A shedding soluble form of interleukin-17 receptor D exacerbates collagen-induced arthritis through facilitating TNF-α-dependent receptor clustering.
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ABSTRACT:
SUBMITTER: Liu S
PROVIDER: S-EPMC8322419 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Tumor necrosis factor is a major pro-inflammatory cytokine which triggers various physiological consequences by binding to and trimerizing its receptors, and has been the single most sought-after drug target for intervening autoimmune diseases such as rheumatoid arthritis and psoriasis. However, current TNF-? blockers, including soluble receptor-Fc fusion and therapeutic antibodies, are all dimeric in structure, whereas their target TNF-? itself is homotrimeric in nature. Here we describe the development of a trivalent soluble TNF receptor and show that it is a more potent than the dimeric TNF receptor decoys in inhibiting TNF-? signaling both in vitro and in vivo. The process involves gene fusion between a soluble receptor TNFRII with a ligand binding domain and a trimerization tag from the C-propeptide of human collagen (Trimer-Tag), which is capable of self-assembly into a covalently linked trimer. We show that the homotrimeric soluble TNF receptor (TNFRII-Trimer) produced with such method is more potent in ligand binding kinetics and cell based bioassays, as well as more efficacious in attenuating collagen-induced arthritis (CIA) in a mouse model than its dimeric TNFRII-Fc counterpart. Thus, this work demonstrates the proof of concept of Trimer-Tag and provides a new platform for rational designs of next generation biologic drugs.
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Project description:The purpose of this study was to evaluate the effects of aerobic exercise in the heat on circulating concentrations of tumor necrosis factor (TNF)-α, soluble TNF receptors (STNFR1&2), and surface expression of TNFR1&2 on monocyte subpopulations. Twelve recreationally active Caucasian men (24.4 ± 3.4 yrs.; 180.0 ± 6.8 cm; 81.5 ± 8.0 kg; 47.2 ± 4.8 mL·kg-1·min-1) completed an exercise protocol in three environmental conditions: high temperature/low humidity [HTLH; 35 °C, 20% relative humidity (RH)]; high temperature/moderate humidity (HTMH; 35 °C, 45%RH); and moderate temperature/moderate humidity (MTMH; 22 °C, 45%RH). Each protocol consisted of a 60-minute cycling trial at 60% VO2max, a 15-minute rest, and a time-to-exhaustion trial at 90% VO2max (TTE). Blood was sampled before (PRE), immediately after (POST) the 60-minute trial, immediately post-TTE (PTTE), and one-hour post-TTE (REC). Circulating TNF-α and STNFR1&2 were assayed. TNFR1&2 expression on monocyte subsets was measured by flow cytometry on a subset of participants (n = 8). TNF-α area under the curve with respect to increase (AUCi) was greater during HTMH compared to MTMH and HTLH. STNFR1 concentration was greater during HTMH compared to MTMH. With all trials combined, STNFR1 concentration increased from PRE to POST, PTTE, and REC. TNFR1 expression on non-classical monocytes was greater during HTMH compared to HTLH while TNFR2 expression was lower during HTLH compared to both MTMH and HTMH. Data suggest that exercise in the heat increases circulating TNF-α and STNFR1 concentration concomitantly. Furthermore, non-classical monocyte expression of TNFRs are impacted by temperature and humidity during exercise.
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Project description:ADAM17, a prominent member of the "Disintegrin and Metalloproteinase" (ADAM) family, is an important regulator of endothelial cell proliferation and cell survival. The protease controls vital cellular functions through cleavage of growth factors, cytokines and their receptors including transforming growth factor-alpha (TGF-α), tumor necrosis factor-alpha (TNF-α) and TNF-α receptor 1 (TNFR1). TNF-α is the major inducer of endothelial cell death in cardiovascular diseases. The latter are also characterized by elevated plasma and tissue levels of extracellular sphingomyelinase (SMase). Whether the SMase affects ADAM activity and thus endothelial cell function has not been addressed to date. Here, we analyzed the effect of SMase on ADAM17-mediated shedding in COS7 cells and in human umbilical vein endothelial cells (HUVECs). Exposure to SMase significantly increased ADAM17-mediated release of alkaline-phosphatase (AP)-tagged TGF-α in COS7 cells and shedding of endogenously expressed TNFR1 in HUVECs. We previously presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase function. We found that SMase treatment led to PS externalization in both cell types. Transient non-apoptotic PS exposure is often mediated by Ca2+-dependent phospholipid scramblases. Accordingly, the Ca2+-chelator EGTA markedly reduced the breakdown of phospholipid asymmetry and shedding of TGF-α and TNFR1. Moreover, sheddase activity was significantly diminished in the presence of the competing PS-headgroup OPLS. SMase-stimulated TNFR1 shedding strikingly diminished TNF-α-induced signalling cascades and endothelial cell death. Taken together, our data suggest that SMase activity might act as protective factor for endothelial cells in cardiovascular diseases.
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Project description:TNF is a highly pro-inflammatory cytokine that contributes not only to the regulation of immune responses but also to the development of severe inflammatory diseases. TNF is synthesized as a transmembrane protein, which is further matured via proteolytic cleavage by metalloproteases such as ADAM17, a process known as shedding. At present, TNF is mainly detected by measuring the precursor or the mature cytokine of bulk cell populations by techniques such as ELISA or immunoblotting. However, these methods do not provide information on the exact timing and extent of TNF cleavage at single-cell resolution and they do not allow the live visualization of shedding events. Here, we generated C-tag TNF as a genetically encoded reporter to study TNF shedding at the single-cell level. The functionality of the C-tag TNF reporter is based on the exposure of a cryptic epitope on the C terminus of the transmembrane portion of pro-TNF on cleavage. In both denatured and nondenatured samples, this epitope can be detected by a nanobody in a highly sensitive and specific manner only upon TNF shedding. As such, C-tag TNF can successfully be used for the detection of TNF cleavage in flow cytometry and live-cell imaging applications. We furthermore demonstrate its applicability in a forward genetic screen geared toward the identification of genetic regulators of TNF maturation. In summary, the C-tag TNF reporter can be employed to gain novel insights into the complex regulation of ADAM-dependent TNF shedding.
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Project description:Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1-dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1-mediated cell adhesion to achieve efficient cell migration on collagen matrices.
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Project description:In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.
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Project description:Very low-density lipoprotein receptor (VLDLR) is a multifunctional transmembrane protein. Beyond the function of the full-length VLDLR in lipid transport, the soluble ectodomain of VLDLR (sVLDLR) confers anti-inflammatory and antiangiogenic roles in ocular tissues through inhibition of canonical Wnt signaling. However, it remains unknown how sVLDLR is shed into the extracellular space. In this study, we present the first evidence that a disintegrin and metalloprotease 17 (ADAM17) is responsible for sVLDLR shedding in human retinal pigment epithelium cells using pharmacological and genetic approaches. Among selected proteinase inhibitors, an ADAM17 inhibitor demonstrated the most potent inhibitory effect on sVLDLR shedding. siRNA-mediated knockdown or CRISPR/Cas9-mediated KO of ADAM17 diminished, whereas plasmid-mediated overexpression of ADAM17 promoted sVLDLR shedding. The amount of shed sVLDLR correlated with an inhibitory effect on the Wnt signaling pathway. Consistent with these in vitro findings, intravitreal injection of an ADAM17 inhibitor reduced sVLDLR levels in the extracellular matrix in the mouse retina. In addition, our results demonstrated that ADAM17 cleaved VLDLR only in cells coexpressing these proteins, suggesting that shedding occurs in a cis manner. Moreover, our study demonstrated that aberrant activation of Wnt signaling was associated with decreased sVLDLR levels, along with downregulation of ADAM17 in ocular tissues of an age-related macular degeneration model. Taken together, our observations reveal the mechanism underlying VLDLR cleavage and identify a potential therapeutic target for the treatment of disorders associated with dysregulation of Wnt signaling.
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Project description:Soluble tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), in contrast to membrane TWEAK and TNF, is only a weak activator of the classical NF?B pathway. We observed that soluble TWEAK was regularly more potent than TNF with respect to the induction of TNF receptor-associated factor 1 (TRAF1), a NF?B-controlled signaling protein involved in the regulation of inflammatory signaling pathways. TNF-induced TRAF1 expression was efficiently blocked by inhibition of the classical NF?B pathway using the IKK2 inhibitor, TPCA1. In contrast, in some cell lines, TWEAK-induced TRAF1 production was only partly inhibited by TPCA1. The NEDD8-activating enzyme inhibitor MLN4924, however, which inhibits classical and alternative NF?B signaling, blocked TNF- and TWEAK-induced TRAF1 expression. This suggests that TRAF1 induction by soluble TWEAK is based on the cooperative activity of the two NF?B signaling pathways. We have previously shown that oligomerization of soluble TWEAK results in ligand complexes with membrane TWEAK-like activity. Oligomerization of soluble TWEAK showed no effect on the dose response of TRAF1 induction, but potentiated the ability of soluble TWEAK to trigger production of the classical NF?B-regulated cytokine IL8. Transfectants expressing soluble TWEAK and membrane TWEAK showed similar induction of TRAF1 while only the membrane TWEAK expressing cells robustly stimulated IL8 production. These data indicate that soluble TWEAK may efficiently induce a distinct subset of the membrane TWEAK-targeted genes and argue again for a crucial role of classical NF?B pathway-independent signaling in TWEAK-induced TRAF1 expression. Other TWEAK targets, which can be equally well induced by soluble and membrane TWEAK, remain to be identified and the relevance of the ability of soluble TWEAK to induce such a distinct subset of membrane TWEAK-targeted genes for TWEAK biology will have to be clarified in future studies.
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Project description:Elevated levels of solTNFR2 are observed in a variety of human pathophysiological conditions but regulation of TNFR2 levels during disease is not well understood. We found that solTNFR2 levels were increased following influenza infection or live-attenuated influenza virus challenge in mice and humans, respectively. As influenza-specific CD8(+) T cells up-regulated expression of TNFR2 after infection in mice, we hypothesized that CD8(+) T cells contributed, in part, to solTNFR2 production after influenza infection and were interested in the mechanisms by which CD8(+) T cells regulate TNFR2 shedding. Activation of these cells by TCR stimulation resulted in enhanced shedding of TNFR2 that required actin remodeling and lipid raft formation and was dependent on MAPK/ERK signaling. Furthermore, we identified ADAM17 as the protease responsible for TNFR2 shedding by CD8(+) T cells, with ADAM17 and TNFR2 required in "cis" for shedding to occur. We observed similar activation thresholds for TNF-? expression and TNFR2 shedding, suggesting that solTNFR2 functioned, in part, to regulate solTNF-? levels. Production of solTNFR2 by activated CD8(+) T cells reduced the availability of solTNF-? released by these cells, and TNFR2 blockade during influenza infection in mice enhanced the levels of solTNF-?, supporting this hypothesis. Taken together, this study identifies critical cellular mechanisms regulating TNFR2 shedding on CD8(+) T cells and demonstrates that TNFR2 contributes, in part, to the regulation of TNF-? levels during infection.
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Project description:Patients with obstructive sleep apnea (OSA) have been described to have increased levels of inflammatory cytokines (particularly TNF-alpha) and have severely disturbed sleep architecture. Serum inflammatory markers, even in normal individuals, have been associated with abnormal sleep architecture. Not much is known about the role the TNF receptor plays in the inflammation of OSA nor if it is associated with changes in sleep architecture or arousals during the night. We hypothesized that the TNF receptor might play an important role in the inflammation as well as sleep architecture changes in patients with OSA.Thirty-six patients with diagnosed (AHI > 15) but untreated OSA were enrolled in this study. Baseline polysomnograms as well as TNF-alpha and soluble TNF receptor I (sTNF-RI) serum levels were obtained on all patients. We evaluated the association between serum levels of TNF-alpha and sTNF-RI with various polysomongraphic characteristics, including sleep stages and EEG arousals.sTNF-RI levels were significantly correlated with snore arousals (r value 0.449, p value 0.009), spontaneous movement arousals (r value 0.378, p value 0.025), and periodic limb movement arousals (r value 0.460, p value 0.008). No statistically significant correlations were observed with TNF-alpha to any polysomnographic variables. To control for statistical significance with multiple comparisons, a MANOVA was performed with TNF-alpha and sTNF-RI as dependent variables and sleep architecture measures and arousals as independent variables. The model for sTNF-RI was statistically significant (F value 2.604, p value 0.03), whereas the model for TNF-alpha was not, suggesting sleep quality significantly affects sTNF-RI. Hierarchal linear regression analysis demonstrated that sTNF-RI was independently associated with spontaneous movement arousal index scores after controlling for age, body mass index, and sleep apnea severity.These findings suggest that sTNF-RI is associated with arousals during sleep, but not with other measures in patients with OSA.
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