The Elastin Receptor Complex Transduces Signals through the Catalytic Activity of Its Neu-1 Subunit
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m609505200
ISSN1083-351X
AutoresLaurent Duca, Charlotte Blanchevoye, Benoît Cantarelli, Christelle Ghoneim, Stéphane Dedieu, Frédéric Delacoux, William Hornebeck, Aleksander Hinek, Laurent Martiny, Laurent Debelle,
Tópico(s)Connective tissue disorders research
ResumoThe binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates. The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates. Elastin is the extracellular matrix protein responsible for the elasticity of tissues. It is more abundant in tissues where resilience is required, such as skin, lung, ligaments, or large arteries (1Kielty C.M. Sherratt M.J. Shuttleworth C.A. J. Cell Sci. 2002; 115: 2817-2828Crossref PubMed Google Scholar). Elastin is constituted of tropoelastin molecules covalently bound to each other by covalent cross-links (2Vrhovski B. Weiss A.S. Eur. J. Biochem. 1998; 258: 1-18Crossref PubMed Scopus (387) Google Scholar) and its hydrophobic and highly cross-linked nature make of it a very durable polymer experiencing essentially no turnover in healthy tissues (3Shapiro S.D. Endicott S.K. Province M.A. Pierce J.A. Campbell E.J. J. Clin. Investig. 1991; 87: 1828-1834Crossref PubMed Scopus (458) Google Scholar).The biological role of elastin was originally thought to be restricted to this mechanical function. However, when Senior et al. (4Senior R.M. Griffin G.L. Mecham R.P. J. Clin. Investig. 1980; 66: 859-862Crossref PubMed Scopus (388) Google Scholar) demonstrated that elastin digestion products were chemotactic for neutrophils and macrophages, it became suddenly apparent that peptides derived from amorphous elastin could modulate cell physiology. In fact, it has been shown because that fibroblasts (5Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 6Grosso L.E. Scott M. Matrix. 1993; 13: 157-164Crossref PubMed Scopus (23) Google Scholar, 7Jacob M.P. Fulop Jr., T. Foris G. Robert L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 995-999Crossref PubMed Scopus (119) Google Scholar, 8Kamoun A. Landeau J.M. Godeau G. Wallach J. Duchesnay A. Pellat B. Hornebeck W. Cell Adhes. Commun. 1995; 3: 273-281Crossref PubMed Scopus (80) Google Scholar, 9Long M.M. King V.J. Prasad K.U. Urry D.W. Biochim. Biophys. Acta. 1988; 968: 300-311Crossref PubMed Scopus (35) Google Scholar, 10Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar, 11Senior R.M. Griffin G.L. Mecham R.P. Wrenn D.S. Prasad K.U. Urry D.W. J. Cell Biol. 1984; 99: 870-874Crossref PubMed Scopus (314) Google Scholar, 12Tajima S. Wachi H. Uemura Y. Okamoto K. Arch. Dermatol. Res. 1997; 289: 489-492Crossref PubMed Scopus (56) Google Scholar), smooth muscle cells (7Jacob M.P. Fulop Jr., T. Foris G. Robert L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 995-999Crossref PubMed Scopus (119) Google Scholar, 13Mochizuki S. Brassart B. Hinek A. J. Biol. Chem. 2002; 277: 44854-44863Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 14Ooyama T. Fukuda K. Oda H. Nakamura H. Hikita Y. Arteriosclerosis. 1987; 7: 593-598Crossref PubMed Google Scholar, 15Wachi H. Seyama Y. Yamashita S. Suganami H. Uemura Y. Okamoto K. Yamada H. Tajima S. FEBS Lett. 1995; 368: 215-219Crossref PubMed Scopus (50) Google Scholar), endothelial cells (16Faury G. Garnier S. Weiss A.S. Wallach J. Fulop Jr., T. Jacob M.P. Mecham R.P. Robert L. Verdetti J. Circ. Res. 1998; 82: 328-336Crossref PubMed Scopus (95) Google Scholar, 17Faury G. Ristori M.T. Verdetti J. Jacob M.P. Robert L. J. Vasc. Res. 1995; 32: 112-119Crossref PubMed Scopus (108) Google Scholar, 18Long M.M. King V.J. Prasad K.U. Freeman B.A. Urry D.W. J. Cell. Physiol. 1989; 140: 512-518Crossref PubMed Scopus (54) Google Scholar, 19Ostuni A. Lograno M.D. Gasbarro A.R. Bisaccia F. Tamburro A.M. Int. J. Biochem. Cell Biol. 2002; 34: 130-135Crossref PubMed Scopus (17) Google Scholar), leukocytes (20Hauck M. Seres I. Kiss I. Saulnier J. Mohacsi A. Wallach J. Fulop Jr., T. Biochem. Mol Biol. Int. 1995; 37: 45-55PubMed Google Scholar, 21Varga Z. Jacob M.P. Robert L. Fulop Jr., T. FEBS Lett. 1989; 258: 5-8Crossref PubMed Scopus (81) Google Scholar), and lymphocytes (22Peterszegi G. Texier S. Robert L. Eur. J. Clin. Investig. 1999; 29: 166-172Crossref PubMed Scopus (54) Google Scholar) were sensitive to the presence of these peptides yielding a broad range of biological activities (see Ref. 23Duca L. Floquet N. Alix A.J. Haye B. Debelle L. Crit. Rev. Oncol. Hematol. 2004; 49: 235-244Crossref PubMed Scopus (152) Google Scholar for a review). A corollary of these observations was that those cells do express a receptor for elastin peptides.The elastin receptor complex is constituted of three subunits, one peripheral 67-kDa subunit, which actually binds elastin, and two membrane-associated proteins of 61 and 55 kDa, respectively (10Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar). The 67-kDa elastin-binding protein (EBP) 2The abbreviations used are: EBP, elastin-binding protein (accession number P16279); PMSF, phenylmethylsulfonyl fluoride; β-Gal, lysosomal β-galactosidase (accession number P16278); PPCA, protective protein/cathepsin A (accession number P10619); Neu-1, lysosomal neuraminidase (accession number Q99519); Neu5Ac, N-acetyl-α-d-neuraminic acid; Muf-NANA, 4-methylumbelliferyl-N-acetyl-neuraminic acid; ddNeu5Ac, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; pro-MMP-1, pro-collagenase-1; ERK, extracellular signal-regulated kinase; kE, κ-elastin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline. 2The abbreviations used are: EBP, elastin-binding protein (accession number P16279); PMSF, phenylmethylsulfonyl fluoride; β-Gal, lysosomal β-galactosidase (accession number P16278); PPCA, protective protein/cathepsin A (accession number P10619); Neu-1, lysosomal neuraminidase (accession number Q99519); Neu5Ac, N-acetyl-α-d-neuraminic acid; Muf-NANA, 4-methylumbelliferyl-N-acetyl-neuraminic acid; ddNeu5Ac, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; pro-MMP-1, pro-collagenase-1; ERK, extracellular signal-regulated kinase; kE, κ-elastin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline. binds the VGVAPG elastin sequence with high affinity. Additionally, EBP can be eluted from elastin affinity column by galactosugars suggesting that the elastin-EBP interaction could be regulated by galactosugars bound on a lectin site on EBP (10Mecham R.P. Hinek A. Entwistle R. Wrenn D.S. Griffin G.L. Senior R.M. Biochemistry. 1989; 28: 3716-3722Crossref PubMed Scopus (160) Google Scholar, 24Hinek A. Wrenn D.S. Mecham R.P. Barondes S.H. Science. 1988; 239: 1539-1541Crossref PubMed Scopus (255) Google Scholar). Consequently, galactosugars such as lactose are commonly used antagonists of EBP.Later, the nature of this subunit was revealed by the work of Privitera et al. (25Privitera S. Prody C.A. Callahan J.W. Hinek A. J. Biol. Chem. 1998; 273: 6319-6326Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) who have shown that EBP is an enzymatically spliced variant of lysosomal β-galactosidase (β-Gal, EC 3.2.1.23). Consequently, it was hypothesized that the 61 and 55 kDa membrane-bound subunits corresponded to the lysosomal companions of β-Gal, respectively neuraminidase-1 (Neu-1, EC 3.2.1.18) and cathepsin A/protective protein (PPCA, EC 3.4.16.1) (26Callahan J.W. Biochim. Biophys. Acta. 1999; 1455: 85-103Crossref PubMed Scopus (99) Google Scholar). This view was strongly supported by the fact that Neu-1 and PPCA are found at the plasma membrane of several cell types (27Lukong K.E. Seyrantepe V. Landry K. Trudel S. Ahmad A. Gahl W.A. Lefrancois S. Morales C.R. Pshezhetsky A.V. J. Biol. Chem. 2001; 276: 46172-46181Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and by the finding that β-Gal-related disorders are associated with elastic fibers abnormalities (28Caciotti A. Donati M.A. Procopio E. Filocamo M. Kleijer W. Wuyts W. Blaumeiser B. d'Azzo A. Simi L. Orlando C. McKenzie F. Fiumara A. Zammarchi E. Morrone A. Hum. Mutat. 2007; 28: 204Crossref PubMed Scopus (25) Google Scholar, 29Caciotti A. Donati M.A. Bardelli T. d'Azzo A. Massai G. Luciani L. Zammarchi E. Morrone A. Am. J. Pathol. 2005; 167: 1689-1698Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Finally, a very recent report has readily identified the elastin receptor complex as the association of these three subunits (30Hinek A. Pshezhetsky A.V. von Itzstein M. Starcher B. J. Biol. Chem. 2006; 281: 3698-3710Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In the lysosome, PPCA protects β-Gal and Neu-1 from intralysosomal digestion independently of its serine protease activity by forming a complex with these enzymes (26Callahan J.W. Biochim. Biophys. Acta. 1999; 1455: 85-103Crossref PubMed Scopus (99) Google Scholar).Neu-1 is a member of the sialidase family and catalyzes the hydrolysis of terminal sialic acid residues of oligosaccharides, glycoproteins, and glycolipids (31Achyuthan K.E. Achyuthan A.M. Comp Biochem. Physiol. B. Biochem. Mol. Biol. 2001; 129: 29-64Crossref PubMed Scopus (111) Google Scholar, 32Seyrantepe V. Poupetova H. Froissart R. Zabot M.T. Maire I. Pshezhetsky A.V. Hum. Mutat. 2003; 22: 343-352Crossref PubMed Scopus (120) Google Scholar). Importantly, it has been shown in vitro that the removal of β-Gal or PPCA hinders the formation of active neuraminidase, indicating that all three components of the complex are required for neuraminidase activity (33van der Horst G.T. Galjart N.J. d'Azzo A. Galjaard H. Verheijen F.W. J. Biol. Chem. 1989; 264: 1317-1322Abstract Full Text PDF PubMed Google Scholar).Beside its role in elastin assembly (30Hinek A. Pshezhetsky A.V. von Itzstein M. Starcher B. J. Biol. Chem. 2006; 281: 3698-3710Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), several studies have pointed out that the elastin complex receptor is able to transduce intracellular signals (23Duca L. Floquet N. Alix A.J. Haye B. Debelle L. Crit. Rev. Oncol. Hematol. 2004; 49: 235-244Crossref PubMed Scopus (152) Google Scholar). Our group has previously demonstrated that elastin hydrolysates and κ-elastin (kE), elastin peptides derived from the alcaline degradation of insoluble elastin, both stimulate the production of pro-MMP-1 in human skin fibroblasts (5Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) by activation of the ERK1/2 pathway (34Duca L. Debelle L. Debret R. Antonicelli F. Hornebeck W. Haye B. FEBS Lett. 2002; 524: 193-198Crossref PubMed Scopus (34) Google Scholar, 35Duca L. Lambert E. Debret R. Rothhut B. Blanchevoye C. Delacoux F. Hornebeck W. Martiny L. Debelle L. Mol. Pharmacol. 2005; 67: 1315-1324Crossref PubMed Scopus (44) Google Scholar).The induction of signaling pathways by the elastin receptor complex is now commonly accepted but remains discussed, notably because a critical issue is undocumented: how is the elastin peptides binding information processed by the receptor, i.e. how does the receptor transduce this signal from the extracellular face of the membrane to the cytoplasmic one? This work aims at answering this important question.We show here that binding of elastin peptides to EBP stimulate Neu-1 sialidase activity. Using pharmacological and genetic techniques, we demonstrate that the induction of this sialidase activity is responsible for elastin receptor complex-dependent signaling assessed by measuring pro-MMP-1 production and ERK1/2 activation. Additionally, we show that the serine protease activity of PPCA is not required for this signaling. Finally, we suggest that the sialic acid released by Neu-1 activity could act as a second messenger and transduce elastin complex receptor signaling.EXPERIMENTAL PROCEDURESMaterials—Elastin peptides were prepared as described previously (5Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Briefly, insoluble elastin was prepared from bovine ligamentum nuchae by hot alkali treatment. Its purity was assessed by comparing its amino acid composition to that predicted from the elastin gene product. Soluble elastin peptides were obtained from insoluble elastin by organo-alkaline hydrolysis. This was achieved using 1 m KOH in 80% aqueous ethanol (36Debelle L. Alix A.J. Jacob M.P. Huvenne J.P. Berjot M. Sombret B. Legrand P. J. Biol. Chem. 1995; 270: 26099-26103Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The obtained mixture of elastin peptides is termed κ-elastin (kE) and exhibits the same biological and physical properties as elastin hydrolysates (5Brassart B. Fuchs P. Huet E. Alix A.J. Wallach J. Tamburro A.M. Delacoux F. Haye B. Emonard H. Hornebeck W. Debelle L. J. Biol. Chem. 2001; 276: 5222-5227Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar).Lactose, aprotinin, phenylmethylsulfonyl fluoride (PMSF), 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid, N-acetylneuraminic acid, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (ddNeu5Ac), proteases inhibitors mixture (reference P8340) and mouse monoclonal anti-β-actin antibody were from Sigma (Saint-Quentin Fallavier, France). Silencer predesigned Neu-1 small interfering RNA (siRNA) and negative control siRNA were purchased from Ambion (distributed by Applied Biosystems, Courtaboeuf, France). Rabbit polyclonal phospho-specific antibodies against active forms of ERK1/2 (phosphorylated on Thr202 and Tyr204) were from Cell Signaling Technology Inc. (Beverly, MA, distributed by Ozyme, Saint Quentin en Yvelines, France). Rabbit polyclonal anti-Neu-1 antibody was purchased from Abcam (Cambridge, UK). The sheep anti-human MMP-1 polyclonal antibody was from Calbiochem (distributed by VWR International, Strasbourg, France). All reagents for cell culture and transfection reagent Lipofectamine 2000 were from Invitrogen (Cergy Pontoise, France). Enhanced chemiluminescence substrate kit was purchased from Amersham Biosciences Inc. (Orsay, France). N-Acetylneuraminic acid (Neu5Ac) was a generous gift of Dr. C. Augé (Laboratoire de Chimie Organique Multifonctionnelle, University of Paris-Sud, France). Others reagents were from Sigma.Expression Plasmids—The –512ColA-luc plasmid encoding luciferase under the control of the MMP-1 promoter was generated by cloning the 5′ upstream sequence of human collagenase-1 (MMP-1) into the pGL3Basic reporter plasmid (Promega, Lyon, France), as described previously (37Rutter J.L. Benbow U. Coon C.I. Brinckerhoff C.E. J. Cell. Biochem. 1997; 66: 322-336Crossref PubMed Scopus (111) Google Scholar). The plasmid encoding Neu-1 neuraminidase (pSCTop-Neu-1) was kindly provided by Dr. A. d'Azzo (Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis).Neu-1-G68V Construct—The Neu-1-G68V mutant was obtained from the wild type plasmid using PCR site-directed mutagenesis. The target site was identified and two complementary oligonucleotides containing the desired mutation were synthesized (Invitrogen, Cergy-Pontoise, France). The sequence of the two oligonucleotides with mutated bases are 5′-CTG TGG GTG AGC GTG AGA CAG ATC GGC 3′ and 5′ GAC ACC CAC TCG CAC TCT GTC TAG CCG-3′. The reaction was performed in a final volume of 50 μl. The sample reaction contained 1 μl of Pfu Turbo DNA polymerase, 5 μl of 10× buffer (20 mm MgSO4), 50 ng of pSCTop-Neu-1 preparation, 125 ng of each oligonucleotide, 10 mm dNTP, and H2O. The reaction was performed using a Mastercycler gradient thermocycler (Eppendorf, Le Pecq, France) and subjected to the following program: denaturation of 30 s at 95 °C, 12 cycles of 30 s at 95 °C, 1 min at 55 °C and 6 min at 68 °C. The sample was digested by 1 μl of DpnI (10 unit/μl) for 3 h at 37 °C and chemically transformed into the XL1 Blue strain. Cells were applied on a LB agar plate containing 50 μg/ml ampicillin and incubated overnight at 37 °C. The obtained clones were sequenced (IBMP, Strasbourg). The plasmid was then concentrated and purified by High Speed Maxi Prep kit (Qiagen, Courtaboeuf, France).Fibroblast Culture and Treatments—Human skin fibroblast strains were established from explants of human adult skin biopsies obtained from informed healthy volunteers (aged 21–49 years). Cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) containing 1 g/liter of glucose, glutamax I and pyruvate, supplemented with 10% fetal calf serum (FCS) and in the presence of 5% CO2. Cells at subcultures 4 to 8 were used. For experiments, fibroblasts were grown to subconfluence in medium containing 10% FCS. Before stimulation, cells were incubated for 18 h in DMEM supplemented with 0.5% FCS, washed twice with PBS, and incubated in serum-free DMEM with or without kE or Neu5Ac for the indicated times. The neuraminidase and serine protease inhibitors, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (ddNeu5Ac) and aprotinin or PMSF respectively, were incubated for 1 h before stimulation, whereas lactose (EBP antagonist) was incubated for 3 h. Inhibitors and antagonists were present in the cell culture media during the stimulation. Cell stimulation was stopped by adding ice-cold PBS containing 50 μm Na3VO4.Measurement of Sialidase Activity at the Plasma Membrane of Fibroblasts—We used the protocol described by Lukong et al. (27Lukong K.E. Seyrantepe V. Landry K. Trudel S. Ahmad A. Gahl W.A. Lefrancois S. Morales C.R. Pshezhetsky A.V. J. Biol. Chem. 2001; 276: 46172-46181Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) with minor modifications. The cells were seeded in 6-well culture dishes and washed several times with prewarmed PBS. They were then incubated in 1 ml of reaction buffer containing 20 mm CH3COONa, pH 6.5, 0.4 mm 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid (Muf-NANA) with or without kE, in the absence or presence of ddNeu5Ac or lactose. After incubation, 400-μl aliquots of medium were added to 3.6 ml of 0.4 m glycine buffer (pH 10.4), and the fluorescent 4-methylumbelliferone product released in the medium was quantitated by exciting its fluorescence at 365 nm and recording its intensity at 445 nm.Western Blotting—106 fibroblasts or COS-7 cells were washed twice in ice-cold PBS containing 50 μm Na3VO4, scrapped, and sonicated in lysis buffer (PBS, pH 7.4, 0.5% Triton X-100, 80 mm glycerophosphate, 50 mm EGTA, 15 mm MgCl2, 1 mm Na3VO4, and protease inhibitor mixture). Insoluble material was removed by centrifugation (20,000 × g, 20 min, 4 °C). Protein concentrations were determined by bicinchoninic acid protein assay (Pierce; distributed by Interchim, Montluçon, France). Equal amounts of proteins were heated for 5 min at 100 °C in Laemmli sample buffer, resolved by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. The membranes were placed in blocking buffer (5% (w/v) nonfat dry milk in Tris-buffered saline/Tween 20 (50 mm Tris, pH 7.5, 150 mm NaCl, and 0.1% (v/v) Tween 20)) for 1 h at room temperature and incubated overnight at 4 °C with anti-phospho-ERK1/2 (1:1000), anti-Neu-1 (1:500), or anti-β-actin (1:5000) antibodies. After five washings with Tris-buffered saline/Tween 20, the membranes were incubated for 1 h at room temperature in the presence of horseradish peroxidase-coupled anti-rabbit or anti-mouse antibodies (1:4,000 and 1:10,000 in blocking buffer, respectively).For detection of pro-MMP-1, the culture media of fibroblasts were harvested and centrifuged (500 × g, 10 min, 4 °C) to remove cellular debris. The supernatants were then concentrated using Microcon YM10 (Millipore, Molsheim, France) and equal amounts of proteins were subjected to SDS-PAGE and blotted as described above using the anti-human MMP-1 antibody (1:500) and a horseradish peroxidase-coupled anti-sheep antibody (1:10,000). Immunocomplexes were detected by chemiluminescence. Blots were quantitated by densitometry using the PhosphorAnalyst software (Bio-Rad, Marne-la-Vallée, France).Luciferase Assay of MMP-1 Promoter Activity—COS-7 cells were grown on 96-well plates to 80% of confluency (16,000 cells) in DMEM containing 4,5 g/liter of glucose, glutamax I, and pyruvate, supplemented with 10% FCS and in the presence of 5% CO2. For transfection experiments they were incubated for 24 h in serum-free DMEM then with a mixture of Lipofectamine 2000 and DNA plasmids during 4 h. The ratio used was 0.2 μl of Lipofectamine 2000 for 0,2 μg of total plasmidic DNA divided in 50% of pSCTop-Neu-1-G68V (or the corresponding empty vector) and 50% of –512ColA-luc plasmid. This proportion was used for 100 μl of DMEM per well. Cells were then incubated with or without kE (150 μg/ml) for 16 h. Elastin peptides stimulation was stopped and 100 μl of SteadyLite reporter lysis buffer were then added (PerkinElmer Life Sciences). Luciferase activities were measured with the PerkinElmer TopCount microplate counter.Neu-1 siRNA Gene Silencing—COS-7 cells were grown on 6-well plates to 70% confluency in DMEM containing 4.5 g/liter of glucose, glutamax I, and pyruvate, supplemented with 10% FCS and in the presence of 5% CO2. For siRNA transfection, we used the silencer predesigned Neu-1 siRNA from Ambion, (ID: 8481), as they are very effective Neu-1 siRNA (38Liang F. Seyrantepe V. Landry K. Ahmad R. Ahmad A. Stamatos N.M. Pshezhetsky A.V. J. Biol. Chem. 2006; 281: 27526-27538Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). After two PBS washes, cells were incubated for 24 h in serum-free DMEM with a mixture of Lipofectamine 2000 (5 μl) and Neu-1 or negative control siRNA (100 pmol). Cells were then washed twice with PBS then stimulated for the indicated time with kE. Cell stimulation was stopped by adding ice-cold PBS containing 50 μm Na3VO4 and ERK1/2 Western blots were conducted as described above.Statistical Analysis—All experiments were performed in triplicate. Results are expressed as mean ± S.E. Comparison between groups were made using Student's t test. The results were considered significantly different at p < 0.05.RESULTSEBP is required for elastin peptide signaling and it has been suggested that tropoelastin recruit EBP to the cell surface where the PPCA and Neu-1 reside thereby leading to the completion of the elastin receptor complex.In the lysosome, Neu-1 activity is highly dependent on the full association of the β-Gal/PPCA/Neu-1 complex. Indeed, it has been shown that the association of PPCA to Neu-1 only results in a weak activity of the sialidase but that the addition of β-Gal to this assembly achieves its full activation.By analogy to what is observed in the lysosome, we supposed that elastin peptide-driven recruitment of EBP to the membrane-bound PPCA/Neu-1 complex could stimulate the enzymatic activities of these sub-units and contribute to signal processing by the elastin receptor. As EBP is devoid of enzymatic activity, we analyzed the possible importance of PPCA and Neu-1 activities on elastin peptide signaling by the elastin receptor complex.Inhibition of PPCA and Neu-1 Activities and Impact on Signaling by the Elastin Receptor Complex—In a previous work, we have shown that the treatment of human skin fibroblasts with elastin peptides resulted in the accumulation of pro-collagenase-1 (pro-MMP-1) in the culture medium (34Duca L. Debelle L. Debret R. Antonicelli F. Hornebeck W. Haye B. FEBS Lett. 2002; 524: 193-198Crossref PubMed Scopus (34) Google Scholar), and that this effect was strictly mediated by ERK1/2 signaling (35Duca L. Lambert E. Debret R. Rothhut B. Blanchevoye C. Delacoux F. Hornebeck W. Martiny L. Debelle L. Mol. Pharmacol. 2005; 67: 1315-1324Crossref PubMed Scopus (44) Google Scholar). As a consequence, we have analyzed the influence of PPCA and Neu-1 inhibition on pro-MMP-1 accumulation and on ERK activation.When cultured fibroblasts were treated with aprotinin, an inhibitor of serine protease, and further stimulated with kE, we observed that PPCA inhibition did not prevent pro-MMP-1 accumulation in the medium following kE treatment (Fig. 1A) nor ERK1/2 induction (Fig. 1B). These results were confirmed using PMSF (Figs. 1, C and D) which is an efficient PPCA inhibitor (39Itoh K. Takiyama N. Kase R. Kondoh K. Sano A. Oshima A. Sakuraba H. Suzuki Y. J. Biol. Chem. 1993; 268: 1180-1186Abstract Full Text PDF PubMed Google Scholar). As a consequence, we concluded that PPCA enzymatic activity was not required for proper signaling by the elastin complex receptor.However, when the cells were treated with elastin peptides in the presence of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (ddNeu5Ac), a sialidase inhibitor (30Hinek A. Pshezhetsky A.V. von Itzstein M. Starcher B. J. Biol. Chem. 2006; 281: 3698-3710Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 40Usuki S. Hoops P. Sweeley C.C. J. Biol. Chem. 1988; 263: 10595-10599Abstract Full Text PDF PubMed Google Scholar), pro-MMP-1 accumulation in the culture medium was reduced in a dose-dependent manner (Fig. 2A). As at 400 μm ddNeu5Ac, the effect of elastin peptides on pro-MMP-1 production was almost totally blocked, we decided to further use this experimental concentration. In the presence of ddNeu5Ac, elastin peptide-treated cells have lost their ability to activate ERK1/2 (Fig. 2B). Indeed, ERK1/2 phosphorylation level in the presence of both the inhibitor and kE is comparable to that of the control in the absence of kE stimulation. These results strongly suggested that a sialidase activity was required for elastin peptide signaling. However, we could not precisely determine which enzyme was involved as ddNeu5Ac is not specific of a sialidase form. As a consequence, we could not link this activity to that of the elastin receptor complex.FIGURE 2Influence of elastin peptide treatment on pro-MMP-1 production and ERK activation in the presence of ddNeu5Ac. A, Western blots analysis of pro-MMP-1 production. Cells were pretreated for 1 h with various doses of ddNeu5Ac then stimulated for 24 h with kE (50 μg/ml). Only the nonglycosylated (53 kDa) form of pro-MMP-1 is detected (see Fig. 1 legend). B, Western blot analysis of cellular extracts. Cells were pretreated with 400 μm ddNeu5Ac prior to stimulation (50 μg kE/ml; 30 min). Membranes were probed with anti-phospho-Thr202/Tyr204-ERK1/2 polyclonal antibodies. To demonstrate equal loading, blots were stripped and reprobed with an anti-β-actin antibody. Blots are representative of three independent experiments with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Sialidase Activity at the Cell Surface of Fibroblast in Culture—The Muf-NANA compound is a neuraminidase substrate, which becomes fluorescent when it is cleaved by the enzyme. The product is detected at 445 nm when it is excited at 365 nm (27Lukong K.E. Seyrantepe V. Landry K. Trudel S. Ahmad A. Gahl W.A. Lefrancois S. Morales C.R. Pshezhetsky A.V. J. Biol. Chem. 2001; 276: 46172-46181Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Using Muf-NANA, we have shown that untreated human skin fibroblasts exhibited a significant sialidase activity at their surface (Fig. 3, open circles). As a consequence the fluorescence level increased with time as the fluorescent product of Muf-NANA processing was released in the culture medium. When they were treated with kE (Fig. 3, open squares), this level was signi
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