ELF3 plays an important role in defining TRAIL sensitivity in breast cancer by modulating the expression of decoy receptor 2 (DCR2).

BACKGROUND: TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. METHODS: Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. RESULTS: Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. CONCLUSION: Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.

Association of RASSF1A, DCR2, and CASP8 Methylation with Survival in Neuroblastoma: A Pooled Analysis Using Reconstructed Individual Patient Data.

Neuroblastoma (NB) is a heterogeneous tumor affecting children. It shows a wide spectrum of clinical outcomes; therefore, development of risk stratification is critical to provide optimum treatment. Since epigenetic alterations such as DNA methylation have emerged as an important feature of both development and progression in NB, in this study, we aimed to quantify the effect of methylation of three distinct genes (RASSF1A, DCR2, and CASP8) on overall survival in NB patients. We performed a systematic review using PubMed, Embase, and Cochrane libraries. Individual patient data was retrieved from extracted Kaplan-Meier curves. Data from studies was then merged, and analysis was done on the full data set. Seven studies met the inclusion criteria. Methylation of the three genes had worse overall survival than the unmethylated arms. Five-year survival for the methylated arm of RASSF1A, DCR2, and CASP8 was 63.19% (95% CI 56.55-70.60), 57.78% (95% CI 47.63-70.08), and 56.39% (95% CI 49.53-64.19), respectively, while for the unmethylated arm, it was 93.10% (95% CI 87.40-99.1), 84.84% (95% CI 80.04-89.92), and 83.68% (95% CI 80.28-87.22), respectively. In conclusion, our results indicate that in NB patients, RASSF1A, DCR2, and CASP8 methylation is associated with poor prognosis. Large prospective studies will be necessary to confirm definitive correlation between methylation of these genes and survival taking into account all other known risk factors. (PROSPERO registration number CRD42017082264).

Upregulated TRAIL and Reduced DcR2 Mediate Apoptosis of Decidual PMN-MDSC in Unexplained Recurrent Pregnancy Loss.

Myeloid-derived suppressor cells (MDSC), especially polymorphonuclear MDSC (PMN-MDSC), accumulate in maternal-fetal interface during pregnancy and are involved in the maintenance of immune tolerance. Decreased PMN-MDSC is associated with pregnancy complications such as unexplained recurrent pregnancy loss (URPL). In the present study we showed decreased PMN-MDSC in the URPL group compared with the normal pregnancy (NP) group, and PMN-MDSC was the major subset of MDSC in human decidua with potent immune suppression activity. We then performed gene expression profile and found that human decidual PMN-MDSC in the NP and URPL groups showed different gene and pathway signature, including apoptosis. Apoptosis of decidual PMN-MDSC was mediated by TNF-related apoptosis-induced ligand (TRAIL) in a Caspase 3 dependent manner. TRAIL was expressed in decidua and upregulated in decidua of the URPL group. Notably, of all the membrane TRAIL receptors, only DcR2 was down-regulated in PMN-MDSC in the URPL group. In vitro experiment demonstrated that DcR2 blockade sensitized PMN-MDSC to TRAIL-mediated apoptosis. Together, these data indicate that increased TRAIL and reduced DcR2 on PMN-MDSC sensitize PMN-MDSC response to TRAIL-induced apoptosis in the URPL group, which is responsible for decreased accumulation of PMN-MDSC in URPL.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and receptors in type 1, type 2 and type 17 inflammation in cross-sectional asthma study.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) reportedly promotes, or conversely, resolves inflammation in asthma. In this study of TRAIL and cell receptors in sputum, bronchoalveolar lavage and biopsy from subjects in the Severe Asthma Research Program at Wake Forest, the high TRAIL group had significant increases in all leucocytes, and was associated with increased type 1, type 2 and type 17 cytokines, but not type 9 interleukin 9. Two variants at loci in the TRAIL gene were associated with higher sputum levels of TRAIL. Increased TRAIL decoy receptor R3/DcR1 was observed on sputum leucocytes compared with death receptor R1/DR4, suggesting reduced apoptosis and prolonged cellular inflammation.

Ischemic Preconditioning Upregulates Decoy Receptors to Protect SH-SY5Y Cells from OGD Induced Cellular Damage by Inhibiting TRAIL Pathway and Agitating PI3K/Akt Pathway.

As ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia, the purpose of the present study is to explore the molecular mechanisms of ischemic preconditioning induced cerebral protective effect. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which induces apoptosis through binding to its death receptors (DR4 and DR5). When TRAIL binds to decoy receptors (DcR1 and DcR2), as DcRs lack intact cytoplasmic death domain, TRAIL fails to induce neuronal apoptosis. In the present study, we demonstrated that ischemic preconditioning upregulated DcR1 and DcR2, which subsequently inhibited oxygen glucose deprivation-induced cellular apoptosis. Then, we investigated the protective molecular mechanism of DcRs after ischemic preconditioning treatment. Results showed that DcR1 could competitively bind to TRAIL and partially inhibit TRAIL-induced cellular apoptosis. On the other hand, DcR2 could disturb DRs-associated death-inducing signaling complex formation (DISC), which further inhibited capase-8 activation. Besides, we also found that ischemic preconditioning activated IPC-induced Akt phosphorylation via regulating DcR2 level. Thus, ischemic preconditioning upregulated decoy receptors, which protected cells from oxygen glucose deprivation-induced cellular damage by inhibiting TRAIL-induced apoptosis and agitating PI3K/Akt pathway. Our data complemented the knowledge of neuroprotective mechanism of ischemic preconditioning and provided new evidence for supporting its clinical application.

Acid/bile exposure triggers TRAIL-mediated apoptosis in esophageal cancer cells by suppressing the decoy receptors and c-FLIPR.

Esophageal adenocarcinoma essentially develops from esophageal inflammation caused by chronic GERD. During GERD episodes, the lower esophageal epithelium is repeatedly exposed to stomach acid, which often contains duodenal bile salts that prompt malignant transformation. TRAIL is one of the cytokines produced in response to such insults and targets the transformed cells exclusively. In this study, we simulated GERD episodes in vitro by exposing the cancer cells to acid or acid/bile combination and found that the cancer cells lived through acid attacks by expression of the decoy receptors and c-FLIPR but died of TRAIL-mediated apoptosis when bile salts were present. Further investigation revealed that acid/bile exposure downregulated the decoy receptors and thereby facilitated TRAIL signaling; meantime, it inhibited protein kinase C activity and thus expedited c-FLIPR degradation, allowing apoptosis to take place.

Dendritic Cells of Mesenteric and Regional Lymph Nodes Contribute to Yersinia enterocolitica O:3-Induced Reactive Arthritis in TNFRp55-/- Mice.

Dendritic cells (DCs) participate in the pathogenesis of several diseases. We investigated DCs and the connection between mucosa and joints in a murine model of Yersinia enterocolitica O:3-induced reactive arthritis (ReA) in TNFRp55-/- mice. DCs of mesenteric lymph nodes (MLN) and joint regional lymph nodes (RLN) were analyzed in TNFRp55-/- and wild-type mice. On day 14 after Y. enterocolitica infection (arthritis onset), we found that under TNFRp55 deficiency, migratory (MHChighCD11c+) DCs increased significantly in RLN. Within these RLN, resident (MHCintCD11c+) DCs increased on days 14 and 21. Similar changes in both migratory and resident DCs were also detected on day 14 in MLN of TNFRp55-/- mice. In vitro, LPS-stimulated migratory TNFRp55-/- DCs of MLN increased IL-12/23p40 compared with wild-type mice. In addition, TNFRp55-/- bone marrow-derived DCs in a TNFRp55-/- MLN microenvironment exhibited higher expression of CCR7 after Y. enterocolitica infection. The major intestinal DC subsets (CD103+CD11b-, CD103-CD11b+, and CD103+CD11b+) were found in the RLN of Y. enterocolitica-infected TNFRp55-/- mice. Fingolimod (FTY720) treatment of Y. enterocolitica-infected mice reduced the CD11b- subset of migratory DCs in RLN of TNFRp55-/- mice and significantly suppressed the severity of ReA in these mice. This result was associated with decreased articular IL-12/23p40 and IFN-γ levels. In vitro FTY720 treatment downregulated CCR7 on Y. enterocolitica-infected bone marrow-derived DCs and purified MLN DCs, which may explain the mechanism underlying the impairment of DCs in RLN induced by FTY720. Taken together, data indicate the migration of intestinal DCs to RLN and the contribution of these cells in the immunopathogenesis of ReA, which may provide evidence for controlling this disease.

DCR2, a Cellular Senescent Molecule, Is a Novel Marker for Assessing Tubulointerstitial Fibrosis in Patients with Immunoglobulin A Nephropathy.

BACKGROUND/AIMS: Stress-induced cell senescence, which contributes to cell cycle arrest and is independent of age, plays an important role in chronic kidney disease (CKD) progression. DcR2, as a senescent marker, exclusively expressed in senescent tubular epithelia. The objective of this study was to examine whether urinary DcR2 (uDcR2) could be a potential biomarker for tubulointerstitial fibrosis (TIF) in patients with immunoglobulin A nephropathy (IgAN). METHODS: This study included 210 IgAN patients and 80 healthy volunteers, with uDcR2 levels measured using enzyme-linked immunosorbent assay. We examined the relationship among uDcR2/Cr levels, renal function, and pathological parameters, using regression analysis to identify risk factors for TIF and the area under the curve (AUC) approach to predict TIF. Renal DcR2 expression was quantified by immunohistochemistry. Co-expression of DcR2 with fibrotic markers (α-smooth muscle actin [α-SMA], collagen III) was analyzed by confocal microscopy. RESULTS: Levels of uDcR2/Cr were significantly higher in IgAN patients and in those with more severe TIF, compared with healthy controls. Serum DcR2 levels were similar across groups. The proportion of IgAN patients with stages 1-2 CKD and T0 was highest among those with uDcR2/Cr <130 ng/g. In contrast, the majority of those with uDcR2/Cr >201 ng/g had stages 4-5 CKD and T2. Levels of uDcR2/Cr were positively associated with urinary albumin to creatinine ratio (ACR), urinary N-acetyl-ß-D-glucosaminidase (uNAG)/Cr, and TIF scores and negatively associated with estimated glomerular filtration rate (eGFR). uDcR2/Cr, uNAG, ACR, and eGFR were independent predictors for TIF, with AUC of 0.907 for uDcR2/Cr. This AUC value was higher than that observed for eGFR, uNAG/Cr, or ACR. The sensitivity and specificity of uDcR2/Cr in predicting TIF were 87.0 and 80.5%, respectively. Moreover, uDcR2/Cr levels were positively associated with the percentage of renal DcR2 expression. Renal DcR2 co-localized with α-SMA and collagen III in the kidneys of IgAN patients. CONCLUSIONS: Levels of uDcR2/Cr were closely associated with the severity of TIF and renal function parameters. uDcR2/Cr represents a potential biomarker for predicting TIF in IgAN patients.

TRAIL-Receptor 4 Modulates γδ T Cell-Cytotoxicity Toward Cancer Cells.

Acquired immune evasion is one of the mechanisms that contributes to the dismal prognosis of cancer. Recently, we observed that different γδ T cell subsets as well as CD8+ αß T cells infiltrate the pancreatic tissue. Interestingly, the abundance of γδ T cells was reported to have a positive prognostic impact on survival of cancer patients. Since γδ T cells utilize TNF-related apoptosis inducing ligand (TRAIL) for killing of tumor cells in addition to granzyme B and perforin, we investigated the role of the TRAIL-/TRAIL-R system in γδ T cell-cytotoxicity toward pancreatic ductal adenocarcinoma (PDAC) and other cancer cells. Coculture of the different cancer cells with γδ T cells resulted in a moderate lysis of tumor cells. The lysis of PDAC Colo357 cells was independent of TRAIL as it was not inhibited by the addition of neutralizing anti-TRAIL antibodies or TRAIL-R2-Fc fusion protein. In accordance, knockdown (KD) of death receptors TRAIL-R1 or TRAIL-R2 in Colo357 cells had no effect on γδ T cell-mediated cytotoxicity. However, KD of decoy receptor TRAIL-R4, which robustly enhanced TRAIL-induced apoptosis, interestingly, almost completely abolished the γδ T cell-mediated lysis of these tumor cells. This effect was associated with a reduced secretion of granzyme B by γδ T cells and enhanced PGE2 production as a result of increased expression level of synthetase cyclooxygenase (COX)-2 by TRAIL-R4-KD cells. In contrast, knockin of TRAIL-R4 decreased COX-2 expression. Importantly, reduced release of granzyme B by γδ T cells cocultured with TRAIL-R4-KD cells was partially reverted by bispecific antibody [HER2xCD3] and led in consequence to enhanced lysis of tumor cells. Likewise, inhibition of COX-1 and/or COX-2 partially enhanced γδ T cell-mediated lysis of TRAIL-R4-KD cells. The combination of bispecific antibody and COX-inhibitor completely restored the lysis of TRAIL-R4-KD cells by γδ T cells. In conclusion, we uncovered an unexpected novel role of TRAIL-R4 in tumor cells. In contrast to its known pro-tumoral, anti-apoptotic function, TRAIL-R4 augments the anti-tumoral cytotoxic activity of γδ T cells.

Hypermethylation of DcR1, DcR2, DR4, DR5 gene promoters and clinical significance in tongue carcinoma.

OBJECTIVE: Tongue squamous cell carcinoma (TSCC) is one of the most common malignancies in the oral cavity, and its incidence and mortality have been constantly increasing these years. A large number of tumor suppressor genes are involved in the development of the TSCC and it has been reported that the aberrant hypermethylation of tumor suppressor genes may play a key role in the process of the TSCC. In this study, we sought to analyze the association of methylation of DcR1, DcR2, DR4 and DR5 gene promoters and clinical significance in the TSCC to evaluate association between methylation of DcR1, DcR2, DR4 and DR5 gene and Clinical Significance in tongue squamous cell carcinoma. METHODS: Methylation-specific PCR(MSP) was used to analyze the methylation of the promoters of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptors in 45 TSCC cases. Real-Time PCR was used to detect the expression of the DcR1, DcR2, DR4 and DR5 gene. RESULTS: All the four genes (DcR1, DcR2, DR4 and DR5) showed different methylation of promoters in TSCC, while methylation of these promoters in paired adjacent normal tissues were almost undetectable. Patients with high methylation index were diagnosed at younger age when compared with the ones with low methylation index. DcR1 and DR4 hypermethylation was correlated significantly with patients' TNM stage. CONCLUSIONS: Methylation of DcR1, DcR2,DR4 and DR5 promoters are found in TSCC and may associate with its occurrence and development. Taking the reversibility of methylation into account，methylation is a potential targeted therapy of TSCC.

Reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55 deficient mice.

Endometriosis is a chronic gynecological disease, characterized by growth of endometrial tissue in ectopic sites due to alteration of peritoneal homeostasis and deregulation of apoptosis. Here we have examined whether TNFRp55 deficiency modulates the pro-inflammatory state and the reinnervation of endometriotic-like lesions in mice. Two-month-old female C57BL/6 mice, eight wild type (WT) and eight TNFRp55-/- (KO) were used in the study. Endometriotic-like lesions were induced experimentally. The right uterine horn was removed from the animal, divided longitudinally, cut in three square pieces and sutured to the intestine mesentery. After 4 weeks, the lesions and the peritoneal fluid were collected. The level of TNFα in the peritoneal fluid was evaluated by enzyme-linked immunosorbent assay (EIA). The expressions of COX2, GRα and GRß were evaluated in the lesions by western blot and immunohistochemistry. ß-III TUBULIN, BDNF and NGF protein concentrations were evaluated in the lesions by western blot. Gene expression of Pgp 9.5, SP and Th was analyzed by RT-PCR, whereas relative concentrations of TRKA, NTRp75, phosphorylated NFκB (pNFκB) and total NFκB in lesions were measured by EIA. Compared with the WT group, the KO mice showed lower TNFα levels in the peritoneal fluid and lower numbers of COX2 immunoreactive cells along with increased expression of GRα, ß-III TUBULIN, Pgp 9.5, SP, Th, BDNF, NGF, NTRp75 and pNFκB in the lesions. Future histological studies will be necessary to confirm the sensory/sympathetic imbalance in the endometriotic-like lesions of the KO mice. Our results suggest that a reduced inflammatory state promotes reinnervation of endometriotic-like lesions in TNFRp55-/- mice. Chronic deregulation of TNF receptors can have serious consequences for women with advanced endometriosis.

Differential expression of senescence tumour markers and its implications on survival outcomes of breast cancer patients.

Breast cancer is a heterogeneous disease displaying different histopathological characteristics, molecular profiling and clinical behavior. This study describes the expression patterns of senescence markers P53, DEC1 and DCR2 and assesses their significance on patient survival as a single or combined marker with P16 or P14 using breast cancer progression series. One thousand and eighty (1080) patients with primary invasive ductal carcinoma, no special type, were recruited through an 11-year retrospective study period. We constructed tissue microarrays of normal, benign hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma from each patient and performed immunohistochemical staining to study the protein expression. Statistical analysis includes Pearson chi-square, Kaplan-Meier log ran test and Cox proportional hazard regression were undertaken to determine the associations and predict the survival outcomes. P53, DEC1 and DCR2 expression correlated significantly with normal, benign, premalignant and malignant tissues with (p<0.05). The expression profile of these genes increases from normal to benign to premalignant and plateaued from premalignant to malignant phenotype. There is a significant association between P53 protein expression and age, grade, staging, lymphovascular invasion, estrogen receptor, progesterone receptor and HER2 whereas DCR2 protein expression significantly correlated with tumour grade, hormone receptors status and HER2 (p<0.05 respectively). P53 overexpression correlated with increased risk of relapse (p = 0.002) specifically in patients who did not receive hormone therapy (p = 0.005) or chemotherapy (p<0.0001). The combination of P53+/P16+ is significantly correlated with poor overall and disease-free survival, whereas a combination of P53+/P14+ is associated with worse outcome in disease-free survival (p<0.05 respectively). P53 overexpression appears to be a univariate predictor of poor disease-free survival. The expression profiles of DEC1 and DCR2 do not appear to correlate with patient survival outcomes. The combination of P53 with P16, rather P53 expression alone, appears to provide more useful clinical information on patient survival outcomes in breast cancer.

Endometriosis progression in tumor necrosis factor receptor p55-deficient mice: Impact on oxidative/nitrosative stress and metallomic profile.

The present study was conducted to investigate whether the deficiency of tumor necrosis factor receptor p55 (TNFRp55) modulates oxidative/nitrosative stress and metallomic profile into the peritoneal cavity during the experimental endometriosis progression in mice. Female C57BL/6 mice, wild-type (WT) and TNFRp55 knockout (KO) of two months were used. Endometriosis was induced experimentally by autotransplanting three pieces of the right uterine horn to the intestinal mesentery. After four weeks, endometriotic-like lesions and peritoneal lavage fluid were collected. The obtained peritoneal fluid was analyzed for nitrite levels using the Griess method and trace elements concentrations by ICP-MS. Both endometriotic-like lesions and cells isolated from peritoneal lavage were analyzed for the following oxidative/nitrosative stress markers: inducible nitric oxide synthase (iNOS) expression by Western Blot; total antioxidant capacity (TAC), the activity of two antioxidant enzymes (CAT and GPX) and thiobarbituric acid-reactive substances (TBARS) concentration, by spectrophotometric method; and protein carbonyl content and nitrotyrosine presence by ELISA. In comparison to WT group, KO mice exhibited larger lesion volume; higher levels of nitrite, copper (Cu) and strontium (Sr) in the peritoneal fluid; increased TAC, CAT, and GPX in peritoneal lavage cells; decreased concentration of TBARS in lesions and protein carbonyl in peritoneal lavage cells. Significant positive correlations between Cu and lesion volume, Sr and lesion volume, and Cu and Sr were obtained. Our results suggest that the TNFRp55 deficiency increases antioxidant protection and promotes high Cu-Sr concentrations in the peritoneal cavity, which favors the progression of experimental endometriosis.

Histone deacetylase inhibitor panobinostat potentiates the anti-cancer effects of mesenchymal stem cell-based sTRAIL gene therapy against malignant glioma.

Human adipose tissue-derived mesenchymal stem cells expressing the secreted form of the tumor necrosis factor-related apoptosis-inducing ligand (hAT-MSC.sTRAIL) have demonstrated therapeutic activity against various tumors in preclinical studies. However, the limited expression of TRAIL death receptors remains a challenge. We evaluated the therapeutic efficacy of panobinostat in enhancing the sensitivity of hAT-MSC.sTRAIL-mediated apoptosis in malignant glioma. Panobinostat effectively inhibited all malignant glioma cells (IC50, 0.03-0.23 µM), enhancing the expression of DRs, but not in hAT-MSCs. Combined treatment with hAT-MSC.sTRAIL and panobinostat significantly suppressed cell viability and enhanced apoptosis. In a diffuse intrinsic pontine glioma (DIPG) mouse model, the combined treatment induced decreases in tumor volume and prolonged survival. Our study demonstrates that panobinostat enhances the expression of TRAIL DRs and potentiates the anti-cancer effects of hAT-MSC.sTRAIL.

Peripheral Neuroimmunoendocrine Interactions: Contribution of TNFRp55 to the Circadian Synchronization of Progesterone and Cytokine Production in Joints of Mice in Late Pregnancy.

OBJECTIVE: Circadian rhythms are generated by the suprachiasmatic nucleus of the hypothalamus and involve rhythmic expression of clock genes and proteins. This rhythmicity is transferred to peripheral tissues by neural and hormonal signals. Late pregnancy is considered a state of inflammation which impacts on peripheral tissues such as joints. Tumor necrosis factor (TNF) mediates inflammatory and circadian responses through its p55 receptor (TNFRp55). Neuroimmunoendocrine interactions in joints have not been studied completely. The purpose of this study was to analyze these interactions, investigating the circadian rhythms of progesterone (Pg) and pro- and anti-inflammatory cytokines in the joints at the end of pregnancy (gestational day 18). Moreover, the impact of TNFRp55 deficiency on these temporal oscillations was explored. METHODS: Wild-type and TNFRp55-deficient (KO) C57BL/6 mice were kept under constant darkness in order to study their endogenous circadian rhythms. The expression of the clock genes Bmal1 and Per1 at circadian time 7 was studied by reverse transcription polymerase chain reaction in the ankle joints of nonpregnant and pregnant (gestational day 18) mice. In late pregnancy, Pg and the cytokines interleukin 17 (IL-17), IL-6, and IL-10 were measured in the joints throughout a 24-h period by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: A significant increase in Bmal1 and Per1 mRNA expression was detected in the joints of pregnant KO mice. Furthermore, KO mice displayed a desynchronization of articular Pg and cytokine production. CONCLUSIONS: Our results show that TNF, via TNFRp55 signaling, modulates articular Pg and cytokine circadian rhythms in late pregnancy. These findings suggest a temporal neuroimmunoendocrine association in peripheral tissues in late pregnancy.

Circadian rhythms of factors involved in luteal regression are modified in p55 tumour necrosis factor receptor (TNFRp55)-deficient mice.

The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.

The p55TNFR-IKK2-Ripk3 axis orchestrates arthritis by regulating death and inflammatory pathways in synovial fibroblasts.

NFκB activation and regulated cell death are important in tissue homeostasis, inflammation and pathogenesis. Here we show the role of the p55TNFR-IKK2l-Ripk3 axis in the regulation of synovial fibroblast homeostasis and pathogenesis in TNF-mediated mouse models of arthritis. Mesenchymal-specific p55TNFR triggering is indispensable for arthritis in acute and chronic TNF-dependent models. IKK2 in joint mesenchymal cells is necessary for the development of cartilage destruction and bone erosion; however, in its absence synovitis still develops. IKK2 deletion affects arthritic and antiapoptotic gene expression leading to hypersensitization of synovial fibroblasts to TNF/Ripk1-mediated death via district mechanisms, depending on acute or chronic TNF signals. Moreover, Ripk3 is dispensable for TNF-mediated arthritis, yet it is required for synovitis in mice with mesenchymal-specific IKK2 deletion. These results demonstrate that p55TNFR-IKK2-Ripk3 signalling orchestrates arthritogenic and death responses in synovial fibroblasts, suggesting that therapeutic manipulation of this pathway in arthritis may require combinatorial blockade of both IKK2 and Ripk3 signals.

Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA.

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells.

Besides its tumor-selective apoptotic activity, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes pro-survival, proliferative or migratory signaling (NF-κB, PI3K/Akt, MAPK and JNK; referred to as 'non-apoptotic' cascades). Indeed, apoptosis and non-apoptotic signaling can be activated in clonal populations of cancer cells in response to treatment and, as a result, only a part of the initial cellular population dies while a fraction survives and develops resistance to TRAIL-induced apoptosis (referred to as 'fractional survival'). Notably, the molecular characterization of the protein platforms streaming into tumoricidal versus tumor-promoting cascades that control fractional survival remained elusive. Here we demonstrate that, in the context of DR4-DR5-DcR2 hetero-oligomeric complexes, a single death receptor (DR5) suffices to assemble composite plasma membrane-proximal pro-apoptotic/pro-survival platforms that propagate TRAIL signaling to both death and survival pathways in clonal populations of cancer cells. Moreover, we show that while all members of TRAIL-induced complexes support survival, none of them acted exclusively pro-apoptotic. Indeed, key apoptotic proteins as FADD and procaspase-8 were also involved in transducing non-apoptotic signaling in response to this cytokine. Collectively, this study reveals the Janus faces of DR5, and the contributions of other death complex components in fractional survival that foster the generation of resistance. Our data highlight a new level of complexity in TRAIL signaling and point to an improved therapeutic rationale in view of hitherto disappointing results.

TRAIL regulatory receptors constrain human hepatic stellate cell apoptosis.

The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.