TACE-mediated Ectodomain Shedding Research Articles

Brain TACE (Tumor Necrosis Factor-α-Converting Enzyme) Contributes to Sympathetic Excitation in Heart Failure Rats.

TNF-α (tumor necrosis factor-α) is initially synthesized as a transmembrane protein that is cleaved by TACE (TNF-α-converting enzyme) to release soluble TNF-α. The elevated level of TNF-α in the brain and circulation in heart failure (HF) suggests an increase in the TACE-mediated ectodomain shedding process. The present study sought to determine whether TACE is upregulated in cardiovascular/autonomic brain regions like subfornical organ and hypothalamic paraventricular nucleus in rats with ischemia-induced HF and whether TACE plays a role in TNF-α-driven sympathetic excitation. We found that TACE was expressed throughout the subfornical organ and paraventricular nucleus, with significantly higher levels in HF than in sham-operated (Sham) rats. Intracerebroventricular injection of recombinant TACE induced a mild increase in blood pressure, heart rate, and renal sympathetic nerve activity that peaked at 15 to 20 minutes in both Sham and HF rats. HF rats had a secondary prolonged increase in these variables that was prevented by the TNF-α inhibitor SPD304. Intracerebroventricular administration of the TACE inhibitor TNF-alpha protease inhibitor 1 decreased blood pressure, heart rate, and renal sympathetic nerve activity in Sham and HF rats, with an exaggerated reduction in heart rate and renal sympathetic nerve activity in the HF rats. Direct microinjection of TACE or TNF-alpha protease inhibitor 1 into paraventricular nucleus or subfornical organ of Sham and HF rats elicited blood pressure, heart rate, and renal sympathetic nerve activity responses similar to intracerebroventricular TACE or TNF-alpha protease inhibitor 1. Intracerebroventricular infusion of Ang II (angiotensin II) and IL (interleukin)-1β increased TACE expression in subfornical organ and paraventricular nucleus of normal rats. These data suggest that a TACE-mediated increase in soluble TNF-α in the brain contributes to sympathetic excitation in HF.

Abstract P290: Central Angiotensin II and Interleukin-1©¬ Upregulate Brain Tumor Necrosis Factor-¥å Converting Enzyme in the Rat

Tumor necrosis factor-α (TNF-α) converting enzyme (TACE/ADAM17) proteolytically cleaves the extracellular domain of transmembrane TNF-α to release the soluble form of TNF-α that contributes to augmented sympathetic nerve activity in heart failure (HF) and hypertension (HTN). Our previous study indicated that TACE is upregulated in the subfornical organ (SFO) and hypothalamic paraventricular nucleus (PVN), two key cardiovascular and autonomic brain centers, in rats with ischemia-induced HF, and is associated with TNF-α –induced sympathetic excitation in that setting. However, the mechanisms regulating TACE activity in the brain remain a mystery. The present study sought to determine whether angiotensin II (ANG II) or interleukin-1β (IL-1β), which increase in SFO and PVN in HF, upregulate TACE expression. Urethane anesthetized male SD rats underwent a 4- hour intracerebroventricular (ICV) infusion of artificial cerebrospinal fluid (aCSF), ANG II (100 ng/hr) or IL-1β (50 ng/hr). Some rats were euthanized to collect SFO and PVN tissues to measure TACE mRNA and protein; others were transcardially perfused with 4% paraformaldehyde to immunostain for TACE in SFO and PVN. ICV infusion of ANG II (n=6) or IL-1β (n=6) significantly (*p<0.05) increased TACE mRNA level (fold change) in SFO (2.54 ± 0.24* and 2.36 ± 0.21*, respectively) and PVN (2.39* ± 0.25 and 2.23 ± 0.20*, respectively) compared with ICV aCSF infusion. Western blot analysis also revealed a significant increase in the TACE protein (normalized to β-actin) in ANG II or IL-1β infused rats in SFO (0.31±0.06* and 0.26±0.05* respectively) and PVN (0.27 ± 0.04*, 0.24 ± 0.03*, respectively) compared with ICV aCSF-infused rats (SFO: 0.12±0.05; PVN: 0.09±0.04). Confocal images exhibited that TACE-like immunoreactivity was markedly higher in all four subdivisions of PVN and in SFO in rats treated with ICV ANG II or IL-1β, compared with aCSF-infused rats. These data indicate that ANG II and IL-1β upregulate TACE expression in SFO and PVN, suggesting that activation of the renin-angiotensin system and augmented inflammation may upregulate TACE expression in the SFO and PVN in HF and HTN. TACE-mediated ectodomain shedding in SFO and PVN may contribute to ANG II- and cytokine-driven sympathoexcitation in HF and HTN.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Abstract IA31: Control of TGF-β responsiveness and epithelial plasticity

Abstract TGF-β signaling inhibits cell growth in epithelial cells and thus acts as tumor suppressor, yet increased TGF-β signaling often promotes cancer progression through effects on the tumor micro-environment and effects on the carcinoma cells that can lead to epithelial plasticity responses and increased invasion. The responsive epithelial or carcinoma cells can regulate their cell surface levels of TGF-β receptors, and thus enhance their responses to TGF-β. In addition to transcription control of TGF-β receptor expression, the cells can rapidly mobilize TβRII and TβRI from a large intracellular receptor pool to the cell surface, and thus enhance their TGF-β responsiveness. For example, high glucose, as seen in hyperglycemia, rapidly induces high levels of cell surface receptors that increase the cell’s TGF-β responsiveness, resulting in extracellular matrix production and increased cell size. Similarly, increased cell surface TGF-β receptor levels and TGF-β responsiveness also occur in response to insulin, which is therapeutically used against diabetes-associated hyperglycemia. The insulin-induced increase in cell surface levels requires activation of Akt, which then phosphorylates the membrane-associated RabGAP AS160, thus alleviating AS160-mediated retention of vesicles containing TβRII and TβRI, and allowing them to reach the cell surface. Additionally, the levels of functional TGF-β receptor complexes at the cell surface is controlled by TACE-mediated ectodomain shedding, which is activated in response to Erk and p38 MAPK activation. We will discuss the physiological implications of these multiple levels of control of TGF-β receptor cell surface presentation and TGF-β responsiveness in cancer cell behavior and cancer progression. Finally, the epithelial plasticity response to TGF-β is also controlled by the direct interaction of activated Smad3 with a methyltransferase, which consequently controls the histone modifications of the gene encoding the transcription factor Snail, a master regulator that drives epithelial-mesenchymal transition. Thus, Smad-mediated changes of histone methylation complement direct Smad-mediated transcription effects in the control of epithelial-mesenchymal transition. Citation Format: Rik Derynck, Erine Budi, Baby-Periyanayaki Muthusamy, Yoko Katsuno, Dan Du. Control of TGF-β responsiveness and epithelial plasticity. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA31.

LPA-producing enzyme PA-PLA1α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.

Direct Activation of TACE-Mediated Ectodomain Shedding by p38 MAP Kinase Regulates EGF Receptor-Dependent Cell Proliferation

Inflammatory stimuli activate ectodomain shedding of TNF-alpha, L-selectin, and other transmembrane proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals, directly activates TACE, a membrane-associated metalloprotease that is also known as ADAM17 and effects shedding in response to growth factors and Erk MAP kinase activation. p38alpha MAP kinase interacts with the cytoplasmic domain of TACE and phosphorylates it on Thr(735), which is required for TACE-mediated ectodomain shedding. Activation of TACE by p38 MAP kinase results in the release of TGF-alpha family ligands, which activate EGF receptor signaling, leading to enhanced cell proliferation. Conversely, depletion of p38alpha MAP kinase activity suppresses EGF receptor signaling and downstream Erk MAP kinase signaling, as well as autocrine EGF receptor-dependent proliferation. Autocrine EGF receptor activation through TACE-mediated ectodomain shedding intimately links inflammation and cancer progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.

Ectodomain shedding of pro-TGF-α is required for COX-2 induction and cell survival in renal medullary cells exposed to osmotic stress

In the renal medulla, cyclooxygenase (COX)-2 is induced by osmotic stress as present in this kidney region during antidiuresis. Increasing evidence suggests that EGF receptor (EGFR) signaling is involved in this process. The aim of the present study was to examine the mechanisms responsible for COX-2 expression and PGE(2) production during hypertonic conditions and to identify potential autocrine/paracrine EGFR ligands. Immunohistochemisty and Western blot analysis revealed abundant expression of the pro-EGFR ligand pro-transforming growth factor (TGF)-alpha in renal medullary cells in vivo and in cultured Madin-Darby canine kidney cells. In Madin-Darby canine kidney cells, hypertonicity rapidly increased TNF-alpha converting enzyme (TACE)-dependent ectodomain shedding of pro-TGF-alpha; phosphorylation of EGFR, p38, and ERK1/2; expression of COX-2; and production of PGE(2). Conversely, TACE inhibition prevented TGF-alpha release; EGFR, p38, and ERK1/2 activation; and COX-2 expression. Furthermore, cell survival was reduced substantially, a response that could be reversed by the addition of PGE(2). Simultaneous addition of recombinant TGF-alpha during TACE inhibition restored EGFR and MAPK phosphorylation, COX-2 expression, PGE(2) production, and cell survival during osmotic stress. These results indicate that hypertonicity induces TACE-mediated ectodomain shedding of pro-TGF-alpha, which subsequently activates COX-2 expression in an autocrine/paracrine fashion, via EGFR and MAPKs. We conclude that tonicity-induced TGF-alpha release is required for COX-2 expression, PGE(2) synthesis, and survival of renal medullary cells during osmotic stress.

Evidence for Regulation of the Tumor Necrosis Factor α-Convertase (TACE) by Protein-tyrosine Phosphatase PTPH1

Tumor necrosis factor alpha-convertase (TACE) is a metalloprotease-disintegrin involved in the ectodomain shedding of several proteins and is critical for proper murine development. TACE-mediated ectodomain shedding is regulated, and the cytoplasmic domain of TACE contains several potential signaling motifs, suggesting that this domain may play a role in regulating the metalloprotease activity. Here we report that the protein-tyrosine phosphatase PTPH1, which contains both a band 4.1 domain and a single PDZ domain, can interact with the cytoplasmic domain of TACE. The interaction was initially observed in a yeast two-hybrid screen and was confirmed using an in vitro binding assay and co-immunoprecipitations from eukaryotic cell extracts. The interaction is mediated via binding of the PDZ domain of PTPH1 to the COOH terminus of TACE. The latter represents a novel group I PDZ binding sequence characterized by a terminal cysteine residue. In co-expression experiments, significantly lower levels of TACE were observed in the presence of catalytically active forms of PTPH1 compared with catalytically inactive forms of PTPH1. Furthermore, phorbol ester-stimulated shedding of the TACE substrate tumor necrosis factor-alpha was decreased in cells expressing catalytically active PTPH1 compared with inactive PTPH1. Taken together, these results suggest that PTPH1 may be a negative regulator of TACE levels and function, and thus provide the first evidence for the regulation of TACE through a cytoplasmic protein.

Inhibition of TNF-alpha processing and TACE-mediated ectodomain shedding by ethanol.

Alcohol (EtOH) is a well-documented immunosuppressant. Acute EtOH-induced immunosuppression is partially due to suppression of tumor necrosis factor alpha (TNF-alpha) secretion. We investigated the mechanism of acute EtOH-induced TNF-alpha suppression in two monocytic cell lines, Mono Mac 6 and DRM. EtOH inhibited TNF-alpha secretion in a dose-dependent manner. However, TNF-alpha transcription was not affected by EtOH. Enzyme-linked immunosorbent assay and confocal microscopy showed that EtOH treatment increased cell-associated TNF-alpha. Ectodomain shedding of TNF-alpha from the cell surface is mediated by TNF-alpha converting enzyme (TACE). In contrast with TNF-alpha, EtOH did not inhibit interleukin-8 (IL-8) secretion, which does not require shedding. Furthermore, TNF p75 receptor shedding, a biomarker for TACE activity, was inhibited by EtOH in both cell lines. EtOH also inhibited TNF p75 receptor shedding in TACE-reconstituted fibroblasts, suggesting that EtOH inhibits the shedding process. These data show that acute EtOH exposure can posttranscriptionally suppress TNF-alpha production, resulting in specific defects in immune defense.

Activation of Tumor Necrosis Factor-α-converting Enzyme-mediated Ectodomain Shedding by Nitric Oxide

Ectodomain shedding of cell surface proteins is an important process in a wide variety of physiological and developmental events. Recently, tumor necrosis factor-alpha-converting enzyme (TACE) has been found to play an essential role in the shedding of several critical surface proteins, which is evidenced by multiple developmental defects exhibited by TACE knockout mice. However, little is known about the physiological activation of TACE. Here, we show that nitric oxide (NO) activates TACE-mediated ectodomain shedding. Using an in vitro model of TACE activation, we show that NO activates TACE by nitrosation of the inhibitory motif of the TACE prodomain. Thus, NO production activates the release of cytokines, cytokine receptors, and adhesion molecules, and NO may be involved in other ectodomain shedding processes.

Intracellular maturation and localization of the tumour necrosis factor α convertase (TACE)

Tumour necrosis factor alpha convertase (TACE) is a metalloprotease/disintegrin involved in the ectodomain shedding of several proteins, a process thought to be important in inflammation, rheumatoid arthritis and murine development. The characterization of the intracellular maturation and subcellular localization of endogenous TACE is decribed in the present study. Similarly to other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the prodomain. Prodomain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin type proprotein convertase in this process. An additional form of TACE, lacking the pro and cytoplasmic domains, is detected when cell lysates are prepared in the presence of EDTA instead of a hydroxamate-based metalloprotease inhibitor or 1,10-phenanthroline. This form appears to be generated by mature TACE cleaving its own cytoplasmic tail and may explain why little mature TACE has been detected in previous studies. In cell-surface labelling experiments, mature TACE was detected on the cell surface but immunofluorescence data indicate that TACE is predominantly localized to a perinuclear compartment similar to that described for tumour necrosis factor (TNF)alpha. This raises the possibility that TACE-mediated ectodomain shedding may occur in an intracellular compartment in addition to the cell surface.