Brendon Richbourgh

Progranulin protects against osteoarthritis through interacting with TNF-α and β-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 β-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.

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains.

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN‐mediated anti‐inflammatory activity in various autoimmune diseases and conditions.

Progranulin Knockout Accelerates Intervertebral Disc Degeneration in Aging Mice

Intervertebral disc (IVD) degeneration is a common degenerative disease, yet much is unknown about the mechanisms during its pathogenesis. Herein we investigated whether progranulin (PGRN), a chondroprotective growth factor, is associated with IVD degeneration. PGRN was detectable in both human and murine IVD. The levels of PGRN were upregulated in murine IVD tissue during aging process. Loss of PGRN resulted in an early onset of degenerative changes in the IVD tissue and altered expressions of the degeneration-associated molecules in the mouse IVD tissue. Moreover, PGRN knockout mice exhibited accelerated IVD matrix degeneration, abnormal bone formation and exaggerated bone resorption in vertebra with aging. The acceleration of IVD degeneration observed in PGRN null mice was probably due to the enhanced activation of NF-κB signaling and β-catenin signaling. Taken together, PGRN may play a critical role in homeostasis of IVD, and may serve as a potential molecular target for prevention and treatment of disc degenerative diseases.

ADAMTS-12: A Multifaced Metalloproteinase in Arthritis and Inflammation

ADAMTS-12 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases, which were known to play important roles in various biological and pathological processes, such as development, angiogenesis, inflammation, cancer, arthritis, and atherosclerosis. In this review, we briefly summarize the structural organization of ADAMTS-12; concentrate on the emerging role of ADAMTS-12 in several pathophysiological conditions, including intervertebral disc degeneration, tumorigenesis and angioinhibitory effects, pediatric stroke, gonad differentiation, trophoblast invasion, and genetic linkage to schizophrenia and asthma, with special focus on its role in arthritis and inflammation; and end with the perspective research of ADAMTS-12 and its potential as a promising diagnostic and therapeutic target in various kinds of diseases and conditions.

The roles of interferon-inducible p200 family members IFI16 and p204 in innate immune responses, cell differentiation and proliferation.

p204 is a member of the interferon-inducible p200 family proteins in mice. The p200 family has been reported to be multifunctional regulators of cell proliferation, differentiation, apoptosis and senescence. Interferon-inducible protein 16 (IFI16) is regarded as the human ortholog of p204 in several studies. This is possibly due to the similarity of their structures. However the consistency of their functions is still elusive. Currently, an emerging focus has been placed upon the role of the p200 proteins as sensors for microbial DNA in innate immune responses and provides new insights into infections as well as autoimmune diseases. This review specially focuses on IFI16 and p204, the member of p200 family in human and murine respectively, and their pathophysiological roles in innate immune responses, cell differentiation and proliferation.

The role of PGRN in musculoskeletal development and disease.

Progranulin (PGRN) is a growth factor that has been implicated in wound healing, inflammation, infection, tumorigenesis, and is most known for its neuroprotective and proliferative properties in neurodegenerative disease. This pleiotropic growth factor has been found to be a key player and regulator of a diverse spectrum of multi-systemic functions. Its critical anti-inflammatory role in rheumatoid arthritis and other inflammatory disease models has allowed for the propulsion of research to establish its significance in musculoskeletal diseases, including inflammatory conditions involving bone and cartilage pathology. In this review, we aim to elaborate on the emerging role of PGRN in the musculoskeletal system, reviewing its particular mechanisms described in various musculoskeletal diseases, with special focus on osteoarthritis and inflammatory joint disease patho-mechanisms and potential therapeutic applications of PGRN and its derivatives in these and other musculoskeletal diseases.

Chondro-protective effects of low intensity pulsed ultrasound

OBJECTIVES Cartilage is a highly mechano-responsive tissue. Chondrocytes undergo a series of complex changes, including proliferation and metabolic alteration as the target of external biomechanical and biochemical stimuli. IL-1β is known to regulate chondrocyte metabolism and plays an important role in the pathogenesis of osteoarthritis (OA). The objective of this study was to employ low-intensity pulsed ultrasound (LIPUS) as a localized mechanical stimulus and assess its effects on chondrocyte migration, proliferation, metabolism, and differentiation, as well as its ability to suppress IL-1β mediated catabolism in cartilage. METHODS Human cartilage explants and chondrocytes were stimulated by LIPUS in the presence and absence of IL-1β to asses cartilage degradation, chondrocytes metabolism, migration, and proliferation. Western blot analyses were conducted to study IL-1β the associated NFκB pathway in chondrocytes. RESULTS LIPUS stimulation increased the proteoglycan content in human cartilage explants and inhibited IL-1β induced loss of proteoglycans. LIPUS stimulation increased rates of chondrocyte migration and proliferation, and promoted chondrogenesis in mesenchymal stem cells (MSC). Further, LIPUS suppressed IL-1β induced activation of phosphorylation of NFκB-p65 and IĸBα leading to reduced expression of MMP13 and ADAMT5 in chondrocytes. CONCLUSIONS Collectively, these data demonstrate the potential therapeutic effects of LIPUS in preventing cartilage degradation and treating OA via a mechanical stimulation that inhibits the catabolic action of IL-1β and stimulates chondrocyte migration, proliferation, and differentiation.

Progranulin inhibition of TNFα

We previously reported that progranulin (PGRN) bound to TNF receptors (TNFR) and was therapeutic against inflammatory arthritis.1 Our report that PGRN inhibits TNF binding and activity as well as the association with TNFR has been independently confirmed and/or supported by other labs recently;2–6 however, Etemadi et al.7 reported in a recent issue of Immunol Cell Biol that the inhibition of PGRN on TNFa activity was not observed in their experiments. In this letter-to-Editor, we respond to Etemadi et al. to emphasize on several important points and explain the possible reasons for their inability to demonstrate the PGRN inhibition of TNFa. Regarding the direct binding of PGRN to TNFR, the finding has been confirmed by Scientists from Atreaon, Inc. using their independent surface plasmon resonance assay and the issue addressed adequately by the Letter-to-Editor (http://www.jneurosci.org/ content/33/21/9202/reply#jneuro_el_111445) and by recent publications.6,8 Thus, this letter specifically focuses on the PGRN inhibition of TNF raised by Etemadi paper. First, PGRN-mediated inhibition of TNFa activity has been well established.2–6,8–10 For instance, PGRN diminishes TNFa-triggered production of reactive oxygen species in neutrophils.9 PGRN ameliorated ischemia reperfusion induced neuronal injury by inhibiting TNFa binding to the neutrophil, and in turn, the suppression of TNFa-induced neutrophil chemotaxis.3 In addition, PGRN also had a protective role in atherosclerosis through suppression of TNFa-induced expression of ICAM-1 and VCAM-1 in endothelial cells.4 PGRN/TNFR2 interaction was found to be crucial for PGRN-mediated protection of lung injury.2 Interestingly, PGRN abrogated TNFa-triggered loss of the primary cilia in mesenchymal stromal cells.5 The inhibition of TNFa activity by PGRN was also supported by a report that PGRN antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis.6 PGRN-antibody-positive patients had more frequent enthesitis or dactylitis, and the protective effects of PGRN were inhibited by serum containing PGRN antibodies in TNFa-induced cytotoxicity assays.6 Second, proper folding and modifications of PGRN are critical for its inhibition of TNFa, which has been well-demonstrated in a recent publication.8 PGRN is a highly cysteine-rich glycoprotein, and contains numerous internal disulfide bonds, which are critical for maintaining the proper folding and conformation of this protein.11 DTT treatment, which is known to disturb the formation of disulfide bonds and in turn affecting protein folding, completely abolished binding of PGRN to TNFR.8 Posttranslational modifications and preparation procedure of PGRN could be other important factors affecting its activity. Indeed, variations in inhibiting TNFa was also observed among different batches purified from the same stable clone or purchased from the same vendor.8 Note that although Adipogen is considered to be reliable sources, one among five batches did not work in the TNFR-binding assays. Variations in inhibiting TNFa activities among purification batches of PGRNs are quite similar to the discrepancy between different purifications of perlecan, a PGRN-binding glycoprotein.12 Perlecans have been shown to have significant variations in glycosylation, and varies dramatically in cell-based assays or in binding assays.13–15 Etemadi et al. acknowledged that they did not know whether the PGRN they used was correctly folded, but they used Atsttrin, an engineered molecule composed of three TNFR-binding domains,1 as an example to justify that higher-order structure of PGRN is not critical for its TNF inhibiting activity.7 This justification is incorrect. High variation in the folding of Atsttrin among purifications was also observed in reverse-phase HPLC assay. The high variation in Atsttrin folding is probably due to the fact that there exists 17 cysterine residues within Atsttrin composed of 154 residues.1 Third, the experimental conditions, including proper dosage of PGRN used, are also important to demonstrate a significant inhibition on TNF binding and activity,1,8 especially in case when the recombinant proteins retain only partial activity. It is noted that only single dose (that is, 250 ng ml 1) of PGRN was reported in Etemadi paper.7 This is clearly lower than the concentrations needed for demonstrating clear inhibition of TNF binding and activity.1,8 In addition, under some pathological conditions the local concentrations of PGRN can be significantly higher than its sera levels, for instance, PGRN levels in synovial fluids in inflammatory arthritis. Etemadi used antibody against TNFR as a positive control in their assays, it is not clear why much higher concentration (2mg ml 1) of antibodies was employed when compared with PGRN. Fourth, PGRN inhibition of TNF binding highly depends on the numbers of cell surface TNFRs. Etemadi et al.7 mentioned that they did not observe the difference in cell death in the presence or absence of PGRN in U937 cells regardless of the TNF-to-PGRN ratio. First, it is not clear whether PGRN itself affects the cell death and survival of U937 cells, which may hide its inhibition on TNF-mediated cell death. Second, selection of the dosages of PGRN based on the ratio of PGRN to TNFa is inappropriate. PGRN inhibition of TNFa closely depends on the availability of TNFR on cell surface, which is clearly different from the regular TNFabinding inhibitors, such as anti-TNFa antibodies. Even if the ratio of PGRN to TNFa is high, as long as the unoccupied TNFRs in cells are still available, TNFa will be able to bind to receptors and activates its signaling. In case of TNFR abundance, TNFa Immunology and Cell Biology (2014) 92, 299–300 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14