Fushimi Kazunari, Nakashima Shigeru, You Fukka, Takigawa Masaharu, Shimizu Katsuji
Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu, Japan.
J Cell Biochem. 2007 Feb 15;100(3):783-93. doi: 10.1002/jcb.21099.
Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.
基质金属蛋白酶-1（MMP-1，胶原酶-1）在退行性关节疾病的关节破坏过程中起关键作用。我们研究了肿瘤坏死因子-α（TNF-α）刺激下人软骨细胞HCS-2/8中MMP-1产生的调节机制。在TNF-α刺激下，MMP-1在HCS-2/8细胞中被诱导并大量释放。MMP-1表达的诱导与ERK1/2、MEK和Raf-1的激活相关，并且被MEK1/2激活的选择性抑制剂U0126有效抑制。相反，选择性p38丝裂原活化蛋白激酶（MAPK）抑制剂SB203580对TNF-α诱导的MMP-1释放没有影响。丝氨酸/苏氨酸激酶Akt在TNF-α刺激的HCS-2/8细胞中未被激活。TNF-α除了刺激HCS-2/8细胞产生MMP-1外，还刺激了前列腺素E2（PGE2）的产生。外源性添加的PGE2有效抑制了TNF-α诱导的MMP-1产生以及ERK1/2的激活。PGE2的作用被选择性EP4受体激动剂ONO-AE1-329模拟，但未被选择性EP2激动剂布他前列素模拟。相反，选择性EP4受体拮抗剂ONO-AE3-208阻断内源性产生的PGE2信号，增强了TNF-α诱导的MMP-1产生。此外，外源性添加的PGE2对MMP-1产生的抑制作用被ONO-AE3-208逆转。EP4受体的激活导致Raf-1在负调控位点Ser259上发生cAMP介导的磷酸化，并阻断了Raf-1/MEK/ERK级联反应的激活。综上所述，这些发现表明Raf-1/MEK/ERK信号通路在HCS-2/8细胞响应TNF-α产生MMP-1的过程中起关键作用，并且产生的PGE2通过EP4-PGE2受体激活阻断TNF-α诱导的Raf-1激活，从而下调MMP-1的表达。
