Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphorylation of GATA-3

GATA-3 plays a critical role in allergic diseases by regulating the release of cytokines from Th2 lymphocytes. However, the molecular mechanisms involved in the regulation of GATA-3 in human T lymphocytes are not yet understood. Using small interfering RNA to knock down GATA-3, we have demonstrated its critical role in regulating IL-4, IL-5, and IL-13 release from a human T cell line. Specific stimulation of T lymphocytes by costimulation of CD3 and CD28 to mimic activation by APCs induces translocation of GATA-3 from the cytoplasm to the nucleus, with binding to the promoter region of Th2 cytokine genes, as determined by chromatin immunoprecipitation. GATA-3 nuclear translocation is dependent on its phosphorylation on serine residues by p38 MAPK, which facilitates interaction with the nuclear transporter protein importin-alpha. This provides a means whereby allergen exposure leads to the expression of Th2 cytokines, and this novel mechanism may provide new approaches to treating allergic diseases.     

Mold allergen, pen C 13, induces IL-8 expression in human airway epithelial cells by activating protease-activated receptor 1 and 2

Allergenic serine proteases are important in the pathogenesis of asthma. One of these, Pen c 13, is the immunodominant allergen produced by Penicillium citrinum. Many serine proteases induce cytokine expression, but whether Pen c 13 does so in human respiratory epithelial cells is not known. In this study, we investigated whether Pen c 13 caused IL-8 release and activated protease-activated receptors (PARs) in airway epithelial cells. In airway-derived A549 cells and normal human airway epithelial cells, Pen c 13 induced IL-8 release in a dose-dependent manner. Pen c 13 also increased IL-8 release in a time-dependent manner in A549 cells. Pen c 13 cleaved PAR-1 and PAR-2 at their activation sites. Treatment with Pen c 13 induced intracellular Ca(2+) mobilization and desensitized the cells to the action of other proteases and PAR-1 and PAR-2 agonists. Moreover, Pen c 13-mediated IL-8 release was significantly decreased in Ca(2+)-free medium and was abolished by the protease inhibitors, PMSF and 4-(2-aminoethyl) benzenesulfonyl fluoride. Blocking Abs against the cleavage sites of PAR-1 and PAR-2, but not of PAR-4, inhibited Pen c 13-induced IL-8 production, as did inhibition of phospholipase C. Pen c 13 induced IL-8 expression via activation of ERK 1/2, and not of p38 and JNK. In addition, treatment of A549 cells or normal human airway epithelial cells with Pen c 13 increased phosphorylation of ERK 1/2 by a Ca(2+)-dependent pathway. These finding show that Pen c 13 induces IL-8 release in airway epithelial cells and that this is dependent on PAR-1 and PAR-2 activation and intracellular calcium.     

Dendritic cells present lytic antigens and maintain function throughout persistent gamma-herpesvirus infection

The activation and maintenance of Ag-specific CD8(+) T cells is central to the long-term control of persistent infections. These killer T cells act to continuously scan and remove reservoirs of pathogen that have eluded the acute immune response. Acutely cleared viral infections depend almost exclusively on dendritic cells (DC) to present Ags to, and to activate, the CD8(+) T cell response. Paradoxically, persistent pathogens often infect professional APCs such as DC, in addition to infecting a broad range of nonprofessional APC, raising the possibility that many cell types could present viral Ags and activate T cells. We addressed whether in persistent viral infection with murine gammaherpesviruses, DC or non-DC, such as B cells and macrophages, were required to maintain the continued activation of Ag-specific CD8(+) T cells. We found that presentation of the surrogate Ag, OVA, expressed under a lytic promoter to CD8(+) T cells during persistent infection was largely restricted to DC, with little contribution from other lymphoid resident cells, such as B cells. This is despite the fact that B cells harbor a very large reservoir of latent virus. Our results support that, during persistent viral infection, continual presentation of lytic Ags by DC leads to T cell activation critical for maintaining CD8(+) T cells capable of limiting persistent viral infection.     

Pathophysiological Roles of Gap Junction in Glomerular Mesangial Cells

Glomerular mesangial cells (MCs) are specialized vascular smooth muscle cells that play a critical role in the control of glomerular hemodynamics. One of the intriguing features of MCs is their extraordinary abundance in gap junctions (GJs). It has long been speculated that GJs may bridge MCs together and provide the mesangium with the characteristics of a functional syncytium. Accumulating scientific evidence supports this idea. GJs are reported to be critically involved in important physiological processes like tubuloglomerular feedback and glomerular filtration. In addition, GJs are implicated in the control of many cellular processes of MCs, including growth, differentiation and survival. This article summarizes the current knowledge on the roles of GJs in glomerular pathophysiology.     

Conformational dynamics of the molecular chaperone Hsp90 in complexes with a co-chaperone and anticancer drugs

The molecular chaperone Hsp90 is essential for the correct folding, maturation and activation of a diverse array of client proteins, including several key constituents of oncogenic processes. Hsp90 has become a focus of cancer research, since it represents a target for direct prophylaxis against multistep malignancy. Hydrogen-exchange mass spectrometry was used to study the structural and conformational changes undergone by full-length human Hsp90beta in solution upon binding of the kinase-specific co-chaperone Cdc37 and two Hsp90 ATPase inhibitors: Radicicol and the first-generation anticancer drug DMAG. Changes in hydrogen exchange pattern in the complexes in regions of Hsp90 remote to the ligand-binding site were observed indicating long-range effects. In particular, the interface between the N-terminal domain and middle domains exhibited significant differences between the apo and complexed forms. For the inhibitors, differences in the interface between the middle domain and the C-terminal domain were also observed. These data provide important insight into the structure of the biologically active form of the protein.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.     

DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur

Cleavage of Notch by furin is required to generate a mature, cell surface heterodimeric receptor that can be proteolytically activated to release its intracellular domain, which functions in signal transduction. Current models propose that ligand binding to heterodimeric Notch (hNotch) induces a disintegrin and metalloprotease (ADAM) proteolytic release of the Notch extracellular domain (NECD), which is subsequently shed and/or endocytosed by DSL ligand cells. We provide evidence for NECD release and internalization by DSL ligand cells, which, surprisingly, did not require ADAM activity. However, losses in either hNotch formation or ligand endocytosis significantly decreased NECD transfer to DSL ligand cells, as well as signaling in Notch cells. Because endocytosis-defective ligands bind hNotch, but do not dissociate it, additional forces beyond those produced through ligand binding must function to disrupt the intramolecular interactions that keep hNotch intact and inactive. Based on our findings, we propose that mechanical forces generated during DSL ligand endocytosis function to physically dissociate hNotch, and that dissociation is a necessary step in Notch activation.     

Complement 3 is involved in changing the phenotype of human glomerular mesangial cells

Complement activation contributes to tissue injury in various forms of glomerulopathy and is characterized by deposition of complement components, which accelerates the progression of chronic renal damage. We recently reported that complement 3 (C3), a critical component of the complement system, is associated with the synthetic phenotype of vascular smooth muscle cells. It is possible that C3 stimulates mesangial cells to assume the synthetic phenotype to, in turn, induce glomerular injury and sclerosis. We investigated the role of C3 in the growth and phenotype of mesangial cells. Cultured human mesangial cells (HMCs) expressed C3 mRNA and protein, and levels were increased in response to IFN-gamma and TNF-alpha. HMCs also expressed C3a receptor mRNA and protein. Exogenous C3a stimulated DNA synthesis in HMCs in a dose-dependent manner. C3a decreased expression h-caldesmon mRNA, a marker of the contractile phenotype, and increased the expression of osteopontin, matrix Gla, and collagen type1 alpha1 (collagen IV) mRNAs, which are markers of the synthetic phenotype. C3a decreased expression of alpha-smooth muscle actin in HMCs. Small interfering RNA (siRNA) targeting C3 reduced the DNA synthesis and proliferation of HMCs, increased expression of h-caldesmon mRNA, and decreased expression of osteopontin, matrix Gla, and collagen IV mRNAs in HMCs. These results indicate that C3 causes HMCs to convert to the synthetic phenotype and stimulates growth of mesangial cells, suggesting that C3 may play an important role in phenotypic regulation of mesangial cells in renal diseases.