Membrane-type 1-matrix metalloproteinase regulates intracellular adhesion molecule-1 (ICAM-1)-mediated monocyte transmigration

We examined the mechanism regulating intercellular cell adhesion molecule-1 (ICAM-1)-dependent monocyte transendothelial migration. Monocyte migration through endothelial cells expressing ICAM-1 alone was comparable to that of tumor necrosis factor-alpha-treated cells. Transmigration was reduced in ICAM-1 lacking the cytoplasmic tail and in tyrosine to alanine substitutions at Tyr-485 and Tyr-474. Tissue inhibitors of matrix metalloproteinases (TIMPs) -2 and -3 blocked transmigration, whereas TIMP-1 was ineffective. This profile suggested a role for membrane-type matrix metalloproteinases (MT-MMPs) in transmigration. Inhibitory antibodies and small interference RNA directed against MT1-MMP blocked transmigration, whereas overexpression of MT1-MMP in endothelial cells or monocytes promoted transmigration. MT1-MMP mediated the ectodomain cleavage of ICAM-1 that was blocked by TIMP-2 and -3. Overexpression of MT1-MMP rescued function in ICAM-1Y485A, and to a lesser extent in the cytoplasmic tail-deleted ICAM-1. In a binding assay, wild-type ICAM-1 bound to purified MT1-MMP while ICAM-1 mutants bound poorly. MT1-MMP co-localized with ICAM-1 at distinct structures in endothelial cells. MT1-MMP localization with cells expressing ICAM-1 mutations was reduced and diffused. These results indicate that the cytoplasmic tail of ICAM-1 regulates leukocyte transmigration through MT1-MMP interaction.     

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.     

Src homology domain 2-containing tyrosine phosphatase 2 associates with intercellular adhesion molecule 1 to regulate cell survival

Intercellular adhesion molecule-1 (ICAM-1) binds to the plasma protein fibrinogen (Fg) to mediate leukocyte/endothelial cell interactions. In our studies, the ligation of Fg to ICAM-1 on tumor necrosis factor-alpha-stimulated endothelial cells resulted in the tyrosine phosphorylation of Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). The ICAM-1 cytoplasmic sequence IKKYRLQ conforms poorly to the concensus immunoreceptor tyrosine-based inhibition motifs found in receptors that bind SHP-2. Nevertheless, the tyrosine phosphorylated sequence (IKKpYRLQ) bound specifically to the SH2 domain proximal to the NH(2)-terminal of SHP-2 (SHP-2-N) but not to the SH2 domain proximal on the COOH-terminal side (SHP-2-C). Phosphorylated ICAM-1 bound SHP-2-N. In immunoprecipitation experiments, SHP-2 associated with phosphorylated ICAM-1. Cells expressing truncated ICAM-1 that lacked the cytoplasmic sequence (ICAM-1(TR)) failed to associate with SHP-2. ICAM-1 containing the tyrosine to alanine substitution at position 485 (ICAM-1(Y485A)) associated weakly with SHP-2. Cells expressing ICAM-1(TR) and ICAM-1(Y485A) underwent apoptosis upon adhesion to Fg, whereas the wild type ICAM-1 maintained cell survival. These results indicate that ICAM-1 interactions with SHP-2 allow better cellular survival mediated through Fg-ICAM-1 ligation.     

Ectodomain shedding of preadipocyte factor 1 (Pref-1) by tumor necrosis factor alpha converting enzyme (TACE) and inhibition of adipocyte differentiation

Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protein found in the preadipocytes, inhibits adipocyte differentiation in vitro and in vivo. Here, we examined the processing of membrane form of Pref-1A to release the 50-kDa soluble form that inhibits adipocyte differentiation. The ectodomain cleavage of Pref-1 is markedly enhanced by phorbol 12-myristate 13-acetate in a dose- and time-dependent manner. The basal and stimulated cleavage is inhibited by the broad metalloproteinase inhibitor GM6001, a fact that suggests that cleavage of membrane Pref-1A is dependent on a metalloproteinase. Next, we showed that release of soluble Pref-1A is inhibited by TAPI-0 and by a tissue inhibitor of metalloproteinase-3, TIMP-3, that can inhibit tumor necrosis factor alpha converting enzyme (TACE), but not by TIMP-1 or TIMP-2. On the other hand, overexpression of TACE increases Pref-1 cleavage to produce the 50-kDa soluble form. Furthermore, this cleavage was not detected in cells with TACE mutation or with TACE small interfering RNA. TACE-mediated shedding of Pref-1 ectodomain inhibits adipocyte differentiation of 3T3-L1 cells and in Pref-1-null mouse embryo fibroblasts transduced with Pref-1A. Identification of TACE as the major protease responsible for conversion of membrane-bound Pref-1 to the biologically active diffusible form provides a new insight into Pref-1 function in adipocyte differentiation.     

Nuclear translocation of cleaved LyGDI dissociated from Rho and Rac during Trp53-dependent ionizing radiation-induced apoptosis of thymus cells in vitro

LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells. Here we report truncation of LyGDI after irradiation in mouse 3SB thymus cells. A 21-kDa fragment of LyGDI, resulting from activated caspase 3-induced cleavage at an N-terminal consensus site following the Asp(18) residue, accumulated at peak quantities between 5 and 12 h after irradiation. Cleavage of LyGDI was inhibited by the caspase inhibitor benzoyloxycarbonyl-Val-Asp-fluoromethylketone. Subcellular fractionation and immunofluorescence revealed the truncated 21-kDa fragment of LyGDI within the nuclear fraction of irradiated 3SB cells, whereas full-length LyGDI was found only in the cytoplasmic fraction. Truncated LyGDI within the nucleus had no association with the Rho family proteins RhoA and Rac1, since these proteins were observed only in the cytoplasmic fractions. These data demonstrate that regulation of Rho family GTPases by LyGDI is disrupted during apoptosis, suggesting that fragmentation of LyGDI implicates the transmission of a signal from the cytoplasm to the nucleus during Trp53-dependent apoptosis of thymus cells after irradiation.     

Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors

Calpain inhibitors induce pertussis toxin (PTx)-sensitive chemotaxis in human neutrophils and monocytes. Here, we show that various calpain inhibitors (PD150606, PD151746, N-acetyl-Leu-Leu-Nle-CHO [ALLN], N-acetyl-Leu-Leu-Met-CHO [ALLM], and calpeptin) and γ-secretase inhibitor I induced PTx-sensitive increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) in human neutrophils and neutrophil migration. HEK-293 cells stably expressing human formyl peptide receptor (hFPR) or hFPR-like 1 (hFPRL1) displayed stimulus-specific increase in [Ca²⁺]_i in response to calpain inhibitors (PD150606, PD151746, ALLN, ALLM, MG-132, and calpeptin), γ-secretase inhibitor I, and N-formyl-Met-Leu-Phe. Parent HEK-293 cells also displayed PTx-sensitive increase in [Ca²⁺]_i in response to calpeptin and γ-secretase inhibitor I, whereas they displayed PTx-resistant increase in [Ca²⁺]_i in response to MG-132. MDL-28170 induced neither an increase in [Ca²⁺]_i in neutrophils and HEK-293 cells nor neutrophil migration. Ionomycin-induced cleavage of talin (a substrate of calpain) in neutrophils was inhibited by all inhibitors used here. These findings suggest that potent calpain inhibitors could stimulate phagocyte functions via activation of hFPR, hFPRL1 and/or other G-protein coupled receptors depending on the inhibitors used.     

Human ether-à-go-go gene potassium channels are regulated by EGFR tyrosine kinase

Human ether á-go-go gene potassium channels (hEAG1 or Kv10.1) are expressed in brain and various human cancers and play a role in neuronal excitement and tumor progression. However, the functional regulation of hEAG channels by signal transduction is not fully understood. The present study was therefore designed to investigate whether hEAG1 channels are regulated by protein tyrosine kinases (PTKs) in HEK 293 cells stably expressing hEAG1 gene using whole-cell patch voltage-clamp, immunoprecipitation, Western blot, and mutagenesis approaches. We found that the selective epidermal growth factor receptor (EGFR) kinase inhibitor AG556 (10 μM), but not the platelet growth factor receptor (PDGFR) kinase inhibitor AG1295 (10 μM) or the Src-family inhibitor PP2 (10 μM), can inhibit hEAG1 current, and the inhibitory effect can be reversed by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of hEAG1 channels was reduced by AG556, and the reduction was significantly countered by orthovanadate. The hEAG1 mutants Y90A, Y344A and Y485A, but not Y376A and Y479A, exhibited reduced response to AG556. Interestingly, the inhibition effect of AG556 was lost in triple mutant hEAG1 channels at Y90, Y344, and Y485 with alanine. These results demonstrate for the first time that hEAG1 channel activity is regulated by EGFR kinase at the tyrosine residues Tyr90, Try344, and Try485. This effect is likely involved in regulating neuronal activity and/or tumor growth.     

Ligand-induced shedding of discoidin domain receptor 1

Tyrosine kinases belonging to the discoidin domain receptor (DDR) family are activated upon stimulation with various types of collagen. In response to collagen treatment, immunoprecipitation of DDR1 with an antibody specific to the juxtamembrane region results in co-purification of a previously unrecognized tyrosine phosphorylated protein of 62 kDa molecular weight. Here, this protein is identified as C-terminal cleavage product of the full-length DDR1 receptor and a DDR1-specific shedding enzyme postulated. Shedding of DDR1 can be partially blocked by the furin inhibitor decanoyl-RVKR-chloromethylketone and completely inhibited by the hydroxamate-based inhibitor batimastat. The characteristic of the DDR1 sheddase to be blocked by batimastat suggests that it belongs to the membrane-bound matrix metalloproteinase or disintegrin and metalloproteinase family of proteases.     

Functional signaling of membrane-bound TL1A induces IFN-γ expression

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4⁺ T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-γ that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-γ secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.     

Calcium Induces Expression of Cytoplasmic Gelsolin in SH-SY5Y and HEK-293 Cells

Gelsolin plays an important role in the regulation of amyloid beta-protein fibrillogenesis. We report here that calcium ionophore A23187 induces the expression of cytoplasmic gelsolin (c-gelsolin), and that protein kinase C (PKC) is involved in the up-regulation of c-gelsolin. In the presence of calcium, both SH-SY5Y and HEK-293 cells upon treatment with A23187 showed an increase in c-gelsolin expression in a concentration-dependent manner. Calcium-mediated up-regulation of c-gelsolin was inhibited by cycloheximide (a general inhibitor of protein synthesis). When cells were pretreated with staurosporine (an inhibitor of a variety of protein kinases including PKC), the up-regulation of c-gelsolin induced by A23187 was inhibited. Calphostin C, an inhibitor of PKC, blocked the up-regulation of c-gelsolin induced by A23187, while inhibitors of mitogen-activated protein kinases had no effect on c-gelsolin expression. In addition, phorbol-12-myristate-13-acetate, an activator of PKC, up-regulated c-gelsolin expression. These results suggest that calcium mediates up-regulation of c-gelsolin in a PKC-dependent manner.     

The cytoplasmic domain of L-selectin participates in regulating L-selectin endoproteolysis

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.     

Molecular analysis of tyrosine-and phenylalanine-mediated repression of the tyrB promoter by the TyrR protein of Escherichia coli

Listeriolysin O (LLO) is a pore-forming cytolysin secreted by the pathogen Listeria monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF-kappaB signalling pathway. In this work, we studied the LLO-induced molecular cascade of NF-kappaB activation with a cellular model extensively used to analyse the signalling pathway of NF-kappaB activation, i.e. the human embryonic kidney HEK-293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK-293 cells expressing IL-1RI were exposed to LLO, a strong NF-kappaB activation was detected, contrasting with other cell lines (HEK-293 wild type, HEK-293.T and COS) expressing a very low level of IL-1RI. Although a delayed kinetics of LLO-dependent NF-kappaB activation suggests an autocrine or paracrine IL-1-dependent pathway, we found that LLO-dependent NF-kappaB activation did not require the IL-1 protein synthesis nor the interaction with the IL-1RI specific receptor. Herein, we demonstrated that LLO-dependent NF-kappaB activation requires the activation of the IkappaB kinase beta (IKKbeta) subunit of IKK complex to phosphorylate and degrade cytoplasmic IkappaBalpha, a natural inhibitor of NF-kappaB. The activation induced by LLO does not require the adapters MyD88 and IL-1R-associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL-1 and its receptor.     

Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain

Membrane-type-1 matrix metalloproteinase (MT1-MMP) has transmembrane and cytoplasmic domains, which target it to invasive fronts. We analyzed the role of the cytoplasmic tail by expressing wild type MT1-MMP and three mutants with progressively truncated C termini in human Bowes melanoma cells. We examined gelatinase A activation and the localization and processing of recombinant proteins in stable cell clones using gelatin zymography, immunoblotting, and immunofluorescence. Cell invasion was analyzed in vitro by Matrigel invasion assays. Gelatinase A was activated in all cell clones. However, the localization of MT1-MMP to the leading edge of migrating cells and cell invasion through Matrigel were strongly enhanced only in cells expressing either wild type or truncated MT1-MMP lacking 6 C-terminal amino acid residues (Delta577). Truncations of 10 or 16 amino acid residues in the cytoplasmic domain (Delta567 and Delta573, respectively) disturbed MT1-MMP localization. The expression of wild type and Delta577 MT1-MMPs induced also their cleavage to 43-kDa cell surface forms and the release of soluble, approximately 20-kDa N-terminal fragments containing the catalytic center. A synthetic MMP inhibitor but not a gelatinase inhibitor prevented the processing, suggesting that autocatalytic cleavage occurs. Purified soluble MT1-MMP was also autoproteolytically processed to 43- and 20-kDa forms in vitro. Our results indicate that the cytoplasmic domain has an important role in cell invasion by controlling both the targeting and degradation/turnover of MT1-MMP.     

Ectodomain shedding of nectin-1alpha by SF/HGF and TPA in MDCK cells

Nectin is a Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecule implicated in the organization of the junctional complex comprised of E-cadherin-based adherens junctions and claudin-based tight junctions in epithelial cells. Scatter factor (SF)/hepatocyte growth factor (HGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor-promoting phorbol ester, induce cell spreading, followed by cell-cell dissociation and cell scattering, in Madin-Darby canine kidney (MDCK) cells. We found here that SF/HGF and TPA induced proteolytic cleavage of nectin-1alpha in the ectodomain, resulting in generation of the 80-kDa extracellular fragment and the 33-kDa fragment composed of the transmembrane and cytoplasmic domains, in MDCK cells. This shedding of nectin-1alpha was inhibited by metalloprotease inhibitors. These results indicate that SF/HGF and TPA induce the ectodomain shedding of nectin-1alpha presumably by a metalloprotease, and have raised the possibility that this shedding is involved in the SF/HGF- and TPA-induced cell-cell dissociation.     

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.     

Shedding of the type II IL-1 decoy receptor requires a multifunctional aminopeptidase, aminopeptidase regulator of TNF receptor type 1 shedding

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.     

The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells

Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1-7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1-7). The shedding of sPTK7-Ig1-7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1-7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1-7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase.     

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP-14) cleaves and releases a 22-kDa extracellular matrix metalloproteinase inducer (EMMPRIN) fragment from tumor cells

Proteolytic shedding is an important step in the functional down-regulation and turnover of most membrane proteins at the cell surface. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a multifunctional glycoprotein that has two Ig-like domains in its extracellular portion and functions in cell adhesion as an inducer of matrix metalloproteinase (MMP) expression in surrounding cells. Although the shedding of EMMPRIN is reportedly because of cleavage by metalloproteinases, the responsible proteases, cleavage sites, and stimulants are not yet known. In this study, we found that human tumor HT1080 and A431 cells shed a 22-kDa EMMPRIN fragment into the culture medium. The shedding was enhanced by phorbol 12-myristate 13-acetate and inhibited by TIMP-2 but not by TIMP-1, suggesting the involvement of membrane-type MMPs (MT-MMPs). Indeed, down-regulation of the MT1-MMP expression in A431 cells using small interfering RNA inhibited the shedding. The 22-kDa fragment was purified, and the C-terminal amino acid was determined. A synthetic peptide spanning the cutting site was cleaved by MT1-MMP in vitro. The cleavage site is located in the linker region connecting the two Ig-like domains. The N-terminal Ig-like domain is important for the MMP inducing activity of EMMPRIN and for cell-cell interactions, presumably through its ability to engage in homophilic interactions, and the 22-kDa fragment retained the ability to augment MMP-2 expression in human fibroblasts. Thus, the MT1-MMP-dependent cleavage eliminates the functional N-terminal domain of EMMPRIN from the cell surface, which is expected to down-regulate its function. At the same time, the released 22-kDa fragment may mediate the expression of MMPs in tumor tissues.     

ADAM10 mediates ectodomain shedding of the betacellulin precursor activated by p-aminophenylmercuric acetate and extracellular calcium influx

Betacellulin belongs to the family of epidermal growth factor-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release a soluble mature growth factor. In this study, we investigated the ectodomain shedding of the betacellulin precursor (pro-BTC) in conditionally immortalized wild-type (WT) and ADAM-deficient cell lines. Sequential ectodomain cleavage of the predominant cell-surface 40-kDa form of pro-BTC generated a major (26-28 kDa) and two minor (20 and 15 kDa) soluble forms and a cellular remnant lacking the ectodomain (12 kDa). Pro-BTC shedding was activated by calcium ionophore (A23187) and by the metalloprotease activator p-aminophenylmercuric acetate (APMA), but not by phorbol esters. Culturing cells in calcium-free medium or with the protein kinase Cdelta inhibitor rottlerin, but not with broad-based protein kinase C inhibitors, blocked A23187-activated pro-BTC shedding. These same treatments were without effect for constitutive and APMA-induced cleavage events. All pro-BTC shedding was blocked by treatment with a broad-spectrum metalloprotease inhibitor (GM6001). In addition, constitutive and activated pro-BTC shedding was differentially blocked by TIMP-1 or TIMP-3, but was insensitive to treatment with TIMP-2. Pro-BTC shedding was functional in cells from ADAM17- and ADAM9-deficient mice and in cells overexpressing WT or catalytically inactive ADAM17. In contrast, overexpression of WT ADAM10 enhanced constitutive and activated shedding of pro-BTC, whereas overexpression of catalytically inactive ADAM10 reduced shedding. These results demonstrate, for the first time, activated pro-BTC shedding in response to extracellular calcium influx and APMA and provide evidence that ADAM10 mediates constitutive and activated pro-BTC shedding.