Apigenin upregulation of CD26/DPPIV on colon epithelial cells requires inhibition of casein kinase 2

Abstract

CD26/DPPIV is a cell surface glycoprotein found on cells of the intestinal epithelium including those of the colon. We have previously shown that the dietary flavone apigenin (4',5,7-trihydroxyflavone) upregulates CD26/DPPIV on colon cells. Flavonoids such as apigenin interfere with the action of multiple cellular protein kinases and have the capacity to modulate the cell exterior and its ability to interface with the local environment through different signaling pathways. We show here that the ability of apigenin to upregulate CD26/DPPIV is exerted through and requires the activity of casein kinase 2 (CK2). Inhibitors of CK2 that are distinct from apigenin (emodin, 6-methyl-1,3,8-trihydroxyanthraquinone; TBB, 4,5,6,7-tetrabromobenzotriazole; and DRB, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside) showed a dose-dependent ability to increase CD26/DPPIV and had the same maximal effect when combined with apigenin at submaximal concentrations. Knockdown of CK2 with siRNA abrogated the ability of apigenin to upregulate CD26/DPPIV. Apigenin treatment of cells had no effect on the levels of CK2 protein, consistent with an inhibition of activity of the enzyme. Apigenin's upregulation of CD26/DPPIV in differentiated human colon epithelial cells depends upon inhibition of CK2 activity. This is a key step in enabling apigenin's ability to regulate the functions of intestinal epithelial cells.

Keywords: 4,5,6,7‐tetrabromobenzotriazole (TBB); 5,6‐dichlorobenzimidazole 1‐β‐D‐ribofuranoside (DRB); 6‐methyl‐1,3,8‐trihydroxyanthraquinone (emodin); CD26; DPPIV; apigenin; casein kinase 2.

FIGURE 1

Apigenin upregulation of CD26/DPPIV in different sources of human colon epithelial cells. Cells were treated for 48 hr in the absence or presence of apigenin (30 µM) and cell surface CD26/DPPIV then measured by radioimmunoassay. Data are corrected for nonspecific binding of anti‐CD26 mAb and expressed based on the number of viable cells. Values are means ± SEM (n = 4); ***, p < .001, increase over respective control; n.s., not significant; #, p < .05, difference between SW620 and SW480 cell lines

FIGURE 2

Apigenin does not substantially elevate total cellular CD26/DPPIV or mobilize CD26/DPPIV from accessible intracellular pools. (a) Detection of CD26 protein. Images from Western blotting for purified recombinant CD26 protein using anti‐human CD26 antibody in the absence (−) or presence (+) of neutralizing CD26 peptide. The position of the 100 kDa protein marker is noted (arrow). (b) Apigenin and related flavonoids do not significantly elevate total cellular immunoreactive CD26/DPPIV. HT‐29 cells were treated with flavonoids at the indicated concentrations, solubilized, and the lysates immunoblotted for CD26/DPPIV. Data are mean values ± SEM from three independent experiments. n.s., not statistically significant. (c) Apigenin does not recruit CD26/DPPIV to the cell surface from accessible pools. HT‐29 cells were treated in the absence or presence of apigenin and accessible CD26/DPPIV measured by radioimmunoassay in whole cells with plasma membranes intact (clear bars) or cells for which the surface membranes had first been permeabilized (hatched bars). Data are means ± SEM (n = 4); *, p < .05 or **, p < .01, increase over control for intact cells; n.s., no significant difference between intact and permeabilized cells

FIGURE 3

Structures of CK2 inhibitors. (a) Emodin (6‐methyl‐1,3,8‐trihydroxyanthraquinone). (b) TBB (4,5,6,7‐tetrabromo‐2‐azabenzimidazole). (c) DRB (5,6‐dichlorobenzimidazole 1‐β‐D‐ribofuranoside). (d) Apigenin (4′,5,7‐trihydroxyflavone; 5,7‐dihydroxy‐2‐(4‐hydroxyphenyl)‐4H‐1‐benzopyran‐4‐one)

FIGURE 4

Inhibition of CK2 activity reproduces the apigenin upregulation of CD26/DPPIV. (a) CK2 inhibitors enhance cell surface CD26/DPPIV levels. HT‐29 cells were treated with CK2 inhibitors and cell surface CD26/DPPIV measured after 48 hr. Values are means ± SEM (n = 4); ANOVA followed by Dunnett's test; **, p < .01, significant enhancement over relevant control. (b) CK2 inhibitors do not extend the increase in CD26/DPPIV above the maximum achieved with apigenin. HT‐29 cells were treated with CK2 inhibitors without or with 60 µM apigenin. Concentrations used were the maximum possible without inducing net toxicity and were as follows: emodin 6 µM; TBB 1 µM; DRB 1 µM. Values are means ± SEM, 5 independent experiments; ***, p < .001, increase due to apigenin; n.s., not significant; #, p < .05 or ##, p < .01, enhancement by CK2 inhibitors relative to control. (c) Lack of effect of apigenin on CK2 protein levels. HT‐29 cells were treated with apigenin (60 µM) for the times noted and Western‐blotted for CK2 or α‐tubulin (Tub). Representative of n = 3 independent experiments

FIGURE 5

Knockdown of CK2 abrogates the apigenin upregulation of CD26/DPPIV. (a) Knockdown of CK2 levels using an siRNA approach. HT‐29 cells were examined at 36 or 84 hr after transfection as indicated and Western‐blotted for CK2 or α‐tubulin (Tub). Representative of n = 3 independent experiments. (b) Knockdown of CK2 abrogates the ability of apigenin to upregulate CD26/DPPIV. Cell surface CD26/DPPIV was evaluated 48 hr following treatment with 60 µM apigenin in the three groups of cells noted in panel A. Means ± SE (n = 4); *, p < .05 or ***, p < .001, increase due to apigenin; n.s., not significant; #, p < .05, enhancement by CK2 knockdown