TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity

Proprotein convertases (PCs) have been proposed to play a role in tumor necrosis factor-alpha converting enzyme (TACE) processing/activation. Using the furin-deficient LoVo cells, as well as the furin-proficient synoviocytes and HT1080 cells expressing the furin inhibitor alpha(1)-PDX, we demonstrate that furin activity alone is not sufficient for effective maturation and activation of the TACE enzyme. Data from in vitro and in vivo cleavage assays indicate that PACE-4, PC5/PC6, PC1 and PC2 can directly cleave the TACE protein and/or peptide. PC inhibition in macrophages reduced the release of soluble TNF-alpha from transmembrane pro-TNF-alpha. We therefore conclude that furin, in addition to other candidate PCs, is involved in TACE maturation and activation.     

Intracellular maturation and transport of tumor necrosis factor alpha converting enzyme

The tumor necrosis factor alpha converting enzyme (TACE) activity is required for the shedding of a variety of biologically active membrane bound precursors. The activation of TACE necessitates the proteolytic cleavage of its prodomain, a process that was suggested to be catalyzed by the proprotein convertase furin. However, the involvement of furin in this activation process has never been experimentally demonstrated. We have shown that the furinlike cleavage site (R-V-K-R(214)) localized between the prodomain and the metalloprotease domain of TACE is the sole site that can be in vitro cleaved by furin. In Cos7 cells, the release of TACE-processed substrates was reduced by the overexpression of the furin-specific proprotein convertase inhibitor Portland alpha1-antitrypsin inhibitor, but the release of TACE-processed substrates was increased by overexpression of furin in LoVo cells (deficient in furin activity) in which a mature form of TACE was identified. The immature form of TACE was detected at the surface of LoVo cells and at the surface of Cos7 and HT29 cells upon proprotein convertase inhibition. These results suggest that furin is the major proprotein convertase involved in the maturation/activation of TACE which is not a prerequisite for its cell-surface expression.     

Opposing effects of phorbol-12-myristate-13-acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The ‘cross‐talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP‐dependent signaling by structurally related human D1‐like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, followed by analysis of dopamine‐mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist‐evoked D1 receptor signaling, whereas constitutive and dopamine‐mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester‐regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ?, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca²⁺‐independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross‐talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1‐like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.     

Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C

Choline acetyltransferase, the enzyme that synthesizes the transmitter acetylcholine in cholinergic neurons, is a substrate for protein kinase C. In the present study, we used mass spectrometry to identify serine 440 in recombinant human 69-kDa choline acetyltransferase as a protein kinase C phosphorylation site, and site-directed mutagenesis to determine that phosphorylation of this residue is involved in regulation of the enzyme's catalytic activity and binding to subcellular membranes. Incubation of HEK293 cells stably expressing wild-type 69-kDa choline acetyltransferase with the protein kinase C activator phorbol 12-myristate 13-acetate showed time- and dose-related increases in specific activity of the enzyme; in control and phorbol ester-treated cells, the enzyme was distributed predominantly in cytoplasm (about 88%) with the remainder (about 12%) bound to cellular membranes. Mutation of serine 440 to alanine resulted in localization of the enzyme entirely in cytoplasm, and this was unchanged by phorbol ester treatment. Furthermore, activation of mutant enzyme in phorbol ester-treated HEK293 cells was about 50% that observed for wild-type enzyme. Incubation of immunoaffinity purified wild-type and mutant choline acetyltransferase with protein kinase C under phosphorylating conditions led to incorporation of [(32)P]phosphate, with radiolabeling of mutant enzyme being about one-half that of wild-type, indicating that another residue is phosphorylated by protein kinase C. Acetylcholine synthesis in HEK293 cells expressing wild-type choline acetyltransferase, but not mutant enzyme, was increased by about 17% by phorbol ester treatment.     

Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.     

P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing

The amyloid precursor protein (APP) is proteolytically processed by beta- and gamma-secretases to release amyloid beta, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid beta domain by alpha-secretase releasing the non-amyloidogenic product sAPP alpha, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate alpha-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPP alpha in a time- and dose-dependent manner. P2Y2 R-mediated sAPP alpha release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPP alpha release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPalpha release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPP alpha release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPP alpha release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPP alpha release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.     

Cell-surface processing of pro-ADAMTS9 by furin

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.     

PMA induces expression from the herpes simplex virus thymidine kinase promoter via the activation of JNK and ERK in the presence of adenoviral E1A proteins

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) promoter contains elements involved in both constitutive and induced expression. We determined that phorbol 12-myristate 13-acetate (PMA) induces the HSV-1 TK promoter in HEK293 cells. However, PMA did not induce expression from the promoter in HeLa cells and did not result in a globally increased gene expression in HEK293 cells. Induction of HSV-1 TK promoter required activation of both of JNK and ERK pathways. However, activation of the two pathways alone was not sufficient for induction of HSV-1 TK promoter. By transiently transfecting into HeLa cells the adenoviral E1A gene, which exists as an integrant in HEK293 genome, we demonstrated that E1A proteins are necessary for induction of HSV-1 TK promoter by PMA. We propose mechanisms by which signaling pathways activated by the tumor-promoter PMA cooperate with the oncogene E1A to stimulate a eukaryotic promoter, namely the HSV-1 TK promoter.     

PI3-K- and PKC-dependent up-regulation of APP processing enzymes by retinoic acid

Retinoic acid stimulates α-secretase processing of amyloid precursor protein (APP) and decreases β-secretase cleavage that leads to amyloid-β formation. Here, we investigated the effect of retinoic acid on the two putative α-secretases, the disintegrin metalloproteinases ADAM10 and TACE, and the β-site cleaving enzyme BACE1, in human neuroblastoma SH-SY5Y cells. Western blot analysis showed that exposure to retinoic acid resulted in significantly increased levels of ADAM10 and TACE, suggesting that regulation of α-secretases causes the effects on APP processing. The presence of the phosphatidylinositol 3-kinase inhibitor LY 294002 selectively reduced the effect on ADAM10 protein levels but not on ADAM10 mRNA levels as determined by RT-PCR. On the other hand, the effect on TACE was shown to be dependent on protein kinase C, since it was completely blocked in the presence of the inhibitor bisindolylmaleimide XI. Our data indicate that different signalling pathways are involved in retinoic acid-induced up-regulation of the secretases.     

Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17)

This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.     

A disintegrin and metalloproteinase (ADAM)-mediated ectodomain shedding of ADAM10

A disintegrin and metalloproteinase (ADAM) 10 is a type I transmembrane glycoprotein responsible for the ectodomain shedding of a range of proteins including the amyloid precursor protein implicated in Alzheimer's disease. In this study we demonstrate that ADAM10 itself is subject to shedding by one or more ADAMs. Expression of epitope-tagged wild-type ADAM10 in SH-SY5Y cells enabled the detection of a soluble ectodomain in conditioned medium. Shedding of the ADAM10 ectodomain was inhibited by a known ADAM inhibitor with a reciprocal accumulation of the full-length mature protein in both cell lysates and extracellular membrane vesicles. Shedding was also stimulated by phorbol ester treatment of cells. A glycosylphosphatidylinositol-anchored form of ADAM10 lacking the cytosolic, transmembrane and α-helical juxtamembrane regions of the wild-type protein was shed in a similar manner. Furthermore, a truncated soluble ADAM10 construct, although correctly post-translationally processed and catalytically active against a synthetic peptide substrate, was incapable of shedding cell-associated amyloid precursor protein. Finally, we show that ADAM9 is, at least in part, responsible for the ectodomain shedding of ADAM10. In conclusion, this is a new mechanism by which levels of ADAM10 are regulated and may have implications in a range of human diseases including Alzheimer's disease.     

Tumor necrosis factor-alpha-converting enzyme controls surface expression of c-Kit and survival of embryonic stem cell-derived mast cells

Transmembrane metalloproteinases of the disintegrin and metalloproteinase (ADAM) family control cell signaling interactions via hydrolysis of protein extracellular domains. Prior work has shown that the receptor tyrosine kinase, c-Kit (CD117), is essential for mast cell survival and that serum levels of c-Kit increase in proliferative mast cell disorders, suggesting the existence of c-Kit shedding pathways in mast cells. In the present work, we report that tumor necrosis factor alpha-converting enzyme (TACE; ADAM-17) mediates shedding of c-Kit. Stimulation of transfected cells with phorbol 12-myristate 13-acetate (PMA) induced metalloproteinase-mediated release of c-Kit ectodomain, which increased further upon TACE overexpression. By contrast, TACE-deficient fibroblasts did not demonstrate inducible release, thus identifying TACE as the metalloproteinase primarily responsible for PMA-induced c-Kit shedding. Surface expression of c-Kit by the human mast cell-1 line decreased upon phorbol-induced shedding, which involved metalloproteinase activity susceptible to inhibition by tissue inhibitor of metalloproteinase (TIMP)-3. To further explore the role of TACE in shedding of c-Kit from mast cells, we compared the behavior of mast cells derived from murine embryonic stem cells. In these studies, PMA decreased surface c-Kit levels on mast cells expressing wild-type (+/+) TACE but not on those expressing an inactive mutant (DeltaZn/DeltaZn), confirming the role of TACE in PMA-induced c-Kit shedding. Compared with TACE(+/+) cells, TACE(DeltaZn/DeltaZn) mast cells also demonstrated decreased constitutive shedding and increased basal surface expression of c-Kit, with diminished apoptosis in response to c-Kit ligand deprivation. These data suggest that TACE controls mast cell survival by regulating shedding and surface expression of c-Kit.     

Signals mediating cleavage of intercellular adhesion molecule-1

ICAM-1, a membrane-bound receptor, is released as soluble ICAM-1 in inflammatory diseases. To delineate mechanisms regulating ICAM-1 cleavage, studies were performed in endothelial cells (EC), human embryonic kidney (HEK)-293 cells transfected with wild-type (WT) ICAM-1, and ICAM-1 containing single tyrosine-to-alanine substitutions (Y474A, Y476A, and Y485A) in the cytoplasmic region. Tyrosine residues at 474 and 485 become phosphorylated upon ICAM-1 ligation and associate with signaling modules. Cleavage was assessed by using an antibody against the cytoplasmic tail of ICAM-1, which recognizes intact ICAM-1 and the 7-kDa membrane-bound fragment remaining after cleavage. Cleavage in HEK-293 WT cells was accelerated by phorbol ester PMA, whereas in EC it was induced by tumor necrosis factor-. In both cell types, a 7-kDa ICAM-1 remnant was detected. Tyrosine phosphatase inhibitors dephostatin and sodium orthovanadate augmented cleavage. PD-98059 (MEK kinase inhibitor), geldanamycin and PP2 (Src kinase inhibitors), and wortmannin (phosphatidylinositol 3-kinase inhibitor) dose-dependently inhibited cleavage in both cell types. SB-203580 (p38 inhibitor) was more effective in EC, and D609 (PLC inhibitor) mostly affected cleavage in HEK-293 cells. Cleavage was drastically decreased in Y474A and Y485A, whereas it was marginally reduced in Y476A. Surprisingly, phosphorylation was not detectable on the 7-kDa fragment of ICAM-1. These results implicate distinct pathways in the cleavage process and suggest a preferred signal transmission route for ICAM-1 shedding in the two cell systems tested. Tyrosine residues Y474 and Y485 within the cytoplasmic sequence of ICAM-1 regulate the cleavage process. ectodomain shedding; signaling; tyrosine phosphorylation     

Furin-mediated processing of Pro-C-type natriuretic peptide

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family that is involved in a variety of homeostatic processes. Here we characterize the processing essential for the conversion of the precursor, human pro-CNP, to the biologically active hormone. In human embryonic kidney 293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a mature peptide intracellularly as detected by Western analysis. Expression of recombinant human corin, a proatrial natriuretic peptide convertase, did not enhance the processing of pro-CNP in these cells. The processing of pro-CNP was inhibited in the presence of an inhibitor of the endoprotease furin but was not affected by inhibitors of matrix metalloproteinases and tumor necrosis factor-alpha convertase. In furin-deficient human colon adenocarcinoma LoVo cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of recombinant human furin in LoVo cells restored the ability of these cells to process pro-CNP. Furthermore, incubation of purified recombinant human furin with LoVo cell lysate containing pro-CNP led to the conversion of the precursor to a mature peptide. The furin-processed CNP was shown to be biologically active in a cell-based cGMP assay. These results demonstrate that furin is a critical enzyme for the processing of human pro-CNP.     

Phorbol ester induces desensitization of PTH-stimulated cyclic AMP production by decreasing the PTH receptor binding in UMR-106 cells

Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with the protein kinase C(PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) results in a time dependent (1-12h) desensitization of PTH-stimulated cAMP production. Compared to controls, PMA-treated cells showed 50% decrease of PTH-stimulated cAMP production. PK-C inhibitor, H-7 significantly blocked this PMA-induced desensitization. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was decreased by about 20% in PMA-treated cells. H-7 treatment completely restored receptor binding in PMA-treated cells. These data suggest that PK-C might act directly on PTH receptor which is coupling to adenylate cyclase, and induce desensitization.     

Attenuation of Focal Adhesion Kinase Signaling Following Depletion of Agonist-Sensitive Pools of Phosphatidylinositol 4,5-Bisphosphate

The effect of phosphoinositide depletion on focal adhesion kinase (FAK) signaling was investigated in two neuronal cell lines. Treatment of either SH-SY5Y neuroblastoma cells or PC12 cells with wortmannin, at a concentration that inhibits phosphatidylinositol 4-kinase activity, led to a selective depletion of phosphatidylinositol 4-phosphate without significantly altering phosphatidylinositol 4,5-bisphosphate (PIP2) content. An enhanced tyrosine phosphorylation of FAK elicited by agonist occupancy of phospholipase C-coupled receptors (muscarinic cholinergic in SH-SY5Y neuroblastoma or bradykinin in PC12 cells) was blocked completely by wortmannin. Under the above conditions, phosphoinositide resynthesis was prevented, and as a consequence, receptor stimulation led to a marked depletion of PIP2. In contrast, the increased tyrosine phosphorylation of FAK elicited by agents that do not activate phospholipase C (phenylarsine oxide, lysophosphatidic acid, or phorbol ester) persisted in the presence of wortmannin. However, the ability of these agents to elicit an increase in FAK phosphorylation was also prevented if PIP2 was depleted by activation of a phospholipase C-coupled receptor in the presence of wortmannin. The results suggest that agonist-sensitive pools of PIP2 must be maintained for FAK signaling to occur in response to a mechanistically diverse range of stimuli.     

Quercetin 3-glucoside protects neuroblastoma (SH-SY5Y) cells in vitro against oxidative damage by inducing sterol regulatory element-binding protein-2-mediated cholesterol biosynthesis

The flavonoid quercetin 3-glucoside (Q3G) protected SH-SY5Y, HEK293, and MCF-7 cells against hydrogen peroxide-induced oxidative stress. cDNA microarray studies suggested that Q3G-pretreated cells subjected to oxidative stress up-regulate the expression of genes associated with lipid and cholesterol biosynthesis. Q3G pretreatment elevated both the expression and activation of sterol regulatory element-binding protein-2 (SREBP-2) only in SH-SY5Y cells subjected to oxidative stress. Inhibition of SREBP-2 expression by small interfering RNA or small molecule inhibitors of 2,3-oxidosqualene:lanosterol cyclase or HMG-CoA reductase blocked Q3G-mediated cytoprotection in SH-SY5Y cells. By contrast, Q3G did not protect either HEK293 or MCF-7 cells via this signaling pathway. Moreover, the addition of isopentenyl pyrophosphate rescued SH-SY5Y cells from the inhibitory effect of HMG-CoA reductase inhibition. Last, Q3G pretreatment enhanced the incorporation of [(14)C]acetate into [(14)C]cholesterol in SH-SY5Y cells under oxidative stress. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection in SH-SY5Y cells involving SREBP-2-mediated sterol synthesis that decreases lipid peroxidation by maintaining membrane integrity in the presence of oxidative stress.     

The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein

We showed previously that PrPc undergoes constitutive and phorbol ester-regulated cleavage inside the 106-126 toxic domain of the protein, leading to the production of a fragment referred to as N1. Here we show by a pharmacological approach that o-phenanthroline, a general zinc-metalloprotease inhibitors, as well as BB3103 and TAPI, the inhibitors of metalloenzymes ADAM10 (A disintegrin and metalloprotease); and TACE, tumor necrosis factor alpha-converting enzyme; ADAM17), respectively, drastically reduce N1 formation. We set up stable human embryonic kidney 293 transfectants overexpressing human ADAM10 and TACE, and we demonstrate that ADAM10 contributes to constitutive N1 production whereas TACE mainly participates in regulated N1 formation. Furthermore, constitutive N1 secretion is drastically reduced in fibroblasts deficient for ADAM10 whereas phorbol 12,13-dibutyrate-regulated N1 production is fully abolished in TACE-deficient cells. Altogether, our data demonstrate for the first time that disintegrins could participate in the catabolism of glycosyl phosphoinositide-anchored proteins such as PrPc. Second, our study identifies ADAM10 and ADAM17 as the protease candidates responsible for normal cleavage of PrPc. Therefore, these disintegrins could be seen as putative cellular targets of a therapeutic strategy aimed at increasing normal PrPc breakdown and thereby depleting cells of the putative 106-126 "toxic" domain of PrPc.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

Furin is an endogenous regulator of alpha-secretase associated APP processing

Constitutive and PKC-regulated alpha-secretase pathways have been reported to produce the secreted form of alpha-secretase-derived APP (sAPPalpha). Here, we examined putative role of furin in the regulation of alpha-secretase activity in vitro and in vivo. Overexpression of the prodomain of furin and infection with a furin-specific inhibitor significantly reduced the levels of sAPPalpha regardless of PKC activity, whereas total APP levels remained unchanged. Furin mRNA levels in the brains of AD patients and Tg2576 mice were significantly lower than those in controls, whereas ADAM10 and TACE mRNA levels were much alike between Tg2576 and littermate mice. Moreover, the injection of furin-adenovirus into Tg2576 mouse brains markedly increased alpha-secretase activity and reduced beta-amyloid protein (Abeta) production in infected brain regions. Our results suggest that furin enhances alpha-secretase activity via the cleavage of ADAM10 and TACE, and that attenuated furin activity is connected to the production of Abeta.