Liu Yan, Su Weidong, Thompson E Aubrey, Leitges Michael, Murray Nicole R, Fields Alan P
Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224, USA.
J Biol Chem. 2004 Oct 29;279(44):45556-63. doi: 10.1074/jbc.M407701200. Epub 2004 Aug 20.
Protein kinase C betaII (PKCbetaII) is induced early during colon carcinogenesis. Transgenic mice expressing elevated PKCbetaII in the colonic epithelium (transgenic PKCbetaII mice) exhibit hyperproliferation and enhanced colon carcinogenesis. Here we demonstrate that nullizygous PKCbeta (PKCbetaKO) mice are highly resistant to azoxymethane (AOM)-induced preneoplastic lesions, aberrant crypt foci. However, reexpression of PKCbetaII in the colon of PKCbetaKO mice by transgenesis restores susceptibility to AOM-induced colon carcinogenesis. Expression of human PKCbetaII in rat intestinal epithelial (RIE) cells induces expression of endogenous rat PKCbetaII mRNA and protein. Induction of PKCbetaII is dependent upon catalytically active PKCbetaII and does not appear to involve changes in alternative splicing of the PKCbeta gene. Two human PKCbeta promoter constructs are activated by expression of PKCbetaII in RIE cells. Both PKCbeta promoter activity and PKCbetaII mRNA levels are inhibited by the MEK1 and -2 inhibitor U0126, but not the Cox-2 inhibitor celecoxib in RIE/PKCbetaII cells. PKCbeta promoter activity correlates directly with expression of endogenous PKCbetaII mRNA and protein in HT29 and HCT116 human colon cancer cell lines. PKCbeta promoter activity and PKCbetaII mRNA expression in HCT116 cells are inhibited by the selective PKCbeta inhibitor LY317615 and by U0126, demonstrating autoregulation of PKCbetaII expression. Transgenic PKCbetaII mice exhibit specific induction of endogenous PKCbetaII, but not its splice variant PKCbetaI, in the colonic epithelium in vivo. Taken together, our results demonstrate that 1) expression of PKCbetaII in the colonic epithelium is both necessary and sufficient to confer susceptibility to AOM-induced colon carcinogenesis in transgenic mice, 2) PKCbetaII regulates its own expression in RIE and human colon cancer cells in vitro and in the colonic epithelium in vivo, and 3) PKCbetaII autoregulation is mediated through a MEK-dependent signaling pathway in RIE/PKCbetaII and HCT116 colon cancer cells.
蛋白激酶CβII（PKCβII）在结肠癌发生早期被诱导。在结肠上皮中表达升高的PKCβII的转基因小鼠（转基因PKCβII小鼠）表现出过度增殖和结肠癌发生增强。在此我们证明，纯合缺失PKCβ（PKCβKO）的小鼠对氧化偶氮甲烷（AOM）诱导的癌前病变——异常隐窝灶具有高度抗性。然而，通过转基因在PKCβKO小鼠的结肠中重新表达PKCβII可恢复对AOM诱导的结肠癌发生的易感性。在大鼠肠上皮（RIE）细胞中表达人PKCβII可诱导内源性大鼠PKCβII mRNA和蛋白的表达。PKCβII的诱导依赖于具有催化活性的PKCβII，并且似乎不涉及PKCβ基因可变剪接的变化。两种人PKCβ启动子构建体在RIE细胞中被PKCβII的表达激活。在RIE/PKCβII细胞中，PKCβ启动子活性和PKCβII mRNA水平均被MEK1和 -2抑制剂U0126抑制，但未被Cox -2抑制剂塞来昔布抑制。PKCβ启动子活性与HT29和HCT116人结肠癌细胞系中内源性PKCβII mRNA和蛋白的表达直接相关。HCT116细胞中的PKCβ启动子活性和PKCβII mRNA表达被选择性PKCβ抑制剂LY317615和U0126抑制，表明PKCβII表达的自动调节。转基因PKCβII小鼠在体内结肠上皮中表现出内源性PKCβII的特异性诱导，但未诱导其剪接变体PKCβI。综上所述，我们的结果表明：1）在转基因小鼠中，结肠上皮中PKCβII的表达对于赋予对AOM诱导的结肠癌发生的易感性既是必要的也是充分的；2）PKCβII在体外的RIE和人结肠癌细胞以及体内结肠上皮中调节其自身的表达；3）PKCβII的自动调节在RIE/PKCβII和HCT116结肠癌细胞中通过MEK依赖的信号通路介导。
