Cook T F, Burke J S, Bergman K D, Quinn C O, Jeffrey J J, Partridge N C
Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, Missouri 63104.
Arch Biochem Biophys. 1994 Jun;311(2):313-20. doi: 10.1006/abbi.1994.1243.
Rat tissue inhibitor of metalloproteinases-2 (TIMP-2) was cloned from a UMR 106-01 rat osteoblastic osteosarcoma cDNA library. The 969-bp full-length clone demonstrates 98 and 86% sequence identity to human TIMP-2 at the amino acid and nucleic acid levels, respectively. Parathyroid hormone (PTH), at 10(-8) M, stimulates an approximately twofold increase in both the 4.2- and 1.0-kb transcripts over basal levels in UMR cells after 24 h of exposure. The PTH stimulation of TIMP-2 transcripts was not affected by the inhibitor of protein synthesis, cycloheximide (10(-5) M), suggesting a primary effect of the hormone. This is in contradistinction to regulation of interstitial collagenase (matrix metalloproteinase-1) by PTH in these same cells. Nuclear run-on assays demonstrate that PTH causes an increase in TIMP-2 transcription that parallels the increase in message levels. Parathyroid hormone, in its stimulation of TIMP-2 mRNA, appears to act through a signal transduction pathway involving protein kinase A (PKA) since the increase in TIMP-2 mRNA is reproduced by treatment with the cAMP analogue, 8-bromo-cAMP (5 x 10(-3) M). The protein kinase C and calcium pathways do not appear to be involved due to the lack of effect of phorbol 12-myristate 13-acetate (2.6 x 10(-6) M) and the calcium ionophore, ionomycin (10(-7) M), on TIMP-2 transcript abundance. In this respect, regulation of TIMP-2 and collagenase in osteoblastic cells by PTH are similar. However, we conclude that since stimulation of TIMP-2 transcription is a primary event, the PKA pathway must be responsible for a direct increase in transcription of this gene.
大鼠金属蛋白酶组织抑制剂-2（TIMP-2）是从UMR 106-01大鼠成骨细胞骨肉瘤cDNA文库中克隆得到的。这个969碱基对的全长克隆在氨基酸和核酸水平上与人TIMP-2的序列同一性分别为98%和86%。甲状旁腺激素（PTH）在10^(-8) M时，在UMR细胞中作用24小时后，可使4.2 kb和1.0 kb转录本相对于基础水平增加约两倍。PTH对TIMP-2转录本的刺激不受蛋白质合成抑制剂环己酰亚胺（10^(-5) M）的影响，提示这是该激素的主要作用。这与PTH对这些相同细胞中间质胶原酶（基质金属蛋白酶-1）的调节形成对比。核转录分析表明，PTH导致TIMP-2转录增加，这与信息水平的增加平行。甲状旁腺激素在刺激TIMP-2 mRNA时，似乎通过涉及蛋白激酶A（PKA）的信号转导途径起作用，因为用cAMP类似物8-溴-cAMP（5×10^(-3) M）处理可重现TIMP-2 mRNA的增加。由于佛波酯12-肉豆蔻酸酯13-乙酸酯（2.6×10^(-6) M）和钙离子载体离子霉素（10^(-7) M）对TIMP-2转录本丰度没有影响，蛋白激酶C和钙途径似乎不参与其中。在这方面，PTH对成骨细胞中TIMP-2和胶原酶的调节是相似的。然而，我们得出结论，由于TIMP-2转录的刺激是一个主要事件，PKA途径必定是该基因转录直接增加的原因。
