Cfps Induces Activation Of Erk12 In Iec6 Cells

Stimulation of cell proliferation and division also depends on multisignal-ing pathways by tyrosine kinases, including MAPK and phosphatidyli-nositol 3-kinase (PI3K) (Khandwala et al., 2000; LeRoith et al., 1995; Rother and Accili, 2000). The MAPK family in mammalian cells includes ERK1/2, the JNK, and p38 MAPK (Paruchuri et al., 2002). To further investigate the mechanism of Cf-PS-induced proliferation, MAPK proteins were used to examine whether Cf-PS activates the EGFR signaling pathway in IEC-6 cells. In the absence of Cf-PS, JNK and p38 were phosphorylated in a time-dependent manner. However, Cf-PS treatment inhibited the activation of JNK and p38 in a time-dependent manner (Fig. 13.4). Further, ERK1/2 phosphorylation increased after treatment with Cf-PS as early as 0 min and reached a maximum 5 min after EGF exposure. JNK and p38 are involved in the cell death pathway and oxida-tive stress. Therefore, Cf-PS-induced cell proliferation appears to be related to the activation of MAPK (ERK1/2, p38, JNK), especially ERK1/2. Therefore, cell proliferation was analyzed after inhibition of ERK1/2 using U0126 to examine the role of ERK1/2 activation during the Cf-PS effect. As shown in Fig. 13.5, exposure to U0126 with Cf-PS inhibited cell proliferation, but viability was higher than that of untreated cells. These results indicate that ERK1/2 plays a role in the proliferation of IEC-6 cells.

Wnt peptides transactivate EGFR, presumably by increasing the availability of EGFR ligands, which in turn leads to strong stimulation of the MAPK pathway (Givenni et al., 2003). Transactivation of EGFR has a specific biological effect: stimulation of cyclinD1 expression (Givenni et al., 2003). Therefore, there might be cross talk between the MAPK

500 ig/ml Cf-PS pERK1/2

pp38 pJNK ß-actin EGF

500 ig/ml Cf-PS pERK1/2

pp38 pJNK ß-actin EGF

FIGURE 13.4 Cf-PS induces activation of ERK1/2. MAPK protein expression was measured by Western blotting. One representative gel from three separate experiments is shown.

FIGURE 13.4 Cf-PS induces activation of ERK1/2. MAPK protein expression was measured by Western blotting. One representative gel from three separate experiments is shown.

250 r

8 100

Control

Cf-PS

Control

Cf-PS

pERK1/2

ERK1/2

pERK1/2

ERK1/2

FIGURE 13.5 ERK1/2 phosphorylation affects Cf-PS-induced cell proliferation.

(A) MTS assay was used to assess ERK1/2 activation on Cf-PS-induced cell proliferation.

(B) Inhibition of phospho-ERK1/2 level by U0126. One representative gel from three separate experiments is shown.

FIGURE 13.5 ERK1/2 phosphorylation affects Cf-PS-induced cell proliferation.

(A) MTS assay was used to assess ERK1/2 activation on Cf-PS-induced cell proliferation.

(B) Inhibition of phospho-ERK1/2 level by U0126. One representative gel from three separate experiments is shown.

signaling pathway and the Wnt signaling pathway during Cf-PS-induced proliferation of IEC-6 cells. To investigate this hypothesis, the activation of ERK1/2 was inhibited using U0126 and the effects of this inhibition on the Wnt signaling pathway was investigated.

Inhibition of ERK1/2 led to a decrease in the level of cytosolic p-catenin (Fig. 13.6). This decrease was also observed when cells were treated with Cf-PS or U0126 only (Fig. 13.3), but to a greater degree after U0126 treatment (Fig. 13.6). Further, p-catenin in the nucleus, which increased when treated with Cf-PS alone (Fig. 13.3), also decreased in the presence of U0126 (Fig. 13.6). These results suggest that translocation of p-catenin from the cytosol to the nucleus was inhibited, and the resulting accumulation of p-catenin in the cytosol was also inhibited by inhibition of ERK1/2. Moreover, we have made a hypothesis model of the cross talk between Wnt signaling and MAPK signaling pathways and given in Fig.13.7.

8 100

Nuclear ß-catenin

Cytosol ß-catenin

Cytosol ß-actin CyclinD1

c-myc

Nuclear ß-catenin

Cytosol ß-catenin

Cytosol ß-actin CyclinD1

c-myc

1

1.8

0.2

1

0.7

0.2

1

2.2

1.3

mmm mm 1

1

1.7

0.7

FIGURE 13.6 Effect of ERK1/2 activation on Wnt signaling. Expression of Wnt signaling proteins was measured by Western blotting. One representative gel from three separate experiments is presented. Each number is quantitative analysis based on densitometry.
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