Lengyel E, Gum R, Stepp E, Juarez J, Wang H, Boyd D
Department of Tumor Biology/Head and Neck Surgery, M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Cell Biochem. 1996 Jun 1;61(3):430-43. doi: 10.1002/(sici)1097-4644(19960601)61:3<430::aid-jcb10>3.0.co;2-n.
The urokinase-type plasminogen activator contributes to tissue remodeling by controlling the synthesis of the extracellular matrix-degrading plasmin. We undertook a study to determine the role of the extracellular signal-regulated kinases (ERKs) in the regulation of urokinase-type plasminogen activator expression in a squamous cell carcinoma cell line (UM-SCC-1) that contains a transcriptionally activated urokinase-type plasminogen activator gene. Transient transfection studies using a CAT reporter driven by the urokinase-type plasminogen activator promoter, which had progressive 5' deletions or which had been point-mutated, indicated the requirement of binding sites for AP-1 (-1967) and PEA3 (-1973) for its maximal activation. Expression of a mutant jun protein, which lacks the transactivation domain, caused a dose-dependent repression of a CAT reporter driven by either the urokinase-type plasminogen activator promoter or three tandem AP-1 repeats upstream of a thymidine kinase minimal promoter indicating the importance of AP-1-binding transcription factor(s) in the regulation of urokinase-type plasminogen activator synthesis. Mobility shift assays with UM-SCC-1 nuclear extract revealed binding of fos and junD proteins to an oligonucleotide spanning the AP-1 site at -1967. In-gel kinase assays indicated the constitutive activation of ERK1, which regulates fos synthesis via phosphorylation of p62TCF, but not ERK2, in UM-SCC-1 cells. Moreover, the expression of a dominant-negative ERK1, but not ERK2, repressed urokinase-type plasminogen activator promoter activity. Similarly, interfering with the function of the c-raf serine-threonine kinase, which lies upstream of ERK1, by the expression of a kinase-inactive c-raf repressed the activity of a CAT reporter driven by either the urokinase-type plasminogen activator promotor or tandem AP-1 repeats. These data suggest that urokinase-type plasminogen activator expression in UM-SCC-1 cells is regulated partly by an ERK1, but not ERK2, -dependent signaling pathway.
尿激酶型纤溶酶原激活剂通过控制细胞外基质降解性纤溶酶的合成来促进组织重塑。我们进行了一项研究，以确定细胞外信号调节激酶（ERK）在含有转录激活的尿激酶型纤溶酶原激活剂基因的鳞状细胞癌细胞系（UM-SCC-1）中对尿激酶型纤溶酶原激活剂表达的调节作用。使用由尿激酶型纤溶酶原激活剂启动子驱动的CAT报告基因进行瞬时转染研究，该启动子具有渐进性5'缺失或点突变，结果表明AP-1（-1967）和PEA3（-1973）的结合位点对其最大激活是必需的。缺乏反式激活结构域的突变型jun蛋白的表达导致由尿激酶型纤溶酶原激活剂启动子或胸苷激酶最小启动子上游的三个串联AP-1重复序列驱动的CAT报告基因的剂量依赖性抑制，这表明AP-1结合转录因子在尿激酶型纤溶酶原激活剂合成调节中的重要性。用UM-SCC-1核提取物进行的迁移率变动分析显示fos和junD蛋白与跨越-1967处AP-1位点的寡核苷酸结合。凝胶内激酶分析表明，在UM-SCC-1细胞中，ERK1通过p62TCF的磷酸化调节fos合成，但不调节ERK2，处于组成性激活状态。此外，显性负性ERK1而非ERK2的表达抑制了尿激酶型纤溶酶原激活剂启动子活性。同样，通过表达激酶无活性的c-raf来干扰位于ERK1上游的c-raf丝氨酸-苏氨酸激酶的功能，可抑制由尿激酶型纤溶酶原激活剂启动子或串联AP-1重复序列驱动的CAT报告基因的活性。这些数据表明，UM-SCC-1细胞中尿激酶型纤溶酶原激活剂的表达部分受ERK1而非ERK2依赖性信号通路的调节。
