IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-kappa B ligand (RANKL)/receptor activator of NF-kappa B/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-kappa B, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.     

Burrower bugs (Heteroptera: Cydnidae) in peanut: seasonal species abundance, tillage effects, grade reduction effects, insecticide efficacy, and management

Pitfall traps placed in South Carolina peanut, Arachis hypogaea (L.), fields collected three species of burrower bugs (Cydnidae): Cyrtomenus ciliatus (Palisot de Beauvois), Sehirus cinctus cinctus (Palisot de Beauvois), and Pangaeus bilineatus (Say). Cyrtomenus ciliatus was rarely collected. Sehirus cinctus produced a nymphal cohort in peanut during May and June, probably because of abundant henbit seeds, Lamium amplexicaule L., in strip-till production systems. No S. cinctus were present during peanut pod formation. Pangaeus bilineatus was the most abundant species collected and the only species associated with peanut kernel feeding injury. Overwintering P. bilineatus adults were present in a conservation tillage peanut field before planting and two to three subsequent generations were observed. Few nymphs were collected until the R6 (full seed) growth stage. Tillage and choice of cover crop affected P. bilineatus populations. Peanuts strip-tilled into corn or wheat residue had greater P. bilineatus populations and kernel-feeding than conventional tillage or strip-tillage into rye residue. Fall tillage before planting a wheat cover crop also reduced burrower bug feeding on peanut. At-pegging (early July) granular chlorpyrifos treatments were most consistent in suppressing kernel feeding. Kernels fed on by P. bilineatus were on average 10% lighter than unfed on kernels. Pangaeus bilineatus feeding reduced peanut grade by reducing individual kernel weight, and increasing the percentage damaged kernels. Each 10% increase in kernels fed on by P. bilineatus was associated with a 1.7% decrease in total sound mature kernels, and kernel feeding levels above 30% increase the risk of damaged kernel grade penalties.     

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.     

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than na?ve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among na?ve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.     

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.     

Phosphorylation of a carboxyl-terminal serine within the kappa-opioid receptor produces desensitization and internalization

G-protein receptor kinase and beta-arrestin mediated desensitization of the rat kappa-opioid receptor (KOR) was previously shown using Xenopus oocyte expression to require serine 369 within the C terminus of KOR. To define the effects of phosphorylation of this residue in desensitization and internalization processes in mammalian expression systems, wild-type KOR-green fluorescent protein (KOR-GFP) and KOR(S369A)-GFP were stably expressed in AtT-20 and HEK293 cells. Using whole-cell patch clamp recording in transfected AtT-20 cells, agonist activation of either kappa receptor form produced equivalent activation of the intrinsic G-protein-gated inwardly rectifying potassium channel. Incubation for 60 min with the kappa agonist U50,488 (100 nm) desensitized the response in cells expressing wild-type KOR-GFP by 86% but had no effect on KOR(S369A)-GFP-expressing cells. Phosphorylation of serine 369 was detected using a phosphospecific antibody (KOR-P) able to distinguish the phosphorylated form of the receptor. The agonist-induced increase in KOR-P labeling was dose-dependent, blocked by co-treatment with the kappa antagonist norbinaltorphimine, and prevented by co-expression of the dominant negative form of the G-protein receptor kinase, GRK2(K220R). In contrast, agonist-induced increase in KOR-P labeling was not evident in KOR(S369A) expressing cells. Prolonged activation resulted in receptor internalization that was also blocked by KOR(S369A) substitution, but interestingly, KOR-P labeling was evident at lower agonist concentrations than required to induce internalization. Following the removal of agonist, receptor dephosphorylation detected by loss of KOR-P labeling was complete within 60 min, could be blocked by okadaic acid, and was not blocked by sucrose inhibition of receptor internalization. These results demonstrate that GRK-mediated phosphorylation of serine 369 mediates rat KOR desensitization and internalization.     

Inhibition of receptor-mediated endocytosis demonstrates generation of amyloid beta-protein at the cell surface

Sequential cleavages of the amyloid beta-protein precursor (APP) by the beta- and gamma-secretases generate the amyloid beta-protein (A beta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in A beta generation. Here, we used HeLa cells stably expressing a tetracycline-regulated dominant-negative dynamin I (dyn K44A), which selectively inhibits receptor-mediated endocytosis, and analyzed the effects on the processing of endogenous APP. Upon induction of dyn K44A, levels of mature APP rose at the cell surface, consistent with retention of APP on the plasma membrane. The alpha-secretase cleavage products of APP were increased by dyn K44A, in that alpha-APPs in medium and the C83 C-terminal stub in the membrane both rose. The beta-secretase cleavage of APP, C99, also increased modestly. The use of specific gamma-secretase inhibitors to study the accumulation of alpha- and beta-cleavage products independent of their processing by gamma-secretase confirmed that retention of APP on the plasma membrane results in increased processing by both alpha- and beta-secretases. Unexpectedly, endogenous A beta secretion was significantly increased by dyn K44A, as detected by three distinct methods: metabolic labeling, immunoprecipitation/Western blotting, and enzyme-linked immunosorbent assay. Levels of p3 (generated by sequential alpha- and gamma-cleavage) also rose. We conclude that endogenous A beta can be produced directly at the plasma membrane and that alterations in the degree of APP endocytosis may help regulate its production. Our findings are consistent with a role for the gamma-secretase complex in the processing of numerous single-transmembrane receptors at the cell surface.