ȐΜΒȑġ Gelatin zymography
5. Gelatin gelǺ
2.5% Triton-100] ࠻ྕΠϸᔈ 30 ϩដǴӆܫΕ Tris-HCl (50mMǴ PH=7.5)ύమࢱٿԛǴԛ 15 ϩដǴҞӦӧܭࡠൺ MMPs ޑࢲ܄Ƕ
ஒጤܫΕ develop buffer composition buffer ύܭ 370CǴ50 rpm ޑ
ᎦጃϣǴऊ 15 λਔࡕǶጤҔ coomassie blue ࢉՅऊλਔࡕӆ٬Ҕ
coomassie blue destain buffer ଏډёа࣮ޑډ band ջё࠾ጤǶ
ȐΜΟȑġ ीϩ
ೀಔᆶڋಔޑኧᏵ໔Ǵ߯௦Ҕ Student’s t-testǴp<0.05Ǵղۓ
ࢂցԖीৡ౦ǴኧᏵ่݀а Mean±S.M. ߄ҢӚኧᏵǶ
ಃѤക ่݀
ǵġ CCN3 ߦΓᜪ೬ମԺዦಒझޑ౽܄
ᕎੱಒझӃԖ౽ՉޑૈΚϐࡕǴωՉᙯ౽բҔ (Metastasis)Ǵҗܭ CCN3 ڀԖፓಒझମࢎޑख़ಔᆶߦځдᕎ ಒझޑ౽ૈΚǴӢԜ CCN3 ёૈׯᡂಒझޑ౽Չ܄Ǵჴ ᡍޑҞӦࣁೀόӕޑᐚࡋޑ CCN3 (0Ǵ10Ǵ30Ǵ100 ng/ml) ࡕǴ ճҔ transwell migration assay ӧڰۓਔ໔ (16 λਔ) ᢀჸ CCN3 ჹܭΓᜪ೬ମԺዦಒझԖค౽܄Ǵ่݀วӧೀ 30 ng/ml ޑ ᐚࡋਔಒझޑ౽നࣁᡉ (Fig. 6A)ǶԶԖЎࡰр CCN3
ڋᕎಒझғߏ (Poliferation)ǴӢԜӧҁჴᡍύ٬Ҕ MTT assay ѐୀෳ CCN3 ჹܭΓᜪ೬ମԺዦಒझޑቚғำࡋаϷዴᇡ ಒझޑ౽܄ǴԶҗჴᡍ่݀วόӕޑ CCN3 ᐚࡋჹܭΓᜪ೬ ମԺዦಒझޑӸࢲ٠ؒԖीޑཀကǴ߄ҢΑ CCN3 ߦ
Γᜪ೬ମԺዦಒझޑ౽܄ǴՠόቹៜಒझӸࢲ(Fig. 6B)Ƕ
Βǵġ CCN3ԋΓᜪ೬ମԺዦಒझϐ MMP-13 ޑεໆ߄
ಒझޑᙯ౽܄کಒझ܌ϩݜޑ MMPs ӸԖࡐεޑᜢᖄ܄Ǵ܌
а ӧ ჴ ᡍ ύ ٬ Ҕ Α RT-PCR ǵ Western blotting а Ϸ gelatin-zymographyϩಒझ܌ϩݜϐ MMP ޑ߄ໆаϷࢲ܄Ǵ
ᢀჸӧೀ CCN3 ࡕࢂցቹៜಒझϩݜ MMP ϷᅿᜪǶԖЎ
ࡰрӧΓᜪޑᕎੱಒझύځ MMP -1ǵ-2ǵ-3ǵ-9 ک-13 ޑ߄ᆶ ဍዦൾ܄ำࡋϷᙯ౽բҔԖᜢ (Egeblad and Werb, 2002; Tan et al., 2009)Ƕ܌аჴᡍӃа RT-PCR ѐᑔᒧځ MMP -1ǵ-2ǵ-3ǵ-9 ک-13 ޑ߄Ǵวа CCN3 ೀΓᜪ೬ମᕎ 24 λਔϐࡕǴа MMP-13 mRNA ޑ߄ໆനࣁᡉ (Fig. 7A)Ƕௗೀ CCN3 όӕਔ໔ࡕ (0ǵ6ǵ12ǵ24 λਔ) ࡕǴ٬Ҕ Western blotting Ϸ gelatin-zymography ޑБݤᢀჸ MMP-13 ೈқ፦߄ᆶࢲ܄Ǵ่݀ࡰр MMP-13 Ԗ time-dependent ޑຝ (Fig. 7B)ǶନԜϐѦǴךॺ׳ޑ٬
Ҕ MMP-13 si-RNA ٰຓܴ MMP-13 ࢂЬाቹៜΓᜪ೬ମԺዦಒ झޑ౽܄ (Fig. 7C)Ǵௗ٬ҔՋБᏀᗺݤຓܴǴᙯࢉ MMP-13 si-RNA Ԗԋф (Fig. 7D)Ƕҗ่݀ࡰр CCN3 ёаԋΓᜪ೬ମ Ժዦಒझౢғ MMP-13 Զԋಒझޑ౽܄Ƕ
Οǵġ CCN3ၸ Įvȕ3 Ϸ Įvȕ5 integrin receptor ԋΓᜪ೬ମԺዦ ಒझ౽Չ
Ўࡰр CCN3 ၸ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 integrin receptor
ำύǶ२Ӄ٬Ҕ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 ޑלᡏ 30 ϩដϐࡕǴӆу Ε CCN3Ǵ24 λਔࡕԏಒझճҔ Western blotting ᢀჸ MMP-13 ޑೈқ፦߄ǴวಒझऩӃೀ Įvȕ3 Ϸ Įvȕ5 ޑלᡏࡕڋ CCN3 ౢғ MMP-13 (Fig 8A)ǶќБय़٬Ҕ migration assay Ϸ RT-PCR ϩǴӕኬӃӧಒझύೀ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 ޑלᡏ 30 ϩដϐࡕǴӆуΕ CCN3Ǵӧ migration assay ޑ่݀ύวӃ
ೀ Įvȕ3 Ϸ Įvȕ5 לᡏޑಒझ౽ՉૈΚܴᡉΠफ़ (Fig. 8B)Ǵ ќѦ RT-PCR ޑ่݀ΨᡉҢಒझೀ Įvȕ3 Ϸ Įvȕ5 לᡏࡕǴ
ԋ MMP-13 ޑ߄ໆܴᡉΠफ़ (Fig. 8C)ǴќѦҗܭ integrin Ьा
ᙖҗᒣ ligand ޑ RGD (Arg-Gly-Asp) ׇӈԶ่ӝǴᚐѦ
٬Ҕ RGD peptide уаዴᇡǴҗܭѬёаߔᘐ integrin ᆶ ligand ޑ่ӝǶԶ RGD ёаߔᘐ CCN3 ܌ቚу MMP-13 ߄Ǵՠ RAD
ؒԖԜբҔ (Fig. 8C)Ƕᆕӝа่݀ளޕ CCN3 ߦΓᜪ೬ମ Ժዦಒझ౽Չᆶϩݜ MMP-13 ࢂၸ Įvȕ3 Ϸ Įvȕ5 integrin receptorǶ
Ѥǵġ FAKୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ
Ўࡰр Focal adhesion kinase (FAK) ࢂᅿӸܭಒझ፦ύ ޑ non-receptor protein tyrosine kiaseǴϩηໆεऊࣁ 125kDaǴ
ಒझҗ integrin ߕܭಒझѦ୷፦ೈқޑਔࡕǴFAK ࡐזޑ
ᐟࢲǴԶᆶځдૻ৲ϩηբҔǴӢԜ FAK ᇡࣁӧ integrin
܌ፓޑૻ৲ሀၸำύǴתᄽख़ाޑفՅǶ٬Ҕ Western blotting ᢀჸ๏ϒ CCN3 ڈᐟၸόӕޑਔ໔ (0ǵ10ǵ15ǵ30ǵ60ǵ120 ϩដ) ځ FAK (tyr397) ޑࢲ܄Ǵว FAK ޑᕗለϯࢂอኩ܄ޑǴ εऊӧ 10 ϩដډ 30 ϩដϐ໔ၲډଯঢ়ࡕǴӧλਔߡזೲΠफ़ (Fig. 9A)ǶԜѦᙯࢉΓᜪ೬ମԺዦಒझ FAK si-RNA ک FAK mutant 24 λਔࡕуΕ CCN3 ڈᐟǴճҔ migration assay аϷ RT-PCR ޑ Бݤϩ (Fig. 9Bǵ9C)ǴёᢀჸډΓᜪ೬ମԺዦಒझޑ౽Չૈ
Κᆶ MMP-13 ޑౢғԖܴᡉޑڋբҔǶவ่݀ύளޕ FAK ޑࢲϯୖᆶӧ CCN3 ޑբҔύǶ
ϖǵġ PI3K/AktୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ
җ integrin ܌ፓޑૻ৲ሀၡ৩Ԗ PI3K/Akt ܌ୖᆶǴ܈ࢂ
ځдၡ৩ӵǺERKǵJNK Ϸ MEK/MAPKǶ܌а໒ۈךॺӃ٬
Ҕ Western blotting ᢀჸӧ๏ϒಒझ CCN3 ࡕځၡ৩ೈқޑᕗለϯ ߄Ƕ๏ϒΓᜪ೬ମᕎಒझ CCN3Ǵ10 ϩដϐࡕǴPI3K ޑᕗለ ϯԖᡉޑቚу (Fig. 10A)ǴӢԜӃख़ӧ PI3K/Akt ೭చၡ৩Ƕ
Ϸ RT-PCRǴёᢀჸډΓᜪ೬ମԺዦಒझޑ౽ՉૈΚᆶ MMP-13 ޑౢғԖܴᡉޑڋբҔ (Fig. 10Bǵ10Cǵ10Dǵ10E)ǶӢԜ PI3K ࢲϯୖᆶӧ CCN3 ϐբҔύǶௗΠٰ Akt ࢂցΨୖᆶӧ ځύǴ๏ϒಒझ Akt ޑڋᏊ (Akt inhibitor) 30 ϩដǴ܈
ࢂᙯࢉ Akt mutant 24 λਔࡕǴ٬Ҕ migration assay Ϸ RT-PCR ᢀ ჸډΓᜪ೬ମԺዦಒझޑ౽ՉૈΚᆶ MMP-13 ޑౢғԖܴᡉޑ
ڋբҔ (Fig. 11Bǵ11Cǵ11Dǵ11E)ǶӢԜ Akt ࢲϯୖᆶӧ CCN3 ϐբҔύǶ
Ϥǵġ NF-țB ୖᆶӧҗ CCN3 ፓቚуΓᜪ೬ମԺዦಒझ౽Չύ
ϐޑࣴزࡰр CCN family ӧᇨวಒझᙯ౽ਔၸ NF-țB ᙯᒵӢη (Lin et al., 2004)ǶӧԜࣁΑΓᜪ೬ମԺዦ ಒझࢂցჹ CCN3 ܌ᇨวޑ NF-țB ౢғፓբҔǴ२Ӄ٬Ҕ Western blotting ᢀჸӧ๏ϒಒझ CCN3 ڈᐟၸόӕޑਔ໔ (0ǵ10ǵ15ǵ30ǵ60ǵ120 ϩដ) ࡕځ IKKĮ/ȕǵIțB ೈқ፦ޑᕗ ለϯ߄Ǵჴᡍ่݀ࡰрځᕗለϯޑ߄Ԗև time-dependent ޑຝ (Fig. 12C)Ƕௗ๏ϒಒझ NF-țB inhibitor (PDTCǵTPCK Ϸ NF-țB inhibitor peptide) 30 ϩដǴ܈ࢂ٬Ҕᙯࢉ IKKĮ Ϸ IKKȕ mutant 24 λਔࡕǴճҔ migration assay Ϸ RT-PCR ᢀჸǴว
ೀ NF-țB ڋᏊᆶ mutant ਔǴΓᜪ೬ମԺዦಒझޑ౽ՉૈΚ ᆶ MMP-13 ޑౢғԖܴᡉޑڋբҔ (Fig. 12Aǵ12Bǵ12Dǵ 12E)Ƕ׳ஒΓᜪ೬ମԺዦಒझᙯࢉΕ țB-luciferase բ NF-țB ࢲ܄ޑࡰӢηǴҗჴᡍ่݀ᡉҢǴಒझೀ PI3K inhibitor (Ly294002 Ϸ wortmannin) ǵ Akt inhibitor ک NF-țB inhibitor (PDTCǵTPCK Ϸ NF- țB inhibitor peptide) ૈफ़եҗ CCN3 ܌ᇨวޑ NF-țB ࢲ܄ (Fig. 13Aǵ13B)ǶќѦǴஒΓᜪ೬ ମዦಒझӕᙯࢉΕ p85ǵAktǵIKKDǵIKKȕ mutant Ψૈڋ NF-țB ᙯᒵӢηޑࢲ܄Ƕᆕӝаޑ่݀ளޕ NF-țB ୖᆶӧ
CCN3 ፓΓᜪ೬ମԺዦಒझޑၡ৩ύǶ
ಃϖക ፕ
ဍዦಒझᙖҗϩݜMMPsǴ٬ளဍዦಒझऀၸಒझѦ୷፦Ǵ ᒿՈన܈రЃسߟΕيᡏځдՏޑಔᙃ܈Ꮤ۔ (Bremnes et al., 2002; Page-McCaw et al., 2007) ࡕǴဍዦಒझεໆቚғԋ҅தಒ झคݤளډкىޑᎦϩԶᏤԿ҅தಔᙃᏔ۔ޑфૈ෧১Ǵ೭Ψࢂԋ ᕎੱੰΓԝΫޑख़ाӢનϐǶ೬ମԺዦڀԖᙯ౽ޑወӧૈΚǴу
೬ମԺዦჹܭϯᏢ܈ܫᕍޑݯᕍਏ݀٠όӳǴаϷલЮԖਏޑᇶշᕍ ݤǴ٬ளεӭኧޑ೬ମԺዦੰΓႣࡕૈΚৡǵ׳ԖൺวϷᙯ౽ޑё
ૈ܄ǶӢԜऩૈவύᕕှ೬ମԺዦᙯ౽ၸำޑϩηᐒڋǴ܈ёаග ٮ҂ٰᜢܭݯᕍ೬ମԺዦᙯ౽ϐԖਏБݤ (Fong et al., 2007)ǶЎࡰ
рӧΓᜪޑᕎੱಒझύځMMP-1ǵ-2ǵ-3ǵ-9ک-13ޑ߄ᆶဍዦൾ܄
ำࡋϷ࣬ᜢᙯ౽բҔԖᜢǶӢԜҁჴᡍޑҞӦӧܭΓᜪ೬ମԺዦ ಒझᙯ౽ޑᐒڋၡ৩Ǵ׳ޑፕࢂٗᅿޑMMPୖᆶӧځ ύǴҗჴᡍ่݀ளޕǴCCN3ԋΓᜪ೬ମԺዦಒझౢғεໆޑ MMP-13ǶMMP-13ࢂᜢ੯ੰύᏤԿ೬ମଏϯޑख़ाӢηϐǴԶ
೬ମрୢᚒਔǴёૈԋӭᅿޑମᓝ੯ੰǴԶځύϣғ܄೬ମ ዦᗋԖёૈൾ܄ᙯϯࣁ೬ମԺዦ (Yu et al., 2003)Ƕ܌аךॺᇡࣁ MMP-13ޑфૈନΑૈߦᕎಒझޑ౽ѦǴᗋёૈԋڬൎ҅
தޑ೬ମಒझᖿܭൾϯԋᕎಒझǶԶࣴزMMPନΑёаᔅշᕕှ
ဍዦวғޑၸำѦǴ׆ఈёаவύѐࣴز໒วрڋMMPբҔޑᛰ ނٰݯᕍᕎੱੰΓǴ܈೭ࢂफ़եੰΓᕎੱൺวϷᙯ౽ޑёૈ܄Ƕ
ӧӃޑࣴزύวCCN3߄ӧ҅தಔᙃǴхࡴǴઓس
ǴޤǴԼԺǴ೬ମǴՠӧဍዦಒझύޑ߄ᗲϿࣁΓޕ (Manara et al., 2002)ǶуԖࣽᏢৎȐRudolf Virchowȑ ࡰрᕎಒझନΑԾيڀ ഢޑ೭٤ૈΚѦǴΨڙډຼᎁಒझϩݜޑಒझᐟનǵϯᏢᐟન܈ғ ߏӢηٰᇨ٬Ѭॺӛᕎಒझᆫ (Coussens and Werb, 2002; DeNardo and Coussens, 2007)Ǵ٠٬Ѭॺ߄߄рԖճܭဍዦғߏǵᙯ౽ޑ
ᄊǴԶബрঁӝဍዦғߏޑ༾ᕉნ (Joyce and Pollard, 2009)ǶҗܭCCN fmailyёаintegrin receptorޑligandǴ่ӝϐࡕ ёаॄೢፓಒझ໔܈ಒझᆶಒझѦ୷፦ޑբҔǴௗቹៜಒझମࢎ
ᄊǵፓಒझғߏǵϩϯǵ౽ǵߕϷαޑঅൺǴӢԜךॺགྷ ޕၰCCN3ࢂցၸintegrin receptorٰԋΓᜪ೬ମԺዦಒझޑ౽
܄аϷCCN3ᆶintegrin receptorޑᜢ߯Ƕ२ӃךॺӃࢂаtranswell
ٰዴۓӧೀCCN3ࡕΓᜪ೬ମԺዦಒझޑ౽܄Ǵ٠ճҔೀ
integrin receptorޑלᡏளډޑ่݀ࣁCCN3ၸĮvȕ3ϷĮvȕ5 integrin
sarcomaکosteosarcomaύǴႣࡕၨৡޑࡰӢη (Perbal et al., 2009;
Perbal et al., 2008)ǴନԜϐѦӧChronic Myeloid Leukaemia (CML) Ϸ
Յનዦ (Melanoma) ύǴCCN3Ԗdown-regulatedޑຝǴځфૈ
ӧܭफ़եဍዦಒझޑߟҍૈΚᆶϩݜMMPsೈқ፦܈ࢂቚуΫբ ҔǴԖᗺᜪ՟ဍዦڋӢηޑفՅ(Fukunaga-Kalabis et al., 2008;
McCallum et al., 2009)ǴฅԶӧମᕎಒझٯӵEwing's sarcomaύᢀჸ ډCCN3ڀԖߦဍዦޑ౽ՉϷߟҍૈΚǴᆕӝаޑፕᗺёаޕ p85 mutantǵAkt mutantǵIKKD mutant Ϸ IKKȕ mutantࡕǴڋ CCN3ڈᐟΓᜪ೬ମԺዦಒझޑ౽ՉૈΚᆶMMP-13ޑౢғǶќБ
य़٬ҔRGD peptide ϷRAG peptideวRGDڋMMP-13ޑౢ
ғǴՠ๏ϒRAG peptideਔؒԖᡉޑৡ౦Ǵ٠ଛӝ٬ҔհӀሇનࢲ
܄ෳۓᢀჸڋᏊᆶmutantჹܭಒझϣNF-țBޑࢲ܄Ǵวೀ
ڋᏊᆶmutantਔफ़եNF-țBޑࢲ܄Ƕ೭٤่݀ࡰрCCN3ၸ Įvȕ3ϷĮvȕ5 integrin receptorፓΓᜪ೬ମԺዦಒझޑ౽Չᆶϩݜ MMP-13ǴࢂၸPI3K/Akt/NF-țB೭చၡ৩ (Fig. 14)Ƕ
ಃϤക ่ፕ
ᆕӝаޑჴᡍ่݀ǴёаளډCCN3җĮvȕ3ϷĮvȕ5 integrin receptor۳Πሀૻ৲ǴࢲϯΠෞPI3K/Akt/NF-țB೭చၡ৩ፓΓᜪ ೬ମԺዦಒझϩݜMMP-13ǴԶፓΓᜪ೬ମዦಒझޑ౽ՉૈΚǶ
ୖԵЎ
Aigner, T., Soeder, S., and Haag, J. (2006). IL-1beta and BMPs--interactive players of cartilage matrix degradation and regeneration. Eur Cell Mater 12, 49-56; discussion 56.
Benini, S., Perbal, B., Zambelli, D., Colombo, M.P., Manara, M.C., Serra, M., Parenza, M., Martinez, V., Picci, P., and Scotlandi, K. (2005). In Ewing's sarcoma CCN3(NOV) inhibits proliferation while promoting migration and invasion of the same cell type.
Oncogene 24, 4349-4361.
Blood, C.H., and Zetter, B.R. (1990). Tumor interactions with the vasculature:
angiogenesis and tumor metastasis. Biochim Biophys Acta 1032, 89-118.
Cary, L.A., Chang, J.F., and Guan, J.L. (1996). Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J Cell Sci 109 ( Pt 7), 1787-1794.
Chen, C.C., and Lau, L.F. (2009). Functions and mechanisms of action of CCN matricellular proteins. Int J Biochem Cell Biol 41, 771-783.
Chow, W.A. (2007). Update on chondrosarcomas. Curr Opin Oncol 19, 371-376.
Cockett, M.I., Murphy, G., Birch, M.L., O'Connell, J.P., Crabbe, T., Millican, A.T., Hart, I.R., and Docherty, A.J. (1998). Matrix metalloproteinases and metastatic cancer.
Biochem Soc Symp 63, 295-313.
Coussens, L.M., Fingleton, B., and Matrisian, L.M. (2002). Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295, 2387-2392.
Coussens, L.M., and Werb, Z. (2002). Inflammation and cancer. Nature 420, 860-867.
Curran, S., and Murray, G.I. (1999). Matrix metalloproteinases in tumour invasion and metastasis. J Pathol 189, 300-308.
Desgrosellier, J.S., and Cheresh, D.A. (2010). Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer 10, 9-22.
Duffy, M.J. (1996). Proteases as prognostic markers in cancer. Clin Cancer Res 2, 613-618.
Egeblad, M., and Werb, Z. (2002). New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161-174.
Fong, Y.C., Yang, W.H., Hsu, S.F., Hsu, H.C., Tseng, K.F., Hsu, C.J., Lee, C.Y., and Scully, S.P. (2007). 2-methoxyestradiol induces apoptosis and cell cycle arrest in human chondrosarcoma cells. J Orthop Res 25, 1106-1114.
Fukunaga-Kalabis, M., Martinez, G., Telson, S.M., Liu, Z.J., Balint, K., Juhasz, I., Elder, D.E., Perbal, B., and Herlyn, M. (2008). Downregulation of CCN3 expression as a potential mechanism for melanoma progression. Oncogene 27, 2552-2560.
Guo, W., and Giancotti, F.G. (2004). Integrin signalling during tumour progression.
Nat Rev Mol Cell Biol 5, 816-826.
Holbourn, K.P., Acharya, K.R., and Perbal, B. (2008). The CCN family of proteins:
structure-function relationships. Trends Biochem Sci 33, 461-473.
Hood, J.D., and Cheresh, D.A. (2002). Role of integrins in cell invasion and migration.
Nat Rev Cancer 2, 91-100.
Hynes, R.O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion.
Cell 69, 11-25.
Joyce, J.A., and Pollard, J.W. (2009). Microenvironmental regulation of metastasis.
Nat Rev Cancer 9, 239-252.
Kanner, S.B., Reynolds, A.B., Vines, R.R., and Parsons, J.T. (1990). Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A 87, 3328-3332.
Knauper, V., Lopez-Otin, C., Smith, B., Knight, G., and Murphy, G. (1996).
Biochemical characterization of human collagenase-3. J Biol Chem 271, 1544-1550.
Kuzuya, M., and Iguchi, A. (2003). Role of matrix metalloproteinases in vascular remodeling. J Atheroscler Thromb 10, 275-282.
Lin, C.G., Chen, C.C., Leu, S.J., Grzeszkiewicz, T.M., and Lau, L.F. (2005).
Integrin-dependent functions of the angiogenic inducer NOV (CCN3): implication in wound healing. J Biol Chem 280, 8229-8237.
Lin, M.T., Chang, C.C., Chen, S.T., Chang, H.L., Su, J.L., Chau, Y.P., and Kuo, M.L.
(2004). Cyr61 expression confers resistance to apoptosis in breast cancer MCF-7 cells by a mechanism of NF-kappaB-dependent XIAP up-regulation. J Biol Chem 279, 24015-24023.
Lochter, A., Sternlicht, M.D., Werb, Z., and Bissell, M.J. (1998). The significance of matrix metalloproteinases during early stages of tumor progression. Ann N Y Acad Sci 857, 180-193.
Maillard, M., Cadot, B., Ball, R.Y., Sethia, K., Edwards, D.R., Perbal, B., and Tatoud, R. (2001). Differential expression of the ccn3 (nov) proto-oncogene in human prostate cell lines and tissues. Mol Pathol 54, 275-280.
Manara, M.C., Perbal, B., Benini, S., Strammiello, R., Cerisano, V., Perdichizzi, S., Serra, M., Astolfi, A., Bertoni, F., Alami, J., et al. (2002). The expression of ccn3(nov) gene in musculoskeletal tumors. Am J Pathol 160, 849-859.
Matrisian, L.M. (1992). The matrix-degrading metalloproteinases. Bioessays 14, 455-463.
McCallum, L., Lu, W., Price, S., Lazar, N., Perbal, B., and Irvine, A.E. (2009). CCN3:
a key growth regulator in Chronic Myeloid Leukaemia. J Cell Commun Signal 3, 115-124.
McCawley, L.J., and Matrisian, L.M. (2000). Matrix metalloproteinases:
multifunctional contributors to tumor progression. Mol Med Today 6, 149-156.
the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8, 221-233.
Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987). Extended and globular protein domains in cartilage proteoglycans. Biochem J 245, 763-772.
Perbal, B. (2004). CCN proteins: multifunctional signalling regulators. Lancet 363, 62-64.
Perbal, B., Lazar, N., Zambelli, D., Lopez-Guerrero, J.A., Llombart-Bosch, A., Scotlandi, K., and Picci, P. (2009). Prognostic relevance of CCN3 in Ewing sarcoma.
Hum Pathol 40, 1479-1486.
Perbal, B., Zuntini, M., Zambelli, D., Serra, M., Sciandra, M., Cantiani, L., Lucarelli, E., Picci, P., and Scotlandi, K. (2008). Prognostic value of CCN3 in osteosarcoma.
Clin Cancer Res 14, 701-709.
Poole, A.R., Rizkalla, G., Ionescu, M., Reiner, A., Brooks, E., Rorabeck, C., Bourne, R., and Bogoch, E. (1993). Osteoarthritis in the human knee: a dynamic process of cartilage matrix degradation, synthesis and reorganization. Agents Actions Suppl 39, 3-13.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12, 697-715.
Scatena, M., and Giachelli, C. (2002). The alpha(v)beta3 integrin, NF-kappaB, osteoprotegerin endothelial cell survival pathway. Potential role in angiogenesis.
Trends Cardiovasc Med 12, 83-88.
Sin, W.C., Tse, M., Planque, N., Perbal, B., Lampe, P.D., and Naus, C.C. (2009).
Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization. J Biol Chem 284, 29935-29944.
Sporn, M.B. (1996). The war on cancer. Lancet 347, 1377-1381.
Steeg, P.S. (2003). Metastasis suppressors alter the signal transduction of cancer cells.
Nat Rev Cancer 3, 55-63.
Stetler-Stevenson, W.G. (1996). Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix. Am J Pathol 148, 1345-1350.
Tan, T.W., Yang, W.H., Lin, Y.T., Hsu, S.F., Li, T.M., Kao, S.T., Chen, W.C., Fong, Y.C., and Tang, C.H. (2009). Cyr61 increases migration and MMP-13 expression via alphavbeta3 integrin, FAK, ERK and AP-1-dependent pathway in human chondrosarcoma cells. Carcinogenesis 30, 258-268.
Van Wart, H.E., and Birkedal-Hansen, H. (1990). The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc Natl Acad Sci U S A 87, 5578-5582.
Webb, D.J., Donais, K., Whitmore, L.A., Thomas, S.M., Turner, C.E., Parsons, J.T., and Horwitz, A.F. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6, 154-161.
Westhoff, M.A., Serrels, B., Fincham, V.J., Frame, M.C., and Carragher, N.O. (2004).
SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling. Mol Cell Biol 24, 8113-8133.
Yang, G.P., and Lau, L.F. (1991). Cyr61, product of a growth factor-inducible immediate early gene, is associated with the extracellular matrix and the cell surface.
Cell Growth Differ 2, 351-357.
Yu, C., Le, A.T., Yeger, H., Perbal, B., and Alman, B.A. (2003). NOV (CCN3) regulation in the growth plate and CCN family member expression in cartilage neoplasia. J Pathol 201, 609-615.
Zeng, Z.J., Yang, L.Y., Ding, X., and Wang, W. (2004). Expressions of cysteine-rich61, connective tissue growth factor and Nov genes in hepatocellular carcinoma and their clinical significance. World J Gastroenterol 10, 3414-3418.
Zhang, Z., Vuori, K., Wang, H., Reed, J.C., and Ruoslahti, E. (1996). Integrin activation by R-ras. Cell 85, 61-69.
კ߄
Fig 6. CCN3 induced the migration activity of human chondrosarcoma cells.
JJ012 cells were incubated with various concentrations of CCN3, and in vitro migration activities measured with the Transwell after 24 h showed that CCN3 (30 ng/ml) increased cell migration significantly (A). JJ012 cells sere stimulated by indicated concentraction intervals (0 ,10, 30 and 100 ng/ml) and proliferation was determined by MTT assay (B). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control.
Fig 7. CCN3-directed migration activity of human chondrosarcoma cells involves upregulation of MMP-13.
JJ012 cells were incubated with CCN3 (30 ng/ml) for 24h or for indicated time intervals, cell lysates were then collected and the mRNA level of MMP-1, -2, -3, -9 and -13 was determined using qPCR (A). Cells were incubated with CCN3 (30 ng/ml) for indicated time intervals .The cultured medium and cell lysates were then collected. Both the protein level of MMP-13 in cell lysates determined by Western blot analysis and the enzyme activity of MMP-13 in cell lysates and supernatant determined using zymography were increased in a time-dependent manner (B). Cells were transfected with MMP-13 or control siRNA for 24 h,and in vitro migration was measured with the Transwell after 24 h (C) Cells were transfected with MMP-13 or control siRNA for 24 h, and the mRNA and protein levels of MMP-13 were examined using Western blot analysis (D). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control. #p<0.05 was compared with CCN3.
Fig 8. CCN3 increased human chondrosarcoma cells migration and MMP-13 expression through Įvȕ3 and Įvȕ5 integrin receptor.
JJ012 cells were pretreated with Į5ȕ1ǵĮvȕ3 or Įvȕ5 antibody (5 ȝg/ml) after treating with CCN3 (30 ng/ml) for 24h, and MMP-13 expression was determined by Western blot analysis (A). JJ012 cells were pretreated with Įvȕ3 mAb (5 ȝg/ml), Įvȕ5 mAb (5 ȝg/ml) cyclic RGD (10 nM) and cyclic RAD (10 nM) for 30 min followed by stimulation with CCN3 (30 ng/ml). The in vitro migration activity measured after 24 h showed that Įvȕ3 mAbǵĮvȕ5 mAb could inhibit the cell migration (C). The qPCR result show that Įvȕ3 mAb, Įvȕ5 mAb and cyclic RGD but not Į5ȕ1 mAb and cyclic RAD could inhibit the MMP-13 expression (C). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control.
#p<0.05 was compared with CCN3.
Fig 9. Involvement of FAK-signaling pathway in response to CCN3 in chondrosarcoma cells.
(A) JJ012 cells were incubated with CCN3(30 ng/ml) for indicated time intervals, and p-FAK expression was determined by Western blot analysis.
Note that CCN3 activated the FAK pathway in JJ012 cells. (B) Cells were transfected with mutant and siRNA of FAK for 24 h followed by stimulation with CCN3 (30 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (C) JJ012 cells were transfected with mutant of FAK for 24 h followed by stimulation with CCN3 (30 ng/ml), and the mRNA level of MMP-13 were determined by using qPCR.
Results are presented as mean±S.E. (n=3). * p<0.05 was compared with control.#p <0.05 was compared with CCN3.
Fig 10. PI3K is involved in CCN3-mediated human chondrosarcoma migration and MMP-13 expression.
JJ012 cells were incubated with CCN3 (30 ng/ml) for indicated time intervals, and p-PI3K expression was determined by Western blot analysis (A). JJ012 cells were pretreated with Ly294002 (10PM) and wortmannin (10PM) for 30 min followed by stimulation with CCN3 (30 ng/ml) for 24 h, and in vitro migration was measured with the Transwell after 24 h (B). JJ012 cells were pretreated with Ly294002 (10PM) and wortmannin (10PM) for 30 min followed by stimulation with CCN3 (30 ng/ml) for 24 h, and the mRNA level of MMP-13 were determined by using qPCR (C). Cells were transfected with mutant of PI3K (p85) for 24 h followed by stimulation with CCN3 (30 ng/ml), and in vitro migration was measured with the Transwell after 24 h (D). JJ012 cells were transfected with mutant of PI3K (p85) for 24 h followed by stimulation with CCN3 (30 ng/ml), and the mRNA of MMP-13 were determined by using qPCR (E). Results are presented as mean±S.E. (n=3). * p<0.05 was compared with control. #p <0.05 was compared with CCN3.
Fig 11. Akt is involved in CCN3-mediated human chondrosarcoma migration and MMP-13 expression.
JJ012 cells were incubated with CCN3 (30 ng/ml) for indicated time intervals, and p- Akt expression was determined by Western blot analysis (A). JJ012 cells were pretreated with Akt inhibitor (10 PM) for 30 min followed by stimulation with CCN3 (30 ng/ml) for 24 h, and in vitro migration was measured with the Transwell after 24 h (B). JJ012 cells were pretreated with Akt inhibitor (10PM) for 30 min followed by stimulation with CCN3 (50 ng/ml) for 24 h, and the mRNA level of MMP-13 were determined by using qPCR (C). Cells were transfected with p85 mutant for 24 h followed by stimulation with CCN3 (30 ng/ml), and in vitro migration was measured with the Transwell after 24 h (D).
JJ012 cells were transfected with p85 mutant for 24 h followed by stimulation with CCN3 (30 ng/ml), and the mRNA of MMP-13 were determined by using qPCR (E). Results are presented as Mean±S.E. (n=3).
* p<0.05 was compared with control. #p<0.05 was compared with CCN3.
Fig 12. NF-țB involved in CCN3-mediated human chondrosarcoma migration and MMP-13 expression.
JJ012 cells were pretreated with NF-țB inhibitor [PDTC (10 PM)ǵTPCK (3 PM), and NF-țB inhibitor peptide (10 PM)] for 30 min followed by stimulation with CCN3 (30 ng/ml) for 24 h, and in vitro migration was measured with the Transwell after 24 h (A). JJ012 cells were pretreated with NF-țB inhibitor [PDTC (10 PM)ǵTPCK (3 PM), and NF-țB inhibitor peptide (10 PM)] for 30 min followed by stimulation with CCN3 (30 ng/ml) for 24 h, and the mRNA level of MMP-13 were determined by using qPCR (B). JJ012 cells were incubated with CCN3 (30 ng/ml) for indicated time intervals, and pIKKĮ/ȕ, p-IțB and p-p65 expression was determined by Western blot analysis (C). Cells were transfected with mutant of IKKĮ and IKKȕ for 24 h followed by stimulation with CCN3 (30 ng/ml), and in vitro migration was measured with the Transwell after 24 h (D). JJ012 cells were transfected with mutant of IKKĮ and IKKȕ for 24 h followed by stimulation with CCN3 (30 ng/ml), and the mRNA of MMP-13 were determined by using qPCR (E). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control. #p<0.05 was compared with CCN3.
Fig 13. NF-țB involved in CCN3-mediated human chondrosarcoma migration and MMP-13 expression.
JJ012 cells were transfected with țB-luciferase expression vector and then pretreated with inhibitor of Ly294002 (10 ȝM) wortmannin (10 ȝM)ǵAkt (10 ȝM)ǵPDTC (10 ȝM)ǵTPCK (10 ȝM) and NF-țB inhibitor peptide (10 ȝM) for 30 min, before incubation with CCN3 (30 ng/ml) for 24 h (A). JJ012 cells were co-transfected with țB-luciferase expression vector and FAK mutantǵp85 mutantǵAkt mutantǵIKKĮ mutantǵIKKȕ mutant for 24 h, before incubation with CCN3 (30 ng/ml) for 24 h (B).
Luciferase activity were determined. Results are presented as mean±S.E.
(n=3). * p<0.05 was compared with control. #p <0.05 was compared with CCN3.
Fig 14. Schematic presentation of the signaling pathways involved in CCN3-induced migration and MMP-13 expression of human chondrosarcoma cells.
CCN3 activates PI3K and Akt pathway, which in turn induces IKKĮ/ȕ phosphorylation, p65 Ser536 phosphorylation, whch leads to MMP-13 expresion and increases the migration through Įvȕ3 and Dvȕ5 integrin.