Gene expression profiles of TNF-like cytokine 1A (TL1A) and its receptors death receptor 3 (DR3) and decoy receptor 3 (DcR3) in multiple sclerosis.

TL1A/DR3/DcR3 pathway is an important mediator of inflammatory responses and contributes to the pathogenesis of several chronic inflammatory diseases. Therefore, we analysed PBMC gene expression of these molecules in 30 relapsing-remitting multiple sclerosis (RRMS) patients, 8 secondary progressive MS (SPMS), 9 primary progressive MS (PPMS), 11 clinically isolated syndrome (CIS) patients, and 16 healthy controls (HCs), to evaluate their biomarker potential in MS. The results showed significant decrease in TL1A expression in RRMS compared to other study groups. TL1A as a marker of inflammation, we found its higher expression among treatment näive RRMS patients as compared to HCs and among patients who were treated with DMTs. Moreover, TL1A expression was found to be associated with the clinical and MRI findings of MS patients suggesting its possible involvement in the establishment or preservation of immune system homeostasis or in the regulation of inflammatory activity. Taken together, these findings suggest the TL1A should be evaluated further for its potential as a candidate biomarker of inflammatory activity and the marker of therapeutic response to immunomodulatory treatments in MS.

Epigenetic modulators hydralazine and sodium valproate act synergistically in VEGI-mediated anti-angiogenesis and VEGF interference in human osteosarcoma and vascular endothelial cells.

Vascular endothelial growth inhibitor (VEGI; also referred to as TNFSF15 or TL1A) is involved in the modulation of vascular homeostasis. VEGI is known to operate via two receptors: Death receptor­3 (DR3) and decoy receptor­3 (DcR3). DR3, which is thus far the only known functional receptor for VEGI, contains a death domain and induces cell apoptosis. DcR3 is secreted as a soluble protein and antagonizes VEGI/DR3 interaction. Overexpression of DcR3 and downregulation of VEGI have been detected in a number of cancers. The aim of the present study was to investigate the effects of sodium valproate (VPA), a histone deacetylase inhibitor, in combination with hydralazine hydrochloride (Hy), a DNA methylation inhibitor, on the expression of VEGI and its related receptors in human osteosarcoma (OS) cell lines and human microvascular endothelial (HMVE) cells. Combination treatment with Hy and VPA synergistically induced the expression of VEGI and DR3 in both OS and HMVE cells, without inducing DcR3 secretion. In addition, it was observed that the combination of VPA and Hy significantly enhanced the inhibitory effect on vascular tube formation by VEGI/DR3 autocrine and paracrine pathways. Furthermore, the VEGI/VEGF­A immune complex was pulled down by immunoprecipitation. Taken together, these findings suggest that DNA methyltransferase and histone deacetylase inhibitors not only have the potential to induce the re­expression of tumor suppressor genes in cancer cells, but also exert anti­angiogenic effects, via enhancement of the VEGI/DR3 pathway and VEGI/VEGF­A interference.

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

TL1-A can engage death receptor-3 and activate NF-kappa B in endothelial cells.

BACKGROUND: Death receptors (DRs) play an important role in renal pathology. We have shown that DR3 is inducibly expressed on renal tubular epithelial cells in the setting of inflammatory injuries. In this study we investigate the expression of DR3 in renal endothelial cells and their response to TL1A, the only known ligand of DR3. METHODS: We did RT-PCR, flow cytometry and subcellular immunoblotting to examine the expression and function of DR3 in cells in vitro. We did organ culture of human and mouse tissue to examine expression and signal of DR3 in vivo. RESULTS: DR3 is expressed in some interstitial vascular endothelial cells (EC) in human kidney in situ; these EC also respond to its ligand TL1A by activating NF-κB. Very low levels of DR3 can be detected on the cell surface of cultured human umbilical vein (HUV) EC, which do not respond to TL1A. HUVEC transfected to overexpress DR3 become responsive to TL1A, assessed by IκBα degradation and E-selectin induction, indicating that the signaling components needed for DR3 responsiveness are expressed. TL1A induces NF-κB activation in EC in renal and cardiac tissue from wild type but not DR3 knock-out mice. CONCLUSION: TL1A and DR3 activate NF-κB in vascular endothelial cells, and can be an important regulator of renal interstitial vascular injury.

DR3 signaling protects against cisplatin nephrotoxicity mediated by tumor necrosis factor.

The expression of death receptor 3 (DR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is up-regulated in human tubular epithelial cells (TECs) during renal injury, but its function in this setting remains unknown. We used cisplatin to induce renal injury in wild-type (DR3(+/+)) or congenitally deficient DR3(-/-) mice to examine the in vivo role of DR3. Cisplatin induced the expression of DR3, its ligand, TNF-like ligand 1A (TL1A), and TNF in TECs, as observed in human renal injury. Cisplatin increased apoptotic death of DR3(-/-) TECs by twofold compared with DR3(+/+) TECs, whereas it reduced the number of tubules expressing phospho-NF-κBp65(Ser276) by 50% at 72 hours. Similar degrees of induction of DR3, TL1A, and TNF, and changes in apoptosis and phospho-NF-κBp65(Ser276), were obtained in mouse kidney organ cultures treated with cisplatin for 3 hours, suggesting a direct effect on TECs. TNF was implicated in mediating cisplatin-induced tubular damage given that the in vivo co-administration of GM6001, an inhibitor of TNF maturation and release, significantly reduced TNF production and tubular damage. Moreover, TNF exacerbated, whereas TL1A reduced, cisplatin-induced apoptosis in the DR3(+/+) mouse proximal tubule cell line, TKPTS. Our data demonstrate that cisplatin-induced nephrotoxicity is mitigated by DR3 signaling, suggesting that this occurs by antagonizing pro-apoptotic signals induced by TNF. Therefore, activating DR3 may be beneficial in reducing acute kidney injury.

Interleukin-32 enhances cytotoxic effect of natural killer cells to cancer cells via activation of death receptor 3.

Studies have demonstrated that the anti-tumour effect of natural killer (NK) cells is successful for patients with several cancers. Although interleukin-32 (IL-32) is endogenously expressed in NK cells, cytolytic function of NK cells against cancer cells has not been fully demonstrated. In the present study, we found that the growth of cancer cells was suppressed when colon cancer cells or prostate cancer cells were co-cultured with NK-92 cells, an NK cell line. We also found that the expression of tumour necrosis factor receptor 2 and death receptor 3 (DR3) was increased in PC3 cells, and the expression of FAS and DR3 was increased in SW620 cells by co-culture with NK-92 cells. However, cancer cell growth inhibition and IL-32 expression were abolished when cancer cells were co-cultured with NK cells transfected with small interfering (si) RNA of IL-32. DR3 expression was also diminished by co-culture with IL-32-specific siRNA-transfected NK-92 cells. Expression of APO3L, a ligand of DR3, was elevated in NK cells that were co-cultured with cancer cells. It was also found that expression of apoptosis-related proteins such as cleaved caspase-3 and bax was increased in cancer cells co-cultured with NK-92 cells, but their expression was abolished by co-culture with IL-32 siRNA-transfected NK-92 cells. Moreover, knockdown of DR3 in co-culture of NK-92 cells with cancer cells by siRNA or antibodies of DR3 and APO3L reversed the growth inhibitory effect of NK-92 cells. In conclusion, our study showed that IL-32 enhanced the cytotoxic effect of NK-92 cells on the cancer cells through activation of DR3 and caspase-3.

Interspecies comparison of prostate cancer gene-expression profiles reveals genes associated with aggressive tumors.

Prostate cancer (PC) is a heterogeneous disease whose aggressive phenotype is the second leading cause of cancer-related death in men. The identification of key molecules and pathways that play a pivotal role in PC progression towards an aggressive form is crucial. A major effort towards this end has been taken by global analyses of gene expression profiles. However, the large body of data did not provide a definitive idea about the genes which are associated with the aggressive growth of PC. In order to identify such genes, we performed an interspecies comparison between several human data sets and high quality microarray data that we generated from the transgenic adenocarcinoma of mouse prostate (TRAMP) strain. The TRAMP PC mimics the histological and pathological appearance as well as the aggressive phenotype of human PC (huPC). Analysis of the microarray data, derived from microdissected TRAMP specimens removed at different stages of the disease yielded genetic signatures delineating the TRAMP PC development and progression. Comparison of the TRAMP data with a set of genes representing the core expression signature of huPC yielded a limited set genes. Some of these genes are known predictors of poor prognosis in huPC. Interestingly, the modulation of genes responsible for the invasive phenotype of huPC occurs in TRAMP already during the transition to prostate intraepithelial neoplasia (PIN) and onwards to localized tumors. We therefore suggest that critical oncogenic events leading to an aggressive phenotype of huPC can be studied in the PIN stage of TRAMP.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

BACKGROUND: Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. METHODS: We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. RESULTS: Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. CONCLUSIONS: We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.