Effects Of Cfps On Wnt Signaling Components

Promotion of cell proliferation and division depends on intracellular signaling pathways such as the Wnt signal transduction pathway. In the absence of Wnt signaling, b-catenin is bound to the GSK3b/Auxin/APC complex in the cytosol; GSK3b phosphorylates b-catenin and consequently induces degradation of b-catenin through the ubiquitin proteo-some pathway (Akiyama, 2000). Receptors for Wnt proteins are members of the frizzled family of transmembrane proteins, and the Wnt signal is converted to a cytoplasmic protein called disheveled (Dvl). Upon activation by the Wnt signal, GSK3b activity is inhibited by Dvl (Akiyama, 2000). This causes b-catenin to accumulate after it is translocated from the cytosol to the nucleus. In turn, b-catenin associates with TCF/LEF transcription factors and alters the expression of the Wnt signaling target genes cyclinD1 and c-myc. We believe that Cf-PS-induced cell proliferation was involved in the Wnt signaling pathway.

To identify the mechanism of Cf-PS-induced proliferation in IEC-6 cells, the effects of Cf-PS were evaluated on canonical Wnt signaling pathway proteins. Further, time-course experiment was conducted to assess the effect of Cf-PS on cyclinD1 and c-myc expression, which plays major roles in the cell cycle and in the proliferation of eukaryotic cells (He et al., 1998; Sun and Jin, 2008; Tetsuo and McCormick, 1999; van de Wetering et al., 2002; Willert et al., 2002; Yamaguchi et al., 2004).

250 500

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500 mg/ml Cf-PS PCNA

250 500

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500 mg/ml Cf-PS PCNA

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With

Without

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FIGURE 13.2 Effect of Cf-PS on the IEC-6 cell proliferation. (A) Effect of Cf-PS treatment on cell proliferation was assayed by MTS assay. (B) Microscopy analysis of the cells (200 x). (C) Effect of Cf-PS on PCNA protein expression. One representative gel from three separate experiments is shown.

500 ig/ml Cf-PS c-myc

CyclinDI ß-actin Nuclear ß-catenin Cytosol ß-catenin Cytosol ß-actin

500 ig/ml Cf-PS c-myc

CyclinDI ß-actin Nuclear ß-catenin Cytosol ß-catenin Cytosol ß-actin

FIGURE 13.3 Effect of Cf-PS on Wnt signaling in IEC-6 cells. Expression of Wnt signaling pathway proteins was measured by Western blotting. One representative gel from three separate experiments is shown.

FIGURE 13.3 Effect of Cf-PS on Wnt signaling in IEC-6 cells. Expression of Wnt signaling pathway proteins was measured by Western blotting. One representative gel from three separate experiments is shown.

Expression of cyclinD1 increased 6 and 24 h but decreased 12 h after Cf-PS exposure. In contrast, expression of c-myc increased at every time point after Cf-PS exposure (Fig. 13.3). The decreased expression of cyclinD1 at 12 h was likely an experimental error. Expression of Wnt signaling target genes is needed to transfer p-catenin from the cytosol to the nucleus. To confirm translocation of p-catenin from the cytosol to the nucleus, cytosolic-enriched fractions of Cf-PS-treated or untreated cells were used. Cytosolic p-catenin protein significantly decreased in the presence of Cf-PS as early as 12 h after exposure, and this reached a maximum by 24 h after exposure. In contrast, nuclear p-catenin increased only 24 h after Cf-PS exposure. There were no changes of nuclear p-catenin 12 h after exposure, but this may have been due to experimental error. These demonstrate that Cf-PS induced p-catenin translocation from the cytosol to the nucleus, which may increase cyclinD1 and c-myc expression. c-myc plays a major regulatory role in the cell cycle and the growth of eukaryotic cells (McMahon and Monroe, 1992; Wang et al., 1993). The c-myc gene codes for a nuclear protein that functions as a transcription factor controlling cell division, differentiation, and apopto-sis (Marcu et al., 1992; Varmus, 1984). Moreover, c-myc modulates cell proliferation and governs cell cycle progression in the intestinal mucosa (Yamaguchi et al., 2004). D-type cyclins are cell cycle regulators, among which cyclinD1 is a major regulator of the progression of cells into the proliferative stage of the cell cycle. CyclinD1 has a positive effect on cell cycle progression (Koseoglu et al., 2009). Cf-PS-induced expression of cyclinD1 and c-myc may play a critical role in the stimulation of normal intestinal epithelial cell proliferation.

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