CMyc and Polyamine Induced Transcription of the ECadherin Gene

The c-Myc protein is a nuclear transcription factor, and its gene expression absolutely requires polyamines (41,42). Recently, E-cadherin was identified as a c-Myc target gene and activation of c-Myc enhances E-cadherin gene transcription in a cell type-dependent manner (43). Our recent study provides new evidence showing that polyamines regulate transcription of the E-cadherin gene through c-Myc in normal intestinal epithelial cells.

Depletion of cellular polyamines dramatically inhibits c-myc gene transcription and decreased levels of c-Myc protein (39,41,42), which is associated with a significant reduction of E-cadherin promoter activity in IEC-6 cells (40). We have successfully constructed the adenoviral vector containing c-Myc cDNA (AdMyc) under the control of the human cytomegalovirus immediate early gene promoter. Infection of IEC-6 cells with AdMyc vector increased c-Myc protein by approx 20-fold, and this forced expression of the c-Myc stimulated transcription of the E-cadherin gene as indicated by a significant increase in E-cadherin promoter activity. The level of E-cadherin-promoter activity was increased by approx threefold when cells were infected with the AdMyc at

Cmyc Cancer

Fig. 4. Effects of manipulating intracellular Ca2+ concentration, either decreased by removal of extracellular Ca2+ or increased by the Ca2+ ionophore ionomycin (Iono), on E-cadherin protein expression. (A) Levels of E-cadherin protein in control cells exposed to Ca2+-free medium: a, representative autoradiograms of Western blots; b, quantitative analysis of Western immunoblots by densitometry from cells described in a, Open bar, no exposure to Ca2+-free

Fig. 4. Effects of manipulating intracellular Ca2+ concentration, either decreased by removal of extracellular Ca2+ or increased by the Ca2+ ionophore ionomycin (Iono), on E-cadherin protein expression. (A) Levels of E-cadherin protein in control cells exposed to Ca2+-free medium: a, representative autoradiograms of Western blots; b, quantitative analysis of Western immunoblots by densitometry from cells described in a, Open bar, no exposure to Ca2+-free the dose of 50 pfu/cell. These results clearly show that activation of c-Myc enhances E-cadherin gene expression in intestinal epithelial cells.

3.5. Polyamines Also Regulate Tight Junction Proteins

A recent study has shown that polyamines are required for normal function of tight junctions and those polyamines regulate expression of various tight junction proteins through different mechanisms (26). In this study, differentiated IEC-Cdx2L1 cells were used, because this line of cells has well-developed tight junctions. Depletion of cellular polyamines by DFMO treatment decreased protein levels of tight junctions occludin, ZO-1, ZO-2, claudin-2, and claudin-3 in differentiated IEC-Cdx2L1 cells. The levels of occludin protein in the cells exposed to DFMO for 4, 6, and 8 d were decreased by more than 80% (Fig. 5). Although there was no inhibition of ZO-1 and ZO-2 expression in undifferentiated parental IEC-6 cells treated with DFMO, levels of ZO-1 and ZO-2 proteins in differentiated IEC-Cdx2L1 cells decreased significantly after polyamine depletion (25). The levels of ZO-1 and ZO-2 proteins in differentiated IEC-Cdx2L1 cells exposed to DFMO for 4, 6, and 8 d were decreased by approx 55 and approx 40%, respectively. Treatment with DFMO for 4 d did not alter expression of claudin-2, but its levels were decreased by approx 50% on d 6 and by approx 80% on d 8, respectively (Fig. 5). Changes in claudin-3 expression were similar to those observed in claudin-2 after polyamine depletion. In the presence of DFMO, decreased levels of occludin, ZO-1, ZO-2, claudin-2, and claudin-3 proteins were completely abolished by addition of exogenous spermidine. Interestingly, depletion of cellular polyamines did not inhibit expression of occludin, ZO-1, and ZO-2 mRNAs, although it significantly decreased levels of their proteins. On the other hand, polyamine depletion inhibited expression of claudin-2 and claudin-3 mRNAs, which was completely prevented by exogenous spermidine. These findings suggest that polyamines are involved in expression of various tight junction proteins through different signaling pathways in intestinal epithelial cells.

To determine whether polyamines regulate occludin at the translation level, the level of newly synthesized occludin protein was examined. Polyamine depletion by DFMO significantly decreased the occludin protein synthesis in differentiated IEC-Cdx2L1 cells. The level of newly synthesized occludin protein was decreased by approx 70% in cells exposed to DFMO for 6 d. We also examined changes in occludin protein stability after polyamine depletion by measurement of the half-life of occludin protein by pulse-chase analysis. In control cells (without DFMO), the levels of occludin protein declined gradually after protein synthesis was inhibited by cycloheximide, with a half-life of approx 120 min. On the other hand, levels of occludin protein in polyamine-deficient medium; filled bars, exposure to Ca2+-free medium. Values are means ± SE; relative E-cadherin protein levels were corrected for loading as measured by densitometry of actin. *p < 0.05 vs 0 h group. (B) Levels of E-cadherin protein in polyamine-deficient cells exposed to ionomycin for 6 h: a, representative autoradiograms of Western blots; b, quantitative analysis of Western immunoblots by densitometry from cells described in a. Open bars, control; gray bars, DFMO; filled bars, DFMO + Iono. Values are means ± SE. *p < 0.05 vs control group; + p < 0.05 vs DFMO-treated cells.

Fig. 5. Changes in expression of tight junction proteins occludin, ZO-1, ZO-2, claudin-2, and claudin-3 in differentiated IEC-Cdx2L1 cells treated with DFMO alone or DFMO + spermidine (SPD). Before experiments, stable IEC-Cdx2L1 cells were grown in DMEM containing 4 mM isopropyl ß-D-thiogalactopyranoside for 16 d to induce cell differentiation. These differentiated IEC-Cdx2L1 cells were grown in DMEM containing DFMO or DFMO plus SPD for 4, 6, and 8 d. (A) Representative immunoblots of Western analysis. (B) Quantitative analysis derived from densitometric analysis of immunoblots of occludin from cells described in (A). Values are means ± SE of data from three separate experiments; relative levels of proteins were corrected for loading as measured by densitometry of actin. *p < 0.05 compared with the corresponding control and DFMO + SPD.

Fig. 5. Changes in expression of tight junction proteins occludin, ZO-1, ZO-2, claudin-2, and claudin-3 in differentiated IEC-Cdx2L1 cells treated with DFMO alone or DFMO + spermidine (SPD). Before experiments, stable IEC-Cdx2L1 cells were grown in DMEM containing 4 mM isopropyl ß-D-thiogalactopyranoside for 16 d to induce cell differentiation. These differentiated IEC-Cdx2L1 cells were grown in DMEM containing DFMO or DFMO plus SPD for 4, 6, and 8 d. (A) Representative immunoblots of Western analysis. (B) Quantitative analysis derived from densitometric analysis of immunoblots of occludin from cells described in (A). Values are means ± SE of data from three separate experiments; relative levels of proteins were corrected for loading as measured by densitometry of actin. *p < 0.05 compared with the corresponding control and DFMO + SPD.

cells decreased quickly, with a half-life of approx 75 min, indicating that occludin protein degradation was faster after polyamine depletion. Spermidine given together with DFMO not only totally overcame the decrease in occludin protein synthesis, but also completely prevented the instability of occludin. These findings clearly indicate that polyamines are implicated in regulation of occludin protein synthesis and stability in intestinal epithelial cells.

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