Tanaka Kumi, Yokosaki Yasuyuki, Higashikawa Fumiko, Saito Yuji, Eboshida Akira, Ochi Mitsuo
Department of Orthopedics, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan.
Matrix Biol. 2007 Jul;26(6):409-18. doi: 10.1016/j.matbio.2007.04.005. Epub 2007 Apr 19.
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.
可溶性鸟苷三磷酸（GTP）结合转谷氨酰胺酶2（TG2）以β1整合素依赖性方式诱导软骨细胞培养物中的肥大分化。β1整合素亚家族由12种异二聚体组成，具有12种不同的α亚基和1个β1亚基。为了确定负责此过程的特定整合素异二聚体，我们在肥大分化过程中特异性阻断了CH-8永生化人软骨细胞上的单个β1整合素。阻断α5β1可使基质金属蛋白酶13（MMP-13）、X型胶原表达、碱性磷酸酶活性和基质钙化降低30%-50%，同时抗α3β1和抗α4β1的作用较弱。抗α1β1、抗α2β1和抗α6β1则无作用。为了研究整合素α5β1的主要作用是否是由于与TG2的直接相互作用，我们将软骨细胞接种在包被有GTP结合TG2的平板上。固定化的GTP结合TG2诱导肥大分化的程度与可溶性GTP结合TG2相同，且也被抗α5β1抑制。在包被有GTP结合TG2的平板上生长的CH-8细胞表现出与粘着斑激酶磷酸化相关的粘附。这些特性被抗α5β1抑制。此外，通过抗α5β1抗体使CH-8细胞上的α5β1结合，实际上确实诱导了分化。尽管CH-8细胞通过整合素α5β1粘附于无GTP的TG2，但细胞未能发生肥大分化。因此，整合素α5β1对于GTP结合TG2诱导的软骨细胞肥大分化至关重要，且这种诱导是配体依赖性的。
