Protective role of macrophage-derived ceruloplasmin in inflammatory bowel disease.

OBJECTIVE: Intestinal microflora and inflammatory cell infiltrates play critical roles in the pathogenesis of acute colitis. Ceruloplasmin is an acute-phase plasma protein produced by hepatocytes and activated macrophages, and has ferroxidase with bactericidal activities. The goal is to understand the role of ceruloplasmin in colitis progression in a genetically modified murine model. DESIGN: Experimental colitis was induced in ceruloplasmin null (Cp(-/-)) and wild-type (WT) mice by dextran sulphate sodium administration. The role of ceruloplasmin was further evaluated by transplantation of WT macrophages into Cp(-/-) mice. RESULTS: Cp(-/-) mice rapidly lost weight and were moribund by day 14, while WT mice survived at least 30 days. Colon culture supernatants from Cp(-/-) mice exhibited elevated levels of TNFα, KC and MCP-1, indicative of increased inflammation and neutrophil and macrophage infiltration. Elevated leucocytes and severe histopathology were observed in Cp(-/-) mice. Elevated protein carbonyl content was detected in colons from Cp(-/-) mice suggesting ceruloplasmin antioxidant activity might contribute to its protective function. Unexpectedly, intraperitoneal administration of human ceruloplasmin into Cp(-/-) mice did not afford protection. Bone marrow transplantation from WT mice or injection of isolated peripheral blood monocytes markedly reduced severity of colitis and morbidity in Cp(-/-) mice. CONCLUSION: Macrophage-derived ceruloplasmin contributes importantly to protection against inflammation and tissue injury in acute and chronic experimental colitis. The findings suggest that defects in ceruloplasmin expression or processing may influence the onset or progression of inflammatory bowel disease in patients.

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion.

The activation of heterodimeric (α/ß) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin ß cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) - a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic "pull-push" process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic "steric clash" model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.

Autoimmunity to uroplakin II causes cystitis in mice: a novel model of interstitial cystitis.

BACKGROUND: The pathophysiology of interstitial cystitis (IC) is unknown. Deficits in urothelial cell layers and autoimmune mechanisms may play a role. OBJECTIVE: To examine whether immunization of mice with recombinant mouse uroplakin II (rmUPK2), a bladder-specific protein, would provoke an autoimmune response sufficient to create an IC phenotype. DESIGN, SETTING, AND PARTICIPANTS: RmUPK2 complementary DNA was generated, transferred into a bacterial expression vector, and the generated protein was purified. Eight-week-old SWXJ female mice were immunized with rmUPK2 protein via subcutaneous injection of 200µg of rmUPK2 protein in 200µl of an emulsion. MEASUREMENTS: Mice were euthanized 5 wk after immunization. Axillary and inguinal lymph node cells were tested for antigen-specific responsiveness and cytokine production, serum isotype antibody titers against rmUPK2 were determined, and gene expression of inflammatory mediators was measured in the bladder and other organs. For functional analysis, mice were placed in urodynamic chambers for 24-h micturition frequency and total voided urine measurements. RESULTS AND LIMITATIONS: Immunization with rmUPK2 resulted in T-cell infiltration of the bladder urothelium and increased rmUPK2-specific serum antibody responses in the experimental autoimmune cystitis (EAC) mice models compared with controls. The ratio of bladder to body weight was increased in EAC mice. Quantitative reverse transcriptase polymerase chain reaction analysis showed elevated gene expression of tumor necrosis factor α, interferon γ, interleukin (IL)-17A, and IL-1ß in bladder urothelium but not in other organs. Evaluation of 24-h micturition habits of EAC mice showed significantly increased urinary frequency (p<0.02) and significantly decreased urine output per void (p<0.021) when compared with control mice. CONCLUSIONS: Our study showed that a bladder-specific autoimmune response sufficient to induce inflammation and EAC occurs in mice following immunization with rmUPK2. EAC mice displayed significant evidence of urinary frequency and decreased urine output per void. Further phenotype characterization of EAC mice should include evidence for pain and/or afferent hypersensitivity, and evidence of urothelial cell layer damage.

Structural basis for the autoinhibition of talin in regulating integrin activation.

Activation of heterodimeric (alpha/beta) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin beta membrane-proximal cytoplasmic tails (beta-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin beta-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.