THE AP-1 SEQUENCE IS NECESSARY BUT NOT SUFFICIENT FOR PHORBOL INDUCTION OF COLLAGENASE IN FIBROBLASTS
THE AP-1 SEQUENCE IS NECESSARY BUT NOT SUFFICIENT FOR PHORBOL INDUCTION OF COLLAGENASE IN FIBROBLASTS
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DOI：
10.1021/bi00232a039
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发表时间：
1991-05-07
期刊：
影响因子：
2.9
通讯作者：
BRINCKERHOFF, CE
中科院分区：
文献类型：
--
作者：
AUBLE, DT;BRINCKERHOFF, CE
胶原酶，唯一的酶活性在中性pH值，启动胶原蛋白降解，是一个主要的基因产物的成纤维细胞，已被刺激与各种药剂，包括佛波醇酯。 为了研究控制胶原酶基因表达的机制，我们用含有与氯霉素乙酰转移酶基因（CAT）连接的高达1.2 kb的兔胶原酶5 '侧翼DNA的嵌合构建体瞬时转染兔滑膜成纤维细胞。 我们的数据表明，佛波醇响应的大小直接与启动子片段的大小有关，并且赋予佛波醇诱导的最小的启动子DNA片段是127 bp。 对该片段的缺失和突变分析表明，单独的AP-1序列不足以诱导佛波醇，需要至少两个额外的序列（PEA 3样元件和包括5 '-TTCA-3'的序列）的存在。 此外，当转染含有182 bp的5 ′-侧翼DNA的片段时，观察到应答性的显著增加，暗示位于-182和-149之间的36 bp区域作为增强子。 我们的结论是：（1）AP-1序列是必需的，但不足以在成人成纤维细胞中表达胶原酶，（2）佛波醇诱导依赖于胶原酶启动子内的几个序列元件之间的合作，和（3）该启动子的调控比以前描述的更复杂。
Collagenase, the only enzyme active at neutral pH that initiates collagen degradation, is a major gene product of fibroblasts that have been stimulated with a variety of agents, including phorbol esters. To study mechanisms controlling collagenase gene expression, we transiently transfected rabbit synovial fibroblasts with chimeric constructs containing up to 1.2 kb of the rabbit collagenase 5'-flanking DNA linked to the chloramphenicol acetyltransferase gene (CAT). Our data indicate that the magnitude of the phorbol response is directly linked to the size of the promoter fragment and that the smallest piece of promoter DNA conferring phorbol inducibility is 127 bp. Deletional and mutational analysis of this fragment revealed that the AP-1 sequence alone is insufficient for phorbol inducibility and the presence of at least two additional sequences (a PEA3-like element and a sequence that includes 5'-TTCA-3') is required. In addition, a substantial increase in responsiveness is seen when a fragment containing 182 bp of 5'-flanking DNA is transfected, implicating a 36 bp region located between -182 and -149 as an enhancer. We conclude (1) that the AP-1 sequence is necessary but insufficient for expression of collagenase in adult fibroblasts, (2) that phorbol inducibility depends on cooperation among several sequence elements within the collagenase promoter, and (3) that regulation of this promoter is more complex than previously described.