Faculty Bibliography

To investigate the effects and mechanisms of irisin, a newly discovered myokine, in cartilage development, osteoarthritis (OA) pathophysiology and its therapeutic potential for treating OA we applied the following five strategical analyses using (1) murine joint tissues at different developmental stages; (2) human normal and OA pathological tissue samples; (3) experimental OA mouse model; (4) irisin gene knockout (KO) and knock in (KI) mouse lines and their cartilage cells; (5) in vitro mechanistic experiments. We found that Irisin was involved in all stages of cartilage development. Both human and mouse OA tissues showed a decreased expression of irisin. Intra-articular injection of irisin attenuated ACLT-induced OA progression. Irisin knockout mice developed severe OA while irisin overexpression in both irisin KI mice and intraarticular injection of irisin protein attenuated OA progression. Irisin inhibited inflammation and promoted anabolism in chondrogenic ADTC5 cells. Proliferative potential of primary chondrocytes from KI mice was found to be enhanced, while KO mice showed an inhibition under normal or inflammatory conditions. The primary chondrocytes from irisin KI mice showed reduced expression of inflammatory factors and the chondrocytes isolated from KO mice showed an opposite pattern. In conclusion, it is the first time to show that irisin is involved in cartilage development and OA pathogenesis. Irisin has the potential to ameliorate OA progression by decreasing cartilage degradation and inhibiting inflammation, which could lead to the development of a novel therapeutic target for treating bone and cartilage disorders including osteoarthritis.

Articular cartilage injury, as a hallmark of arthritic diseases, is difficult to repair and causes joint pain, stiffness, and loss of mobility. Over the years, the most significant problems for the drug-based treatment of arthritis have been related to drug administration and delivery. In recent years, much research has been devoted to developing new strategies for repairing or regenerating the damaged osteoarticular tissue. The RNA interference (RNAi) has been suggested to have the potential for implementation in targeted therapy in which the faulty gene can be edited by delivering its complementary Short Interfering RNA (siRNA) at the post-transcriptional stage. The successful editing of a specific gene by the delivered siRNA might slow or halt osteoarthritic diseases without side effects caused by chemical inhibitors. However, cartilage siRNA delivery remains a challenging objective because cartilage is an avascular and very dense tissue with very low permeability. Furthermore, RNA is prone to degradation by serum nucleases (such as RNase H and RNase A) due to an extra hydroxyl group in its phosphodiester backbone. Therefore, successful delivery is the first and most crucial requirement for efficient RNAi therapy. Nanomaterials have emerged as highly advantage tools for these studies, as they can be engineered to protect siRNA from degrading, address barriers in siRNA delivery to joints, and target specific cells. This review will discuss recent breakthroughs of different siRNA delivery technologies for cartilage diseases.

β-Cell dysfunction and reduction in β-cell mass are hallmark events of diabetes mellitus. Here we show that β-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca²⁺ release in β-cells. Kindlin-2 loss activates GSK-3β and downregulates β-catenin, leading to reduced β-cell proliferation and mass. Kindlin-2 loss reduces the percentage of β-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of β-catenin in β-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of β-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.

BACKGROUND AND AIMS/OBJECTIVE:Recent studies have unveiled an association between ADAMTS7 gene variation and coronary artery disease (CAD) caused by atherosclerosis. We investigated if the ADAMTS7 Serine214-to-Proline substitution arising from a CAD-associated variant affected angiogenesis, since neovascularization plays an important role in atherosclerosis. METHODS AND RESULTS/RESULTS:ADAMTS7 knockdown in vascular endothelial cells (ECs) attenuated their angiogenesis potential, whereas augmented ADAMTS7-Ser214 expression had the opposite effect, leading to increased ECs migratory and tube formation ability. Proteomics analysis showed an increase in thrombospondin-1, a reported angiogenesis inhibitor, in culture media conditioned by ECs with ADAMTS7 knockdown and a decrease of thrombospondin-1 in media conditioned by ECs with ADAMTS7-Ser214 overexpression. Cleavage assay indicated that ADAMTS7 possessed thrombospondin-1 degrading activity, which was reduced by the Ser214-to-Pro substitution. The pro-angiogenic effect of ADAMTS7-Ser214 diminished in the presence of a thrombospondin-1 blocking antibody. CONCLUSIONS:The ADAMTS7 Ser217-to-Pro substitution as a result of ADAMTS7 polymorphism affects thrombospondin-1 degradation, thereby promoting atherogenesis through increased EC migration and tube formation.

OBJECTIVES/OBJECTIVE:This study aims to investigate the role and mechanism of FGFR3 in macrophages and their biological effects on the pathology of arthritis. METHODS:Mice with conditional knockout of FGFR3 in myeloid cells (R3cKO) were generated. Gait behaviours of the mice were monitored at different ages. Spontaneous synovial joint destruction was evaluated by digital radiographic imaging and μCT analysis; changes of articular cartilage and synovitis were determined by histological analysis. The recruitment of macrophages in the synovium was examined by immunostaining and monocyte trafficking assay. RNA-seq analysis, Western blotting and chemotaxis experiment were performed on control and FGFR3-deficient macrophages. The peripheral blood from non-osteoarthritis (OA) donors and patients with OA were analysed. Mice were treated with neutralising antibody against CXCR7 to investigate the role of CXCR7 in arthritis. RESULTS:R3cKO mice but not control mice developed spontaneous cartilage destruction in multiple synovial joints at the age of 13 months. Moreover, the synovitis and macrophage accumulation were observed in the joints of 9-month-old R3cKO mice when the articular cartilage was not grossly destructed. FGFR3 deficiency in myeloid cells also aggravated joint destruction in DMM mouse model. Mechanically, FGFR3 deficiency promoted macrophage chemotaxis partly through activation of NF-κB/CXCR7 pathway. Inhibition of CXCR7 could significantly reverse FGFR3-deficiency-enhanced macrophage chemotaxis and the arthritic phenotype in R3cKO mice. CONCLUSIONS:Our study identifies the role of FGFR3 in synovial macrophage recruitment and synovitis, which provides a new insight into the pathological mechanisms of inflammation-related arthritis.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by insulin deficiency, and patients with diabetes have an increased risk of bone fracture and significantly impaired fracture healing. Proinflammatory cytokine tumor necrosis factor-alpha is significantly upregulated in diabetic fractures and is believed to underlie delayed fracture healing commonly observed in diabetes. Our previous genetic screen for the binding partners of progranulin (PGRN), a growth factor-like molecule that induces chondrogenesis, led to the identification of tumor necrosis factor receptors (TNFRs) as the PGRN-binding receptors. In this study, we employed several in vivo models to ascertain whether PGRN has therapeutic effects in diabetic fracture healing. Here, we report that deletion of PGRN significantly delayed bone fracture healing and aggravated inflammation in the fracture models of mice with T1DM. In contrast, recombinant PGRN effectively promoted diabetic fracture healing by inhibiting inflammation and enhancing chondrogenesis. In addition, both TNFR1 proinflammatory and TNFR2 anti-inflammatory signaling pathways are involved in PGRN-stimulated diabetic fracture healing. Collectively, these findings illuminate a novel understanding concerning the role of PGRN in diabetic fracture healing and may have an application in the development of novel therapeutic intervention strategies for diabetic and other types of impaired fracture healing.

Our recent studies demonstrate that the focal adhesion protein Kindlin-2 is critical for chondrogenesis and early skeletal development. Here, we show that deleting Kindlin-2 from osteoblasts using the 2.3-kb mouse Col1a1-Cre transgene minimally impacts bone mass in mice, but deleting Kindlin-2 using the 10-kb mouse Dmp1-Cre transgene, which targets osteocytes and mature osteoblasts, results in striking osteopenia in mice. Kindlin-2 loss reduces the osteoblastic population but increases the osteoclastic and adipocytic populations in the bone microenvironment. Kindlin-2 loss upregulates sclerostin in osteocytes, downregulates β-catenin in osteoblasts, and inhibits osteoblast formation and differentiation in vitro and in vivo. Upregulation of β-catenin in the mutant cells reverses the osteopenia induced by Kindlin-2 deficiency. Kindlin-2 loss additionally increases the expression of RANKL in osteocytes and increases osteoclast formation and bone resorption. Kindlin-2 deletion in osteocytes promotes osteoclast formation in osteocyte/bone marrow monocyte cocultures, which is significantly blocked by an anti-RANKL-neutralizing antibody. Finally, Kindlin-2 loss increases osteocyte apoptosis and impairs osteocyte spreading and dendrite formation. Thus, we demonstrate an important role of Kindlin-2 in the regulation of bone homeostasis and provide a potential target for the treatment of metabolic bone diseases.

Background/UNASSIGNED:Cartilage defects account for substantial economic and humanistic burdens and pose a significant clinical problem. The efficacy of clinical approaches to cartilage repair is often inadequate, in part, owing to the restricted proliferative capacity of chondrocytes. Molecules have the capacity to promote the differentiation of multipotent mesenchymal stem cells into chondrocytes and may also gain the ability to repair the damaged cartilage. Objective/UNASSIGNED:This study aimed to investigate the role of Atsttrin (progranulin-derived engineered protein) in cartilage repair as well as the signaling pathway involved. Methods/UNASSIGNED:. Real-time polymerase chain reaction and Western blot analysis were used to monitor the effect of Atsttrin on the transcriptional and protein levels, respectively, of key anabolic and catabolic signaling molecules. Results/UNASSIGNED:In addition, Atsttrin-mediated cartilage repair occurred primarily through tumor necrosis factor receptor 2-initiated Akt signaling and downstream JunB transcription factor. Conclusion/UNASSIGNED:Atsttrin might serve as a promising therapeutic modality for cartilage regeneration.

The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell-extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb shortening. Collectively, our findings demonstrate critical roles for Pinch1/2 and a functional redundancy of both factors in the control of chondrogenesis and bone mass through distinct mechanisms.