Loss of JunB Activity Enhances Stromelysin 1 Expression in a Model of the Epithelial-to-Mesenchymal Transition of Mouse Skin Tumors

Chemical carcinogenesis in mouse skin has been useful in delineating the molecular events that underlie squamous cell carcinoma progression. A late event in this progression, the epithelial-to-mesenchymal transition (EMT), is characterized by the loss of epithelial markers and the presence of mesenchymal markers. One mesenchymal marker associated with this transition is the matrix metalloproteinase stromelysin 1 (Str-1). To examine the molecular mechanisms regulating the expression of Str-1 during the EMT, genetically related mouse skin tumor cell lines representing the epithelial (B9(SQ)) and mesenchymal (A5(SP)) phenotypes were studied. As expected, B9(SQ) cells did not make Str-1, while A5(SP) cells did. B9(SQ)-A5(SP) somatic hybrids did not make Str-1, suggesting that a critical regulatory factor was a B9(SQ)-specific repressor. Str-1 promoter analysis revealed that a canonical AP-1 site was sufficient to maintain differential reporter gene activity. This result correlated with the observed loss of binding of the transcriptionally inactive JunB–Fra-2 AP-1 complex from B9(SQ) cells, being replaced primarily by the more active JunD–Fra-2 complex in A5(SP) cells. The higher level of JunB binding to both DNA and Fra-2 correlated with its hyperphosphorylation by Jun N-terminal kinase, an activity that was significantly higher in B9(SQ) cells. In the somatic hybrids, JunB gene expression was highly upregulated, a condition that also was sufficient to repress the expression of the endogenous Str-1 gene in A5(SP) cells. These data suggested that alterations in JunB activity, by changes in either phosphorylation or gene expression, contributed to the phenotypic differences that occur in this model of the EMT.

小鼠皮肤化学致癌作用模型长期以来均被用于解析鳞状细胞癌进展背后的分子事件。该进展中的晚期事件——上皮间质转化（epithelial-to-mesenchymal transition，EMT），其特征为上皮标志物的丢失与间质标志物的表达。与该转化相关的一种间质标志物为基质金属蛋白酶溶基质素1（matrix metalloproteinase stromelysin 1，Str-1）。为探究上皮间质转化过程中调控Str-1表达的分子机制，本研究针对分别携带上皮型（B9(SQ)）与间质型（A5(SP)）表型的遗传背景一致的小鼠皮肤肿瘤细胞系展开研究。正如预期，B9(SQ)细胞不表达Str-1，而A5(SP)细胞可表达Str-1。B9(SQ)-A5(SP)体细胞杂种细胞无法表达Str-1，这提示存在一类由B9(SQ)细胞特异性表达的关键调控阻遏因子。对Str-1启动子的分析结果显示，经典激活蛋白1（AP-1）结合位点足以维持差异的报告基因活性。该结果与观测到的现象一致：B9(SQ)细胞中，转录失活的JunB–Fra-2 AP-1复合物的结合活性丧失，而A5(SP)细胞中则主要被活性更强的JunD–Fra-2复合物所取代。JunB与DNA及Fra-2的结合水平更高，这与其经Jun氨基末端激酶（Jun N-terminal kinase）过度磷酸化密切相关，而B9(SQ)细胞内该激酶的活性显著更高。在体细胞杂种细胞中，JunB基因的表达显著上调，该条件同样足以阻遏A5(SP)细胞内源性Str-1基因的表达。上述数据表明，通过改变磷酸化水平或基因表达来调控JunB活性，可促成该上皮间质转化模型中出现的表型差异。