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Treatment for acute kidney injury (AKI) is suboptimal. A better understanding of the pathogenesis of AKI may lead to new therapeutic approaches. Kidney transcriptomics of folic acid-induced AKI (FA-AKI) in mice identified Runx1 as the most upregulated RUNX family gene. We then examined the expression of RUNX1 in FA-AKI, in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI), and in human AKI. In cultured mouse tubule cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. A chemical inhibitor of RUNX1 (Ro5-3335) was used in animal models of AKI to test its potential as a therapeutic target. RUNX1 overexpression in FA-AKI was validated at the mRNA and protein levels and localized mainly to tubule cell nuclei. CS-AKI also upregulated kidney RUNX1. Increased tubule and interstitial RUNX1 expression were also observed in human AKI. In cultured mouse tubule cells, the pro-inflammatory cytokine TWEAK and LPS increased RUNX1 and IL-6 expression. Mechanistically, RUNX1 bound to the Il6 gene promoter and RUNX1 targeting with the chemical inhibitor Ro5-3335, or a specific small interfering RNA (siRNA), prevented the TWEAK- and LPS-induced upregulation of IL6 through a RUNX1/NFκB1 p50 pathway. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factor mRNAs such as Yap1 and Trp53 as key targets of Ro5-3335 in CS-AKI. In conclusion, RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Allergic conjunctivitis (AC) affects people's daily life and work, especially the health of children. Although there are few relevant studies, Th17/Treg imbalance plays an important role in AC development. The aim of this study was to elucidate the effect of TWEAK/Fn14 on AC and Th17/Treg balance., Methods: Ovalbumin induced AC mouse model was utilized to observe the mechanism of TWEAK/Fn14 in vivo. Conjunctivitis was evaluated by hematoxylin-eosin staining, toluidine blue staining and AC clinical score. Flow cytometry was used to measure Th17 and Treg cell ratios. The level of Th17/Treg balance related factors and Nrf2/HO-1 signal was detected by ELISA, WB, qRT-PCR and immunohistochemistry., Results: In the AC state, disruption of Th17/Treg cell balance, increased TWEAK/Fn14 signaling level and conjunctival inflammation were observed. After TWEAK knockdown, Th17 cell differentiation was inhibited, Treg cell differentiation was promoted, and AC symptoms were alleviated in AC mice. Moreover, TWEAK knockdown caused an enhancement of the Nrf2/HO-1 signaling pathway in the AC models. Treatment with Nrf2 inhibitor reversed these changes induced by TWEAK knockdown. Therefore, TWEAK/Fn14 regulated the Nrf2/HO-1 pathway to affect Th17/Treg cell balance and conjunctivitis in AC mouse models., Conclusion: In summary, TWEAK/Fn14 caused Th17/Treg imbalance by inhibiting Nrf2/HO-1 pathway, which might be one potential mechanism of the exacerbation of AC., Competing Interests: Declarations. Ethics approval: All animal experiments were conducted with the approval of the Experimental Animal Ethics Committee of The Second Affiliated Hospital of Nanchang University. Consent for publication: The data used in this study has never been published before. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Amyotrophic lateral sclerosis (ALS) is a devastating and incurable neurodegenerative disease. Accumulating evidence strongly suggests that intrinsic muscle defects exist and contribute to disease progression, including imbalances in whole-body metabolic homeostasis. We have previously reported that tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor inducible 14 (Fn14) are significantly upregulated in skeletal muscle of the SOD1 G93A ALS mouse model. While antagonising TWEAK did not impact survival, we did observe positive effects in skeletal muscle. Given that Fn14 has been proposed as the main effector of the TWEAK/Fn14 activity and that Fn14 can act independently from TWEAK in muscle, we suggest that manipulating Fn14 instead of TWEAK in the SOD1 G93A ALS mice could lead to differential and potentially improved benefits., Methods: We thus investigated the contribution of Fn14 to disease phenotypes in the SOD1 G93A ALS mice. To do so, Fn14 knockout mice (Fn14 -/- ) were crossed onto the SOD1 G93A background to generate SOD1 G93A ;Fn14 -/- mice. Investigations were performed on both unexercised and exercised (rotarod and/or grid test) animals (wild type (WT), Fn14 -/- , SOD1 G93A and SOD1 G93A ;Fn14 -/- )., Results: Here, we firstly confirm that the TWEAK/Fn14 pathway is dysregulated in skeletal muscle of SOD1 G93A mice. We then show that Fn14-depleted SOD1 G93A mice display increased lifespan, myofiber size, neuromuscular junction endplate area as well as altered expression of known molecular effectors of the TWEAK/Fn14 pathway, without an impact on motor function. Importantly, we also observe a complex interaction between exercise (rotarod and grid test), genotype, disease state and sex that influences the overall effects of Fn14 deletion on survival, expression of known molecular effectors of the TWEAK/Fn14 pathway, expression of myosin heavy chain isoforms and myofiber size., Conclusions: Our study provides further insights on the different roles of the TWEAK/Fn14 pathway in pathological skeletal muscle and how they can be influenced by age, disease, sex and exercise. This is particularly relevant in the ALS field, where combinatorial therapies that include exercise regimens are currently being explored. As such, a better understanding and consideration of the interactions between treatments, muscle metabolism, sex and exercise will be of importance in future studies., (© 2024. Crown.)

Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from limited targeted treatment options. Following recent reports correlating Fibroblast growth factor-inducible 14 (Fn14) receptor overexpression in Estrogen Receptor (ER)-negative breast cancers with metastatic events, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of Nicotinamide phosphoribosyltransferase (NAMPT), which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients., (© 2024. The Author(s).)

Background: Reports suggest that lipid profiles may be linked to the likelihood of developing skin cancer, yet the exact causal relationship is still unknown., Objective: This study aimed to examine the connection between lipidome and skin cancers, as well as investigate any possible mediators., Methods: A two-sample Mendelian randomization (MR) analysis was conducted on 179 lipidomes and each skin cancer based on a genome-wide association study (GWAS), including melanoma, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Then, Bayesian weighted MR was performed to verify the analysis results of two-sample MR. Moreover, a two-step MR was employed to investigate the impact of TNF-like weak inducer of apoptosis (TWEAK)-mediated lipidome on skin cancer rates., Results: MR analysis identified higher genetically predicted phosphatidylcholine (PC) (17:0_18:2) could reduce the risk of skin tumors, including BCC (OR = 0.9149, 95% CI: 0.8667-0.9658), SCC (OR = 0.9343, 95% CI: 0.9087-0.9606) and melanoma (OR = 0.9982, 95% CI: 0.9966-0.9997). The proportion of PC (17:0_18:2) predicted by TWEAK-mediated genetic prediction was 6.6 % in BCC and 7.6% in SCC. The causal relationship between PC (17:0_18:2) and melanoma was not mediated by TWEAK., Conclusion: This study identified a negative causal relationship between PC (17:0_18:2) and keratinocyte carcinomas, a small part of which was mediated by TWEAK, and most of the remaining mediating factors are still unclear. Further research on other risk factors is needed in the future., (© 2024 The Author(s). Skin Research and Technology published by John Wiley & Sons Ltd.)

Current data on the molecular mechanisms of liver fibrosis and cirrhosis fail to fully explain all stages of their development. Interactions between individual genes and signaling pathways are known to play an important role in their functions. However, data on their relationships are insufficient and often contradictory. For the first time, mRNA expression of Notch1, Notch2, Yap1, Tweak (Tnfsf12), Fn14 (Tnfrsf12a), Ang, Vegfa, Cxcl12 (Sdf), Nos2, and Mmp-9 was studied in detail at several stages of thioacetamide-induced liver fibrosis in Wistar rats. A factor analysis isolated three factors, which combined highly correlated target genes. The first factor included four genes: Cxcl12 (r = 0.829, p < 0.05), Tweak (r = 0.841, p < 0.05), Notch1 (r = 0.848, p < 0.05), and Yap1 (r = 0.921, p < 0.05). The second factor described the correlation between Mmp-9 (r = 0.791, p < 0.05) and Notch2 (r = 0.836, p < 0.05). The third factor included Ang (r = 0.748, p < 0.05) and Vegfa (r = 0.679, p < 0.05). The Nos2 and Fn14 genes were not included in any of the factors. The gene grouping by mRNA expression levels made it possible to assume a pathogenetic relationship between their products in the development of fibrotic changes due to liver toxicity.

Background Finding effective and safe therapeutic drugs for atrial fibrillation (AF) is an important concern for clinicians. Proteome-wide Mendelian randomization analysis provides new ideas for finding potential drug targets. Methods and Results Using a proteome-wide Mendelian randomization approach, we assessed the genetic predictive causality between thousands of proteins and AF risk and found that genetically predicted plasma levels of phosphomevalonate kinase, tumor necrosis factor ligand superfamily member 12, sulfhydryl oxidase 2, interleukin-6 receptor subunit alpha, and low-affinity immunoglobulin gamma Fc region receptor II-b might decrease AF risk, while genetically predicted plasma levels of beta-mannosidase, collagen alpha-1(XV) chain, ANXA4 (annexin A4), COF2 (cofilin-2), and RAB1A (Ras-related protein Rab-1A) might increase AF risk ( P <3.4×10 -5 ). By using different Mendelian randomization methods and instrumental variable selection thresholds, we performed sensitivity analyses in 30 scenarios to test the robustness of positive findings. Replication analyses were also performed in independent samples to further avoid false-positive findings. Drugs targeting tumor necrosis factor ligand superfamily member 12, interleukin-6 receptor subunit alpha, low-affinity immunoglobulin gamma Fc region receptor II-b, and annexin A4 are approved or in development. The results of the phenome-wide Mendelian randomization analysis showed that changing the plasma levels of phosphomevalonate kinase, cofilin-2, annexin A4, Ras-related protein Rab-1A, sulfhydryl oxidase 2, and collagen alpha-1(XV) chain did not increase the risk of other diseases while decreasing the risk of AF. Conclusions We found a significant causal association between genetically predicted levels of 10 plasma proteins and AF risk. Four of these proteins have drugs targeting them that are approved or in development, and our results suggest the potential for these drugs to treat AF or cause AF. Sulfhydryl oxidase 2, low-affinity immunoglobulin gamma Fc region receptor II-b, and beta-mannosidase have not been suggested by previous laboratory or epidemiological studies to be associated with AF and may reveal new pathophysiological pathways as well as therapeutic targets for AF.

Keloids represent a fibrotic disorder characterized by the excessive deposition of extracellular matrix (ECM). However, the mechanisms through which ECM deposition in keloids is regulated remain elusive. In this study, we found that the expression of both TWEAK and its cognate receptor Fn14 was significantly downregulated in keloids and that TWEAK/Fn14 signaling repressed the expression of ECM-related genes in keloid fibroblasts. The IRF1 gene was essential for this repression, and the TWEAK/Fn14 downstream transcription factor p65 directly bound to the promoter of the IRF1 gene and induced its expression. Furthermore, in patients with keloid, the expression of TWEAK and Fn14 was negatively correlated with that of ECM genes and positively correlated with that of IRF1. These observations indicate that relief of TWEAK/Fn14/IRF1-mediated ECM deposition repression contributes to keloid pathogenesis, and the identified mechanism and related molecules provide potential targets for keloid treatment in the future., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Draw upon research into the serum concentration, mRNA expression, and DNA methylation of TNF-like weak inducer of apoptosis (TWEAK) in the peripheral blood of systemic lupus erythematosus patients and healthy controls in an attempt to investigate the epigenetics associated with TWEAK in the pathogenesis of systemic lupus erythematosus (SLE)., Methods: A total of 178 SLE patients (SLE group) and 131 sex-age matched healthy controls (HC group) were recruited. Enzyme-linked immunosorbent assays (ELISA) was used to detect serum protein concentration of TWEAK. TWEAK mRNA expression was analyzed by Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Methylation levels of the promotor of TWEAK were measured using quantitative DNA methylation analysis on the MassARRAY spectrometry., Results: Serum TWEAK concentrations were not statistically significant in SLE patients and HCs. Nevertheless, serum TWEAK concentrations were significantly lower in patients with renal involvement when compared to those without it. Serum TWEAK concentrations were reduced in clinically active patients (SLEDAI ≥ 10) compared with clinically stable patients (SLEDAI < 10). It was also significantly associated with SLEDAI. Compared with the HC group, the TWEAK mRNA expression in the SLE group was significantly lower. The global DNA methylation levels of TWEAK in the SLE group were observed to be significantly higher than the HC group. SLE patients with renal involvement, and the clinically active patients had higher TWEAK global methylation as well as exhibited variation in certain CpG island methylation. Furthermore, TWEAK methylation negatively correlated with TWEAK mRNA expression., Conclusion: This study suggests that TWEAK DNA methylation is a valuable as a focus for epigenetic studies because of it potentially influencing TWEAK gene expression in SLE patients. Aberrant DNA methylation of TWEAK may be involved in the initiation and development of SLE.

Background: Lung cancer remains the leading cause of cancer death worldwide, and the most subtype is lung adenocarcinoma (LUAD). Tumor-infiltrating immune cells (TIICs) greatly impact the prognosis of LUAD. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), signal via its receptor fibroblast growth factor-inducible 14 (Fn14), dysregulates immune cell recruitment within tumor environment, thus promoting the progression of autoimmune diseases and cancer. We aimed to explore its role in LUAD., Methods: The expression level of TWEAK was explored in Tumor Immune Estimation Resource 2.0 (TIMER2.0) and Oncomine databases. The Tumor Immune Dysfunction and Exclusion (TIDE) and Lung Cancer Explorer (LCE) databases were applied to evaluate the survival in correlation to TWEAK expression. TIICs were assessed with TIMER2.0 and TIDE datasets. The expression of TWEAK protein was detected in LUAD cell lines and also in tissue samples from LUAD patients via western blotting or combination with immunochemistry., Results: Our results showed that TWEAK was downregulated in LUAD tumors compared to normal tissues in TIMER2.0, Oncomine, cell lines, and clinical specimens. Poor survival was uncovered in lower TWEAK expression of LUAD patients in LCE (meta - HR = 0.84 [95% CI, 0.76-0.92]) and TCGA (Continuous Z = -1.97, p = 0.0486) and GSE13213@PRECOG (Continuous Z = -4.25, p = 2.12e - 5) in TIDE. Multiple tumor-infiltrating immune cells (TIICs) were found closely correlated with TWEAK expression in LUAD, especially hematopoietic stem cell (Rho = 0.505, p = 2.78e - 33), common lymphoid progenitor (Rho = -0.504, p = 3.79e - 33), and myeloid-derived suppressor cells (MDSCs) (Rho = -0.615, p = 1.36e - 52)., Conclusion: Lower level of TWEAK was linked with poor survival and aberrant recruitment and phenotype of TIICs in LUAD, which might motivate immune escape and weaken the effects of immunotherapy., Competing Interests: The authors report no conflicts of interest in this work., (Copyright © 2022 Zhengxi He et al.)

Objective: To explore the relationship between tumor necrosis factor like weak inducer of apoptosis ( TWEAK ) gene and the pathogenesis of rheumatoid arthritis (RA) by detecting the DNA methylation level, mRNA expression level and serum protein concentration of TWEAK gene in peripheral blood., Methods: The MassARRAY method was used to detect the DNA methylation level of the TWEAK gene in the peripheral blood of 112 RA patients and 86 matched healthy volunteers. The real-time quantitative polymerase chain reaction method was used to detect the mRNA expression level of the TWEAK gene in the peripheral blood of the subjects. The enzyme-linked immunosorbent assay method was used to detect the serum TWEAK protein concentration of the subjects. The TWEAK gene DNA methylation level, mRNA expression level and serum protein concentration between the RA group and the healthy control group were compared, and the relationship between it and the degree of disease activity analyzed., Results: The overall DNA methylation level of TWEAK gene and the DNA methylation levels of CpG&#95;11, CpG&#95;17.18.19.20, CpG&#95;40.41.42 site in the RA group were higher than those in the healthy control group ( P =0.002, P =0.01, P =0.006, P =0.002, respectively). The DNA methylation level of CpG&#95;55.56 site in the high disease activity group was higher than that in the medium and low disease activity group ( P =0.041). The expression level of TWEAK gene mRNA in the peripheral blood of the RA group was lower than that of the healthy control group ( P =0.023). The expression level of TWEAK gene mRNA in the high disease activity group was lower than that in the medium and low disease activity group ( P =0.035). The serum TWEAK protein concentration of the RA group was not significantly different from that of the healthy control group ( P =0.508), but it was positively correlated with the mRNA expression level ( r =0.482, P < 0.001)., Conclusion: The TWEAK gene is closely related to the onset and progression of RA, and its hypermethylation state may be one of the epigenetic mechanisms regulating its low mRNA expression, and it can be used as one of the important indicators for clinical monitoring and evaluation of RA.

Cutaneous melanoma (CM) is the most aggressive form of skin cancer, and its worldwide incidence is rapidly increasing. Early stages can be successfully treated by surgery, but once metastasis has occurred, the prognosis is poor. However, some 5-10% of thick (≥2 mm) melanomas do not follow this scenario and run an unpredictable course. Little is known about the factors that contribute to metastasis in some patient with thick melanomas and the lack thereof in thick melanoma patients who never develop metastatic disease. We were therefore interested to study differential gene expression and pathway analysis and compare non-metastatic and metastatic thick melanomas. We found that the TNF-like weak inducer of apoptosis (TWEAK) pathway was upregulated in thick non-metastasizing melanomas. MAP3K14 (NIK1), BIRC2 (cIAP1), RIPK1, CASP7, CASP8, and TNF play an important role in inhibiting proliferation and invasion of tumor cells via the activation of the non-canonical NF-κB signaling pathway. In particular, this pathway sensitizes melanoma cells to TNF-alpha and activates the apoptosis module of the TWEAK pathway in thick non-metastasizing melanomas. Hence, our study suggests a potential role of the TWEAK pathway in inhibiting thick melanoma from metastasis. Exploitation of these genes and the pathway they control may open future therapeutic avenues.

Background: In autosomal dominant polycystic kidney disease (ADPKD), cyst development and enlargement lead to ESKD. Macrophage recruitment and interstitial inflammation promote cyst growth. TWEAK is a TNF superfamily (TNFSF) cytokine that regulates inflammatory responses, cell proliferation, and cell death, and its receptor Fn14 (TNFRSF12a) is expressed in macrophage and nephron epithelia., Methods: To evaluate the role of the TWEAK signaling pathway in cystic disease, we evaluated Fn14 expression in human and in an orthologous murine model of ADPKD. We also explored the cystic response to TWEAK signaling pathway activation and inhibition by peritoneal injection., Results: Meta-analysis of published animal-model data of cystic disease reveals mRNA upregulation of several components of the TWEAK signaling pathway. We also observed that TWEAK and Fn14 were overexpressed in mouse ADPKD kidney cysts, and TWEAK was significantly high in urine and cystic fluid from patients with ADPKD. TWEAK administration induced cystogenesis and increased cystic growth, worsening the phenotype in a murine ADPKD model. Anti-TWEAK antibodies significantly slowed the progression of ADPKD, preserved renal function, and improved survival. Furthermore, the anti-TWEAK cystogenesis reduction is related to decreased cell proliferation-related MAPK signaling, decreased NF- κ B pathway activation, a slight reduction of fibrosis and apoptosis, and an indirect decrease in macrophage recruitment., Conclusions: This study identifies the TWEAK signaling pathway as a new disease mechanism involved in cystogenesis and cystic growth and may lead to a new therapeutic approach in ADPKD., (Copyright © 2021 by the American Society of Nephrology.)

Neuropsychiatric manifestations of systemic lupus erythematosus (SLE), specifically cognitive dysfunction and mood disorders, are widely prevalent in SLE patients, and yet poorly understood. TNF-like weak inducer of apoptosis (TWEAK) has previously been implicated in the pathogenesis of neuropsychiatric lupus (NPSLE), and we have recently shown its effects on the transcriptome of the cortex of the lupus-prone mice model MRL/lpr. As the hippocampus is thought to be an important focus of NPSLE processes, we explored the TWEAK-induced transcriptional changes that occur in the hippocampus, and isolated several genes ( Dnajc28 , Syne2 , transthyretin ) and pathways (PI3K-AKT, as well as chemokine-signaling and neurotransmission pathways) that are most differentially affected by TWEAK activation. While the functional roles of these genes and pathways within NPSLE need to be further investigated, an interesting link between neuroinflammation and neurodegeneration appears to emerge, which may prove to be a promising novel direction in NPSLE research.

An imbalance of TNF signalling in the inflammatory milieu generated by meningeal immune cell infiltrates in the subarachnoid space in multiple sclerosis (MS), and its animal model may lead to increased cortical pathology. In order to explore whether this feature may be present from the early stages of MS and may be associated with the clinical outcome, the protein levels of TNF, sTNF-R1 and sTNF-R2 were assayed in CSF collected from 122 treatment-naïve MS patients and 36 subjects with other neurological conditions at diagnosis. Potential correlations with other CSF cytokines/chemokines and with clinical and imaging parameters at diagnosis (T0) and after 2 years of follow-up (T24) were evaluated. Significantly increased levels of TNF (fold change: 7.739; p < 0.001), sTNF-R1 (fold change: 1.693; p < 0.001) and sTNF-R2 (fold change: 2.189; p < 0.001) were detected in CSF of MS patients compared to the control group at T0. Increased TNF levels in CSF were significantly ( p < 0.01) associated with increased EDSS change (r = 0.43), relapses (r = 0.48) and the appearance of white matter lesions (r = 0.49). CSF levels of TNFR1 were associated with cortical lesion volume (r = 0.41) at T0, as well as with new cortical lesions (r = 0.56), whilst no correlation could be found between TNFR2 levels in CSF and clinical or MRI features. Combined correlation and pathway analysis (ingenuity) of the CSF protein pattern associated with TNF expression (encompassing elevated levels of BAFF, IFN-γ, IL-1β, IL-10, IL-8, IL-16, CCL21, haptoglobin and fibrinogen) showed a particular relationship to the interaction between innate and adaptive immune response. The CSF sTNF-R1-associated pattern (encompassing high levels of CXCL13, TWEAK, LIGHT, IL-35, osteopontin, pentraxin-3, sCD163 and chitinase-3-L1) was mainly related to altered T cell and B cell signalling. Finally, the CSF TNFR2-associated pattern (encompassing high CSF levels of IFN-β, IFN-λ2, sIL-6Rα) was linked to Th cell differentiation and regulatory cytokine signalling. In conclusion, dysregulation of TNF and TNF-R1/2 pathways associates with specific clinical/MRI profiles and can be identified at a very early stage in MS patients, at the time of diagnosis, contributing to the prediction of the disease outcome.

Endothelial-mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts (CAFs), which facilitates tumour progression. PDAC is characterised by abundant CAFs and tumour necrosis factor-α (TNF-α). Here, we show that TNF-α strongly induces human endothelial cells to undergo EndMT. Interestingly, TNF-α strongly downregulates the expression of the endothelial receptor TIE1, and reciprocally TIE1 overexpression partially prevents TNF-α-induced EndMT, suggesting that TNF-α acts, at least partially, through TIE1 regulation in this process. We also show that TNF-α-induced EndMT is reversible. Furthermore, TNF-α treatment of orthotopic mice resulted in an important increase in the stroma, including CAFs. Finally, secretome analysis identified TNFSF12, as a regulator that is also present in PDAC patients. With the aim of restoring normal angiogenesis and better access to drugs, our results support the development of therapies targeting CAFs or inducing the EndMT reversion process in PDAC.

Background and Aim: As a proinflammatory cytokine, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) participates in the progression of renal fibrosis by binding to its receptor, fibroblast growth factor-inducible 14 (Fn14). However, the effect of Fn14 inhibition on tubular epithelial cell-mediated tubulointerstitial fibrosis remains unclear. This study aimed to elucidate the role of TWEAK/Fn14 interaction in the development of experimental tubulointerstitial fibrosis as well as the protective effect of Fn14 knockdown on proximal tubular epithelial cells., Methods: A murine model of unilateral ureteral obstruction was constructed in both wild-type and Fn14-deficient BALB/c mice, followed by observation of the tubulointerstitial pathologies., Results: Fn14 deficiency ameliorated the pathological changes, including inflammatory cell infiltration and cell proliferation, accompanied by reduced production of profibrotic factors and extracellular matrix deposition. In vitro experiments showed that TWEAK dose-dependently enhanced the expression of collagen I, fibronectin, and α-smooth muscle actin in proximal tubular epithelial cells. Interestingly, TWEAK also upregulated the expression levels of Notch1/Jagged1. Fn14 knockdown and Notch1/Jagged1 inhibition also mitigated the effect of TWEAK on these cells., Conclusions: In conclusion, TWEAK/Fn14 signals contributed to tubulointerstitial fibrosis by acting on proximal tubular epithelial cells. Fn14 inhibition might be a therapeutic strategy for protecting against renal interstitial fibrosis.

Cognitive dysfunction and mood changes are prevalent and especially taxing issues for patients with systemic lupus erythematosus (SLE). Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its cognate receptor Fn14 have been shown to play an important role in neurocognitive dysfunction in murine lupus. We profiled and compared gene expression in the cortices of MRL/+, MRL/ lpr (that manifest lupus-like phenotype) and MRL/ lpr -Fn14 knockout (Fn14ko) adult female mice to determine the transcriptomic impact of TWEAK/Fn14 on cortical gene expression in lupus. We found that the TWEAK/Fn14 pathway strongly affects the expression level, variability and coordination of the genomic fabrics responsible for neurotransmission and chemokine signaling. Dysregulation of the Phosphoinositide 3-kinase (PI3K)-AKT pathway in the MRL/ lpr lupus strain compared with the MRL/+ control and Fn14ko mice was particularly prominent and, therefore, promising as a potential therapeutic target, although the complexity of the transcriptomic fabric highlights important considerations in in vivo experimental models.

The tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor ligand family and has been shown to be overexpressed in tumoral cells together with the fibroblast growth factor-inducible 14 (Fn14) receptor. TWEAK-Fn14 interaction triggers a set of intracellular pathways responsible for tumour cell invasion and migration, as well as proliferation and angiogenesis. Hence, modulation of the TWEAK-Fn14 interaction is an important therapeutic goal. The targeting of protein-protein interactions by external agents, e.g., drugs, remains a substantial challenge. Given their intrinsic features, as well as recent advances that improve their pharmacological profiles, peptides have arisen as promising agents in this regard. Here, we report, by in silico structural design validated by cell-based and in vitro assays, the discovery of four peptides able to target TWEAK. Our results show that, when added to TWEAK-dependent cellular cultures, peptides cause a down-regulation of genes that are part of TWEAK-Fn14 signalling pathway. The direct, physical interaction between the peptides and TWEAK was further elucidated in an in vitro assay which confirmed that the bioactivity shown in cell-based assays was due to the targeting of TWEAK. The results presented here are framed within early pre-clinical drug development and therefore these peptide hits represent a starting point for the development of novel therapeutic agents. Our approach exemplifies the powerful combination of in silico and experimental efforts to quickly identify peptides with desirable traits.

Astrocytic dysfunction has been implicated in Parkinson's disease (PD) pathogenesis. While the Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 signaling axis is known to play a role in PD-like neuropathology, the molecular mechanisms that govern this process remain poorly understood. Herein, we show that TWEAK levels are elevated in PD serum compared to controls. Moreover, using both U373 human astrocyte cells and primary mouse astrocytes, we demonstrate that TWEAK induces mitochondrial oxidative stress as well as protein kinase C delta (PKCδ) and signal transducer and activator of transcription 3 (STAT3) activation, accompanied by NLRC4 inflammasome activation and upregulation and release of proinflammatory cytokines, including IL-1β, TNF-α, and IL-18. Mechanistically, TWEAK-induced PKCδ activation enhances the STAT3/NLRC4 signaling pathway and other proinflammatory mediators through a mitochondrial oxidative stress-dependent mechanism. We further show that PKCδ knockdown and mito-apocynin, a mitochondrial antioxidant, suppress TWEAK-induced proinflammatory NLRC4/STAT3 signaling and cellular oxidative stress response. Notably, we validated our in vitro findings in an MPTP mouse model of PD and in mice receiving intrastriatal administration of TWEAK. These results indicate that TWEAK is a key regulator of astroglial reactivity and illustrate a novel mechanism by which mitochondrial oxidative stress may influence dopaminergic neuronal survival in PD.

Cardiovascular diseases (CVD) are the leading cause of mortality in Western countries. CVD include several pathologies, such as coronary artery disease, stroke, peripheral artery disease, and aortic aneurysm, among others. All of them are characterized by a pathological vascular remodeling in which inflammation plays a key role. Interaction between different members of the tumor necrosis factor superfamily and their cognate receptors induce several biological actions that may participate in CVD. The cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its functional receptor, fibroblast growth factor-inducible 14 (Fn14), are abundantly expressed during pathological cardiovascular remodeling. The TWEAK/Fn14 axis controls a variety of cellular functions, such as proliferation, differentiation, and apoptosis, and has several biological functions, such as inflammation and fibrosis that are linked to CVD. It has been demonstrated that persistent TWEAK/Fn14 activation is involved in both vessel and heart remodeling associated with acute and chronic CVD. In this review, we summarized the role of the TWEAK/Fn14 axis during pathological cardiovascular remodeling, highlighting the cellular components and the signaling pathways that are involved in these processes., Competing Interests: The authors declare no conflict of interest.

Autophagy plays an important role in protecting the host against pathogens. Mycobacterium tuberculosis can suppress autophagy and then remain dormant and survive within the host for an extended period, which is responsible for latent tuberculosis infection (LTBI). Here, we explored the role of microRNAs (miRNAs) in LTBI. The miRNA profiles were explored using the next-generation sequencing approach, followed by quantitative reverse transcription-PCR validation. The biological function of candidate miRNA was evaluated using immunoblotting, immunofluorescence techniques, and enzyme-linked immunosorbent assay in an in vitro human TB granuloma model. An increased miR-889 expression was observed in patients with LTBI compared with that in patients without infection. The reporter assay identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as the target of miR-889. Mycobacterial infection induced TWEAK upregulation in the early phase. TWEAK induced autophagy and promoted mycobacterial autophagosome maturation through activation of AMP-activated protein kinase (AMPK). Upon entry to LTBI status, elevated miR-889 levels were associated with TNF alpha (TNF-α) and granuloma formation/maintenance. MiR-889 inhibited autophagy via posttranscriptional suppression of TWEAK expression to maintain mycobacterial survival in granulomas. Adalimumab (anti-TNF-α monoclonal antibody) treatment reduced levels of both TNF-α and miR-889 and caused granuloma destruction and LTBI reactivation. The circulating miR-889 and TWEAK levels were correlated with LTBI and subsequently associated with anti-TNF-α-related LTBI reactivation in patients. We propose that miR-889 and TWEAK can act as targets that can be manipulated for antimycobacterial therapeutic purposes and act as candidate biomarkers for LTBI and LTBI reactivation, respectively. IMPORTANCE TB remains a leading cause of morbidity and mortality worldwide. Approximately one-quarter of the world's population has latent TB infection. TWEAK is a multiple-function cytokine and may be used as a target for the treatment of rheumatic diseases, cardiovascular diseases, and renal diseases. Here, we demonstrated a novel relationship between TWEAK and activation of the autophagic machinery which promotes antimycobacterial immunity. Additionally, TB infection is highly dynamic and determined by the interaction between the host and mycobacterium. We demonstrated a mechanism of fine-tuned balance between the mycobacterium and host for granuloma formation and/or maintenance in LTBI status. Once patients entered LTBI status, the upregulation of miR-889 was associated with TNF-α levels and granuloma formation to maintain mycobacterial survival. Adalimumab (a TNF-α inhibitor) reduced both TNF-α and miR-889 levels and caused LTBI reactivation and, thus, TWEAK enhancement. MiR-889 and TWEAK may become potential diagnostic biomarkers or therapeutic targets for LTBI and LTBI reactivation, respectively., (Copyright © 2020 Chen et al.)

Peste des petits ruminants virus (PPRV) has emerged as a significant threat to the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and induces in the hosts a transient but severe immunosuppression, especially innate immunity. However, it remains largely unknown how NK cells respond and are regulated at the earliest time points after an acute viral PPRV infection in goats. In this study, we revealed that multiple immune responses of goat peripheral NK cells were compromised during PPRV infection, including the cytolytic effector molecule expression and cytokine production. Importantly, we demonstrated that PPRV infection stimulated the expression of TWEAK, a negative regulator of cytotoxic function of NK cells, which may be involved in the suppression of cytotoxicity as well as cytokine production in infected goat NK cells. Furthermore, we found that PPRV infection induced TWEAK expression in goat NK cells involving post-transcription by suppressing miR-1, a novel negative miRNA directly targeting the TWEAK gene. Moreover, replication of virus is required for inhibition of miR-1 expression during PPRV infection, and the non-structural V protein of PPRV plays an important role in miR-1 mediated TWEAK upregulation. Additionally, we revealed that the regulation of NK cell immune responses by TWEAK is mediated by MyD88, SOCS1, and STAT3. Taken together, our results demonstrated that TWEAK may play a key role in regulating goat peripheral NK cell cytotoxicity and cytokine expression levels during PPRV infection., (Copyright © 2020 Qi, Li, Li, Wang, Zhang and Wang.)

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) acts through its receptor fibroblast growth factor inducible 14 (Fn14), and participates in skin inflammation. Both TWEAK and Fn14 are highly expressed in skin lesions of patients with atopic dermatitis. The purpose of this study was to further explore the effect of Fn14 inhibition on experimental atopic dermatitis. Experimental atopic dermatitis was induced in the wild-type and Fn14 knock-out BALB/c mice. The effect of TWEAK/Fn14 interaction on keratinocytes was studied in an in-vitro model of atopic dermatitis. Fn14 deficiency ameliorates skin lesions in the mice model, accompanied by less infiltration of inflammatory cells and lower local levels of proinflammatory cytokines, including TWEAK, TNF-α and interleukin (IL)-17. Fn14 deficiency also attenuates the up-regulation of TNFR1 in skin lesions of atopic dermatitis. Moreover, topical TWEAK exacerbates skin lesion in the wild-type but not in the Fn14 knock-out mice. In vitro, TWEAK enhances the expressions of IL-17, IL-18 and IFN-γ in keratinocytes under atopic dermatitis-like inflammation. These results suggest that Fn14 deficiency protects mice from experimental atopic dermatitis, involving the attenuation of inflammatory responses and keratinocyte apoptosis. In the context of atopic dermatitis-like inflammation, TWEAK modulates keratinocytes via a TNFR1-mediated pathway., (© 2019 British Society for Immunology.)

The interaction between tumor necrosis factor- (TNF-) like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible 14 (Fn14) regulates the fate of keratinocytes, depending on the relative expression of TNF receptor (TNFR) 1 or TNFR2. However, the precise mechanism underlying this TWEAK-mediated regulation remains unclear. The aim of this study was to provide comprehensive insight into the roles of Fn14, TNFR1/2, and other relevant molecules in the fate of keratinocytes. Further, we sought to elucidate the structural basis for the interaction of TWEAK and Fn14 in regulating cellular outcomes. Normal keratinocytes (mainly expressing TNFR1) and TNFR2-overexpressing keratinocytes were stimulated with TWEAK. Through immunoprecipitation and Western blotting of keratinocyte lysates, we elucidated the associations between Fn14, TNFR-associated factor 2 (TRAF2), cellular inhibitor of apoptosis protein 1 (cIAP1), and TNFR1/2 molecules. Additionally, we found that TRAF2 exhibited binding to Fn14, cIAP1, and TNFR1/2. Our data suggest that TWEAK induces apoptosis in normal keratinocytes and proliferation in TNFR2-overexpressing keratinocytes in a TNF- α -independent manner; however, inhibition of TRAF2 appears to reverse this effect. Interestingly, the interaction between TWEAK and Fn14 increased TNFR1-associated death domain protein and caspase-8 expression in normal keratinocytes and promoted cytoplasmic import of cIAP1 in TNFR2-overexpressing keratinocytes. In conclusion, we found that the Fn14-TRAF2-TNFR signaling axis mediates TWEAK's regulation of the fate of keratinocytes, possibly in a manner involving the TNF- α -independent TNFR signal transduction., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2019 Xuening Wang et al.)

LncRNA small nucleolar RNA host gene 3 (Snhg3) has been involved in cell proliferation and migration in malignant cells. However, its role in regulating functions of non-malignant cells has been hardly reported. Here, we found Snhg3 expression was sharply induced in primary brain microvascular endothelial cells (BMVECs) treated with oxygen-and-glucose-deprivation (OGD) plus hemin, an in vitro model of intracerebral hemorrhage (ICH). Downregulation of Snhg3 by siRNA transfection improved cell proliferation and migration abilities and reduced cell apoptosis and monolayer permeability in BMVECs under treatment with OGD plus hemin. Snhg3 overexpression suppressed cell proliferation and migration and increased cell apoptosis and monolayer permeability under normal condition. In ICH rats, downregulation of Snhg3 by siRNA injection improved behavioral and histological manifestations, including number of right turns, limb placement score, integrity of blood-brain barrier (BBB), brain water content and cell apoptosis in vivo. In the mechanism exploration, we found that, TWEAK and Snhg3 displayed a positive correlation with each other. Snhg3 overexpression increased expression of TWEAK protein and its receptor Fn14, that were also induced by OGD plus hemin, activating the downstream neuroinflammatory pathway STAT3 and enhancing the secretion of MMP-2/9. Finally, the TWEAK-siRNA, the Fn14 inhibitor ATA and the STAT3 blocker AG490 were respectively used to treat BMVECs under treatment with OGD plus hemin. Our results showed either TWEAK downregulation, Fn14 inhibition, or STAT3 blockade, could rescue Snhg3-induced impairment of BMVEC functions. In conclusion, the lncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in ICH rats by activating the TWEAK/Fn14/STAT3 pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Novel, effective, and safe drugs are warranted for treatment of ischemic stroke. Circulating protein biomarkers with causal genetic evidence represent promising drug targets, but no systematic screen of the proteome has been performed., Methods: First, using Mendelian randomization (MR) analyses, we assessed 653 circulating proteins as possible causal mediators for 3 different subtypes of ischemic stroke: large artery atherosclerosis, cardioembolic stroke, and small artery occlusion. Second, we used MR to assess whether identified biomarkers also affect risk for intracranial bleeding, specifically intracerebral and subarachnoid hemorrhages. Third, we expanded this analysis to 679 diseases to test a broad spectrum of side effects associated with hypothetical therapeutic agents for ischemic stroke that target the identified biomarkers. For all MR analyses, summary-level data from genome-wide association studies (GWAS) were used to ascertain genetic effects on circulating biomarker levels versus disease risk. Biomarker effects were derived by meta-analysis of 5 GWAS (N≤20 509). Disease effects were derived from large GWAS analyses, including MEGASTROKE (N≤322 150) and UK Biobank (N≤408 961) studies., Results: Several biomarkers emerged as causal mediators for ischemic stroke. Causal mediators for cardioembolic stroke included histo-blood group ABO system transferase, coagulation factor XI, scavenger receptor class A5 (SCARA5), and tumor necrosis factor-like weak inducer of apoptosis (TNFSF12). Causal mediators for large artery atherosclerosis included ABO, cluster of differentiation 40, apolipoprotein(a), and matrix metalloproteinase-12. SCARA5 (odds ratio [OR]=0.78; 95% CI, 0.70-0.88; P=1.46×10 -5 ) and TNFSF12 (OR=0.86; 95% CI, 0.81-0.91; P=7.69×10 -7 ) represent novel protective mediators of cardioembolic stroke. TNFSF12 also increased the risk of subarachnoid (OR=1.53; 95% CI, 1.31-1.78; P=3.32×10 -8 ) and intracerebral (OR=1.34; 95% CI, 1.14-1.58; P=4.05×10 -4 ) hemorrhages, whereas SCARA5 decreased the risk of subarachnoid hemorrhage (OR=0.61; 95% CI, 0.47-0.81; P=5.20×10 -4 ). Multiple side effects beyond stroke were identified for 6 of 7 biomarkers, most (75%) of which were beneficial. No adverse side effects were found for coagulation factor XI, apolipoprotein(a), and SCARA5., Conclusions: Through a systematic MR screen of the circulating proteome, causal roles for 5 established and 2 novel biomarkers for ischemic stroke were identified. Side-effect profiles were characterized to help inform drug target prioritization. In particular, SCARA5 represents a promising target for treatment of cardioembolic stroke, with no predicted adverse side effects.

Background: Mutations in Melanoma Antigen-encoding Gene D2 (MAGED2) promote tubular dysfunction, suggesting that MAGE proteins may play a role in kidney pathophysiology. We have characterized the expression and regulation of MAGE genes in normal kidneys and during kidney disease., Methods: The expression of MAGE genes and their encoded proteins was explored by systems biology multi-omics (kidney transcriptomics and proteomics) in healthy adult murine kidneys and following induction of experimental acute kidney injury (AKI) by a folic acid overdose. Changes in kidney expression during nephrotoxic AKI were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry. Factors regulating gene expression were studied in cultured tubular cells., Results: Five MAGE genes (MAGED1, MAGED2, MAGED3, MAGEH1, MAGEE1) were expressed at the mRNA level in healthy adult mouse kidneys, as assessed by RNA-Seq. Additionally, MAGED2 was significantly upregulated during experimental AKI as assessed by array transcriptomics. Kidney proteomics also identified MAGED2 as upregulated during AKI. The increased kidney expression of MAGED2 mRNA and protein was confirmed by qRT-PCR and western blot, respectively, in murine folic acid- and cisplatin-induced AKI. Immunohistochemistry located MAGED2 to tubular cells in experimental and human kidney injury. Tubular cell stressors [serum deprivation and the inflammatory cytokine tumour necrosis factor-like weak inducer of apoptosis (TWEAK)] upregulated MAGED2 in cultured tubular cells., Conclusions: MAGED2 is upregulated in tubular cells in experimental and human kidney injury and is increased by stressors in cultured tubular cells. This points to a role of MAGED2 in tubular cell injury during kidney disease that should be dissected by carefully designed functional approaches., (© The Author(s) 2018. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Previously, we found that endogenously produced pro-inflammatory molecules, advanced glycation end products (AGEs), interact with tumor necrosis factor-like weak inducer of apoptosis (TWEAK), and attenuate its immunomodulatory function. In the present study, to elucidate the mechanism by which AGEs attenuate TWEAK function, we searched for regions responsible for TWEAK-AGE interaction using TWEAK deletion mutants. Pull-down assays with the TWEAK mutants and AGEs revealed that the C-terminal half of TWEAK, which is the region essential for receptor stimulation, was required for this interaction. On the other hand, the N-terminal deletion mutants did not exhibit a significant decrease in AGE binding. Moreover, a moderate decrease in the AGE binding by double-deletion in quartered C-terminal half regions and a substantial decrease by triple-deletion in this region were observed. In addition, full-length TWEAK stimulated IL-8 gene expression in endothelial EA.hy.926 cells, whereas the triple-deletion mutant lost much of this activity, suggesting that the TWEAK-AGE interaction sites overlap with the region needed to exert normal function of TWEAK. Our present findings may help to elucidate the pathophysiological roles of the TWEAK-AGE interaction for prevention and treatment of AGE-related inflammatory diseases., (© 2018 International Union of Biochemistry and Molecular Biology, Inc.)

Background: Interferon gamma (IFNγ) and tumor necrosis factor-related weak inducer of apoptosis (TWEAK) molecules seem to have a potential effect on angiogenic factors such as vascular endothelial growth factor (VEGF). The aim of this study was to assess a possible interplay between IFNγ and TWEAK cytokines and VEGF machinery in the different steps of colorectal carcinogenesis., Methods: A total of 92 subjects with colonic adenoma or cancer who underwent screening colonoscopy or surgery were prospectively enrolled. Polypoid lesion tissue samples were collected and frozen. Real-time reverse transcription polymerase chain reaction for IFNγ, TWEAK, and VEGF-A mRNA expression was performed. Immunoassays for VEGF-A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 were also performed. Nonparametric statistics, receiver operating characteristic curve analysis, and logistic multiple regression analysis were used., Results: IFNγ and TWEAK mRNA expression was higher in patients with T2 or more advanced colorectal cancer than in those with adenomas or T1 cancer (p < 0.001 and p = 0.01, respectively). IFNγ and TWEAK mRNA expression levels directly correlated with VEGF-A mRNA expression levels (rho = 0.44, p < 0.001 and rho = 0.29, p = 0.004, respectively). On the contrary, IFNγ and TWEAK mRNA expression levels inversely correlated with VEGF-C protein levels (rho = -0.29, p = 0.04 and rho = -0.31, p = 0.03, respectively). Similarly, IFNγ and TWEAK mRNA expression levels inversely correlated with VEGFR2 protein levels (rho = -0.38, p = 0.033 and rho = -0.40, p = 0.025, respectively)., Conclusion: This study showed that in colorectal polypoid lesions, IFNγ and TWEAK expressions are directly correlated to VEGF-A expression but inversely correlated with VEGFR2 levels, suggesting a possible feedback mechanism in the regulation of VEGF-A expression., (© 2019 S. Karger AG, Basel.)

Background & Aims: Crohn's disease (CD) is a debilitating inflammatory disorder that affects more than 1.6 million people in North America alone. Members of the tumor necrosis factor superfamily are key regulators of intestinal inflammation; specifically, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor, fibroblast growth factor-inducible 14 (Fn14), are involved in normal and pathologic tissue remodeling. Our aim was to determine the role of TWEAK/Fn14 in CD and a murine model of CD-like ileitis (ie, SAMP1/YitFc [SAMP] strain)., Methods: SAMP mice deficient in Fn14 (SAMP × Fn14 -/- ) were developed and a detailed time-course study was performed evaluating ileal tissues by histology and stereomicroscopy, as well as quantitative polymerase chain reaction and NanoString technology (Seattle, WA). Reciprocal bone marrow chimeras were generated to assess the relevance of Fn14 in hematopoietic vs nonhematopoietic compartments. Surgically resected intestinal tissues and mucosal biopsy specimens from patients with CD, ulcerative colitis, and healthy controls were analyzed for the expression of TWEAK/Fn14 by quantitative polymerase chain reaction, Western blot, immunohistochemistry, and immunofluorescence., Results: SAMP × Fn14 -/- showed a marked decrease in ileitis severity at 20 weeks of age compared with SAMP WT controls. Bone marrow chimeras showed that Fn14 was required in both hematopoietic and nonhematopoietic compartments for ileitis to develop. Transcriptome data showed multiple cellular pathways regulated by Fn14 signaling. Finally, increased expression of TWEAK and Fn14 was observed in tissue lesions from CD patients compared with ulcerative colitis and healthy controls., Conclusions: TWEAK/Fn14 are up-regulated in CD, and also mediate experimental CD-like ileitis, by regulation of multiple innate and adaptive cellular pathways. Therefore, TWEAK/Fn14 may represent a novel therapeutic target for the treatment of small intestinal inflammation in CD., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), along with its receptor fibroblast growth factor-inducible (Fn)14, is associated with various biological activities including inflammation. However, its role in the pathogenesis of Graves' orbitopathy (GO) is unknown. In this study, we investigated the mechanism by which TWEAK regulates inflammatory signaling in orbital fibroblasts from GO patients. We found that TWEAK and tumor necrosis factor-α (TNFA) mRNA levels were upregulated in GO as compared to non-GO tissue samples. TWEAK, TNF receptor (TNFR)1, TNFR2, and TNFR superfamily member 12A mRNA, and TWEAK and Fn14 protein levels were increased by interleukin (IL)-1β and TNF-α treatment. Treatment with exogenous recombinant TWEAK increased the transcript and protein expression of the pro-inflammatory cytokines IL-6, IL-8, and monocyte chemoattractant protein-1 to a greater extent in GO than in non-GO cells, while treatment with the anti-Fn14 antibody ITEM4 suppressed TWEAK-induced pro-inflammatory cytokine release and hyaluronan production. Additionally, the serum level of TWEAK was higher in Graves' disease patients with (341.86 ± 86.3 pg/ml) as compared to those without (294.09 ± 41.44 pg/ml) GO and healthy subjects (255.33 ± 39.38 pg/ml), and was positively correlated with clinical activity score (r = 0.629, P < 0.001) and thyroid binding immunoglobulin level (r = 0.659, P < 0.001). These results demonstrate that TWEAK/Fn14 signaling contributes to GO pathogenesis. Moreover, serum TWEAK level is a potential diagnostic biomarker for inflammatory GO, and modulating TWEAK activity may be an effective therapeutic strategy for suppressing inflammation and tissue remodeling in GO., Competing Interests: The authors have declared that no competing interests exist.

Exposure to prenatal hypoxia in rats leads to intrauterine growth restriction (IUGR), decreases fetal cardiomyocyte proliferation and increases the risk to develop cardiovascular diseases (CVD) later in life. The tumor necrosis factor-related weak inducer of apoptosis (TWEAK) induces cardiomyocyte proliferation through activation of the fibroblast growth factor-inducible molecule 14 (Fn-14) receptor. The TWEAK/Fn-14 pathway becomes quiescent shortly after birth, however, it becomes upregulated with CVD; suggesting that it could be a link between the increased susceptibility to CVD in pregnancies complicated by hypoxia/IUGR. We hypothesized that offspring exposed to prenatal hypoxia will exhibit reduced cardiomyocyte proliferation due to reduced Fn-14 expression and that the TWEAK/Fn-14 pathway will be expressed in those adult offspring. We exposed pregnant Sprague Dawley rats to control (21% oxygen) or hypoxic (11% oxygen) conditions from gestational days 15 to 21. Ventricular cardiomyocytes were isolated from male and female, control and hypoxic offspring at postnatal day 1. Proliferation was assessed in the presence or absence of r-TWEAK (72 h, 100 ng/ml). Prenatal hypoxia was not associated with differences in Fn-14 protein expression in either male or female offspring. Cardiomyocytes from prenatal hypoxic male, but not female, offspring had decreased proliferation compared with controls. Addition of r-TWEAK increased cardiomyocyte proliferation in all offspring. In adult offspring of all groups, the TWEAK/Fn-14 pathway was not detectable. Cardiomyocyte proliferation was reduced in only male offspring exposed to prenatal hypoxia but this was not due to changes in the Fn-14 pathway. Studies addressing other pathways associated with CVD and prenatal hypoxia are needed.

TWE-PRIL is a naturally occurring fusion protein of components of two TNF superfamily members: the extracellular domain of APRIL; and the intracellular and transmembrane domains of TWEAK with no known function. Here, we show that April -/- mice (which lack APRIL and TWE-PRIL) exhibited overgrowth of sympathetic fibres in vivo , and sympathetic neurons cultured from these mice had significantly longer axons than neurons cultured from wild-type littermates. Enhanced axon growth from sympathetic neurons cultured from April -/- mice was prevented by expressing full-length TWE-PRIL in these neurons but not by treating them with soluble APRIL. Soluble APRIL, however, enhanced axon growth from the sympathetic neurons of wild-type mice. siRNA knockdown of TWE-PRIL but not siRNA knockdown of APRIL alone also enhanced axon growth from wild-type sympathetic neurons. Our work reveals the first and physiologically relevant role for TWE-PRIL and suggests that it mediates reverse signalling., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Atrial myocyte hypertrophy is one of the most important substrates in the development of atrial fibrillation (AF). The TWEAK/Fn14 axis is a positive regulator of cardiac hypertrophy in cardiomyopathy. This study therefore investigated the effects of Fn14 on atrial hypertrophy and underlying cellular mechanisms using HL-1 atrial myocytes. In patients with AF, Fn14 protein levels were higher in atrial myocytes from atrial appendages, and expression of TWEAK was increased in peripheral blood mononuclear cells, while TWEAK serum levels were decreased. In vitro, Fn14 expression was up-regulated in response to TWEAK treatment in HL-1 atrial myocytes. TWEAK increased the expression of ANP and Troponin T, and Fn14 knockdown counteracted the effect. Inhibition of JAK2, STAT3 by specific siRNA attenuated TWEAK-induced HL-1 atrial myocytes hypertrophy. In conclusion, TWEAK/Fn14 axis mediates HL-1 atrial myocytes hypertrophy partly through activation of the JAK2/STAT3 pathway., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Persistent inflammation promotes development and progression of heart failure (HF). TWEAK (TNF-Related WEAK Inducer Of Apoptosis), a NF-κB- and/or AP-1-responsive proinflammatory cytokine that signals via TWEAK receptor (TWEAKR), is expressed at high levels in human and preclinical models of HF. Since the adapter molecule TRAF3IP2 (TRAF3 Interacting Protein 2) is an upstream regulator of various proinflammatory pathways, including those activated by NF-κB and AP-1, we hypothesized that targeting TRAF3IP2 inhibits TWEAK-induced proinflammatory and pro-fibrotic responses in vitro and in vivo. Consistent with the hypothesis, forced expression of TRAF3IP2 upregulated TWEAK and its receptor expression in cultured adult mouse cardiac fibroblasts (CF). Further, exogenous TWEAK upregulated TRAF3IP2 expression in a time- and dose-dependent manner, suggesting a positive-feedback regulation of TRAF3IP2 and TWEAK. TWEAK also promoted TRAF3IP2 nuclear translocation. Confirming its critical role in TWEAK signaling, silencing TRAF3IP2 inhibited TWEAK autoregulation, TWEAKR upregulation, p38 MAPK, NF-κB and AP-1 activation, inflammatory cytokine expression, MMP and TIMP1 activation, collagen expression and secretion, and importantly, proliferation and migration. Recapitulating these in vitro results, continuous infusion of TWEAK for 7 days increased systolic blood pressure (SBP), upregulated TRAF3IP2 expression, activated p38 MAPK, NF-κB and AP-1, induced the expression of multiple proinflammatory and pro-fibrotic mediators, and interstitial fibrosis in hearts of wild type mice. These proinflammatory and pro-fibrotic changes occurred in conjunction with myocardial hypertrophy and contractile dysfunction. Importantly, genetic ablation of TRAF3IP2 inhibited these TWEAK-induced adverse cardiac changes independent of increases in SBP, indicating that TRAF3IP2 plays a causal role, and thus a therapeutic target, in chronic inflammatory and fibro-proliferative diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Introduction: Corticotherapy is the leading medication worldwide for patients with history of repeated implantation failures (RIF) after IVF/ICSI. Nevertheless, we still do not know its local mechanism of action, hence its precise indication. Our objective is to document the impact of prednisone on the endometrial expression of immune biomarkers (CD56 cells count, IL-18/TWEAK, IL-15/Fn-14 mRNA ratio) at the time of uterine receptivity in a RIF population., Materials and Method: An endometrial biopsy was realized in the mid luteal phase for immune profiling: IL-15/Fn-14 and IL-18/TWEAK mRNA ratios were determined by quantitative RT-PCR and CD56 mobilization per IHC. Fifty-five patients with a RIF history were diagnosed to have local over-immune activation [high IL-18/TWEAK mRNA ratio, and/or high IL-15/Fn-14 mRNA ratio] likely to impair the implantation process. They underwent a second immune profiling with supplementation of prednisone. A paired comparison of the immune profile before and under prednisone was performed in the subset of patients subsequently pregnant under prednisone., Finding: In 54.5% of the cases, both immune biomarkers were normalized and in 16.5%, only one was normalized under prednisone. In 29% we observed a paradoxical increase of both immune biomarkers. The IL-18/TWEAK mRNA ratio reflecting the Th-1/Th-2 local equilibrium was significantly reduced (0.29 versus 0.10, p = .004), through very significant increase of TWEAK expression, in patients who were subsequently pregnant under prednisone., Conclusion: Testing the response to prednisone in a RIF context may be very useful. Less than half of RIF patients with immune deregulation may be prednisone responders and would benefit from its administration., (Copyright © 2018. Published by Elsevier B.V.)

Asthma, an increasingly common chronic disease among children, are characterized by airway remodeling, which is partly attributed to the proliferation and migration of airway smooth muscle cell (ASMC). The purpose of the present study was to investigate potential roles and mechanisms of the tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible molecule 14 (Fn14) axis on cell proliferation and migration in HASMCs. Compared to HASMCs from non-asthmatic patients, those from asthmatic patients showed elevated expression levels of both Fn14 and TWEAK. Additionally, similar to the response triggered by platelet-derived growth factor-BB, stimulation with recombinant TWEAK strongly induced cell proliferation and migration in HASMCs. However, depletion of Fn14 remarkably abrogated the enhancement of TWEAK on the cell proliferation and migration of HASMCs. Furthermore, treatment with TWEAK led to the activation of NF-κB. This effect was eliminated by silencing Fn14, indicating that TWEAK-induced NF-κB signaling was mediated via Fn14. Moreover, the TWEAK/Fn14 interaction promoted cell proliferation and migration. These effects were blocked by NF-κB inhibitor SN50, which suggest that the TWEAK/Fn14 signaling system partially depends on NF-κB activity. Collectively, we demonstrated that the TWEAK/Fn14 axis accelerated HASMC cell proliferation and migration by activating the NF-κB pathway, thereby exacerbating airway remodeling in asthma. Altogether, these findings indicate a novel role for the TWEAK/Fn14/NF-κB pathway as a potent option for limiting airway remodeling in asthma., (© 2017 Wiley Periodicals, Inc.)

Several of the soluble inflammatory molecules such as cytokines and chemokines are involved in the regulation of cancer behaviors. Tumor necrosis factor (TNF)‑like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily and is a ligand of fibroblast growth factor inducible 14 (Fn14). TWEAK/Fn14 signaling pathways promote tumor progression in several types of human cancer. In the present study, we investigated the role of TWEAK through bioinformatic assay, in vitro experiments, and serum levels in patients with non‑small cell lung cancer (NSCLC). Our results indicated that TWEAK expression in normal tissues was higher than that in lung cancer tissues. In contrast, relatively higher Fn14 expression was detected in lung cancer tissues compared to normal tissues. Recombinant TWEAK treatment did not enhance and inhibit the proliferation and migration of human NSCLC cell lines including A549, H1299, CL1‑0 and CL1‑5. In addition, the serum concentration of TWEAK in normal controls was significantly higher than that in NSCLC patients. However, the TWEAK levels did not show significant difference in regards to TNM stage, cell type and metastasis status in the sera of NSCLC patients. In summary, the present study suggests that a low serum level of TWEAK may be a feature of NSCLC, and the role of TWEAK‑mediated pathways warrant further investigation.

TWEAK regulates multiple physio-pathological processes in fibroblasts such as fibrosis. It also induces migration and invasion in tumors and it can activate p38 MAPK in various cell types. Moreover, p38α MAPK promotes migration and invasion in several cancer cells types and in mouse embryonic fibroblasts (MEFs). However, it remains unknown if TWEAK could promote migration in fibroblasts and whether p38α MAPK might play a role. Our results reveal that TWEAK activates ERKs, Akt, and p38α/β MAPKs and reduces secreted Fibulin 3 in MEFs. TWEAK also increases migration and invasion in wt and p38α deficient MEFs, which indicates that p38α MAPK is not required to mediate these effects. In contrast, ERKs inhibition significantly decreases TWEAK-induced migration and Fibulin 3 knock-down mimics TWEAK effect. These results indicate that both ERKs activation and Fibulin 3 down-regulation would contribute to mediate TWEAK pro-migratory effect. In fact, the additional regulation of ERKs and/or p38β as a consequence of Fibulin 3 decrease might be also involved in the pro-migratory effect of TWEAK in MEFs. In conclusion, our studies uncover novel mechanisms by which TWEAK would favor tissue repair by promoting fibroblasts migration., (© 2017 Wiley Periodicals, Inc.)

Immunoglobulin A nephropathy (IgAN) is one of the most common glomerular diseases worldwide. Various studies have identified a host of microRNAs (miRNAs) abnormally expressed in IgAN and might affect the pathogenesis and progression of IgAN. However, miR-200bc/429 cluster in the pathopoiesis of IgAN remains poorly understood. For this study, we found that miR-200bc/429 cluster is downregulated in IgAN tissues and IgAN podocytes and HK2 cells compared with their matched controls respectively. In addition, overexpression of miR-200bc/429 cluster in IgAN podocytes and HK2 cells could attenuate the release of inflammatory cytokines MCP-1, IL-6 and RANTES. Moreover, the 3' untranslated region (UTR) of TNF-like weak inducer of apoptosis (TWEAK) was identified to be a direct target of miR-200bc/429 cluster. Furthermore, our results showed that miR-200bc/429 cluster can inhibit TWEAK mediated NF-κB pathway activation in IgAN. Overall, our findings revealed that miR-200bc/429 cluster alleviates inflammation in IgAN through TWEAK/Fn14 system and might serve as a biomarker as well as a promising therapeutic target for IgAN., (Copyright © 2017. Published by Elsevier B.V.)

Adipose tissue releases bioactive mediators called adipokines. This review focuses on the effects of omentin, vaspin, cardiotrophin-1, Tumor necrosis factor-like Weak Inducer of Apoptosis (TWEAK) and nephroblastoma overexpressed (NOV/CCN3) on obesity and diabetes. Omentin is produced by the stromal-vascular fraction of visceral adipose tissue. Obesity reduces omentin serum concentrations and adipose tissue secretion in adults and adolescents. This adipokine regulates insulin sensitivity, but its clinical relevance has to be confirmed. Vaspin is produced by visceral and subcutaneous adipose tissues. Vaspin levels are higher in obese subjects, as well as in subjects showing insulin resistance or type 2 diabetes. Cardiotrophin-1 is an adipokine with a similar structure as cytokines from interleukin-6 family. There is some controversy regarding the regulation of cardiotrophin-1 levels in obese -subjects, but gene expression levels of cardiotrophin-1 are down-regulated in white adipose tissue from diet-induced obese mice. It also shows anti-obesity and hypoglycemic properties. TWEAK is a potential regulator of the low-grade chronic inflammation characteristic of obesity. TWEAK levels seem not to be directly related to adiposity, and metabolic factors play a critical role in its regulation. Finally, a strong correlation has been found between plasma NOV/CCN3 concentration and fat mass. This adipokine improves insulin actions., Competing Interests: The authors declare no conflict of interest.

Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU, ADAM8, TNSF12, GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.

Atopic dermatitis (AD) and psoriasis are driven by alternate type 2 and type 17 immune responses, but some proteins might be critical to both diseases. Here we show that a deficiency of the TNF superfamily molecule TWEAK (TNFSF12) in mice results in defective maintenance of AD-specific T helper type 2 (Th2) and psoriasis-specific Th17 cells in the skin, and impaired expression of disease-characteristic chemokines and cytokines, such as CCL17 and TSLP in AD, and CCL20 and IL-19 in psoriasis. The TWEAK receptor, Fn14, is upregulated in keratinocytes and dermal fibroblasts, and TWEAK induces these cytokines and chemokines alone and in synergy with the signature T helper cytokines of either disease, IL-13 and IL-17. Furthermore, subcutaneous injection of recombinant TWEAK into naive mice induces cutaneous inflammation with histological and molecular signs of both diseases. TWEAK is therefore a critical contributor to skin inflammation and a possible therapeutic target in AD and psoriasis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK/Tnfsf12) is a cytokine implicated in different steps associated with vascular remodeling. However, the role of TWEAK under hyperglycemic conditions is currently unknown. Using two different approaches, genetic deletion of Tnfsf12 and treatment with a TWEAK blocking mAb, we have analyzed the effect of TWEAK inhibition on atherosclerotic plaque progression and stability in streptozotocin-induced diabetic ApoE deficient mice. Genetic inactivation of Tnfsf12 reduced atherosclerosis extension and severity in diabetic ApoE deficient mice. Tnfsf12 deficient mice display a more stable plaque phenotype characterized by lower lipid and macrophage content within atherosclerotic plaques. A similar phenotype was observed in diabetic mice treated with anti-TWEAK mAb. The proatherosclerotic effects of TWEAK were mediated, at least in part, by STAT1 activation and expression of proinflammatory target genes (CCL5, CXCL10 and ICAM-1), both in plaques of ApoE mice and in cultured vascular smooth muscle cells (VSMCs) under hyperglycemic conditions. Loss-of-function experiments demonstrated that TWEAK induces proinflammatory genes mRNA expression through its receptor Fn14 and STAT1 activation in cultured VSMCs. Overall, TWEAK blockade delay plaque progression and alter plaque composition in diabetic atherosclerotic mice. Therapies aimed to inhibit TWEAK expression and/or function could protect from diabetic vascular complications.

Background: Embryo implantation remains the main limiting factor in IVF/ICSI program. Endometrial immune remodeling events begin before implantation and are a vital process for pregnancy, preparing future maternal immune tolerance and regulating the placentation process., Methods: Between 2012 and 2014, 193 patients (analyzed group) enrolled in our IVF program benefitted of an endometrial immune profiling to determine if their uterus was immunologically ready to accept an embryo and, if not, the specific immune mechanisms involved. Subsequently, they had an effective embryo transfer (ET) with personalization of their treatments if an immune deregulation has been diagnosed. Each analyzed patient was paired to the closest patient included in the IVF program according to biological criteria (age, number of mature oocytes, stage and number of transferred embryo), which had no endometrial immune profiling (193 patients, non-analyzed group)., Finding: 78% of analyzed patients had a uterine immune dysregulation and therefore care personalization. Their corresponding live birth rate (LBR) was twice higher than observed in the matched control group with conventional cares (30.5% versus 16.6%, OR: 2.2 [1.27-3.83] p=0.004) with a simultaneous drastic reduction of miscarriages per initiated pregnancy (17.9% versus 43.2%, OR: 0.29 [0.12-0.71], p=0.005). 22% of analyzed patients had no dysregulation. They did not differ from their matched controls for LBR and miscarriages., Conclusion: Uterine immune profiling enables an integrated approach of infertility that includes endometrial immunity as a key factor in planning personalized IVF/ICSI treatments. Personalization of treatment according to the woman's uterine immune balance produced a very significantly higher LBR., (Copyright © 2016. Published by Elsevier B.V.)

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that binds with high affinity to a plasma membrane-anchored receptor named Fn14. Both TWEAK and Fn14 expression has been detected in human cancer tissue, and studies have shown that TWEAK/Fn14 signaling can promote either "pro-cancer" or "anti-cancer" cellular effects in vitro, depending on the cancer cell line under investigation. In this study, we engineered murine B16 melanoma cells to secrete high levels of soluble TWEAK and examined their properties. TWEAK production by B16 cells preferentially activated the non-canonical NF-κB signaling pathway and increased the expression of several previously described TWEAK-inducible genes, including Fn14. TWEAK overexpression in B16 cells inhibited both cell growth and invasion in vitro. The TWEAK-mediated reduction in B16 cell invasive capacity was dependent on activation of the non-canonical NF-κB signaling pathway. Finally, we found that this same signaling pathway was also important for TWEAK-stimulated human DU145 prostate cancer cell invasion. Therefore, even though TWEAK:Fn14 binding activates non-canonical NF-κB signaling in both melanoma and prostate cancer cells, this shared cellular response can trigger a very different downstream outcome (inhibition or stimulation of cell invasiveness, respectively).