The Role Of Progranulin And Its Derived Engineered Protein In Titanium Particie Induced Inflammatory Osteolysis And Osteoarthritis

Osteoarthritis(OA) is a common degenerative disease, which is characterized by cartilage destruction, synovitis, subchondral bone sclerosis and osteophyte formation. Nowdays, OA affects more than 15% population of the world. Unforturnately, so far, there is no method for preventing or treating OA. Existing drugs are provided to reduce joint inflammation and pain. However, they are just symptom-modifying drugs instead of disease-modifying drugs. In present study, we investigated the role of growth factor progranulin-derived engineered protein, Atsttrin, in the pathogenesis of OA.First of all, we found that as progranuin-derived engineered protein, Atsttrin could rescue progranulin-deficiency caused accelerated OA.Secondly, in the surgically-induced OA mouse model, we found that Atsttrin could effectively prevent articular cartilage loss. Additionally, Atsttrin significantly reduced synovium inflammation in OA development. Furthermore, Atsttrin effectively reduced osteoclasts formation in subchondral bone, maintaining the thickness of subchondral bone to support cartilage. Most importantly, Atsttrin reduced OA-related pain by abserving mice behavior and testing the pain-related inflammatory gene markers.Besides preventative effect, we found that in the non-invasive rat OA model, Atsttrin exhibited an therapeutic effect. Given the importance that Atsttrin selectively binding to TNFRs, to further determine the mechanism of Atsttrin-mediated protective effect in OA progression, we established surgically-induced OA model in wild type(WT), TNFR2-deficient(TNFR1-/-) and TNFR2-deficient(TNFR2-/-) mice. After the model was established, we locally deliver Atsttrin and interestingly, we found in WT mice, Atsttrin effectively protected articular cartilage. However, in TNFR1-/- mice, Atsttrin still protected cartilage, but this protective effect was moderately decreased compared to the WT group, indicating TNFR1 also contributed partially to this process. In contrast, Atsttrin-mediated chondro-protective effect was largely lost in TNFR2-/-mice. Above all, Atsttrin-mediated protective effect depends on both TNFR1 and TNFR2 pathways.To further determine the mechanism involved, we found that Atsttrin could regulate chondrocyte metabolism. Interestingly, we found Atsttrin could promote chondrocyte anabolic effect. Specifically, Atsttrin could increase the expression of chondrocyte anabolic gene markers, such as type II collagen(Col II) and aggrecan(ACN). In searching for the Atsttrin-activated signaling pathways, we took advantage of the cocktail western assay and found Atsttrin strongly active Akt signaling and slightly active Erkl/2 signaling. To further determine the specificity of this activation, we applied specific signling inhibitor to block these signaling pathways. In detail, we used U0126 to block Erkl/2 siganling and we used wortamannin to block Akt signaling. Interestingly, after blocking Akt or Erkl/2 siganling, Atsttrin-mediated anabolic gene markers, such as Col II and ACN were dramatically decreased. In addition, Atsttrin’s anabolic effect was almost lost in presenc of both inhibitors. These data indicate Atsttrin-mediated anabolic effect depends on Akt and Erkl/2 signaling. To investigate which receptor is important for Atsttrin-mediated anabolism, we isolated primary chondrocytes from WT, TNFR1-/- and TNFR2-/- mice and cultured in absence or presence of Atsttrin. In WT and TNFR1-/- chondrocytes, Atsttrin significantly increased expression of Col II and ACN. However, in TNFR2-/-chondrocytes, Atsttrin-mediated expression of Col II and ACN was almost abolished. Overall, Atsttrin-mediated anabolism depends on TNFR2-Akt/Erkl/2 signaling.Regardless of anabolism, Atsttrin effectively inhibited TNF α-induced inflammatory catabolism. It is known that TNF a is increased in OA progression, and TNF a attracts attention duing to its peak position of inflammatory cascade. Previous studies indicated blocking TNF a is beneficial for OA in multiple animal models. In addition, a clinical study demonstrated that TNF a inhibitor could significantly improve OA symptom. Since Atsttrin exerts its role by binding to TNFRs and TNF a plays an important role in OA, we further used human primary chondrocytes to determine whether Atsttrin could effectively inhibit TNF α-induced inflammatory catabolism. To address this issue, we isolated human primary cartilage and cultured in absence or presence of TNF a with or without Atsttrin. To further investigate the involved mechanism, we isolated human primary chondrocyte. We found Atsttrin could significantly reduce TNF α-induced inflammatory catabolic markers, including MMP-3,MMP-13,ADAMTS-5 and NOS-2. To further confirm this finding, we also extracted RNA and analyzed the inflammatory gene markers’expression. Real-time PCR indicated Atsttrin significantly reduced gene expression of MMP-3,MMP-13,ADAMTS-5 and NOS-2.Besides these, it is well-established that TNF a strongly active MAPK and NF-KB signaling, consequently up-regulate the expression of downstream pro-inflammatory transcriptional factors, leading to articular cartilage destruction. In present study, Atsttrin effectively inhibited TNF α-induced activation of MAPK and NF-KB signaling. As a consequent, the expression of inflammatory catabolic biomarkers, including MMP-3,MMP-13,ADAMTS-5 and NOS-2 were dramatically decreased.It is known that TNF α/NF-KB is a classic signaling pathway. NF-KB is compromised by two subunits:p50(IKB) and p65. Under unstimulated condition, p50 and p65 form a complication, staying in cytoplasm. Once TNF a stimulates cell, NF-KB signaling is activated. In detail,IKB is phorysphorylated and p-IKB stays in cytoplasm. In contrast, phorysphorylated p65 translocated into nuclear, followed by activation of downstream molecule.In present study, we found Atsttrin inhibited IKB phoryspholation in human primary chondrocytes. Additionally, Atsttrin effectively inhibited p65 translocation. To further determine whether Atsttrin could affect activity of NF-KB, we transfected NF-KB plasmid into chondrocytes. And we found Attstrin could significantly reduce NF-KB activity. Overall, Atsttrin effectively inhibit TNF a-induced inflammatory catabolism.In summary, Atsttrin protects against osteoarthritis through at least two pathways:1) directly binding to TNFR2, active Akt and Erkl/2 signaling, followed by activation of chondrocyte anabolism.2) competitively binding to TNFR1, antagonize TNF a-induced inflammatory catabolism. The finding not only provides the insight of Atsttrin in arthritis homostasis, but also provide Atsttrin as an alternative potential for treating osteoarthritis.Total joint arthroplasty (TJA) is one of the most widely performed elective surgeries for the treatment of severe joint diseases such as osteoarthritis and rheumatoid arthritis, providing effective pain relief and functional improvement in patients’ daily lives 1. Annually, approximate 1.5 million hip and knee arthroplasties are performed worldwide, and the demand for these procedures is increasing2. Despite several recent advances, revision of TJAs remains a major concern. Aseptic loosening and periprosthetic osteolysis are the leading causes of arthroplasty failure, which occurs as a result of the biological response to particulate wear debris such as titanium particles. Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFa. Since TNF level is increased, and it is known that TNF plays a critical role in titanium particles-induced inflammatory osteolysis, additionally, TNF inhibitors exhibit a therapeutic effect in the titanium particles-induced inflammatory osteolysis animal models. Thus, in present study, we hypothesize that progranulin, with potent anti-inflammatory effect, could suppress titanium particles-induced inflammatory osteolysis through TNF signaling.To determine whether progranulin expression is changed in the pathogenesis of titanium particles-induced inflammatory osteolysis, we used both in vitro and in vivo assays. For the in vitro assay, we took advantage of RAW cell line. After titanium particles stimulation, we collected RNA and protein. Data indicate that titanium particles could induce progranulin expression. For the in vivo data, we used immunochemistry staining and found in the titanium particles-induced air pouch model, progranulin expression was dramatically induced. Thus, titianium particles could induce expression of progranulin both in vitro and in vivo. However, this elevation could not neutralize titanium particles-induced inflammation.To determine the endogenous role of progranulin in titanium particles-induced inflammatory osteolysis, we established the titanium particles-induced air pouch model in progranulin-deficient mice and their control littermates. After the model is established, we collect the tissues as well as the blood. Histology demonstrated progranulin-deficient mice exhibited much severer inflammation. Additionally, we homogenesis the fresh tissue, and we collected the RNA and protein. Real-time PCR indicated that the pro-inflammatory cytokines were dramatically increased in the progranulin-dificient mice compared to their control littermates. Furthermore, ELISA assay demonstrated that the pro-inflammatroy cytokines release, such as IL-1 and IL-6 were significantly increased in the progranulin-dificient mice. Above all, the endogenous of progranulin contributed to the titanium particles-induced inflammatory osteolysis.To determine whether recombinant progranulin has a therapeutic effect in the titanium particles-induced inflammatory osteolysis, we established the titanium particles-induced air pouch model in wild type mice and followed by PBS or recombinant progranulin treatment. After the model is established, we collect the tissues as well as the blood. Histology demonstrated progranulin-treated mice exhibited much less inflammation. Additionally, we homogenesis the fresh tissue, and we collected the RNA and protein. Real-time PCR indicated that the pro-inflammatory cytokines were dramatically decreased in the progranulin-treated mice compared to the PBS-treated group. Furthermore, ELISA assay demonstrated that the pro-inflammatroy cytokines release, such as IL-1 and IL-6 were significantly decreased in the progranulin-treated mice. Above all, the recombinant progranulin effectively suppress titanium particles-induced inflammatory osteolysis.To further confirm this finding, we used ex vivo model. Specifically, we isolated the mice clavia and cultured in the regular medium or conditional medium. The conditional medium is compromised by regular medium and titanium particles stimulated medium with ratio of 1:1. After incubation, the tissues underwent fixation, decalcification and section. TRAP staining indicated titanium particles could enhance osteoclastogenesis, in contrast, recombinant progranulin could effectively inhibit this process. Additionally, we extracted RNA from the cultured clavia and we found progranulin significantly reduced the osteoclast gene markers.To examine the role of progranulin in titanium particles-induced inflammatory osteolysis in vivo, we established the titanium particles-induced clavia osteolysis model. MicroCT indicated that titanium particles enhanced osteolysis while progranulin effectively inbitied this process. TRAP staining demonstrated that progranulin largely suppressed the titanium particles-induced osteoclastogenesis. In addition to that, progranulin effectively promoted collagen synthesis. Furthermore, we isolated bone marrow stem cells and differentiatied them into osteoclasts. Interestingly, titanium particles dramatically enhanced osteoclast formation, while recombinant progranulin significantly reduced the osteoclast number. Collectively, progranulin may exerts its effect through inhibiting osteoclastogenesis.To further study the involved mechanism, we cultured the RAW cell line in absence or presence of titanium particles without or with recombinant progranulin. Data indicated that in RAW cell line, titanium particles could induce expression of pro-inflammatory cytokines. However, progranulin significantly reduced titanium particles-indced expression of these cytokines. Given the importance that TNF-NF/KB signaliing plays an important role in the pathogenesis of titanium particles-induced inflammatory osteolysis, we attempted to figure out whether progranulin-mediated anti-inflammatory effect is through, or at least partially through targeting TNF-NF/KB signaling. To address this issue, we examined the phorysphorylation of IKB in the titanium particles-induced air pouch model. And we found progranulin could dramatically inhibit IKB acitivation. In addition, we cultured RAW cell line in absence or presence of titanium particles without or with progranulin, western blotting demonstrated progranulin effectively inhibited p65 translocate into nuclear. Moreover, we transfected RAW cell line with NF-KB plasmid and measured the fluresent density to test NF-KB activity. Data indicated titanium particles could significantly increase the NF-KB activity and recombinant progranulinn significantly suppressed titanium particles-induced inflammation.Above all, progranulin plays an important role in the titanium particles-induced inflammatory osteolysis. Progranulin-mediated protective effect is through, at least partially through, inhibiting NF-KB signaling.