Sampieri Clara L, Nuttall Robert K, Young David A, Goldspink Deborah, Clark Ian M, Edwards Dylan R
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Matrix Biol. 2008 Mar;27(2):128-38. doi: 10.1016/j.matbio.2007.09.004. Epub 2007 Oct 4.
The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.
人类基质金属蛋白酶（MMP）基因家族包含24个基因，其表达受到调控，与四种金属蛋白酶组织抑制剂（TIMP）共同作用，在组织重塑和细胞信号传导中至关重要。采用定量实时聚合酶链反应（qPCR）分析，评估蛋白激酶C（PKC）激活剂佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）作用下，人类MRC-5和WI-38成纤维细胞中这两个基因家族共同的和独特的调控模式。使用三种蛋白质合成抑制剂茴香霉素、环己酰亚胺和依米丁分析持续翻译的需求。PMA在4-8小时后诱导MMP1、3、8、9、10、12、13、14以及TIMP1和TIMP3的RNA表达，除MMP9和TIMP3外，所有蛋白质合成抑制剂均能阻断其他基因的诱导表达。然而，尽管所有抑制剂均有效阻断翻译，但依米丁可阻断PMA对MMP9和TIMP3的诱导作用，而环己酰亚胺和茴香霉素对此无影响。单独使用茴香霉素可诱导MMP9、TIMP3以及MMP25和MMP19的表达。细胞外信号调节激酶（ERK）-1/2被PMA强烈激活，而茴香霉素激活c-Jun氨基末端激酶（JNK）和p38信号通路，环己酰亚胺激活p38，但依米丁对应激激活的丝裂原活化蛋白激酶（MAPK）信号通路无影响。使用药理学抑制剂证实了p38和JNK信号通路参与茴香霉素和环己酰亚胺对MMP/TIMP表达的选择性作用。这些数据证实，大多数可诱导的MMP和TIMP1在成纤维细胞中表现为“晚期”激活的、蛋白质合成依赖性基因。然而，蛋白质合成对PMA诱导MMP和TIMP的需求并非普遍存在，因为应激激活的MAPK信号通路的刺激可消除MMP9和TIMP3对蛋白质合成的需求。确定这两个基因家族中共同调控基因的簇将有助于对调控机制进行生物信息学剖析。
