Serge Przedborski, Miquel Vila, Vernice Jackson-Lewis
Todd E. Golde
John D. Minna
Using physiological, pharmacological, and gene disruption approaches, we demonstrate that proteinase-activated receptor-2 (PAR-2) plays a pivotal role in mediating chronic inflammation. Using an adjuvant monoarthritis model of chronic inflammation, joint swelling was substantially inhibited in PAR-2–deficient mice, being reduced by more than fourfold compared with wild-type mice, with virtually no histological evidence of joint damage. Mice heterozygous for PAR-2 gene disruption showed an intermediate phenotype. PAR-2 expression, normally limited to endothelial cells in small arterioles, was substantially upregulated 2 weeks after induction of inflammation, both in synovium and in other periarticular tissues. PAR-2 agonists showed potent proinflammatory effects as intra-articular injection of ASKH95, a novel synthetic PAR-2 agonist, induced prolonged joint swelling and synovial hyperemia. Given the absence of the chronic inflammatory response in the PAR-2–deficient mice, our findings demonstrate a key role for PAR-2 in mediating chronic inflammation, thereby identifying a novel and important therapeutic target for the management of chronic inflammatory diseases such as rheumatoid arthritis.
William R. Ferrell, John C. Lockhart, Elizabeth B. Kelso, Lynette Dunning, Robin Plevin, Stephen E. Meek, Andrew J.H. Smith, Gary D. Hunter, John S. McLean, Frances McGarry, Robert Ramage, Lu Jiang, Toru Kanke, Junichi Kawagoe
Nonmelanoma skin cancer is one of the most common malignancies in humans. Different therapeutic strategies for the treatment of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the wide tissue distribution of the two subtypes of cannabinoid receptors (CB1 and CB2), we studied the potential utility of these compounds in anti–skin tumor therapy. Here we show that the CB1 and the CB2 receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic death of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CB1/CB2 agonist WIN-55,212-2 or the selective CB2 agonist JWH-133 induced a considerable growth inhibition of malignant tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by altered blood vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth factor, and angiopoietin 2). Abrogation of EGF-R function was also observed in cannabinoid-treated tumors. These results support a new therapeutic approach for the treatment of skin tumors.
M. Llanos Casanova, Cristina Blázquez, Jesús Martínez-Palacio, Concepción Villanueva, M. Jesús Fernández-Aceñero, John W. Huffman, José L. Jorcano, Manuel Guzmán
The adhesion receptors known as integrins perform key functions for hematopoietic cells. The platelet integrin αIIbβ3 is critical in hemostasis, and the β1 and β2 integrins on leukocytes have many roles in cell-mediated immunity. Mutations in the β2 subunit lead to integrin nonexpression and to an immune deficiency, leukocyte adhesion deficiency-1. Mutations in either the α or β subunit of αIIbβ3 usually lead to integrin nonexpression and a bleeding tendency termed Glanzmann thrombasthenia. Here we describe a unique patient with clinical features of both Glanzmann thrombasthenia and leukocyte adhesion deficiency-1. The patient has normal expression of β1, β2, and β3 integrins, but all are dysfunctional. The key findings are that “inside-out” signaling pathways leading to integrin activation are defective and that this is associated with abnormal integrin clustering. The integrins themselves are intact and capable of function following extracellular stimulation. T cell motility is normal, as are the expression levels and electrophoretic characteristics of all cytoskeletal and signaling proteins tested, except PKC-α, which has enhanced expression in the patient’s cells. To our knowledge, this is the first description of a dysfunction affecting three classes of integrins. We propose that it is caused by a lesion in an intracellular factor or signaling pathway essential for integrin activation in hematopoietic cells and results in lack of regulation of clustering, an essential component of integrin-mediated adhesion.
Alison McDowall, David Inwald, Birgit Leitinger, Alison Jones, Ri Liesner, Nigel Klein, Nancy Hogg
Group B streptococcus (GBS) is an important human pathogen. In this study, we sought to identify mechanisms that may protect GBS from host defenses in addition to its capsular polysaccharide. A gene encoding a cell-surface–associated protein (cspA) was characterized from a highly virulent type III GBS isolate, COH1. Its sequence indicated that it is a subtilisin-like extracellular serine protease homologous to streptococcal C5a peptidases and caseinases of lactic acid bacteria. The wild-type strain cleaved the α chain of human fibrinogen, whereas a cspA mutant, TOH121, was unable to cleave fibrinogen. We observed aggregated material when COH1 was incubated with fibrinogen but not when the mutant strain was treated similarly. This suggested that the product(s) of fibrinogen cleavage have strong adhesive properties and may be similar to fibrin. The cspA gene was present among representative clinical isolates from all nine capsular serotypes, as revealed by Southern blotting. A cspA– mutant was ten times less virulent in a neonatal rat sepsis model of GBS infections, as measured by LD50 analysis. In addition, the cspA– mutant was significantly more sensitive than the wild-type strain to opsonophagocytic killing by human neutrophils in vitro. Taken together, the results suggest that cleavage of fibrinogen by CspA may increase the lethality of GBS infection, potentially by protecting the bacterium from opsonophagocytic killing.
Theresa O. Harris, Daniel W. Shelver, John F. Bohnsack, Craig E. Rubens
Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration.
Susan M. Majka, Kathyjo A. Jackson, Kirsten A. Kienstra, Mark W. Majesky, Margaret A. Goodell, Karen K. Hirschi
Tobacco-related diseases such as lung cancer cause over 4.2 million deaths annually, with approximately 400,000 deaths per year occurring in the US. Genotoxic effects of tobacco components have been described, but effects on signaling pathways in normal cells have not been described. Here, we show activation of the serine/threonine kinase Akt in nonimmortalized human airway epithelial cells in vitro by two components of cigarette smoke, nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Activation of Akt by nicotine or NNK occurred within minutes at concentrations achievable by smokers and depended upon α3-/α4-containing or α7-containing nicotinic acetylcholine receptors, respectively. Activated Akt increased phosphorylation of downstream substrates such as GSK-3, p70S6K, 4EBP-1, and FKHR. Treatment with nicotine or NNK attenuated apoptosis caused by etoposide, ultraviolet irradiation, or hydrogen peroxide and partially induced a transformed phenotype manifest as loss of contact inhibition and loss of dependence on exogenous growth factors or adherence to ECM. In vivo, active Akt was detected in airway epithelial cells and lung tumors from NNK-treated A/J mice, and in human lung cancers derived from smokers. Redundant Akt activation by nicotine and NNK could contribute to tobacco-related carcinogenesis by regulating two processes critical for tumorigenesis, cell growth and apoptosis.
Kip A. West, John Brognard, Amy S. Clark, Ilona R. Linnoila, Xiaowei Yang, Sandra M. Swain, Curtis Harris, Steven Belinsky, Phillip A. Dennis
AMP-activated protein kinase (AMPK) is viewed as a fuel sensor for glucose and lipid metabolism. To better understand the physiological role of AMPK, we generated a knockout mouse model in which the AMPKα2 catalytic subunit gene was inactivated. AMPKα2–/– mice presented high glucose levels in the fed period and during an oral glucose challenge associated with low insulin plasma levels. However, in isolated AMPKα2–/– pancreatic islets, glucose- and L-arginine–stimulated insulin secretion were not affected. AMPKα2–/– mice have reduced insulin-stimulated whole-body glucose utilization and muscle glycogen synthesis rates assessed in vivo by the hyperinsulinemic euglycemic clamp technique. Surprisingly, both parameters were not altered in mice expressing a dominant-negative mutant of AMPK in skeletal muscle. Furthermore, glucose transport was normal in incubated isolated AMPKα2–/– muscles. These data indicate that AMPKα2 in tissues other than skeletal muscles regulates insulin action. Concordantly, we found an increased daily urinary catecholamine excretion in AMPKα2–/– mice, suggesting altered function of the autonomic nervous system that could explain both the impaired insulin secretion and insulin sensitivity observed in vivo. Therefore, extramuscular AMPKα2 catalytic subunit is important for whole-body insulin action in vivo, probably through modulation of sympathetic nervous activity.
Benoit Viollet, Fabrizio Andreelli, Sebastian B. Jørgensen, Christophe Perrin, Alain Geloen, Daisy Flamez, James Mu, Claudia Lenzner, Olivier Baud, Myriam Bennoun, Emmanuel Gomas, Gaël Nicolas, Jørgen F.P. Wojtaszewski, Axel Kahn, David Carling, Frans C. Schuit, Morris J. Birnbaum, Erik A. Richter, Rémy Burcelin, Sophie Vaulont
Cholesterol-loaded macrophage foam cells are a central component of atherosclerotic lesions. ABCA1, the defective molecule in Tangier disease, mediates the efflux of phospholipids and cholesterol from cells to apoA-I, reversing foam cell formation. In ABCA1, we identified a sequence rich in proline, glutamic acid, serine, and threonine (PEST sequence) that enhances the degradation of ABCA1 by calpain protease and thereby controls the cell surface concentration and cholesterol efflux activity of ABCA1. In an apparent positive feedback loop, apoA-I binds ABCA1, promotes lipid efflux, inhibits calpain degradation, and leads to increased levels of ABCA1. ApoA-I infusion also increases ABCA1 in vivo. These studies reveal a novel mode of regulation of ABCA1 by PEST sequence–mediated calpain proteolysis that appears to be reversed by apolipoprotein-mediated phospholipid efflux. Inhibition of ABCA1 degradation by calpain could represent a novel therapeutic approach to increasing macrophage cholesterol efflux and decreasing atherosclerosis.
Nan Wang, Wengen Chen, Patrick Linsel-Nitschke, Laurent O. Martinez, Birgit Agerholm-Larsen, David L. Silver, Alan R. Tall
Th2 cells are generated from naive CD4 T cells upon T cell receptor (TCR) recognition of antigen and IL-4 stimulation and play crucial roles in humoral immunity against infectious microorganisms and the pathogenesis of allergic and autoimmune diseases. A tyrosine phosphatase, SHP-1, that contains src homology 2 (SH2) domains is recognized as a negative regulator for various intracellular signaling molecules, including those downstream of the TCR and the IL-4 receptor. Here we assessed the role of SHP-1 in Th1/Th2 cell differentiation and in the development of Th2-dependent allergic airway inflammation by using a natural SHP-1 mutant, the motheaten mouse. CD4 T cells appear to develop normally in the heterozygous motheaten (me/+) thymus even though they express decreased amounts of SHP-1 (about one-third the level of wild-type thymus). The me/+ naive splenic CD4 T cells showed enhanced activation by IL-4 receptor–mediated signaling but only marginal enhancement of TCR-mediated signaling. Interestingly, the generation of Th2 cells was increased and specific cytokine production of mast cells was enhanced in me/+ mice. In an OVA-induced allergic airway inflammation model, eosinophilic inflammation, mucus hyperproduction, and airway hyperresponsiveness were enhanced in me/+ mice. Thus, SHP-1 may have a role as a negative regulator in the development of allergic responses, such as allergic asthma.
Tohru Kamata, Masakatsu Yamashita, Motoko Kimura, Kaoru Murata, Masamichi Inami, Chiori Shimizu, Kaoru Sugaya, Chrong-Reen Wang, Masaru Taniguchi, Toshinori Nakayama
Steroidal anti-inflammatory drugs induce proteins that inhibit phospholipase A2 (PLA2), including uteroglobin and lipocortin-1 (annexin I). Uteroglobin and lipocortin-1 retain several conserved sequences. Based on these sequences, several nonapeptides (antiflammins) were synthesized. These nonapeptides were shown to have anti-inflammatory effects in vitro and in vivo, possibly by inhibiting PLA2. Subsequent research showed that PLA2 is activated by transglutaminase 2 (TGase 2). We hypothesize here that TGase 2 inhibitors may increase the anti-inflammatory efficacy of inhibiting PLA2 activity. To test this theory, we constructed recombinant peptides containing sequences from pro-elafin (for inhibition of TGase 2), and from lipocortin-1, lipocortin-5, and uteroglobin (for inhibition of PLA2). The recombinant peptides, which had dual inhibitory effects on purified TGase 2 and PLA2, reversed the inflammation of allergic conjunctivitis to ragweed in a guinea pig model. The present work suggests that novel recombinant peptides may be safe and effective agents for the treatment of various inflammatory diseases.
Joonhong Sohn, Tae-Im Kim, Young-Hee Yoon, Joo-Yong Kim, Soo-Youl Kim
Acute liver failure caused by viral hepatitis or toxic damage involves both apoptotic and necrotic pathways. IGF binding protein-1 (IGFBP-1), a hepatocyte-derived secreted protein, is required for normal liver regeneration. To determine whether IGFBP-1 could prevent liver injury that entails direct stimulation of hepatocyte apoptosis, IGFBP-1–/– mice, IGFBP-1+/+ mice, and mice pretreated with Ab’s against IGFBP-1 were treated with a normally sublethal dose of Fas agonist. IGFBP-1 deficiency was associated with massive hepatocyte apoptosis and caspase activation within 3 hours of Fas agonist treatment, which could be corrected by pretreatment with IGFBP-1. IGFBP-1–deficient livers had enhanced signaling via the integrin receptor at early times (0.5 to 1 hour) after Fas agonist treatment accompanied by elevated activated matrix metalloproteinase-9 (MMP-9), a known target of fibronectin signaling and activator of TGF-β. Within 3 hours of Fas agonist treatment, elevated expression of active TGF-β1, a hepatocyte apoptogen, was observed in IGFBP-1–deficient livers that correlated with the appearance of the apoptotic process. Both MMP-9 and TGF-β1 expression were suppressed by IGFBP-1 treatment, supporting their role in the apoptotic process. IGFBP-1–/– mice also displayed increased injury in a toxic hepatic injury model caused by CCl4. These findings indicate that IGFBP-1 functions as a critical hepatic survival factor in the liver by reducing the level of proapoptotic signals.
Julia I. Leu, Mary Ann S. Crissey, Rebecca Taub