Edward A. Lin

The Growth Factor Progranulin Binds to TNF Receptors and Is Therapeutic Against Inflammatory Arthritis in Mice

A growth factor protects against arthritis in mice by blocking tumor necrosis factor–dependent signaling. The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling, and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.

miR-199a*, a Bone Morphogenic Protein 2-responsive MicroRNA, Regulates Chondrogenesis via Direct Targeting to Smad1

MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a* is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-untranslated region of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3′-untranslated region of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor.

Granulin‐epithelin precursor binds directly to ADAMTS‐7 and ADAMTS‐12 and inhibits their degradation of cartilage oligomeric matrix protein

OBJECTIVE To determine 1) whether a protein interaction network exists between granulin-epithelin precursor (GEP), ADAMTS-7/ADAMTS-12, and cartilage oligomeric matrix protein (COMP); 2) whether GEP interferes with the interactions between ADAMTS-7/ADAMTS-12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects tumor necrosis factor alpha (TNFalpha)-mediated induction of ADAMTS-7/ADAMTS-12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis. METHODS Yeast two-hybrid, in vitro glutathione S-transferase pull-down, and coimmunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS-7/ADAMTS-12, and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS-7/ADAMTS-12. Immunofluorescence cell staining was performed to visualize the subcellular localization of GEP and ADAMTS-7/ADAMTS-12. An in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS-7/ADAMTS-12-mediated digestion of COMP. The role of GEP in inhibiting TNFalpha-induced ADAMTS-7/ADAMTS-12 expression and COMP degradation in cartilage explants was also analyzed. RESULTS GEP bound directly to ADAMTS-7 and ADAMTS-12 in vitro and in chondrocytes, and the 4 C-terminal thrombospondin motifs of ADAMTS-7/ADAMTS-12 and each granulin unit of GEP mediated their interactions. Additionally, GEP colocalized with ADAMTS-7 and ADAMTS-12 on the cell surface of chondrocytes. More importantly, GEP inhibited COMP degradation by ADAMTS-7/ADAMTS-12 in a dose-dependent manner through 1) competitive inhibition through direct protein-protein interactions with ADAMTS-7/ADAMTS-12 and COMP, and 2) inhibition of TNFalpha-induced ADAMTS-7/ADAMTS-12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis. CONCLUSION Our observations demonstrate a novel protein-protein interaction network between GEP, ADAMTS-7/ADAMTS-12, and COMP. Furthermore, GEP is a novel specific inhibitor of ADAMTS-7/ADAMTS-12-mediated COMP degradation and may play a significant role in preventing the destruction of joint cartilage in arthritis.

Natural Transformation of Helicobacter pylori Involves the Integration of Short DNA Fragments Interrupted by Gaps of Variable Size

Helicobacter pylori are gram-negative bacteria notable for their high level of genetic diversity and plasticity, features that may play a key role in the organisms ability to colonize the human stomach. Homeologous natural transformation, a key contributor to genomic diversification, has been well-described for H. pylori. To examine the mechanisms involved, we performed restriction analysis and sequencing of recombination products to characterize the length, fragmentation, and position of DNA imported via natural transformation. Our analysis revealed DNA imports of small size (1,300 bp, 95% confidence limits 950–1850 bp) with instances of substantial asymmetry in relation to selectable antibiotic-resistance markers. We also observed clustering of imported DNA endpoints, suggesting a possible role for restriction endonucleases in limiting recombination length. Additionally, we observed gaps in integrated DNA and found evidence suggesting that these gaps are the result of two or more separate strand invasions. Taken together, these observations support a system of highly efficient short-fragment recombination involving multiple recombination events within a single locus.

The role of ADAMTSs in arthritis

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family consists of 19 proteases. These enzymes are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. This review briefly summarizes the structural organization and functional roles of ADAMTSs in normal and pathological conditions, focusing on members that are known to be involved in the degradation of extracellular matrix and loss of cartilage in arthritis, including the aggrecanases (ADAMTS-4 and ADAMTS-5), ADAMTS-7 and ADAMTS-12, the latter two are associated with cartilage oligomeric matrix protein (COMP), a component of the cartilage extracellular matrix (ECM). We will discuss the expression pattern and the regulation of these metalloproteinases at multiple levels, including their interaction with substrates, induction by pro-inflammatory cytokines, protein processing, inhibition (e.g., TIMP-3, alpha-2-macroglobulin, GEP), and activation (e.g., syndecan-4, PACE-4).

Plastic cells and populations: DNA substrate characteristics in Helicobacter pylori transformation define a flexible but conservative system for genomic variation

Helicobacter pylori, bacteria that colonize the human gastric mucosa, are naturally competent for transformation by exogenous DNA, and show a panmic‐tic population structure. To understand the mechanisms involved in its horizontal gene transfer, we sought to define the interval required from exposure to substrate DNA until DNA uptake and expression of a selectable phenotype, as well as the relationship of transforming fragment length, concentration, homol‐ogy, symmetry, and strandedness, to the transformation frequency. We provide evidence that natural transformation in H. pylori differs in efficiency among wild‐type strains but is saturable and varies with substrate DNA length, symmetry, strandedness, and species origin. We show that H. pylori cells can be transformed within one minute of contact with DNA, by DNA fragments as small as 50 bp, and as few as 5 bp on one flank of a selectable single nucleotide mutation is sufficient substrate for recombination of a transforming fragment, and that double‐stranded DNA is the preferred (1000‐fold > single‐stranded) substrate. The high efficiency of double‐stranded DNA as transformation substrate, in conjunction with strain‐specific restriction endonucle‐ases suggests a model of short‐fragment recombination favoring closest relatives, consistent with the observed H. pylori population biology.—Levine S. M., Lin, E. A., Emara, W., Kang, J., DiBenedetto, M., Ando, T., Falush, D., Blaser M. J. Plastic cells and populations: DNA substrate characteristics in Helicobacter pylori transformation define a flexible but conservative system for genomic variation. FASEB J. 21, 3458–3467 (2007)

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.

Addition of pregabalin to multimodal analgesic therapy following ankle surgery: a randomized double-blind, placebo-controlled trial.

Background and Objectives Pregabalin is often used as a perioperative analgesic adjunct; some studies show benefit, but others do not. Adverse effects, such as confusion and sedation, have been attributed to perioperative use of pregabalin. We tested the hypothesis that pregabalin, when used as part of a multimodal analgesic regimen, reduces the duration of moderate to severe pain in the first 24 hrs following foot or ankle surgery. Secondary outcomes included measures of opioid and pregabalin adverse effects. Methods Sixty patients scheduled for hospital admission after foot or ankle surgery entered this randomized, double-blind, placebo-controlled trial. Patients received a neuraxial anesthetic, a popliteal fossa sciatic nerve block using 30 mL 0.375% bupivacaine with clonidine 100 µg and epinephrine, a saphenous nerve block, postoperative hydromorphone intravenous patient-controlled analgesia, and oral analgesics (oxycodone/acetaminophen). Patients were randomized to receive pregabalin (100 mg preoperatively, then 50 mg every 12 hrs) or a placebo for 3 days. The primary outcome was the number of hours that patients reported moderate to severe pain. Results Both groups reported a similar number of hours of moderate to severe pain during the first 24 hrs: 4.1 (SD, 4.1) hrs (pregabalin) versus 4.5 (SD, 3.5) hrs (placebo). Pain scores, opioid use, and adverse effects were also similar in both groups. Conclusions No clinical benefit was obtained from perioperative administration of pregabalin (100 mg preoperative, then 50 mg every 12 hrs) as part of a multimodal postoperative analgesic regimen following foot and ankle surgery.

XBP1U inhibits the XBP1S-mediated upregulation of the iNOS gene expression in mammalian ER stress response

Upregulation of the inducible nitric oxide synthase (iNOS) gene is associated with many pathological conditions such as endoplasmic reticulum (ER) stress, and X-box binding protein 1 (XBP1) is critical in mediating ER-stress responsive genes, including iNOS. Nonetheless, the mechanism by which XBP1 regulates iNOS during ER stress remains unexplored. Here we show that the active/spliced form of XBP1 protein, XBP1S, directly binds to the AABS (A-activator-binding site) in the iNOS promoter in vitro and in living cells. XBP1S exhibits dose-dependent activation of iNOS-specific reporter gene activity and endogenous iNOS expression. XBP1S is elevated whereas the unspliced form of XBP1, XBP1U, reduced in ER stress in HepG2 cells. In addition, XBP1U binds to XBP1S and this complex is associated with the iNOS promoter in response to ER stress. Furthermore, XBP1U acts as a negative mediator and suppresses XBP1S-mediated induction of iNOS. Collectively, we present the first evidence demonstrating the regulation of iNOS gene induction by the interaction between the spliced and unspliced forms of XBP1 in response to ER stress.

Prevention of atrophic nonunion by the systemic administration of parathyroid hormone (PTH 1-34) in an experimental animal model.

Objectives: Recombinant human parathyroid hormone (PTH 1–34) has been previously shown to enhance fracture healing in animal models. Here, we sought to determine whether the systemic administration of PTH 1–34 is effective in preventing atrophic fracture nonunion in a murine, surgical nonunion model. Methods: We used an established reproducible long-bone murine fracture nonunion model by generating a midshaft femur fracture, followed by fracture distraction using an intramedullary pin and custom metallic clip to maintain a fracture gap of 1.7 mm. Mice were randomized to receive either daily intraperitoneal injections of 30 &mgr;g/kg PTH 1–34 for 14 days or saline injections. At 6 weeks after the procedure, radiographic and histologic assessment of fracture healing was performed. Results: At 6 weeks after surgery, the group treated with PTH showed higher rates of bony union (50% vs 8%; P < 0.05) as assessed by radiographic analysis. Mean gap size was also significantly lower in the PTH group (1.42 vs 0.36 mm in the control group; P < 0.05). Histologic analysis of atrophic nonunions in the control group revealed a persistent fracture gap with intervening fibrous tissue. In contrast, healed subjects in the PTH-treated group had cortical bridging with mature bone and relatively little callus, which is consistent with primary intramembranous ossification. Conclusions: Daily systemic administration of recombinant PTH 1–34 increased the rate of union in a mouse atrophic nonunion model. This may have important implications for the potential clinical role of PTH 1–34 in the treatment of atrophic fracture nonunions.