A cell-penetrating CD40-TRAF2,3 blocking peptide diminishes inflammation and neuronal loss after ischemia/reperfusion.

While the administration of anti-CD154 mAbs in mice validated the CD40-CD154 pathway as a target against inflammatory disorders, this approach caused thromboembolism in humans (unrelated to CD40 inhibition) and is expected to predispose to opportunistic infections. There is a need for alternative approaches to inhibit CD40 that avoid these complications. CD40 signals through TRAF2,3 and TRAF6-binding sites. Given that CD40-TRAF6 is the pathway that stimulates responses key for cell-mediated immunity against opportunistic pathogens, we examined the effects of pharmacologic inhibition of CD40-TRAF2,3 signaling. We used a model of ischemia/reperfusion (I/R)-induced retinopathy, a CD40-driven inflammatory disorder. Intravitreal administration of a cell-penetrating CD40-TRAF2,3 blocking peptide impaired ICAM-1 upregulation in retinal endothelial cells and CXCL1 upregulation in endothelial and Müller cells. The peptide reduced leukocyte infiltration, upregulation of NOS2/COX-2/TNF-α/IL-1ß, and ameliorated neuronal loss, effects that mimic those observed after I/R in Cd40-/- mice. While a cell-penetrating CD40-TRAF6 blocking peptide also diminished I/R-induced inflammation, this peptide (but not the CD40-TRAF2,3 blocking peptide) impaired control of the opportunistic pathogen Toxoplasma gondii in the retina. Thus, inhibition of the CD40-TRAF2,3 pathway is a novel and potent approach to reduce CD40-induced inflammation, while likely diminishing the risk of opportunistic infections that would otherwise accompany CD40 inhibition.

Proinflammatory responses induced by CD40 in retinal endothelial and Müller cells are inhibited by blocking CD40-Traf2,3 or CD40-Traf6 signaling.

PURPOSE: The cell surface receptor CD40 is required for the development of retinopathies induced by diabetes and ischemia/reperfusion. The purpose of this study was to identify signaling pathways by which CD40 triggers proinflammatory responses in retinal cells, since this may lead to pharmacologic targeting of these pathways as novel therapy against retinopathies. METHODS: Retinal endothelial and Müller cells were transduced with vectors that encode wild-type CD40 or CD40 with mutations in sites that recruit TNF receptor associated factors (TRAF): TRAF2,3 (ΔT2,3), TRAF6 (ΔT6), or TRAF2,3 plus TRAF6 (ΔT2,3,6). Cells also were incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. We assessed intercellular adhesion molecule-1 (ICAM-1), CD40, monocyte chemoattractant protein-1 (MCP-1), VEGF, and prostaglandin E2 (PGE2) by fluorescence-activated cell sorting (FACS), ELISA, or mass spectrometry. Mice (B6 and CD40(-/-)) were made diabetic using streptozotocin. The MCP-1 mRNA was assessed by real-time PCR. RESULTS: The CD40-mediated ICAM-1 upregulation in endothelial and Müller cells was markedly inhibited by expression of CD40 ΔT2,3 or CD40 ΔT6. The CD40 was required for MCP-1 mRNA upregulation in the retina of diabetic mice. The CD40 stimulation of endothelial and Müller cells enhanced MCP-1 production that was markedly diminished by CD40 ΔT2,3 or CD40 ΔT6. Similar results were obtained in cells incubated with CD40-TRAF2,3 or CD40-TRAF6 blocking peptides. The CD40 ligation upregulated PGE2 and VEGF production by Müller cells, that was inhibited by CD40 ΔT2,3 or CD40 ΔT6. All cellular responses tested were obliterated by expression of CD40 ΔT2,3,6. CONCLUSIONS: Blockade of a single CD40-TRAF pathway was sufficient to impair ICAM-1, MCP-1, PGE2, and VEGF upregulation in retinal endothelial and/or Müller cells. Blockade of CD40-TRAF signaling may control retinopathies.

CD40 and tumour necrosis factor-α co-operate to up-regulate inducuble nitric oxide synthase expression in macrophages.

Regulation of inducible nitric oxide synthase (NOS2) expression is important given the role of this enzyme in inflammation, control of infections and immune regulation. In contrast to tumour necrosis factor-α (TNF-α) alone or CD40 stimulation alone, simultaneous stimulation of mouse macrophages through CD40 ligation and TNF-α led to up-regulation of NOS2 and nitric oxide production. This response was of functional relevance because CD40/TNF-α-stimulated macrophages acquired nitric oxide-dependent anti-Leishmania major activity. CD40 plus TNF-α up-regulated NOS2 independently of interferon-γ, interferon-α/ß and interleukin-1. TNF receptor-associated factor 6 (TRAF6), an adapter protein downstream of CD40, appears to be required for NOS2 up-regulation because a CD40-TRAF6 blocking peptide inhibited up-regulation of NOS2 in CD40/TNF-α-stimulated macrophages. CCAAT/enhancer-binding protein-ß (C/EBPß), a transcription factor activated by TNF-α but not CD40, was required for NOS2 up-regulation because this enzyme was not up-regulated when C/EBPß(-/-) macrophages received CD40 plus TNF-α stimulation. These results indicate that CD40 and TNF-α co-operate to up-regulate NOS2, probably via the effect of TRAF6 and C/EBPß.

Fem1b antigen in the stool of ApcMin mice as a biomarker of early Wnt signaling activation in intestinal neoplasia.

BACKGROUND: Colorectal cancer is preventable by early detection and removal of precursor lesions. Central to early stages of colorectal neoplasia is activation of Wnt signaling, usually due to inactivation of the Apc tumor suppressor gene for which there is an established animal model, the Apc(Min) mouse. Immunodetection in stool of proteins up-regulated by aberrant Wnt signaling, within intestinal epithelial cells shed into the lumen, could be a rational approach to identify biomarkers of early intestinal neoplasia. Fem1b gene expression is up-regulated, following inactivation of Apc, in mouse intestinal epithelium. METHODS: We initially screened pooled random stool samples by immunoblotting and found that we could detect, in Apc(Min) mice but not wild-type mice, a fragment of Fem1b protein with an antibody (Li-50) directed against an epitope near the middle of the protein, but not with antibodies directed against N-terminus or C-terminus epitopes. We then evaluated freshly voided individual stool samples collected on four consecutive days from four each of male and female Apc(Min) mice and their wild-type littermates. RESULTS: The Fem1b antigen was detected with the Li-50 antibody in 15/16 samples from male Apc(Min) mice compared to 0/16 samples from male wild-type mice, and in 5/16 samples from female Apc(Min) mice compared to 0/16 samples from female wild-type mice. CONCLUSIONS: This study provides proof-of-principle that fragments of proteins, whose expression is increased by aberrant Wnt signaling early in intestinal neoplasia, can be immunodetected in stool. Excreted Fem1b protein fragments may be a useful biomarker for epithelial Wnt signaling and early intestinal neoplasia.

Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

RACK1 downregulates levels of the pro-apoptotic protein Fem1b in apoptosis-resistant colon cancer cells.

Evasion of apoptosis plays an important role in colon cancer progression. Following loss of the Apc tumor suppressor gene in mice, the gene encoding Fem1b is upregulated early in neoplastic intestinal epithelium. Fem1b is a pro-apoptotic protein that interacts with Fas, TNFR1 and Apaf-1, and increased expression of Fem1b induces apoptosis of cancer cells. Fem1b is a homolog of FEM-1, a protein in Caenorhabditis elegans that is negatively regulated by ubiquitination and proteasomal degradation. To study Fem1b regulation in colon cancer progression, we used apoptotis-sensitive SW480 cells, derived from a primary colon cancer, and their isogenic, apoptosis-resistant counterparts SW620 cells, derived from a subsequent metastatic lesion in the same patient. Treatment with proteasome inhibitor increased Fem1b protein levels in SW620 cells, but not in SW480 cells. In SW620 cells we found that endogenous Fem1b co-immunoprecipitates in complexes with RACK1, a protein known to mediate ubiquitination and proteasomal degradation of other pro-apoptotic proteins and to be upregulated in colon cancer. Full-length Fem1b, or the N-terminal region of Fem1b, associated with RACK1 when co-expressed in HEK293T cells, and RACK1 stimulated ubiquitination of Fem1b. RACK1 overexpression in SW620 cells led to downregulation of Fem1b protein levels. Conversely, downregulation of RACK1 led to upregulation of Fem1b protein levels, associated with induction of apoptosis, and this apoptosis was inhibited by blocking Fem1b protein upregulation. In conclusion, RACK1 downregulates levels of the pro-apoptotic protein Fem1b in metastatic, apoptosis-resistant colon cancer cells, which may promote apoptosis-resistance during progression of colon cancer.

Evaluation of metastatic and angiogenic potentials of human colon carcinoma cells in chick embryo model systems.

Increased metastatic and angiogenic potentials of aggressive human colon carcinoma cells were verified in independent chick embryo models by comparing in vivo highly metastatic SW620 colon carcinoma cell line with its isogenic, non-metastatic SW480 cell variant. In the experimental metastasis model, both cell types rapidly arrested in the chorioallantoic membrane (CAM) vasculature as demonstrated by quantitative PCR and immunohistochemistry. Live cell imaging also indicated that both SW620 and SW480 cells efficiently extravasated from the CAM capillary system. However, only few SW480 cells were present in the CAM tissue after 24-48 h. In contrast, the numbers of SW620 cells increased exponentially, indicating proliferative and survival advantages of metastatic colon carcinoma cells in vivo. Multicellular SW620 foci were identified in close proximity to CAM blood vessels. A positive correlation between increased metastatic ability and VEGF-expression of colon carcinoma SW620 cells was demonstrated by the substantial inhibitory effects of anti-VEGF treatment on the levels of metastatic colonization and density of blood vessels adjacent to tumor cell foci. Furthermore, the chick embryo angiogenesis model confirmed high levels of VEGF-dependent angiogenesis induced by SW620 cells, but not SW480 cells. Thus, chick embryo experimental metastasis and CAM angiogenesis models appear to coordinately reflect critical features of advanced colon carcinomas, i.e., the acquisition of enhanced survival and increased angiogenic potentials, both constituting critical determinants of colon cancer progression. The use of rapid and quantitative chick embryo models might provide alternative approaches to conventional mammalian model systems for screening anti-cancer agents.

Vinculin controls PTEN protein level by maintaining the interaction of the adherens junction protein beta-catenin with the scaffolding protein MAGI-2.

PTEN is a frequently mutated tumor suppressor in malignancies. Interestingly, some malignancies exhibit undetectable PTEN protein without mutations or loss of PTEN mRNA. The cause(s) for this reduction in PTEN is unknown. Cancer cells frequently exhibit loss of cadherin, beta-catenin, alpha-catenin and/or vinculin, key elements of adherens junctions. Here we show that F9 vinculin-null (vin(-/-)) cells lack PTEN protein despite normal PTEN mRNA levels. Their PTEN protein expression was restored by transfection with vinculin or by inhibition of PTEN degradation. F9 vin(-/-) cells express PTEN protein upon transfection with a vinculin fragment (amino acids 243-1066) that is capable of interacting with alpha-catenin but unable to target into focal adhesions. On the other hand, disruption of adherens junctions with an E-cadherin blocking antibody reduced PTEN protein to undetectable levels in wild-type F9 cells. PTEN protein levels were restored in F9 vin(-/-) cells upon transfection with an E-cadherin-alpha-catenin fusion protein, which targets into adherens junctions and interacts with beta-catenin in F9 vin(-/-) cells. beta-Catenin is known to interact with MAGI-2. MAGI-2 interaction with PTEN in the cell membrane is known to prevent PTEN protein degradation. Thus, MAGI-2 overexpression in F9 vin(-/-) cells restored PTEN protein levels. Moreover, expression of vinculin mutants that reinstated the disrupted interactions of beta-catenin with MAGI-2 in F9 vin(-/-) cells also restored PTEN protein levels. These studies indicate that PTEN protein levels are dependent on the maintenance of beta-catenin-MAGI-2 interaction, in which vinculin plays a critical role.

Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility.

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin-/-) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal-regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin-FAK interactions. A vinculin fragment (amino acids 811-1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin-/- cells. Both vinY822F and vin-/- cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin-/- and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.