Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease

TL1A is a novel TNF-like factor that acts as a costimulator of IFN-gamma secretion through binding to the death domain-containing receptor, DR3. The aim of this study was to test the hypothesis that TL1A may play an important role in inflammatory bowel disease (IBD) by functioning as a Th1-polarizing cytokine. The expression, cellular localization, and functional activity of TL1A and DR3 were studied in intestinal tissue specimens as well as isolated lamina propria mononuclear cells from IBD patients and controls. TL1A mRNA and protein expression was up-regulated in IBD, particularly in involved areas of Crohn's disease (CD; p < 0.03 vs control). TL1A production was localized to the intestinal lamina propria in macrophages and CD4(+) and CD8(+) lymphocytes from CD patients as well as in plasma cells from ulcerative colitis patients. The amount of TL1A protein and the number of TL1A-positive cells correlated with the severity of inflammation, most significantly in CD. Increased numbers of immunoreactive DR3-positive T lymphocytes were detected in the intestinal lamina propria from IBD patients. Addition of recombinant human TL1A to cultures of PHA-stimulated lamina propria mononuclear from CD patients significantly augmented IFN-gamma production by 4-fold, whereas a minimal effect was observed in control patients. Our study provides evidence for the first time that the novel cytokine TL1A may play an important role in a Th1-mediated disease such as CD.     

Dendritic cells process and present antigens across a range of maturation states

We isolated dendritic cells (DC) from lymphoid organs of mice bearing a transgene for a membrane-bound form of the model protein hen egg white lysozyme (HEL). DC from the spleen had a lower representation of costimulatory molecules and class II MHC molecules than those isolated from lymph nodes and thymi. Splenic DC were capable of further maturation by in vivo treatment of mice with LPS. The immature DC from spleen processed HEL and displayed the chemically dominant epitope as evidenced by FACS analysis. These immature DC also presented this epitope to CD4(+) T cells. Splenic DC from another transgenic mouse (ML-5) containing serum HEL also showed the ability to process and present Ag despite low levels of circulating HEL. In vitro-derived DC from the bone marrow (bone marrow-derived DC) of mHEL mice also displayed immature to mature features and in both cases displayed HEL peptides as well as SDS-stable MHC class II molecules. Immature bone marrow-derived DC also processed exogenous HEL. We conclude that the DC sets normally found in tissue show a scale of maturation features but even the most immature process and present peptides by MHC class II molecules.     

Cyclin T: three forms for different roles in physiological and pathological functions

Cyclins are members of family of proteins involved in the cell cycle regulation. They are regulatory subunits of complexes with proteins called cyclin-dependent kinases (CDKs). There are three forms of cyclin T: cyclin T1, cyclin T2a, and T2b. All cyclin T contain an N-terminal "cyclin homology box," the most conserved region among different members of the cyclin family that serves to bind CDK9. In addition to the N-terminal cyclin domain, cyclin T contains a putative coiled-coil motif, a His-rich motif, and a C-terminal PEST sequence. The CDK9/cyclin T complex is able to activate gene expression in a catalytic-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. In addition, only cyclin T1 supports interactions between Tat and TAR. The interaction of Tat with cyclin T1 alters the conformation of Tat to enhance the affinity and specificity of the Tat:TAR interaction. On the other hand, CDK9/cyclin T2 complexes are involved in the regulation of terminal differentiation in muscle cells.     

Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting

Ex-FABP, an extracellular fatty acid binding lipocalin, is physiologically expressed by differentiating chicken chondrocytes and myoblasts. Its expression is enhanced after cell treatment with inflammatory stimuli and repressed by anti-inflammatory agents, behaving as an acute phase protein. Chicken liver fragments in culture show enhanced protein expression after bacterial endotoxin treatment. To investigate the biological role of Ex-FABP, we stably transfected proliferating chondrocytes with an expression vector carrying antisense oriented Ex-FABP cDNA. We observed a dramatic loss of cell viability and a strong inhibition of cell proliferation and differentiation. When chondrocytes were transfected with the antisense oriented Ex-FABP cDNA we observed that Ex-FABP down-modulation increased apoptotic cell number. Myoblasts transfected with the same expression vector showed extensive cell death and impaired myotube formation. We suggest that Ex-FABP acts as a constitutive survival protein and that its expression and activation are fundamental to protect chondrocytes from cell death.     

Enlargement and contracture of C2-ceramide channels

Ceramides are known to play a major regulatory role in apoptosis by inducing cytochrome c release from mitochondria. We have previously reported that ceramide, but not dihydroceramide, forms large and stable channels in phospholipid membranes and outer membranes of isolated mitochondria. C(2)-ceramide channel formation is characterized by conductance increments ranging from <1 to >200 nS. These conductance increments often represent the enlargement and contracture of channels rather than the opening and closure of independent channels. Enlargement is supported by the observation that many small conductance increments can lead to a large decrement. Also the initial conductances favor cations, but this selectivity drops dramatically with increasing total conductance. La(+3) causes rapid ceramide channel disassembly in a manner indicative of large conducting structures. These channels have a propensity to contract by a defined size (often multiples of 4 nS) indicating the formation of cylindrical channels with preferred diameters rather than a continuum of sizes. The results are consistent with ceramides forming barrel-stave channels whose size can change by loss or insertion of multiple ceramide columns.     

The proton pump of the mitochondrial bc1 complex

The molecular mechanism of the proton pump activity by the respiratory chain bc1 complex is still unknown. This group has proposed since long time that protonation/deprotonation events in the apoproteins of the complex are cooperatively linked to the oxido-reduction reactions at the quinone catalytic centre. Protolytic residues in the apoproteins can thus provide proton transfer pathways between the bulk aqueons phases and the redox centre. A series of experiments has been carried out aimed at demonstrating a role of particular complex subunits in the pump process. In this paper recent results are reviewed which have evidenced a definite role of polypeptide carboxyl residues in the proton pump mechanism. In particular, experiments carried out with both the bovine and P. denitrificans purified enzymes have indicated a specific involvement of aspartic residue(s) in the Rieske Fe/S protein in the proton pump function.     

Dimerization of the type 4 cAMP-specific phosphodiesterases is mediated by the upstream conserved regions (UCRs)

The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.