBA1/Gba1 mutation-related pathologies, as well as a clinically relevant mouse model for mechanistic and potential therapeutics studies for nGD and PD. Importantly, a BBB penetrant PGRN-derived biologic was developed that may provide treatment for rare lysosomal storage diseases and common neurodegenerative disorders, particularly nGD and PD.

Digoxin targets low density lipoprotein receptor-related protein 4 and protects against osteoarthritis.

OBJECTIVES: Dysregulated chondrocyte metabolism is closely associated with the pathogenesis of osteoarthritis (OA). Suppressing chondrocyte catabolism to restore cartilage homeostasis has been extensively explored, whereas far less effort has been invested toward enhancing chondrocyte anabolism. This study aimed to repurpose clinically approved drugs as potential stimulators of chondrocyte anabolism in treating OA. METHODS: Screening of a Food and Drug Administration-approved drug library; Assays for examining the chondroprotective effects of digoxin in vitro; Assays for defining the therapeutic effects of digoxin using a surgically-induced OA model; A propensity-score matched cohort study using The Health Improvement Network to examine the relationship between digoxin use and the risk of joint OA-associated replacement among patients with atrial fibrillation; identification and characterisation of the binding of digoxin to low-density lipoprotein receptor-related protein 4 (LRP4); various assays, including use of CRISPR-Cas9 genome editing to delete LRP4 in human chondrocytes, for examining the dependence on LRP4 of digoxin regulation of chondrocytes. RESULTS: Serial screenings led to the identification of ouabain and digoxin as stimulators of chondrocyte differentiation and anabolism. Ouabain and digoxin protected against OA and relieved OA-associated pain. The cohort study of 56 794 patients revealed that digoxin use was associated with reduced risk of OA-associated joint replacement. LRP4 was isolated as a novel target of digoxin, and deletion of LRP4 abolished digoxin's regulations of chondrocytes. CONCLUSIONS: These findings not only provide new insights into the understanding of digoxin's chondroprotective action and underlying mechanisms, but also present new evidence for repurposing digoxin for OA.

Pharmacological and Therapeutic Applications of Esculetin.

Esculetin is a coumarin compound, which belongs to the class of benzopyrone enriched in various plants such as Sonchus grandifolius, Aesculus turbinata, etc. Free radicals lead to the development of oxidative stress causing inflammation, arthritis, cancer, diabetes, fatty liver disease, etc. These further reduce the efficacy of anticancer drugs, activate inflammatory signaling pathways, degrade joints and cartilage, and disrupt the glycemic index and normal function of liver enzymes. For instance, the current treatment modalities used in arthritis such as non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatoid drugs, and lipoxygenase inhibitors present limited efficacy and adverse effects. Thus, there is a constant need to find newer and safer alternatives. Esculetin has an immense antioxidative potential thereby alleviating arthritis, diabetes, malignancies, and hepatic disorders. Structurally, esculetin contains two hydroxyl groups, which enhance its ability to function as an antioxidant by inhibiting oxidative stress in pathological conditions. Leukotriene B4 synthesis, NF-κB and MPAK pathway activation, and inflammatory cytokine production are the main causes of bone and joint deterioration in arthritis, whereas esculetin treatment reverses these factors and relieves the disease condition. In contrast, lipid peroxidation caused by upregulation of TGF-ß-mediated expression and dysfunction of antioxidant enzymes is inhibited by esculetin therapy, thus reducing liver fibrosis by acting on the PI3K/FoxO1 pathway. Therefore, targeting NF-κB, pro-inflammatory cytokines, TGF-ß and oxidative stress may be a therapeutic strategy to alleviate arthritis and liver fibrosis.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in the pathogenesis of OA. TNFR1 primarily mediates inflammation, whereas emerging evidences demonstrate that TNFR2 plays an anti-inflammatory and protective role in several diseases and conditions. This study aims to decipher TNFR2 signalling in chondrocytes and OA. METHODS: Biochemical copurification and proteomics screen were performed to isolate the intracellular cofactors of TNFR2 complex. Bulk and single cell RNA-seq were employed to determine 14-3-3 epsilon (14-3-3ε) expression in human normal and OA cartilage. Transcription factor activity screen was used to isolate the transcription factors downstream of TNFR2/14-3-3ε. Various cell-based assays and genetically modified mice with naturally occurring and surgically induced OA were performed to examine the importance of this pathway in chondrocytes and OA. RESULTS: Signalling molecule 14-3-3ε was identified as an intracellular component of TNFR2 complexes in chondrocytes in response to progranulin (PGRN), a growth factor known to protect against OA primarily through activating TNFR2. 14-3-3ε was downregulated in OA and its deficiency deteriorated OA. 14-3-3ε was required for PGRN regulation of chondrocyte metabolism. In addition, both global and chondrocyte-specific deletion of 14-3-3ε largely abolished PGRN's therapeutic effects against OA. Furthermore, PGRN/TNFR2/14-3-3ε signalled through activating extracellular signal-regulated kinase (ERK)-dependent Elk-1 while suppressing nuclear factor kappa B (NF-κB) in chondrocytes. CONCLUSIONS: This study identifies 14-3-3ε as an inducible component of TNFR2 receptor complex in response to PGRN in chondrocytes and presents a previously unrecognised TNFR2 pathway in the pathogenesis of OA.

Progranulin deficiency exacerbates spinal cord injury by promoting neuroinflammation and cell apoptosis in mice.

PURPOSE: Spinal cord injury (SCI) often results in significant and catastrophic dysfunction and disability and imposes a huge economic burden on society. This study aimed to determine whether progranulin (PGRN) plays a role in the progressive damage following SCI and evaluate the potential for development of a PGRN derivative as a new therapeutic target in SCI. METHODS: PGRN-deficient (Gr-/-) and wild-type (WT) littermate mice were subjected to SCI using a weight-drop technique. Local PGRN expression following injury was evaluated by Western blotting and immunofluorescence. Basso Mouse Scale (BMS), inclined grid walking test, and inclined plane test were conducted at indicated time points to assess neurological recovery. Inflammation and apoptosis were examined by histology (Hematoxylin and Eosin (H&E) staining and Nissl staining, TUNEL assays, and immunofluorescence), Western blotting (from whole tissue protein for iNOS/p-p65/Bax/Bcl-2), and ex vivo ELISA (for TNFα/IL-1ß/IL-6/IL-10). To identify the prophylactic and therapeutic potential of targeting PGRN, a PGRN derived small protein, Atsttrin, was conjugated to PLGA-PEG-PLGA thermosensitive hydrogel and injected into intrathecal space prior to SCI. BMS was recorded for neurological recovery and Western blotting was applied to detect the inflammatory and apoptotic proteins. RESULTS: After SCI, PGRN was highly expressed in activated macrophage/microglia and peaked at day 7 post-injury. Grn-/- mice showed a delayed neurological recovery after SCI at day 21, 28, 35, and 42 post-injury relative to WT controls. Histology, TUNEL assay, immunofluorescence, Western blotting, and ELISA all indicated that Grn-/- mice manifested uncontrolled and expanded inflammation and apoptosis. Administration of control-released Atsttrin could improve the neurological recovery and the pro-inflammatory/pro-apoptotic effect of PGRN deficiency. CONCLUSION: PGRN deficiency exacerbates SCI by promoting neuroinflammation and cellular apoptosis, which can be alleviated by Atsttrin. Collectively, our data provide novel evidence of using PGRN derivatives as a promising therapeutic approach to improve the functional recovery for patients with spinal cord injury.

Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against inflammatory arthritis.

OBJECTIVE: Tumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis. METHODS: In vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine's anti-TNF activity on cPLA2. RESULTS: Serial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine's anti-TNF activity. CONCLUSION: Collectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

Foxo4- and Stat3-dependent IL-10 production by progranulin in regulatory T cells restrains inflammatory arthritis.

Progranulin (PGRN) restrains inflammation and is therapeutic against inflammatory arthritis; however, the underlying immunological mechanism remains unknown. In this study, we demonstrated that anti-inflammatory cytokine IL-10 was a critical mediator for PGRN-mediated anti-inflammation in collagen-induced arthritis by using PGRN and IL-10 genetically modified mouse models. IL-10 green fluorescent protein reporter mice revealed that regulatory T (Treg) cells were the predominant source of IL-10 in response to PGRN. In addition, PGRN-mediated expansion and activation of Treg cells, as well as IL-10 production, depends on JNK signaling, but not on known PGRN-activated ERK and PI3K pathways. Furthermore, microarray and chromatin immunoprecipitation sequencing screens led to the discovery of forkhead box protein O4 and signal transducer and activator of transcription 3 as the transcription factors required for PGRN induction of IL-10 in Treg cells. These findings define a previously unrecognized signaling pathway that underlies IL-10 production by PGRN in Treg cells and present new insights into the mechanisms by which PGRN resolves inflammation in inflammatory conditions and autoimmune diseases, particularly inflammatory arthritis.-Fu, W., Hu, W., Shi, L., Mundra, J. J. Xiao, G., Dustin, M. L., Liu, C. Foxo4- and Stat3-dependent IL-10 production by progranulin in regulatory T cells restrains inflammatory arthritis.

Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.-Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

Selective oral ROCK2 inhibitor down-regulates IL-21 and IL-17 secretion in human T cells via STAT3-dependent mechanism.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.

Progranulin protects against osteoarthritis through interacting with TNF-α and ß-Catenin signalling.

OBJECTIVE: Progranulin (PGRN) was previously isolated as an osteoarthritis (OA)-associated growth factor. Additionally, PGRN was found to play a therapeutic role in inflammatory arthritis mice models through antagonising tumour necrosis factor α (TNF-α). This study was aimed at investigating the role of PGRN in degradation of cartilage and progression of OA. METHODS: Progression of OA was analysed in both spontaneous and surgically induced OA models in wild type and PGRN-deficient mice. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry and ELISA. Additionally, mRNA expression of degenerative factors and catabolic markers known to be involved in cartilage degeneration in OA were analysed. Furthermore, the anabolic effects and underlying mechanisms of PGRN were investigated by in vitro experiments with primary chondrocytes. RESULTS: Here, we found that deficiency of PGRN led to spontaneous OA-like phenotype in 'aged' mice. Additionally, PGRN-deficient mice exhibited exaggerated breakdown of cartilage structure and OA progression, while local delivery of recombinant PGRN protein attenuated degradation of cartilage matrix and protected against OA development in surgically induced OA models. Furthermore, PGRN activated extracellular signal-regulated kinases (ERK) 1/2 signalling and elevated the levels of anabolic biomarkers in human chondrocyte, and the protective function of PGRN was mediated mainly through TNF receptor 2. Additionally, PGRN suppressed inflammatory action of TNF-α and inhibited the activation of ß-Catenin signalling in cartilage and chondrocytes. CONCLUSIONS: Collectively, this study provides new insight into the pathogenesis of OA, and also presents PGRN as a potential target for the treatment of joint degenerative diseases, including OA.

Overexpression of ADAMTS-7 leads to accelerated initiation and progression of collagen-induced arthritis in mice.

The aim of the present study is to determine whether ADAMTS-7 contributes to the onset and severity of joint inflammation in the pathogenesis of inflammatory arthritis. ADAMTS-7 was found to be elevated in the course of collagen-induced arthritis (CIA). ADAMTS-7 transgenic (TG) mice were more susceptible to the induction of CIA. The onset of CIA was accelerated and the arthritic severity was increased in TG mice compared to wild-type mice. The overall incidence was also significantly higher in TG mice. In addition, arthritic TG mice displayed significantly higher clinical and histological arthritis scores. The COMP degradative fragments were significantly elevated in articular cartilage and sera in CIA models of TG mice. Furthermore, the production of tumor necrosis factor-alpha and interleukin-17 was also increased in serum and draining lymph nodes of arthritic TG mice. Therefore, these data provided the in vivo evidence, suggesting that ADAMTS-7 may play an important role in the pathogenesis of inflammatory arthritis, and that inhibition of ADAMTS-7 may be a potential target to ameliorate the severity of inflammatory arthritis.

ADAMTS-7 forms a positive feedback loop with TNF-α in the pathogenesis of osteoarthritis.

OBJECTIVE: To examine the expression of ADAMTS-7 during the progression of osteoarthritis (OA), defining its role in the pathogenesis of OA, and elucidating the molecular events involved. METHODS: ADAMTS-7 expression in cartilage of a rat OA model was assayed using immunohistochemistry. Cartilage-specific ADAMTS-7 transgenic mice and ADAMTS-7 small interfering (si)RNA knockdown mice were generated and used to analyse OA progression in both spontaneous and surgically induced OA models. Cartilage degradation and OA was evaluated using Safranin-O staining, immunohistochemistry, ELISA and western blotting. In addition, mRNA expression of tumour necrosis factor (TNF)-α and metalloproteinases known to be involved in cartilage degeneration in OA was analysed. Furthermore, the transactivation of ADAMTS-7 by TNF-α and its downstream NF-κB signalling was measured using reporter gene assay. RESULTS: ADAMTS-7 expression was elevated during disease progression in the surgically induced rat OA model. Targeted overexpression of ADAMTS-7 in chondrocytes led to chondrodysplasia characterised by short-limbed dwarfism and a delay in endochondral ossification in 'young mice' and a spontaneous OA-like phenotype in 'aged' mice. In addition, overexpression of ADAMTS-7 led to exaggerated breakdown of cartilage and accelerated OA progression, while knockdown of ADAMTS-7 attenuated degradation of cartilage matrix and protected against OA development, in surgically induced OA models. ADAMTS-7 upregulated TNF-α and metalloproteinases associated with OA; in addition, TNF-α induced ADAMTS-7 through NF-κB signalling. CONCLUSIONS: ADAMTS-7 and TNF-α form a positive feedback loop in the regulation of cartilage degradation and OA progression, making them potential molecular targets for prevention and treatment of joint degenerative diseases, including OA.

Progranulinopathy: A diverse realm of disorders linked to progranulin imbalances.

Progranulin (PGRN), encoded by the GRN gene in humans, was originally isolated as a secreted growth factor that implicates in a multitude of processes ranging from regulation of tumorigenesis, inflammation to neural proliferation. Compelling evidence indicating that GRN mutation can lead to various common neuronal degenerative diseases and rare lysosomal storage diseases. These findings have unveiled a critical role for PGRN as a lysosomal protein in maintaining lysosomal function. The phenotypic spectrum of PGRN imbalance has expanded to encompass a broad spectrum of diseases, including autoimmune diseases, metabolic, musculoskeletal and cardiovascular diseases. These diseases collectively referred to as Progranulinopathy- a term encompasses the wide spectrum of disorders influenced by PGRN imbalance. Unlike its known extracellular function as a growth factor-like molecule associated with multiple membrane receptors, PGRN also serves as an intracellular co-chaperone engaged in the folding and traffic of its associated proteins, particularly the lysosomal hydrolases. This chaperone activity is required for PGRN to exert its diverse functions across a broad range of diseases, encompassing both the central nervous system and peripheral systems. In this comprehensive review, we present an update of the emerging role of PGRN in Progranulinopathy, with special focus on elucidating the intricate interplay between PGRN and a diverse array of proteins at various levels, ranging from extracellular fluids and intracellular components, as well as various pathophysiological processes involved. This review seeks to offer a comprehensive grasp of PGRN's diverse functions, aiming to unveil intricate mechanisms behind Progranulinopathy and open doors for future research endeavors.

Dietary pyruvate targets cytosolic phospholipase A2 to mitigate inflammation and obesity in mice.

Obesity has a multifactorial etiology and is known to be a state of chronic low-grade inflammation, known as meta-inflammation. This state is associated with the development of metabolic disorders such as glucose intolerance and nonalcoholic fatty liver disease. Pyruvate is a glycolytic metabolite and a crucial node in various metabolic pathways. However, its role and molecular mechanism in obesity and associated complications are obscure. In this study, we reported that pyruvate substantially inhibited adipogenic differentiation in vitro and its administration significantly prevented HFD-induced weight gain, white adipose tissue inflammation, and metabolic dysregulation. To identify the target proteins of pyruvate, drug affinity responsive target stability was employed with proteomics, cellular thermal shift assay, and isothermal drug response to detect the interactions between pyruvate and its molecular targets. Consequently, we identified cytosolic phospholipase A2 (cPLA2) as a novel molecular target of pyruvate and demonstrated that pyruvate restrained diet-induced obesity, white adipose tissue inflammation, and hepatic steatosis in a cPLA2-dependent manner. Studies with global ablation of cPLA2 in mice showed that the protective effects of pyruvate were largely abrogated, confirming the importance of pyruvate/cPLA2 interaction in pyruvate attenuation of inflammation and obesity. Overall, our study not only establishes pyruvate as an antagonist of cPLA2 signaling and a potential therapeutic option for obesity, but it also sheds light on the mechanism of its action. Pyruvate's prior clinical use indicates that it can be considered a safe and viable alternative for obesity, whether consumed as a dietary supplement or as part of a regular diet.

Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Cartilage oligomeric matrix protein inhibits vascular smooth muscle calcification by interacting with bone morphogenetic protein-2.

RATIONALE: Vascular calcification is a significant contributor to cardiovascular morbidity and mortality. We recently reported that cartilage oligomeric matrix protein (COMP) is pivotal for maintaining the homeostasis of vascular smooth muscle cells (VSMCs). Whether COMP affects the process of vascular calcification is unknown. OBJECTIVE: We aimed to test whether COMP modulates vascular calcification. METHODS AND RESULTS: VSMC calcification in vitro was induced by calcifying media containing high inorganic phosphate or calcium. In vivo medial vessel calcification was induced in rats by 5/6 nephrectomy with a high-phosphate diet or by periadventitial application of CaCl(2) to the abdominal aorta. COMP protein level was markedly reduced in both calcified VSMCs and arteries. COMP deficiency remarkably exacerbated VSMC calcification, whereas ectopic expression of COMP greatly reduced calcification. Furthermore, COMP knockdown facilitated osteogenic markers expression by VSMCs even in the absence of calcifying media. By contrast, COMP overexpression significantly inhibited high phosphate- or high calcium-induced VSMC osteochondrogenic transition. Induction of osteogenic marker expression by COMP silencing was reversed by a soluble form of bone morphogenetic protein (BMP)-2 receptor IA, which suggests a BMP-2-dependent mechanism. Our data revealed that COMP bound directly to BMP-2 through the C terminus, inhibited BMP-2 receptor binding, and blocked BMP-2 osteogenic signaling, indicating COMP inhibits osteochondrogenic transition of VSMCs at least partially through inhibiting BMP-2. CONCLUSIONS: Our data strongly suggest that COMP is a novel inhibitor of vascular calcification. The imbalance between the effects of COMP and BMP-2 may provide new insights into the pathophysiology of vascular calcification.

GEP constitutes a negative feedback loop with MyoD and acts as a novel mediator in controlling skeletal muscle differentiation.

Granulin-epithelin precursor (GEP) is an autocrine growth factor that has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation. Here we report that GEP was expressed in skeletal muscle tissue and its level was differentially altered in the course of C2C12 myoblast fusion. The GEP expression during myoblast fusion was a consequence of MyoD transcription factor binding to several E-box (CANNTG) sequences in the 5'-flanking regulatory region of GEP gene, followed by transcription. Recombinant GEP potently inhibited myotube formation from C2C12 myoblasts whereas the knockdown of endogenous of GEP via a siRNA approach accelerated the fusion of myoblasts to myotubes. Interestingly, the muscle fibers of GEP knockdown mice were larger in number but noticeably smaller in size when compared to the wild-type. Mechanistic studies revealed that during myoblast fusion, the addition of GEP led to remarkable reductions in the expressions of muscle-specific transcription factors, including MyoD. In addition, the regulation of myotube formation by GEP is mediated by the anti-myogenic factor JunB, which is upregulated following GEP stimulation. Thus, GEP growth factor, JunB, and MyoD transcription factor form a regulatory loop and act in concert in the course of myogenesis.

Unraveling the mechanisms behind joint damage.

A subtype of myeloid monocyte mediates the transition from autoimmunity to joint destruction in rheumatoid arthritis.

Tau deficiency inhibits classically activated macrophage polarization and protects against collagen-induced arthritis in mice.

BACKGROUND: Tau protein serves a pro-inflammatory function in neuroinflammation. However, the role of tau in other inflammatory disorders such as rheumatoid arthritis (RA) is less explored. This study is to investigate the role of endogenous tau and the potential mechanisms in the pathogenesis of inflammatory arthritis. METHODS: We established collagen-induced arthritis (CIA) model in wild-type and Tau-/- mice to compare the clinical score and arthritis incidence. Micro-CT analysis was used to evaluate bone erosion of ankle joints. Histological analysis was performed to assess inflammatory cell infiltration, cartilage damage, and osteoclast activity in the ankle joints. Serum levels of pro-inflammatory cytokines were measured by ELISA. The expression levels of macrophage markers were determined by immunohistochemistry staining and quantitative real-time PCR. RESULTS: Tau expression was upregulated in joints under inflammatory condition. Tau deletion in mice exhibited milder inflammation and protected against the progression of CIA, evidenced by reduced serum levels of pro-inflammatory cytokines and attenuated bone loss, inflammatory cell infiltration, cartilage damage, and osteoclast activity in the ankle joints. Furthermore, tau deficiency led to the inhibition of classically activated type 1 (M1) macrophage polarization in the synovium. CONCLUSION: Tau is a previously unrecognized critical regulator in the pathogenesis of RA and may provide a potential therapeutic target for autoimmune and inflammatory joint diseases.