Cross-Talk Between RhoGTPases and Stress Activated Kinases for Matrix Metalloproteinase-9 Induction in Response to Keratinocytes Injury
2003; Elsevier BV; Volume: 121; Issue: 6 Linguagem: Inglês
10.1111/j.1523-1747.2003.12627.x
ISSN1523-1747
AutoresIsabelle Bourget, Emmanuel Lemichez, Laurent Turchi, Anne‐Amandine Chassot, Christine Baldescchi, Jean Paul Ortonne, Guerrino Meneguzzi, Gilles Ponzio,
Tópico(s)Signaling Pathways in Disease
ResumoCell migration and extracellular matrix remodeling are two essential processes of wound healing, regulated by extracellular metalloproteinases such as matrix metalloproteinase-2 (Gelatinase A) and matrix metalloproteinase-9 (Gelatinase B). Expression of matrix metalloproteinase-9 is deregulated in numerous wound healing pathologies. To date the mechanisms regulating matrix metalloproteinase-9 during normal wound healing are poorly documented. Using both primary cultures of normal human keratinocytes and a wounding device especially designed to dissect the molecular events during the healing process in vitro, we show that matrix metalloproteinase-9 is stimulated by injury in normal human keratinocytes. This upregulation results from the mechanical stress created by injury and not from a soluble factor, secreted by wounded normal human keratinocytes. We also demonstrate that the Rho family of small GTPases, p38[MAPK] and JNK together play a key part in the signaling pathways controlling the stimulation of matrix metalloproteinase-9 in wounded cells. We provide lines of evidence indicating that in wounded keratinocytes, upregulation of matrix metalloproteinase-9 depends on two distinct pathways. The first involves Rac1 and/or Cdc42 that control the activation of p38[MAPK]. The second depends on RhoA activation that is required for stimulation of JNK. Cell migration and extracellular matrix remodeling are two essential processes of wound healing, regulated by extracellular metalloproteinases such as matrix metalloproteinase-2 (Gelatinase A) and matrix metalloproteinase-9 (Gelatinase B). Expression of matrix metalloproteinase-9 is deregulated in numerous wound healing pathologies. To date the mechanisms regulating matrix metalloproteinase-9 during normal wound healing are poorly documented. Using both primary cultures of normal human keratinocytes and a wounding device especially designed to dissect the molecular events during the healing process in vitro, we show that matrix metalloproteinase-9 is stimulated by injury in normal human keratinocytes. This upregulation results from the mechanical stress created by injury and not from a soluble factor, secreted by wounded normal human keratinocytes. We also demonstrate that the Rho family of small GTPases, p38[MAPK] and JNK together play a key part in the signaling pathways controlling the stimulation of matrix metalloproteinase-9 in wounded cells. We provide lines of evidence indicating that in wounded keratinocytes, upregulation of matrix metalloproteinase-9 depends on two distinct pathways. The first involves Rac1 and/or Cdc42 that control the activation of p38[MAPK]. The second depends on RhoA activation that is required for stimulation of JNK. 4-(2-aminoethyl) benzenesulfonyl fluoride matrix metalloproteinase NH2 terminal c-Jun kinase extracellular matrix stress activated protein kinases mitogen activated protein kinase extracellular signal-regulated kinase normal human keratinocytes glutathione S transferase phosphatidylinositol 3 phosphate kinase toxin A of Clostridium difficile exoenzyme C3 of Clostridium botulinum Matrix metalloproteinases (MMP) are a family of extracellular endoproteinases that degrade extracellular matrix (ECM) proteins, such as laminin, fibronectin, and collagen. These enzymes are involved in several physiologic processes such as development and tissue remodeling but they also contribute to pathologic processes including tumorigenesis and arthritis (Werb, 1997Werb Z. ECM and cell surface proteolysis: Regulating cellular ecology.Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar;Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar). The different MMPs have been classified into five groups including gelatinases (MMP-2 and MMP-9), collagenases (MMP-1, MMP-8, MMP-17, and MMP-13), membrane-type MMP (MT1–MMP), stromelysins (MMP-3, MMP-10, and MMP-7), and unclassified MMP (MMP-11, 12, 19, 26, etc.) (Werb, 1997Werb Z. ECM and cell surface proteolysis: Regulating cellular ecology.Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). These proteases, which are either secreted or membrane bound, are produced as inactive forms (pro-enzyme) that are activated in the pericellular environment via an autocatalytic cleavage or by proteolytic cleavage mediated by other proteinases (Werb, 1997Werb Z. ECM and cell surface proteolysis: Regulating cellular ecology.Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar;John and Tuszynski, 2001John A. Tuszynski G. The role of matrix metalloproteinases in tumor angiogenesis and tumor metastasis.Pathol Oncol Res. 2001; 7: 14-23Crossref PubMed Scopus (546) Google Scholar). MMPs display a conserved Zn2+ binding catalytic site and can be inhibited by specific physiologic inhibitors (Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar). Cutaneous wound healing is a complex process characterized by the co-ordinated re-epithelization of the epidermis and restoration of injured connective tissue. During wound healing the keratinocytes, the endothelial cells, and the fibroblasts proliferate and migrate into the wound bed (Singer and Clark, 1999Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4642) Google Scholar). Cell migration and tissue remodeling require the controlled degradation of the ECM and the activation of growth factors (Vassalli and Saurat, 1996Vassalli J.D. Saurat J.H. Cuts and scrapes? Plasmin Heals!.Nat Med. 1996; 2: 284-285Crossref PubMed Scopus (14) Google Scholar). These processes are generally mediated by serine proteases and MMP family members (Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar). Specifically it has been shown that MMP-2 (72–68 kDa Gelatinase A) and MMP-9 (92–82 kDa Gelatinase B) play an essential part during healing of wounded skin. MMP-2, which localizes in the connective tissue fibroblasts and endothelial cells, is thought to have an important function during granulation and the early remodeling phases of repair. MMP-9 is stimulated in response to injury and is mostly expressed in the migrating keratinocytes sheet (Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar;Munaut et al., 1999Munaut C. Salonurmi T. Kontusaari S. Reponen P. Morita T. Foidart J.M. Tryggvason K. Murine matrix metalloproteinase 9 gene. 5′-upstream region contains cis-acting elements for expression in osteoclasts and migrating keratinocytes in transgenic mice.J Biol Chem. 1999; 274: 5588-5596Crossref PubMed Scopus (51) Google Scholar;Soo et al., 2000Soo C. Shaw W.W. Zhang X. Longaker M.T. Howard E.W. Ting K. Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair.Plast Reconstr Surg. 2000; 105: 638-647Crossref PubMed Scopus (156) Google Scholar). MMP-9 catalyzes cleavage of type IV collagen and other basement membrane components (Sternlicht and Werb, 2001Sternlicht M.D. Werb Z. How matrix metalloproteinases regulate cell behavior.Annu Rev Cell Dev Biol. 2001; 17: 463-516Crossref PubMed Scopus (3226) Google Scholar). Recently, the fibrinogen (Lelongt et al., 2001Lelongt B. Bengatta S. Delauche M. Lund L.R. Werb Z. Ronco P.M. Matrix metalloproteinase 9 protects mice from anti-glomerular basement membrane nephritis through its fibrinolytic activity.J Exp Med. 2001; 193: 793-802Crossref PubMed Scopus (119) Google Scholar), interleukin (IL)-1 (Schonbeck et al., 1998Schonbeck U. Mach F. Libby P. Generation of biologically active IL-1 beta by matrix metalloproteinases: A novel caspase-1-independent pathway of IL-1 beta processing.J Immunol. 1998; 161: 3340-3346PubMed Google Scholar), transforming growth factor-β (Yu and Stamenkovic, 2000Yu Q. Stamenkovic I. Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis.Genes Dev. 2000; 14: 163-176PubMed Google Scholar), and the hemidesmosomal type XVII collagen (Liu et al., 2000Liu Z. Zhou X. Shapiro S.D. et al.The serpin alpha1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo.Cell. 2000; 102: 647-655Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar) have been also identified as MMP-9 substrates. In wounded tissues, expression of MMP-9 parallels the reabsorption of the provisional matrix and assembly of new structures of the epidermal/dermal junction; then, the expression decreases to basal level (Soo et al., 2000Soo C. Shaw W.W. Zhang X. Longaker M.T. Howard E.W. Ting K. Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair.Plast Reconstr Surg. 2000; 105: 638-647Crossref PubMed Scopus (156) Google Scholar). In contrast, fluids from chronic wound persistently contain high levels of MMP-9 that probably contribute to abnormal healing of the lesion (Wysocki et al., 1993Wysocki A.B. Staiano-Coico L. Grinnell F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9.J Invest Dermatol. 1993; 101: 64-68Abstract Full Text PDF PubMed Google Scholar;Yager et al., 1996Yager D.R. Zhang L.Y. Liang H.X. Diegelmann R.F. Cohen I.K. Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids.J Invest Dermatol. 1996; 107: 743-748Crossref PubMed Scopus (298) Google Scholar). Indeed, excessive proteinase activity may degrade growth factors and ECM components required for the restoration of ECM bed that support migration of the cells colonizing the wound. Therefore, the control of molecular pathways involved in MMP-9 regulation is an essential requirement for the healing process and its deregulation may result in abnormal healing. The molecular pathways involved in regulation of MMP-9 have been widely investigated in variety of physiologic or physiopathologic systems; however, the mechanisms of MMP-9 regulation during wound healing are poorly documented. In other contexts, MMP-9 has been shown to be regulated, at least partially, at the transcriptional level. Several consensus DNA sequences for transcription factors have been identified within the promoter of MMP-9. Activation of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) appears to be essential for the induction of MMP-9 by IL-1 (Yokoo and Kitamura, 1996Yokoo T. Kitamura M. Dual regulation of IL-1 beta-mediated matrix metalloproteinase-9 expression in mesangial cells by NF-kappa B and AP-1.Am J Physiol. 1996; 270: F123-F130PubMed Google Scholar) or 12-O-tetradecanoyl-phorbol-13-acetate (Simon et al., 2001Simon C. Simon M. Vucelic G. Hicks M.J. Plinkert P.K. Koitschev A. Zenner H.P. The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation.Exp Cell Res. 2001; 271: 344-355Crossref PubMed Scopus (105) Google Scholar). Several consensus binding sites, including those for Sp1, PEA3, AP-1, Ets, NF-κB, and a retinoblastoma binding element, are involved in the transcriptional activation of MMP-9 in H-ras and c-myc transformed rat embryo cell lines (Gum et al., 1997Gum R. Wang H. Lengyel E. Juarez J. Boyd D. Regulation of 92 kDa type IV collagenase expression by the jun aminoterminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades.Oncogene. 1997; 14: 1481-1493Crossref PubMed Scopus (223) Google Scholar;Himelstein et al., 1997Himelstein B.P. Lee E.J. Sato H. Seiki M. Muschel R.J. Transcriptional activation of the matrix metalloproteinase-9 gene in an H-ras and v-myc transformed rat embryo cell line.Oncogene. 1997; 14: 1995-1998Crossref PubMed Scopus (98) Google Scholar). In the transformed cell systems studied up to date, MMP-9 promoter is driven by signal transduction pathways involving stress- and mitogen-activated protein kinases (p38[MAPK], JNK, and ERK1/2), which are stimulated either by growth factors, mitogen, cytokines, or environmental changes (Robinson and Cobb, 1997Robinson M.J. Cobb M.H. Mitogen-activated protein kinase pathways.Curr Opin Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2282) Google Scholar). We have recently developed an in vitro wounding system that allows the detection and the quantification of the molecular events occurring in keratinocytes and fibroblasts in response to injury. Using this device we have reported that injury of normal human keratinocytes (NHK) stimulated the activation of early signaling events such as tyrosine kinases, MAP kinases (ERK, p38[MAPK], and JNK), AP-1 binding activity, and late signaling events such as laminin-5, MMP-9, fibronectin EDA, IL-8, β4 integrin expression (Turchi et al., 2002Turchi L. Chassot A.A. Rezzonico R. et al.Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63Crossref PubMed Scopus (55) Google Scholar). The specific molecular pathways linking these early and late events, however, remained to be elucidated. In this study, we dissect one of these pathways, by demonstrating that the mechanical injury of NHK monolayers stimulates expression of MMP-9 at mRNA and protein levels, through a mechanism not described previously, and involving the Rho family of guanosine triphosphate (GTP) binding proteins and the stress kinases JNK and p38[MAPK]. This stimulation likely results from the mechanical stress created by the injury and does not involve a soluble factor secreted by wounded NHK. Trypsin, ethylenediamine tetraacetic acid, HEPES, penicillin, and streptomycin were purchased from Invitrogen Corp, Carlsbad, CA. Dulbecco minimal Eagle's medium, HamF12, and fetal calf serum were obtained from Hyclone (Northumberland, UK). Insulin, hydrocortisone, cholera toxin, human recombinant epidermal growth factor, triiodothyronine, adenine, mitomycin, actinomycin D, and gelatin were purchased from Sigma (Sigma Aldrich, St. Louis, MO). RNAble, RNA preparation kit, was from Eurobio (Les Ulis, France). LY294002 SB203580, PD 98059, and AG-1478 were purchased from Calbiochem (Calbiochem/France, Meudon, France). BB94 is a kind tift of British Biotech (Oxford, UK). Clostridium difficile toxin A and Clostridium botulinum C3 exoenzyme were generous tifts of Drs I. Just (Hanover, Germany) and P. Boquet (Nice, France), respectively. The cDNA encoding the dominant negative allele of JNK1 (JNK1(APF)) was described previously (Derijard et al., 1994Derijard B. Hibi M. Wu I.H. et al.JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2954) Google Scholar;Lin et al., 1995Lin A. Minden A. Martinetto H. et al.Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2.Science. 1995; 268: 286-290Crossref PubMed Scopus (709) Google Scholar) and was a generous tift of Dr B. Derijard (Nice, France). NHK were isolated from healthy neonatal foreskin as described (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3900) Google Scholar). Keratinocytes were seeded on mitomycin C (10 μg per mL) treated 3T3-J2 fibroblasts feeder layers (2×105 cells per cm2) and grown in "Green" medium (2/3 Dulbecco minimal Eagle's medium, 1/3 HamF12 supplemented with 10% fetal calf serum, HEPES 20 mM, penicillin (1000 U per mL), streptomycin (1000 U per mL), insulin (5 μg per mL), hydrocortisone (0.4 μm M), cholera toxin (10 μM), recombinant human epidermal growth factor (10 μM), triiodothyronine (2 μM), and adenine (18 μM). Confluent NHK cells were wounded and incubated at 37°C/5% CO2. Twenty-four hours after injury, cells were washed with phosphate-buffered saline, pH 7.4 and fixed in 2% paraformaldehyde. Cells were then incubated with monoclonal anti-MMP-9 antibody (1/50) for 1 h at room temperature and with a secondary fluorescein isothiocyanate conjugated goat anti-mouse antibody (1/50). For nuclei labeling, cells were incubated for 10 min at room temperature with a 10 ng per mL Hoechst reagent solution. Cells wounded or not for the indicated times were solubilized at 4°C in 500 μL RIPA buffer (Tris/HCl 10 mM pH 7.5, NaCl 150 mM, sodium deoxycholate 1%, nonidet P-40 1%, sodium dodecyl sulfate (SDS) 0.1%, 1 mM phenylmethylsulfonyl fluoride, 50 μg aprotinin per mL, 50 μg leupeptin per mL, 1 μg pepstatin A per mL, 20 mM NaF, 1 mM NaVO4, 1 mM β glycerophosphate). Proteins (100 μg per lane) were resolved on SDS–polyacrylamide gel electrophoresis and transferred on to Immobilon-P membrane (Millipore) as described previously (Turchi et al., 2002Turchi L. Chassot A.A. Rezzonico R. et al.Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63Crossref PubMed Scopus (55) Google Scholar). The membrane was saturated with phosphate-buffered saline supplemented with 3% bovine serum albumin, 0.5% gelatin, and 0.1% Tween 20, and incubated with either anti-MMP-9 (1/200), anti-phospho/ACTIVE p38[MAPK] or JNK (1/2000), phospho-specific anti-CREB (1/1000) or -c-Jun (1/1000) or anti-pan p38[MAPK] (1/1000°) antibodies, for 2 h at room temperature. The membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (1/4000) and blots were revealed using the Amersham (Roche/Amersham, Les ULIS, France) ECL detection system. Monoclonal anti-MMP-9 antibody was obtained from Research Diagnostic, Inc. (Flanders, NJ) Polyclonal p38[MAPK] (pTPpY) antibody was purchased from Promega Corporation (Madison, WI) Anti-phospho-CREB (Ser133) antibody (used to detect phospho-ATF1) was from Biolabs Inc. (Beverly, MA). Anti-Rho, Rac, Cdc42 monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies were purchased from Dako. Anti-pan p38[MAPK] antibody was a generous tift of Dr B. Dérijard (Nice, France). The activation of cellular Rho, Rac, and Cdc42 was determined by precipitation with the glutathione S transferase (GST) protein coupled to the Rho binding domain of Rhotekin (Ren et al., 1999Ren X.D. Kiosses W.B. Schwartz M.A. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton.Embo J. 1999; 18: 578-585Crossref PubMed Scopus (1361) Google Scholar) or to Rac/Cdc42 binding domain of PAK (Manser et al., 1998Manser E. Loo T.H. Koh C.G. et al.PAK kinases are directly coupled to the PIX family of nucleotide exchange factors.Mol Cell. 1998; 1: 183-192Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar), according to the procedure described byHerrmann et al., 1995Herrmann C. Martin G.A. Wittinghofer A. Quantitative analysis of the complex between p21ras and the Ras-binding domain of the human Raf-1 protein kinase.J Biol Chem. 1995; 270: 2901-2905Crossref PubMed Scopus (318) Google Scholar. Briefly, wounded (or intact) keratinocytes were lyzed in (1) Rho-specific lysis buffer (Tris/HCl 50 mM pH 7.2, NaCl 500 mM, MgCl2 10 mM, Triton X-100 1%, sodium deoxycholate 0.5%, SDS 0.1%, AEBSF 100 μM, 1 mg leupeptin per mL, Pepstatine A 1 mM), or (2) Rac/Cdc42 specific lysis buffer (HEPES 25 mM pH 7.3, NaCl 150 mM, MgCl2 5 mM, EGTA 0.5 mM, Triton X-100 0.5%, glycerol 4%, 20 mM β-glycerophosphate, 10 mM NaF, AEBSF 100 μM, leupeptin 1 mg per mL, Pepstatine A 1 mM). Fifteen milligrams of clarified protein extracts were incubated with glutathione-Sepharose precoupled with GST-Rho-BD or GST-Rac-BD protein (30 μg) for 45 to 60 min at 4°C. Beads were washed four times with Rac/Cdc42 lysis buffer (for Rac and Cdc42) or with Tris buffer (Tris/HCl 50 mM pH 7.2, NaCl 500 mM, MgCl2 10 mM, Triton X-100 1%, AEBSF 100 μM, 1 mg leupeptin per mL, Pepstatine A 1 mM) (for Rho) and denatured in Laemmli buffer. GTP-bound Rho, Rac, and Cdc42 were separated on 12% SDS–polyacrylamide gel electrophoresis and measured by western blot using monoclonal antibodies rise against Rho (1/250), Rac (1/1000°), and Cdc42 (1/250°). Total RNA from NHK were isolated using the RNABle kit according to the manufacturer's instructions. Northern blot analysis were carried out using 20 μg of total RNA following the procedure described previously (Rezzonico et al., 1995Rezzonico R. Ponzio G. Loubat A. Lallemand D. Proudfoot A. Rossi B. Two distinct signalling pathways are involved in the control of the biphasic junB transcription induced by interleukin-6 in the B cell hybridoma 7TD1.J Biol Chem. 1995; 270: 1261-1268Crossref PubMed Scopus (21) Google Scholar). The MMP-9 (458 bp) cDNA probe was obtained by polymerase chain reaction (PCR) amplification from NHK total RNA using specific primers, respectively: forward: 5′-TGGCCCAGGTGACCGGGGCCCTC-3′; reverse: 5′-AAAGGTTAGAGAATCCAAGTTTAT-3′. Aliquots (5 μL) of culture medium corresponding to the different experimental conditions were applied on 10% polyacrylamide gel containing 0.3% gelatin. The gels were incubated in 2.5% Triton X-100 for 2 h, then incubated for 18 h at 37°C in 100 mM Tris/HCl, pH 7.5, 200 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2. The gels were finally stained with Coomassie Brilliant Blue. The full-length open reading frame of the cDNA encoding the dominant negative JNK (APF) allele of JNK (a tift of Dr Derijard, Nice France), was subcloned downstream the mouse Moloney murine leukemia virus (MMLV) long-term repeat (LTR) of Plasmid pLZRS-Ires-Zeo (tift of G.P. Nolan, Standford University, California). Plasmids (pLZRS-Ires-Zeo-JNK(APF) were amplified into Escherichia coli XL10 strain and purified using QIAquick kit (Qiagen Inc., Chatsworth, CA). Infectious recombinant retrovirus were generated by transfecting the recombinant plasmid pLZRS-Ires-Zeo-JNK (APF) into the amphotropic Phoenix packaging cells by the calcium phosphate precipitation method (Michiels et al., 2000Michiels F. van der Kammen R.A. Janssen L. Nolan G. Collard J.G. Expression of Rho GTPases using retroviral vectors.Methods Enzymol. 2000; 325: 295-302Crossref PubMed Google Scholar). The supernatant from these cultures (about 2.5×104 colony forming units (cfu) per mL) were collected after 48 h and applied for 24 h at 32°C to NHK seeded at 3×104 cells per cm2 on a feeder of J2-3T3 fibroblasts. Cells were fed with fresh medium for 2 d and were selected in the presence of 200 μg zeocin per mL. Total NHK RNA (2 μg) was reverse-transcribed at 37°C for 60 min, using Omniscript Kit (Promega, Madison, WI). Two microliters of each cDNA sample were used for PCR using TAQ polymerase (Life Technologies, Inc.). The cDNA encoding MMP-9 and GAPDH were amplified using the specific sets of primers, respectively: forward: 5′-TGGCCCAGGTGACCGGGGCCCTC-3′; reverse: 5′-AAAGGTTAGAGAATCCAAGTTTAT-3′, and forward: 5′-ATGG-GGAAGGTGAAGGTCGG-3; reverse: 5′-TTACTCCTTGGAGGCCATGT-3′. The RNA and proteins were quantitated using two methods: (1) by densitometric scanning using a computerized microscopic image processor Biocom 500 (Biocom, les Ulis France) comprising a PC/AT-compatible microcomputer, a real-time imaging processor, a control monitor, a color high definition monitor, and a Panasonic WV-CD50 camera, and (2) Using the NIH image software available on web site: http://www.rsb.info.nih.gov/nih-image/ To dissect the molecular pathways involved in the regulation of the metalloprotease MMP-9 (Gelatinase B) during epidermal wound healing in vitro, we have used NHK and a new technologic approach specially designed for this purpose and already described (Turchi et al., 2002Turchi L. Chassot A.A. Rezzonico R. et al.Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63Crossref PubMed Scopus (55) Google Scholar). A confluent monolayer of primary human keratinocytes was mechanically injured as described in Materials and Methods. After 24 h, the healing cultures were analyzed by immunofluorescence after labeling with Hoechst reagent, which allows the identification of keratinocytes migrating from the edge (dotted line) into the wound bed (arrows) (Figure 1a). Interestingly, the wound closure was totally impaired when cells were incubated, before injury, with 20 μM of BB94, a wide spectrum metalloproteinases inhibitors (Davies et al., 1993Davies B. Brown P.D. East N. Crimmin M.J. Balkwill F.R. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and prolongs survival of mice bearing human ovarian carcinoma xenografts.Cancer Res. 1993; 53: 2087-2091PubMed Google Scholar), indicating that, in our biologic system, MMP are involved in wound repair. Using a specific anti-MMP-9 antibody, we clearly observed that the protease MMP-9 was upregulated in wounded NHK and was specifically expressed in cells forming the migrating front (Figure 1b, arrows). Upregulation of MMP-9 in the wounded cultures was further demonstrated at the molecular level by gelatinolytic zymography, western and northern blots (Figure 1c). Results demonstrated that MMP-9 increased between 6 h and 24 h after injury of the cell layers (Figure 1c), then was downregulated to the basal level after 48 h (not shown). In zymography and immunoblots, MMP-9 can be detected as a double band identifying the inactive form (pro-enzyme 92 kDa) and the active (82 kDa) enzyme form (Figure 1c). Here we show that, in wounded keratinocyte cultures, MMP-9 is mostly expressed in its active form. This observation and the fact that MMP-9 was exclusively visualized by immunofluorescence in keratinocytes located at the edge of the wound (Figure 1b) support the idea that MMP-9 is involved in the degradation of ECM deposed by the cultured keratinocytes and the 3T3-J2 fibroblasts (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-343Abstract Full Text PDF PubMed Scopus (3900) Google Scholar), and consequently that MMP-9 is active in the healing process. Our data also show a good correlation between the variations in the steady-state levels of MMP-9 mRNA, expression of the protein, and enzymatic activity. This suggests that upregulation of MMP-9 mainly resulted from enhanced expression of MMP-9 mRNA transcripts consequent to mechanical injury of the cell culture. The gelatinolytic activity of the conditioned medium of keratinocyte cultures also revealed that MMP-2 (Gelatinase A) is secreted by unwounded cell cultures. In contrast to MMP-9, however, expression of MMP-2 was not upregulated following injury, confirming the observation reported by others (Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar). MMP-9 is stimulated by chemicals (phorbol myristate acetate), growth factors, and cytokines. Some of which (transforming growth factor-β, IL-1, tumor necrosis factor-α) are upregulated during wound healing (Salo et al., 1994Salo T. Makela M. Kylmaniemi M. Autio-Harmainen H. Larjava H. Expression of matrix metalloproteinase-2 and -9 during early human wound healing.Lab Invest. 1994; 70: 176-182PubMed Google Scholar;Holzheimer and Steinmetz, 2000Holzheimer R.G. Steinmetz W. Local and systemic concentrations of pro- and anti-inflammatory cytokines in human wounds.Eur J Med Res. 2000; 5: 347-355PubMed Google Scholar;Han et al., 2001Han Y.P. Tuan T.L. Hughes M. Wu H. Garner W.L. Transforming growth factor-beta- and tumor necrosis factor-alpha-mediated induction and proteolytic activation of MMP-9 in human skin.J Biol Chem. 2001; 276: 22341-22350Crossref PubMed Scopus (144) Google Scholar;Turchi et al., 2002Turchi L. Chassot A.A. Rezzonico R. et al.Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63Crossref PubMed Scopus (55) Google Scholar). To assess whether induction of MMP-9 could be mediated by a second messenger released by wounded keratinocytes, we tested the ability of spent medium of injured NHK cultures to upregulate the expression of MMP-9 in exponentially growing and quiescent keratinocytes. The MMP-9 gelatinolytic activity present in the conditioned medium of NHK and 3, 6, and 12 h after wounding is presented in Figure 2a, respectively lanes 1, 2, 3, 4. Exposure for 24 h of proliferating NHK to such conditioned medium had no effect on MMP-9 activity (Figure 2b). Indeed, the activity of MMP-9 in the culture medium of exponentially growing cells (Figure 2b) did not increase compared with the activity present in the conditioned medium of wounded cells (Figure 2a). The same results were obtained when the conditioned medium was added to confluent keratinocyte cultures (not shown). To confirm these data we performed reverse transcriptase–PCR experiments using RNA obtained from (1) keratinocytes before and 12 h after injury, and of (2) 50% confluent intact NHK treated for 24 h with the conditioned medium of NHK harvested, 6 and 12 h after wounding. The results indicate that MMP-9 mRNA was stimulated
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