TNF-alpha Converting Enzyme Activity Research Articles

Enhanced expression of TACE contributes to elevated levels of sVCAM-1 in endometriosis.

Are increased sVCAM-1 and sICAM-1 levels associated with tumor necrosis factor-alpha-converting enzyme (TACE) activity in endometriosis? Here we provide the first functional evidence that induced TACE activity in human endometriotic epithelial cells is at least in part responsible for the enhanced release of sVCAM-1 from these cells. We and others have shown that serum-soluble (s)VCAM-1 levels are significantly higher in women with endometriosis, compared to disease-free controls. Experimental evidence exists suggesting a role of sICAM-1 and sVCAM-1 in the pathogenesis of endometriosis. TACE was identified as the protease responsible for phorbol 12-myristate 13-acetate (PMA)-induced VCAM-1 release in murine endothelial cells. Additionally, it has recently been shown that TACE is upregulated in the endometrial luminal epithelium of the mid-secretory phase in infertile women. This study was conducted at the Tertiary Endometriosis Referral Center of the Medical University of Vienna. Samples from a total number of 97 women were collected between July 2013 and September 2014. After complete surgical exploration of the abdominopelvic cavity, 49 women with histologically proven endometriosis and 48 endometriosis-free control women were enrolled. Each participating woman contributed only one sample of eutopic endometrium and normal peritoneum, and some of the women with endometriosis contributed samples of diverse types of endometriotic lesions (in total 52 ectopic samples). Among the 49 women with endometriosis, 36 matched samples of endometriotic lesions and corresponding eutopic endometrium were collected. In order to detect sVCAM-1 and TACE protein by ELISA, peritoneal fluid (PF) samples were collected from 44 cases and 32 controls during surgery. Expression of TACE mRNA was analyzed by qRT-PCR in 111 endometrium tissue samples (28 eutopic control samples, 33 eutopic samples from women with endometriosis, 50 ectopic samples from lesions) and 37 healthy peritoneum samples. Immunohistochemistry was performed in 123 tissue samples (39 eutopic control samples, 42 eutopic samples from women with endometriosis, 42 ectopic samples from lesions) and the relation between tissue TACE protein levels and sVCAM-1 secretion was examined. PMA-induced sVCAM-1 release, and TACE- and VCAM-1-transcripts or proteins were measured in an immortalized endometriotic epithelial cell line (11Z) pre-incubated either with TACE inhibitors or following TACE siRNA knockdown. Here, we demonstrate that TACE protein is overexpressed in epithelium of tissue samples of both eutopic endometrium and ectopic lesions of women with endometriosis compared to disease-free controls (P < 0.001 both) and that the overexpression of the protein in the lesions is due to activation of TACE gene transcription (P < 0.001). Moreover, epithelial TACE protein was significantly higher in ectopic samples than in corresponding eutopic tissue of women with the disease (P < 0.001). High endometrial tissue TACE protein expression correlated with higher serum sVCAM-1 levels (P < 0.05) but not with sICAM-1 levels. Inhibition of TACE either by TACE inhibitors or by TACE siRNA knockdown resulted in decreased PMA-induced shedding of sVCAM-1 in vitro (P < 0.005 or P < 0.01, respectively), but the TACE inhibitors did not affect transcription of TACE or VCAM-1. Additionally, we observed an upregulation of TACE in proliferative endometrial epithelium of infertile (P < 0.005), compared to fertile women. TACE was increased in infertile women with endometriosis (P = 0.051) but not in infertile women without endometriosis. Albeit well characterized, our control population included women with other gynecologic diseases, which may have impacted the levels of sVCAM-1 and tissue TACE expression levels, e.g. benign ovarian cysts or uterine fibroids. Thus, the results of our analysis have to be interpreted carefully and in the context of the current experimental settings. The dysregulation of TACE substrate shedding represents a promising yet relatively unexplored area of endometriosis progression and could serve as a basis for the development of new treatments of the disease. This work was supported by the Ingrid Flick Foundation. The authors have no competing interests to declare.

TACE-dependent TGFα shedding drives triple-negative breast cancer cell invasion

The epidermal growth factor receptor (EGFR) is frequently expressed in triple-negative breast cancer (TNBC) and is a marker of poor prognosis in this patient population. Because activating mutations in this kinase are very rare events in breast cancer, we screened breast tumor gene expression profiles to examine the distribution of EGFR ligand expression. Of the six known EGFR ligands, transforming growth factor alpha (TGFα) was expressed more highly in triple-negative breast tumors than in tumors of other subtypes. TGFα is synthesized as a transmembrane precursor requiring tumor necrosis factor alpha converting enzyme (TACE)/ADAM17-dependent proteolytic release to activate its receptor. In our study, we show that an inhibitor of this proteolytic release blocks invasion, migration and colony formation by several TNBC cell lines. Each of the effects of the drug was reversed upon expression of a soluble TGFα mutant that does not require TACE activity, implicating this growth factor as a key metalloproteinase substrate for these phenotypes. Together, these data demonstrate that TACE-dependent TGFα shedding is a key process driving EGFR activation and subsequent proliferation and invasion in TNBC cell lines.

Broad-Spectrum Matrix Metalloproteinase Inhibition Curbs Inflammation and Liver Injury but Aggravates Experimental Liver Fibrosis in Mice

BackgroundLiver fibrosis is characterized by excessive synthesis of extracellular matrix proteins, which prevails over their enzymatic degradation, primarily by matrix metalloproteinases (MMPs). The effect of pharmacological MMP inhibition on fibrogenesis, however, is largely unexplored. Inflammation is considered a prerequisite and important co-contributor to fibrosis and is, in part, mediated by tumor necrosis factor (TNF)-α-converting enzyme (TACE). We hypothesized that treatment with a broad-spectrum MMP and TACE-inhibitor (Marimastat) would ameliorate injury and inflammation, leading to decreased fibrogenesis during repeated hepatotoxin-induced liver injury.Methodology/Principal FindingsLiver fibrosis was induced in mice by repeated carbon tetrachloride (CCl4) administration, during which the mice received either Marimastat or vehicle twice daily. A single dose of CCl4 was administered to investigate acute liver injury in mice pretreated with Marimastat, mice deficient in Mmp9, or mice deficient in both TNF-α receptors. Liver injury was quantified by alanine aminotransferase (ALT) levels and confirmed by histology. Hepatic collagen was determined as hydroxyproline, and expression of fibrogenesis and fibrolysis-related transcripts was determined by quantitative reverse-transcription polymerase chain reaction. Marimastat-treated animals demonstrated significantly attenuated liver injury and inflammation but a 25% increase in collagen deposition. Transcripts related to fibrogenesis were significantly less upregulated compared to vehicle-treated animals, while MMP expression and activity analysis revealed efficient pharmacologic MMP-inhibition and decreased fibrolysis following Marimastat treatment. Marimastat pre-treatment significantly attenuated liver injury following acute CCl4-administration, whereas Mmp9 deficient animals demonstrated no protection. Mice deficient in both TNF-α receptors exhibited an 80% reduction of serum ALT, confirming the hepatoprotective effects of Marimastat via the TNF-signaling pathway.Conclusions/SignificanceInhibition of MMP and TACE activity with Marimastat during chronic CCl4 administration counterbalanced any beneficial anti-inflammatory effect, resulting in a positive balance of collagen deposition. Since effective inhibition of MMPs accelerates fibrosis progression, MMP inhibitors should be used with caution in patients with chronic liver diseases.

The Study of the Inhibition of the Recombinant TACE Prodomain to Endotoxemia in Mice

Objective:To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-α (TNF-α) converting enzyme (TACE) on TACE activity and to develop an approach to interfere with inflammation processes.Methods:The cDNA encoding the full-length ectodomain (T1300) and prodomain (T591) of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+)-T1300 and pET-28a (+)-T591) were constructed and transformed into E. coli BL21. After Ni2+-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning.Results:The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-α, which mediated the accumulation of TNF-α on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice.Conclusions:The recombinant prodomain of TACE has the ability to inhibit sTNF-α release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.

Effects of biological drug adalimumab on tumour necrosis factor‐α‐converting enzyme activation

Tumour necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE) is a membrane-bound metalloprotease and disintegrin. It is produced by a number of host cells and is known to shed and release cell-bound cytokines, particularly members of the TNF family. No investigations into the regulation of this enzyme by autoantibodies have been reported. In this study, we tested the hypothesis that anti-Ro/SSA autoantibodies, purified from IgG fractions of patients with primary Sjögren's syndrome, are capable to regulate TACE expression and activation in human salivary gland epithelial cells (SGEC). We also evaluated the potential physiological and therapeutic consequences of TNF-alpha blocking by the biological agent adalimumab, the first fully human (100% human peptide sequences) therapeutic anti-TNF-alpha antibody, on post-translational regulation of TACE. Taken together, our results show a dose-dependent increase in TACE expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein. Adalimumab treatment brought TACE expression to levels than those observed in untreated SGEC. These findings, showing the presence of autoantibodies-dependent mechanisms by which TACE levels are regulated in human SGECs, may have implications in the context of current investigations on the pathological role of autoantibodies.

Induction of TNF-alpha-converting enzyme-ectodomain shedding by pathogenic autoantibodies

The release of the soluble form of tumor necrosis factor (TNF)-alpha from the plasma membrane occurs through the activation of the secretase tumor necrosis factor-alpha-converting enzyme (TACE). The current study was designed to examine whether the anti-Ro/SSA autoantibodies (Abs) are capable to regulate TACE expression in non-neoplastic human salivary gland epithelial cells (SGEC) cultures. We investigated the effect of anti-Ro/SSA Abs on the localization and abundance of cell-surface TACE and on TACE pro-domain-shedding and activation. In addition, the potential physiological consequences of TNF-alpha blockage by the biological agent Adalimumab on post-translational regulation of TACE are discussed. Anti-Ro/SSA Abs were purified from IgG fractions of patients with primary Sjögren's syndrome, using Sepharose 4B-Ro/SSA affinity columns. Flow cytometry, reverse transcription-PCR, western blot and immunohistochemistry were used to study TACE expression on SGEC and TACE regulation by Abs. Our study demonstrated a dose-dependent increase of TACE messenger RNA (mRNA) expression in anti-Ro/SSA Abs-treated SGEC, followed by internalization, pro-domain shedding and activation of TACE protein, suggesting that increased TACE activity is necessary for the release of TNF-alpha observed in anti-Ro/SSA Abs-stimulated SGEC. Adalimumab treatment brought TACE mRNA and surface TACE expression to levels than those observed in untreated SGEC. These data suggest that the effect of anti-Ro/SSA Abs on TACE expression and intracellular distribution is exerted by TNF-alpha production.

Anti‐inflammatory Effect of Palmitoylethanolamide on Human Adipocytes

Obesity leads to the appearance of an inflammatory process, which can be initiated even with a moderate weight gain. Palmitoylethanolamide (PEA) is an endogenous lipid, secreted by human adipocytes, that possesses numerous anti-inflammatory properties. The main purpose of this study was to investigate the anti-inflammatory effect of PEA on human adipocytes, as well as in a murine model. The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-treated human subcutaneous adipocytes in primary culture and CF-1 mice was investigated by enzyme-linked immunosorbent assay. The effects of PEA on adipocyte TNF-alpha secretion were explored as well as some suspected PEA anti-inflammatory pathways: nuclear factor-kappaB (NF-kappaB) pathway, peroxisome proliferator-activated receptor-alpha (PPAR-alpha) gene expression, and TNF-alpha-converting enzyme (TACE) activity. The effects of PEA on the TNF-alpha serum concentration in intraperitoneally LPS-treated mice were also studied. We demonstrate that the LPS induced secretion of TNF-alpha by human adipocytes is inhibited by PEA. This action is neither linked to a reduction in TNF-alpha gene transcription nor to the inhibition of TACE activity. Moreover, PPAR-alpha is not implicated in this anti-inflammatory activity. Lastly, PEA exhibits a wide-reaching anti-inflammatory action as the molecule is able to completely inhibit the strong increase in TNF-alpha levels in the serum of mice treated with high doses of LPS. In view of its virtual lack of toxicity, PEA might become a potentially interesting candidate molecule in the prevention of obesity-associated insulin resistance.

Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

BackgroundNuclear localization of proteolytically formed intracellular fragment of ErbB4 receptor tyrosine kinase has been shown to promote cell survival, and nuclear localization of ErbB4 receptor has been described in human breast cancer. Tumor necrosis factor alpha converting enzyme (TACE) initiates the proteolytic cascade leading to ErbB4 intracellular domain formation. Interactions between matrix metalloproteases and heparan sulfate have been described, but the effect of cell surface heparan sulfate on TACE activity has not been previously described.ResultsAs indicated by immunodetection of increased ErbB4 intracellular domain formation and direct enzyme activity analysis, TACE activity was substantially amplified by enzymatic removal of cell surface heparan sulfate but not chondroitin sulfate.ConclusionIn this communication, we suggest a novel role for cell surface heparan sulfate. Removal of cell surface heparan sulfate led to increased formation of ErbB4 intracellular domain. As ErbB4 intracellular domain has previously been shown to promote cell survival this finding may indicate a novel mechanism how HS degradation active in tumor tissue may favor cell survival.

Upregulation of tumor necrosis factor receptor 1 and TNF-α converting enzyme during corneal wound healing

To elucidate tumor necrosis factor (TNF) receptor-1 (TNFR1) and TNF-alpha converting enzyme (TACE) involvement during corneal wound healing. The corneas of BALB/c mice cornea were scarred by alkali burns using filter paper dipped in 1N NaOH solution. TACE and TNFR1 expression in the alkali-burned corneas were evaluated by immunohistochemistry (total, 16 eyes). Using cultured fibroblasts (human) and macrophages (mice), we evaluated the release of soluble TNF-alpha (sTNF-alpha) and soluble TNFR1 (sTNFR1) in the supernatant by enzyme-linked immunosorbent assay after stimulating TACE activity with phorbol myristate acetate (PMA). In alkali-burned corneas, both TACE and TNFR1 expression were observed in the stromal cells after the acute phase of wound healing response. In macrophage-cultured supernatant, both sTNF-alpha and sTNFR1 release were promoted by PMA stimulation. On the other hand, only sTNFR1 released by PMA stimulation was observed in fibroblast-cultured supernatant. TACE and TNFR1 were expressed mainly after the acute phase of corneal wound healing. The TACE-dependent extracellular release of sTNFR1 was recognized in cultured fibroblasts and macrophages.

Interleukin‐10 regulates TNF‐α−converting enzyme (TACE/ADAM‐17) involving a TIMP‐3 dependent and independent mechanism

IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.

The transmembrane domain of TACE regulates protein ectodomain shedding

Numerous membrane proteins are cleaved by tumor necrosis factor-alpha converting enzyme (TACE), which causes the release of their ectodomains. An ADAM (a disintegrin and metalloprotease domain) family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain (TM) of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI)-binding polypeptide failed to restore shedding of transforming growth factor-alpha (TGF-alpha), tumor necrosis factor-alpha (TNF-alpha) and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor (PDGFR) also resulted in severe loss of TGF-alpha shedding, but had no effects on the cleavage of TNF-alpha and L-selectin. Replacement of the TM in TGF-alpha with that of L-selectin enabled TGF-alpha shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.

Inhibition of TACE Activity Enhances the Susceptibility of Myeloma Cells to TRAIL.

TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. Although TRAIL-mediated immunotherapy is as attractive maneuver against MM because of its tumor-specific cytotoxic activity, the susceptibility of MM cells to TRAIL has been demonstrated to vary among MM cells, which limits clinical applications of TRAIL-mediated immunotherapy. TRAIL is known to bind to 2 different proapoptotic receptors, death receptor 4 (DR4) and DR5. The membrane-bound TRAIL receptors have been reported to be subjected to proteolytic cleavage in melanoma cells in a manner inhibitable by tissue inhibitor of metalloproteinases-3 (TIMP-3), an endogenous inhibitor for TNF-alpha converting enzyme (TACE) (Ahonen M, Oncogene. 2003). Although TACE is regarded as a sheddase for TNF-like ligands/receptors, the role for TACE in TRAIL-mediated cytotoxicity in MM cells is largely unknown. In the present study, we therefore investigated the role for TACE in TRAIL and its receptor editing as well as the effect of TACE inhibition on TRAIL-triggered cytotoxicity in MM cells. MM cells constitutively expressed DR4 on their surface as determined in flow cytometry, while the expression of DR5 as well as TRAIL was negligible. TACE mRNA was constitutively overexpressed in all MM cell lines including RPMI8226, MM.1S, KMS12 and U266, whereas its endogenous inhibitor, TIMP-3, was not detected, suggesting enhanced TACE activity along with down-regulation of TIMP-3 in MM cells. TAPI-0, a TACE inhibitor, up-regulated the cell-surface expression of DR4 about 2-fold in RPMI8226 and U266 cells, while DR5 and TRAIL expression remained undetectable. Immunoblot analyses for DR4 revealed that conditioned media from RPMI8226 and U266 cells contained DR4 protein with a molecular size smaller than that observed in their cell lysates. Interestingly, TAPI-0 treatment down-regulated DR4 levels in culture supernatants of these MM cells, while increasing DR4 levels in their cell lysates as well as on their surface, suggesting ectodomain shedding of DR4 by TACE in MM cells. Importantly, TAPI-0 treatment potentiated TRAIL-triggered cytotoxicity against MM cells including RPMI8226 and MM.1S, which was almost completely antagonized by addition of osteoprotegerin, a soluble inhibitor for TRAIL, indicating a TRAIL-dependent mechanism. Taken together, the present observations demonstrate that cell-surface TRAIL receptor expression is post-transcriptionally down-regulated by endogenous TACE activity enhanced in MM cells, which may protect MM cells from TRAIL-mediated apoptosis by surrounding immune cells expressing TRAIL, and that up-regulation of TRAIL receptor expression by inhibiting TACE activity is able to restore the susceptibility of MM cells to TRAIL. Therefore, these preclinical data provide that TACE inhibitors in combination with TRAIL-based immunotherapy may be a novel therapeutic approach against MM which can not be cured by the present chemotherapeutic modalities.

Effects of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in a model of intestinal reperfusion injury in mice

Tumor necrosis factor (TNF)-alpha plays an important role in the mediation of reperfusion-induced tissue injury and lethality. Here, we assessed the effects of PKF242-484 and PKF241-466, two dual inhibitors of TNF-alpha converting enzyme (TACE) and matrix metalloproteinases (MMPs), in a model of ischemia and reperfusion injury in mice. Reperfused animals that received PKF242-484 or PKF241-466 treatment had a dose-dependent reduction of TNF-alpha concentrations in serum. Both drugs delayed and partially inhibited the reperfusion-associated lethality. Maximal inhibition occurred at 10 mg/kg. At this dose, both inhibitors reduced reperfusion-associated local and remote tissue injury, as assessed by changes in vascular permeability, neutrophil recruitment and hemorrhage. In addition, the compounds markedly reduced production of TNF-alpha, CXCL1 (keratinocyte-derived chemokine, KC) and CCL2 (monocyte chemoattractant protein-1, MCP-1) in intestine and lungs of animals which underwent reperfusion. FN-439, an inhibitor of MMPs which possesses no effect on TACE, decreased MMP-2 and MMP-3 activity, but failed to affect tissue injury, TNF-alpha production or lethality. Thus, combined TACE and MMP inhibitors might be effective co-adjuvants in treatments of injuries that follow reperfusion of an ischemic vascular territory. The effects of these drugs on TNF-alpha production appear to be more relevant than their effects on MMP inhibition.

TIMP-3 Ameliorates Hepatic Ischemia/Reperfusion Injury Through Inhibition of Tumor Necrosis Factor-Alpha-Converting Enzyme Activity in Rats

Tumor necrosis factor (TNF)-alpha and its receptors play a critical role in the inflammatory cascade after hepatic ischemia/reperfusion injury. TNF-alpha converting enzyme (TACE) or disintegrin and metalloproteinase (ADAM)-17 is a metalloproteinase disintegrin that specifically cleaves precursor TNF-alpha to its mature form and is involved in the ectodomain shedding of TNF receptors. The regulation of TACE is poorly understood and its role in liver injury and/or regeneration is unknown. Male Wistar rats were subjected to 10 or 30 min of partial warm hepatic ischemia followed by 3 to 24 hr of reperfusion. Serum and/or hepatic TACE, TNF-alpha, TNF receptor 1 (TNFR1), and interleukin (IL)-6 levels were assessed by enzyme-linked immunosorbent assay, real-time reverse-transcriptase polymerase chain reaction, and/or Western blot. Low levels of TACE were detected in normal liver tissue. Both 10 and 30 min warm ischemia resulted in a rise in TACE expression which peaked six hr after reperfusion. TNF-alpha, TNFR1, and IL-6 levels were up-regulated in a pattern similar to TACE messenger RNA (mRNA) levels. Moreover, selective inhibition of TACE activity by specific inhibitor tissue inhibitor of metalloproteinase (TIMP)-3 at dosages of 100 or 1000 ng/kg body weight showed significant decrease of circulating TNF-alpha and serum alanine transferase (ALT) levels and histological improvement of hepatic ischemia/reperfusion injuries. TACE expression and its activity, as measured by increases in TNF-alpha, TNFR1, and IL-6 levels, are increased following hepatic ischemia/reperfusion injury, implying that TACE plays an important role in hepatic ischemia/reperfusion injury. Amelioration of hepatic ischemia/reperfusion injury after inhibition of TACE activity by TIMP-3 suggests that TACE inhibition may play an important role in preventing liver ischemia/reperfusion injury warranting further experimental and clinical study.

Control of Bioamine Metabolism by 5‐HT2B and α1D Autoreceptors through Reactive Oxygen Species and Tumor Necrosis Factor‐α Signaling in Neuronal Cells

Homeostasis of the central nervous system relies on the proper integration of cell-signaling pathways recruited by a variety of neuronal and non-neuronal factors, with the aim of tightly controlling neurotransmitter metabolism, storage, and transport. We took advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to selectively differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, to identify functional targets of serotonin (5-hydroxytryptamine [5-HT]) and norepinephrine (NE) autoreceptors possibly involved in the control of neuronal functions. We demonstrate that 5-HT(2B) and adreno alpha(1D) receptors are coupled to reactive oxygen species (ROS) production through NADPH oxidase activation in 1C11(5-HT) and 1C11(NE) neuronal cells, respectively. In the signaling cascade linking 5-HT(2B) receptors to NADPH oxidase, phospholipase A2-mediated arachidonic acid production is required for ROS synthesis. ROS, in turn, act as second message signals and control the activation of TACE (TNF-alpha converting enzyme), a member of a disintegrin and metalloproteinase family. 5-HT(2B) and alpha(1D) receptor stimulation triggers TACE-dependent TNF-alpha shedding in the surrounding milieu of 1C11(5-HT) and 1C11(NE) cells. In these cells, shed TNF-alpha triggers degradation of 5-HT and NE into 5-HIAA and MHPG, respectively. Finally, we observe that 5-HT(2B) and alpha(1D) receptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete neuronal phenotype. Altogether, our data indicate that couplings of 5-HT(2B) and alpha(1D) autoreceptors to ROS and TNF-alpha signaling control neurotransmitter metabolism in 1C11-derived neuronal cells. Eventually, we might explain the origin of oxidative stress and high level of TNF-alpha in neurodegenerative diseases as a consequence of deviation of normal signaling pathways coupled to neurotransmitters.

Interleukin‐6 receptor shedding is enhanced by interleukin‐1β and tumor necrosis factor α and is partially mediated by tumor necrosis factor α–converting enzyme in osteoblast‐like cells

Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process. Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding. IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells. This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA. Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels. IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation.

Abnormal TNF activity in Timp3-/- mice leads to chronic hepatic inflammation and failure of liver regeneration.

Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.

Epidermal Growth Factor Receptor Transactivation Mediates Tumor Necrosis Factor-induced Hepatocyte Replication

Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor alpha (TGFalpha); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFalpha into the medium through activation of the metalloproteinase TNFalpha-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFalpha and activation of EGFR signaling were inhibited by TNFalpha protease inhibitor-1, an agent that interferes with TNFalpha-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration.

Release of TNF-alpha during myocardial reperfusion depends on oxidative stress and is prevented by mast cell stabilizers.

Our study sought to elucidate the role of oxidative stress for shedding of tumor necrosis factor-alpha (TNF-alpha) and for activating TNF-alpha-converting enzyme (TACE). TNF-alpha, a central inflammatory cytokine, is discussed as one of the mediators of reperfusion injury. Shedding of membrane-bound pro-TNF-alpha is thought to be largely due to TNF-alpha-converting enzyme (TACE). Release of TNF-alpha and TACE dependency were studied in isolated rat hearts and in the human mast cell line HMC-1. In reperfused hearts, interstitial release of TNF-alpha occurred in two phases (2-10 and >45 min). It depended on the presence of oxygen during reperfusion and was attenuated by reduced glutathione. Infusion of the oxidants H(2)O(2) or HOCl elicited release in non-ischemic hearts. TNF-alpha release was inhibited in hearts treated with degranulation inhibitors ketotifen or cromoglycate, suggesting mast cells as major source for myocardial TNF-alpha. This was confirmed by tissue staining. Post-ischemic release of histamine, however, did not parallel that of TNF-alpha. Heart tissue contained mainly mature TACE. HMC-1 expressed abundant pro-TACE and cleaved the pro-TNF-alpha-peptide Ac-SPLAQAVRSSSR-NH(2). However, cleavage was nonspecific and only partly inhibited by TACE inhibitor TAPI-2 (10-100 micromol/l), while it was stimulated by H(2)O(2) and HOCl and fully blocked by the nonspecific metalloprotease inhibitor o-phenanthroline. The mechanism underlying TNF-alpha release from post-ischemic myocardium is oxidation-dependent but largely independent of activation of TACE. Mast cell stabilizers may be useful in preventing TNF-alpha release during reperfusion.

Structure and functions of tumor necrosis factor-alpha converting enzyme.

Tumor necrosis factor-alpha converting enzyme (TACE) is the first described and best characterized secretase. In this review the structure and the possible roles for TACE are summarized. The substrate specificity and the regulation of TACE activity as well as redundancy and possible cooperations of distinct secretases are also discussed.