Polyamines and ECadherin Expression

A series of studies from our laboratory has demonstrated that polyamines are necessary for expression of E-cadherin in intestinal epithelial cells and that polyamine-modulated E-cadherin plays an important role in maintenance of normal epithelial barrier functions (24-26). To determine the involvement of polyamines in regulation of cell-cell adherens junctions and tight junctions, normal intestinal epithelial cells (IEC-6 line) were grown in the presence or absence of a-difluoromethylornithine (DFMO), the highly specific inhibitor of polyamine synthesis. Treatment with DFMO completely depleted putrescine within 48 h, but it took 4 d to totally deplete spermidine and significantly decrease spermine (by >60%). Similar results have been published previously (38,39).

Depletion of cellular polyamines by DFMO selectively and significantly inhibited mRNA expression of E-cadherin, but negligibly affected levels of other adherens proteins such as P-catenin and a-catenin mRNAs. The levels of E-cadherin mRNA in the cells treated with DFMO for 4, 6, and 8 d were decreased by approx 60%. The changes in E-cadherin mRNA were paralleled by those of E-cadherin proteins (Fig. 3). The levels of E-cadherin protein in the cells exposed to DFMO for 4, 6, and 8 d were decreased by approx 80%. Although there was no significant inhibition of P-catenin and a-catenin mRNA expression in DFMO-treated cells, protein levels of these two adherens junction proteins decreased significantly at 6 and 8 d after polyamine depletion (Fig. 3A). The levels of P-catenin and a-catenin proteins were decreased by approx 20 and approx 15%, respectively. In the presence of DFMO, decreased expression of E-cadherin mRNA and protein, P-catenin, and a-catenin proteins was completely prevented by addition of exogenous spermidine (5 |iM). We also examined the effect of polyamine depletion on E-cadherin expression in Caco-2 cells and demonstrated that changes in E-cadherin levels were similar to those observed in IEC-6 cells. Immunofluorescence staining was performed to determine cellular distribution of E-cadherin and P-catenin in IEC-6 cells. In control cells (Fig. 3B), immunoreactivities for E-cadherin and P-catenin were mainly located along the entire cell-cell contact region of adjacent cells. In DFMO-treated cells, these membrane immunoreactivities for E-cadherin and P-catenin markedly were decreased. Exogenous spermidine given together with DFMO reversed the inhibitory effect of polyamine depletion on E-cadherin and P-catenin formation. These results clearly indicate that polyamines are necessary for expression of E-cadherin protein in intestinal epithelial cells.

To test the possibility that the decrease in E-cadherin mRNA level in polyamine-deficient cells results from inhibition of the gene transcription, we examined changes in E-cadherin promoter activity by using luciferase reporter gene assays (40). Consistent with its effect on E-cadherin mRNA expression, polyamine depletion by DFMO decreased E-cadherin promoter activity dramatically. The activity of E-cadherin promoter luciferase reporter was decreased by approx 70% on d 6 after administration of DFMO, which was completely prevented by spermidine given together with DFMO. We also examined changes in E-cadherin protein stability after polyamine depletion by measurement of the half-life of E-cadherin protein by pulse-chase analysis. In control cells, E-cadherin protein decreased at a relatively slow rate, with about 60% remaining after 6 h. On the other hand, levels of E-cadherin protein in polyamine-deficient cells decreased quickly, with a half-life of approx 4 h, indicating that E-cadherin protein degradation was approx 20% faster after polyamine depletion. Changes in E-cadherin mRNA stability and its translation rate were also examined in the presence or absence of polyamines. The stability and translation of E-cadherin mRNA in IEC-6 cells was not affected by polyamine depletion. There were no significant changes in E-cadherin mRNA half-life and its translation rate between control cells and cells exposed to DFMO with or without spermidine for 6 d. Taken together, these findings indicate that polyamines predominantly regulate transcription of the E-cadherin gene, although they also slightly modulate E-cadherin protein stability in intestinal epithelial cells.

Fig. 3. Changes in protein levels of E-cadherin, P-catenin, and a-catenin after polyamine depletion in IEC-6 cells. (A) Representative autoradiograms of Western blots. E-cadherin (~120 kDa), P-catenin (~92 kDa), and a-catenin (~102 kDa) were identified by probing nitrocellulose with specific antibodies. (B) Cellular distribution of E-cadherin and P-catenin: a, control; b, cells treated with a-difluoromethylornithine (DFMO) alone for 6 d; and c, cells treated with DFMO + spermidine for 6 d. Cells were permeabilized and incubated with the specific antibody against E-cadherin or P-catenin and then with anti-immunoglobulin G conjugated with FITC. Original magnification: x1000.

Fig. 3. Changes in protein levels of E-cadherin, P-catenin, and a-catenin after polyamine depletion in IEC-6 cells. (A) Representative autoradiograms of Western blots. E-cadherin (~120 kDa), P-catenin (~92 kDa), and a-catenin (~102 kDa) were identified by probing nitrocellulose with specific antibodies. (B) Cellular distribution of E-cadherin and P-catenin: a, control; b, cells treated with a-difluoromethylornithine (DFMO) alone for 6 d; and c, cells treated with DFMO + spermidine for 6 d. Cells were permeabilized and incubated with the specific antibody against E-cadherin or P-catenin and then with anti-immunoglobulin G conjugated with FITC. Original magnification: x1000.

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