Kim S, Mao P L, Gagliardi M, Bédard P A
Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3.
Mol Cell Biol. 1999 Aug;19(8):5718-31. doi: 10.1128/MCB.19.8.5718.
The p20K gene is induced in conditions of reversible growth arrest in chicken embryo fibroblasts (CEF). This expression is dependent on transcriptional activation and on a region of the promoter designated the quiescence-responsive unit (QRU). In this report, we describe the regulatory elements of the QRU responsible for activation in resting cells and characterize the trans-acting proteins interacting with these elements. We show that the QRU consists of functionally distinct domains including quiescence-specific and weak proliferation-responsive elements. The quiescence responsiveness of the QRU was mapped to two C/EBP binding sites, and the activity of the p20K promoter and its QRU was inhibited by the expression of a dominant negative mutant of C/EBPbeta in nondividing cells. The activation of QRU in response to serum starvation and contact inhibition correlated with the presence of a growth arrest-specific complex in electrophoretic mobility shift assays. This complex was supershifted by antibody for C/EBPbeta. C/EBPbeta accumulated in conditions of contact inhibition as a result of transcriptional activation. Therefore, C/EBPbeta was itself regulated as a growth arrest-specific gene in CEF. Finally, we show that the expression of p20K is regulated by linoleic acid, an essential fatty acid binding to p20K. The addition of linoleic acid to contact-inhibited CEF markedly repressed the synthesis of p20K without inducing mitogenesis. The activity of the QRU was inhibited by linoleic acid or the peroxisome proliferator-activated receptor PPARgamma2 in transient expression assays. Therefore, we have identified C/EBPbeta as a key activator of a growth arrest-specific gene in CEF and implicated an essential fatty acid, linoleic acid, in regulation of the QRU and the p20K lipocalin gene.
p20K基因在鸡胚成纤维细胞（CEF）可逆性生长停滞的条件下被诱导表达。这种表达依赖于转录激活以及启动子中一个被称为静止反应单元（QRU）的区域。在本报告中，我们描述了负责静止细胞中激活的QRU调控元件，并对与这些元件相互作用的反式作用蛋白进行了表征。我们表明，QRU由功能不同的结构域组成，包括静止特异性和弱增殖反应元件。QRU的静止反应性被定位到两个C/EBP结合位点，并且在非分裂细胞中，C/EBPβ显性负突变体的表达抑制了p20K启动子及其QRU的活性。在电泳迁移率变动分析中，QRU对血清饥饿和接触抑制的激活与生长停滞特异性复合物的存在相关。该复合物被C/EBPβ抗体超迁移。由于转录激活，C/EBPβ在接触抑制条件下积累。因此，C/EBPβ自身在CEF中作为生长停滞特异性基因受到调控。最后，我们表明p20K的表达受亚油酸调节，亚油酸是一种与p20K结合的必需脂肪酸。向接触抑制的CEF中添加亚油酸显著抑制了p20K的合成，而不诱导有丝分裂。在瞬时表达分析中，亚油酸或过氧化物酶体增殖物激活受体PPARγ2抑制了QRU的活性。因此，我们确定C/EBPβ是CEF中生长停滞特异性基因的关键激活因子，并表明必需脂肪酸亚油酸参与了QRU和p20K脂质运载蛋白基因的调控。
