Bojović B, Rodrigues N, Dehbi M, Bédard P A
Department of Biology, York University, North York, Ontario M3J 1P3, Canada.
J Biol Chem. 1996 Sep 13;271(37):22528-37. doi: 10.1074/jbc.271.37.22528.
The CEF-4/9E3 cytokine gene is expressed aberrantly in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus. The expression of CEF-4 is dependent on both transcriptional and post-transcriptional mechanisms of regulation. The characterization of the promoter region indicated that three distinct regulatory elements corresponding to an AP-1 binding site (or TRE), a PRDII/kappaB domain, and a CAAT box are involved in the activation by pp60(v-)src. In this report we investigate the signaling pathways controlling the expression of the TRE and PRDII domain. The expression of a dominant negative mutant of p21(ras) reduced the activity of both elements. In contrast a similar mutant of c-Raf-1 affected modestly the activation dependent on the TRE but not PRDII. The stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was important for the activity of PRDII and the TRE but was not markedly stimulated by pp60(v-)src. The addition of calphostin C and the inhibition of protein kinase C (PKC) diminished the accumulation of the CEF-4 mRNA and reduced the activity of a TRE-controlled promoter. Likewise, the depletion of PKC by chronic treatment with phorbol esters inhibited the activation of the TRE. Rous sarcoma virus-transformed CEF treated with calphostin C were also flatter, did not display a high degree of criss-crossing, and appeared morphologically normal. Hence PKC was important for the activation of AP-1 and the morphological transformation of CEF. The constitutive expression of CEF-4 was correlated with transformation only when dependent on the TRE. This was not true for PRDII, which was the only element required for the constitutive activation to the CEF-4 promoter in nontransformed cells treated chronically with phorbol esters.
CEF-4/9E3细胞因子基因在劳氏肉瘤病毒转化的鸡胚成纤维细胞（CEF）中异常表达。CEF-4的表达依赖于转录和转录后调控机制。启动子区域的特征表明，与AP-1结合位点（或TRE）、PRDII/kappaB结构域和CAAT盒相对应的三个不同调控元件参与了pp60（v-）src的激活。在本报告中，我们研究了控制TRE和PRDII结构域表达的信号通路。p21（ras）显性负突变体的表达降低了两个元件的活性。相比之下，c-Raf-1的类似突变体对依赖TRE的激活有适度影响，但对PRDII没有影响。应激激活蛋白激酶（SAPK）/Jun N末端激酶（JNK）通路对PRDII和TRE的活性很重要，但未被pp60（v-）src显著激活。加入钙泊三醇C和抑制蛋白激酶C（PKC）可减少CEF-4 mRNA的积累，并降低TRE控制的启动子的活性。同样，用佛波酯长期处理耗尽PKC可抑制TRE的激活。用钙泊三醇C处理的劳氏肉瘤病毒转化的CEF也更扁平，没有显示出高度的交叉，并且形态上看起来正常。因此，PKC对AP-1的激活和CEF的形态转化很重要。仅当依赖TRE时，CEF-4的组成型表达才与转化相关。对于PRDII则不然，PRDII是在用佛波酯长期处理的未转化细胞中组成型激活CEF-4启动子所需的唯一元件。
