Faculty Bibliography

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.

PURPOSE/OBJECTIVE: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:) 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-1β/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: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/CONCLUSIONS: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.

Mammalian focal adhesion proteins Pinch1 and Pinch2 regulate integrin activation and cell-extracellular matrix adhesion and migration. Here, we show that deleting Pinch1 in osteocytes and mature osteoblasts using the 10-kb mouse Dmp1-Cre and Pinch2 globally (double KO; dKO) results in severe osteopenia throughout life, while ablating either gene does not cause bone loss, suggesting a functional redundancy of both factors in bone. Pinch deletion in osteocytes and mature osteoblasts generates signals that inhibit osteoblast and bone formation. Pinch-deficient osteocytes and conditioned media from dKO bone slice cultures contain abundant sclerostin protein and potently suppress osteoblast differentiation in primary BM stromal cells (BMSC) and calvarial cultures. Pinch deletion increases adiposity in the BM cavity. Primary dKO BMSC cultures display decreased osteoblastic but enhanced adipogenic, differentiation capacity. Pinch loss decreases expression of integrin β3, integrin-linked kinase (ILK), and α-parvin and increases that of active caspase-3 and -8 in osteocytes. Pinch loss increases osteocyte apoptosis in vitro and in bone. Pinch loss upregulates expression of both Rankl and Opg in the cortical bone and does not increase osteoclast formation and bone resorption. Finally, Pinch ablation exacerbates hindlimb unloading-induced bone loss and impairs active ulna loading-stimulated bone formation. Thus, we establish a critical role of Pinch in control of bone homeostasis.

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/CONCLUSIONS: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.

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

Kindlin-2 regulates integrin-mediated cell adhesion to and migration on the extracellular matrix. Our recent studies demonstrate important roles of kindlin-2 in regulation of mesenchymal stem cell differentiation and skeletal development. In this study, we generated adipose tissue-specific conditional knockout of kindlin-2 in mice by using Adipoq-Cre BAC-transgenic mice. The results showed that deleting kindlin-2 expression in adipocytes in mice caused a severe lipodystrophy with drastically reduced adipose tissue mass. Kindlin-2 ablation elevated the blood levels of nonesterified fatty acids and triglycerides, resulting in massive fatty livers in the mutant mice fed with high-fat diet (HFD). Furthermore, HFD-fed mutant mice displayed type II diabetes-like phenotypes, including elevated levels of fasting blood glucose, glucose intolerance, and peripheral insulin resistance. Kindlin-2 loss dramatically reduced the expression levels of multiple key factors, including PPARγ, mTOR, AKT, and β-catenin proteins, and suppressed adipocyte gene expression and differentiation. Finally, kindlin-2 loss drastically reduced leptin production and caused a high bone mass phenotype. Collectively, these studies establish a critical role of kindlin-2 in control of adipogenesis and lipid metabolism as well as bone homeostasis.

Over the past two decades, considerable advances in our understanding of inflammatory and immune pathways have allowed for the growing use of targeted biologic therapy. Most notably, the introduction of tumor necrosis factor (TNF) inhibitors has dramatically changed the management of autoimmune inflammatory disorders, including ankylosing spondylitis (AS). Despite the efficacy of TNF inhibitors documented in multiple clinical trials, anti-TNF therapy in AS is far from foolproof; it is associated with serious adverse effects and limited response to therapy in some patients. Moreover, specific questions regarding the role of TNF as a mediator of AS remain unanswered. Therefore, additional efforts are needed in order to better understand the role of TNF in the pathogenesis of AS and to develop safer and more effective treatment strategies. The purpose of this review is to better the understanding of the physiologic and pathogenic roles of TNF signaling in the course of AS. Relevant TNF biology and novel approaches to TNF blockade in AS are discussed.

Progranulin (PGRN), a widely expressed glycoprotein with pleiotropic function, has been linked to a host of physiological processes and diverse pathological states. A series of contemporary preclinical disease models and clinical trials have evaluated various therapeutic strategies targeting PGRN, highlighting PGRN as a promising therapeutic target. Herein we summarize available knowledge of PGRN targeting in various kinds of diseases, including common neurological diseases, inflammatory autoimmune diseases, cancer, tissue repair, and rare lysosomal storage diseases, with a focus on the functional domain-oriented drug development strategies. In particular, we emphasize the role of extracellular PGRN as a non-conventional, extracellular matrix bound, growth factor-like conductor orchestrating multiple membrane receptors and intracellular PGRN as a chaperone/co-chaperone that mediates the folding and traffic of its various binding partners.

BACKGROUND:Teriparatide, a 1-34 fragment of parathyroid hormone (PTH) that maintains most of the biological activities of PTH, has been employed since 2002 as an anabolic agent for osteoporotic individuals who are at high risk of fracture. The purpose of the present review is to provide a systematic summary and timely update on treatment with teriparatide for fracture prevention. METHODS:Electronic databases, including OVID MEDLINE, OVID Embase, and the Cochrane Library, were searched on February 9, 2018, to identify published systematic reviews and meta-analyses addressing treatment with teriparatide for fracture prevention, and A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR 2) was used to assess the quality of included studies. RESULTS:Seventeen studies were included. Of the 17 eligible studies, 3 were rated as high quality, 3 were rated as moderate quality, 6 were rated as low quality, and 5 were rated as critically low quality. Teriparatide reduced vertebral and overall nonvertebral fractures in osteoporotic patients regardless of the existence of precipitating conditions, including postmenopausal status, glucocorticoid treatment, and chronic kidney disease, as compared with placebo, but not the site-specific nonvertebral fractures of the wrist and hip. Teriparatide did not more effectively reduce fracture risks when compared with other medications, such as bisphosphonates, selective estrogen receptor modulators, RANKL (receptor activator of nuclear factor kappa-beta ligand) inhibitor, or strontium ranelate. CONCLUSIONS:Teriparatide was safe and was not associated with an increased rate of adverse events when compared with other drugs. Teriparatide was effective for the prevention of vertebral and overall nonvertebral fractures in osteoporotic patients but not for the prevention of site-specific nonvertebral fractures at the wrist and hip. LEVEL OF EVIDENCE/METHODS:Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.