The integrin alpha5beta1 regulates chondrocyte hypertrophic differentiation induced by GTP-bound transglutaminase 2.
Soluble GTP-bound transglutaminase 2 (TG2) induces hypertrophic differentiation in chondrocyte cultures in a beta1 integrin-dependent fashion. beta1 integrin subfamily consists of 12 heterodimers with 12 different alpha subunits and a beta1 subunit. To identify the specific integrin heterodimer(s) responsible for this process, we specifically blocked individual beta1 integrins on the CH-8 immortalized human chondrocytes during hypertrophic differentiation. Blockade of alpha5beta1 inhibited matrix metalloproteinase 13 (MMP-13), type X collagen expression, alkaline phosphatase activity and matrix calcification by 30-50% associated with weak effects of anti-alpha3beta1 and -alpha4beta1. Anti-alpha1beta1, -alpha2beta1 and -alpha6beta1 had no effect. To examine whether the dominant effect of integrin alpha5beta1 was due to a direct interaction with TG2, we incubated the chondrocytic cells on plates coated with GTP-bound TG2. The immobilized GTP-bound TG2 induced hypertrophic differentiation to the same extent as the soluble GTP-bound TG2, which was also inhibited by anti-alpha5beta1. CH-8 cells grown on plates coated with GTP-bound TG2 demonstrated adherence associated with focal adhesion kinase phosphorylation. These properties were inhibited by anti-alpha5beta1. Furthermore, engagement of alpha5beta1 on CH-8 cells via anti-alpha5beta1 antibody did, in fact, induce differentiation. Although CH-8 cells adhered to GTP-free TG2 via integrin alpha5beta1, the cells failed to undergo hypertrophic differentiation. Thus, integrin alpha5beta1 is critical for the chondrocyte hypertrophic differentiation induced by GTP-bound TG2, and this induction is ligand dependent.
Tanaka
Kumi
K
Department of Orthopedics, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan.
Yokosaki
Yasuyuki
Y
Higashikawa
Fumiko
F
Saito
Yuji
Y
Eboshida
Akira
A
Ochi
Mitsuo
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D024121:Collagen Type X / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D039001:Integrin alpha Chains / Q000276:immunology / Q000378:metabolism; D039201:Integrin alpha3beta1 / Q000378:metabolism; D039041:Integrin alpha4beta1 / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000502:physiology*; D053509:Matrix Metalloproteinase 13 / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011503:Transglutaminases / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D024043:Collagen Type II; D024121:Collagen Type X; D004800:Enzymes, Immobilized; D039001:Integrin alpha Chains; D039201:Integrin alpha3beta1; D039041:Integrin alpha4beta1; D039081:Integrin alpha5beta1; D006160:Guanosine Triphosphate; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D000469:Alkaline Phosphatase; D053509:Matrix Metalloproteinase 13; D019204:GTP-Binding Proteins