Wnt信号通路对成纤维细胞Rat-1生长及表型的调控

构建Wnt-3a的真核表达拽体并稳定转染人鼠成纤维细胞Rat-1,建立Wnt信号通路持续激活的细胞模型,以探讨Wnt信号通路激活对咳细胞的牛K以及某些表型特征的影响。结果表明：Wnt信号通路持续激活时,Rat-1细胞形态表现为细胞长度的增加,其折光性以及呈绳索状的成束密集排布：MTT以及流式细胞仪检测表明稳定转染后细胞增殖率明显高于正常对照组,进入G2期的细胞增多,细胞增殖分裂能力增强：Transwell小窄迁移实验证实转染组的细胞迁移率略高于对照组,但无显著性差异;体外划痕实验表明,稳定转染后的Rat-1细胞在划痕后伤口愈合时问显著缩短。结果提示：体外Wnt信号通路的激沂能够引起成纤维细胞某些表型改变,并促进细胞增殖,加速体外伤口的修复。     

LDL receptor-related protein LRP6 regulates proliferation and survival through the Wnt cascade in vascular smooth muscle cells

Initial studies have established expression of low-density lipoprotein (LDL) receptor-related protein 6 (LRP6) in vascular smooth muscle cells (VSMCs). We hypothesized that LRP6 is a critical mediator governing the regulation of the canonical Wnt/beta-catenin/T cell factor 4 (Tcf-4) cascade in the vasculature. This hypothesis was based on our previous work demonstrating a role for the beta-catenin/Tcf-4 pathway in vascular remodeling as well as work in other cell systems establishing a role for LRP family members in the Wnt cascade. In line with our hypothesis, LRP6 upregulation significantly increased Wnt-1-induced Tcf activation. Moreover, a dominant interfering LRP6 mutant lacking the carboxyl intracellular domain (LRP6DeltaC) abolished Tcf activity. LRP6-induced stimulation of Tcf was blocked in VSMCs harboring constitutive expression of a dominant negative Tcf-4 transgene lacking the beta-catenin binding domain, suggesting that LRP6-induced activation of Tcf was mediated through a beta-catenin-dependent signal. Expression of the dominant interfering LRP6DeltaC transgene was sufficient to abolish the Wnt-induced survival as well as cyclin D1 activity and cell cycle progression. In conclusion, these findings provide the first evidence of a role for an LDL receptor-related protein in the regulation of VSMC proliferation and survival through the evolutionary conserved Wnt signaling cascade.     

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.     

The tuberin-hamartin complex negatively regulates beta-catenin signaling activity

Tuberous sclerosis complex (TSC) is characterized by the formation of hamartomas in multiple organs resulting from mutations in the TSC1 or TSC2 gene. Their protein products, hamartin and tuberin, respectively, form a functional complex that affects cell growth, differentiation, and proliferation. Several lines of evidence, including renal tumors derived from TSC2+/- animals, suggest that the loss or inhibition of tuberin is associated with up-regulation of cyclin D1. As cyclin D1 can be regulated through the canonical Wnt/beta-catenin signaling pathway, we hypothesize that the cell proliferative effects of hamartin and tuberin are partly mediated through beta-catenin. In this study, total beta-catenin protein levels were found to be elevated in the TSC2-related renal tumors. Ectopic expression of hamartin and wild-type tuberin, but not mutant tuberin, reduced beta-catenin steady-state levels and its half-life. The TSC1-TSC2 complex also inhibited Wnt-1 stimulated Tcf/LEF luciferase reporter activity. This inhibition was eliminated by constitutively active beta-catenin but not by Disheveled, suggesting that hamartin and tuberin function at the level of the beta-catenin degradation complex. Indeed, hamartin and tuberin co-immunoprecipitated with glycogen synthase kinase 3 beta and Axin, components of this complex in a Wnt-1-dependent manner. Our data suggest that hamartin and tuberin negatively regulate beta-catenin stability and activity by participating in the beta-catenin degradation complex.     

Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or beta-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of beta-catenin, and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells.     

Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation

The Wnt signaling pathway is critical in normal development, and mutation of specific components is frequently observed in carcinomas of diverse origins. However, the potential involvement of this pathway in lung tumorigenesis has not been established. In this study, analysis of multiple Wnt mRNAs in non-small cell lung cancer (NSCLC) cell lines and primary lung tumors revealed markedly decreased Wnt-7a expression compared with normal short-term bronchial epithelial cell lines and normal uninvolved lung tissue. Wnt-7a transfection in NSCLC cell lines reversed cellular transformation, decreased anchorage-independent growth, and induced epithelial differentiation as demonstrated by soft agar and three-dimensional cell culture assays in a subset of the NSCLC cell lines. The action of Wnt-7a correlated with expression of the specific Wnt receptor Frizzled-9 (Fzd-9), and transfection of Fzd-9 into a Wnt-7a-insensitive NSCLC cell line established Wnt-7a sensitivity. Moreover, Wnt-7a was present in Fzd-9 immunoprecipitates, indicating a direct interaction of Wnt-7a and Fzd-9. In NSCLC cells, Wnt-7a and Fzd-9 induced both cadherin and Sprouty-4 expression and stimulated the JNK pathway, but not beta-catenin/T cell factor activity. In addition, transfection of gain-of-function JNK strongly inhibited anchorage-independent growth. Thus, this study demonstrates that Wnt-7a and Fzd-9 signaling through activation of the JNK pathway induces cadherin proteins and the receptor tyrosine kinase inhibitor Sprouty-4 and represents a novel tumor suppressor pathway in lung cancer that is required for maintenance of epithelial differentiation and inhibition of transformed cell growth in a subset of human NSCLCs.