Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers

Abstract

ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.

Figure 1. Contrasting effects of EGF on H₂O₂-induced epithelial barrier disruption in under-differentiated and differentiated cell monolayers

(A) Alkaline phosphatase activity was measured in extracts from under-differentiated (UD) 4-day-old and differentiated (D) 14-day-old Caco-2 cell monolayers. Values are means ± S.E.M. (n = 4). *P < 0.05. (B) Extracts from Caco-2 cell monolayers on day 4 or 14 after seeding were immunoblotted for villin and β-actin. The molecular mass in kDa is indicated. Band density was quantified by densitometric analysis. Values are means ± S.E.M. (n = 4). *P < 0.05. (C and D) 14-day-old or (E and F) 4-day-old Caco-2 cell monolayers were pretreated with 10 μM U0126 for 50 min prior to administration of 30 nM EGF. H₂O₂ (20 μM) was administered 10 min after EGF. Control cells without treatments, as indicated, are also shown. (C and E) TER and (D and F) inulin permeability were measured at various times. Values are means ± S.E.M. (n = 6). *P < 0.05 compared with corresponding control values, @P < 0.05 compared with H₂O₂-treated values, #P < 0.05 compared with EGF+H₂O₂ values.

Figure 2. Contrasting effects of EGF on H₂O₂-induced disruption of tight junctions in under-differentiated and differentiated cell monolayers

(A) Differentiated 14-day-old or (B) under-differentiated 4-day-old Caco-2 cell monolayers were pretreated with 10 μM U0126 for 50 min prior to administration of 30 nM EGF. H₂O₂ (20 μM) was administered 10 min after EGF. Control cells without treatments, as indicated, are also shown. Cell monolayers were fixed and immunostained for occludin and ZO-1.

Figure 3. Contrasting effects of knockdown of ERK on epithelial barrier disruption in under-differentiated and differentiated cell monolayers

(A) 3-Day-old under-differentiated (UD) and 7-day-old differentiated (D) Caco-2 cells transfected with control siRNA (NS-RNA) or ERK1/2-specific siRNA (si-RNA) were immunoblotted for ERK, p38-MAPK and β-actin. The molecular mass in kDa is indicated. (B) Densitometric analysis of immunoblots shown in (A). Values are means ± S.E.M. (n = 3). *P < 0.05 compared with NS-RNA. (C) The TER and (D) inulin permeability were measured in cell monolayers transfected with NS-RNA (white bars) or ERK-specific siRNA (grey bars) on day 3 or 7 after seeding on transwell inserts. Values are means ± S.E.M. (n = 6). *P < 0.05 compared with NS-RNA values. Extracts from 3-day-old (E) or 7-day-old (F) cell monolayers transfected with NS-RNA or ERK-specific siRNA (Si-RNA) were fixed and immunostained for occludin and ZO-1.

Figure 4. Contrasting effects of knockdown of ERK on the assembly of tight junctions in under-differentiated and differentiated cell monolayers

(A and B) Under-differentiated 3-day-old Caco-2 cells transfected with NS-RNA (●, white bars) or ERK-specific siRNA (∎, grey bars) were subjected to calcium-switch-mediated tight junction assembly. (A) The TER was measured during EGTA treatment and calcium replacement. (B) Inulin permeability measured at 150 min after calcium replacement. (C and D) Tight junction assembly was also studied in differentiated 7-day-old cells transfected with control siRNA (NS-RNA; ●, white bars) or ERK-specific siRNA (si-ERK; ∎, grey bars). (C) The TER and (D) inulin permeability were measured. Values are means ± S.E.M. (n = 6). *P < 0.05 compared with NS-RNA values.

Figure 5. Effects of expression of active and inactive MEK1 on tight junction integrity at different stages of differentiation

(A and B) Caco-2 cells expressing Tet-on regulator were transfected with GFP or GFP-tagged WT-MEK1, DN-MEK1 or CA-MEK in pTRE2hyg-GFP-N vector, and the expression of GFP and MEK was induced by administering doxycycline on (A) day 3 or (B) day 7. Expression of GFP before and after doxycycline (Dox) administration was visualized by immunofluorescence staining and confocal microscopy. (C) Under-differentiated 3-day-old and (D) differentiated 7-day-old cell monolayers, in which MEK expression was induced by doxycycline, were fixed and stained for occludin and ZO-1.

Figure 6. Differential effects of EGF on cellular distribution of PP2A and PKCζ in under-differentiated and differentiated cell monolayers

(A and B) Extracts from 4-day-old under-differentiated (UD) and 14-day-old differentiated (D) Caco-2 cells were (A) immunoblotted for claudin-4, PKCζ and PP2A-Cα and (B) the bands were quantified by densitometric analysis. The molecular mass in kDa is indicated. Values are means ± S.E.M. (n = 3). (C) Caco-2 cell monolayers were pretreated with 10 μM U0126 for 50 min prior to administration of 30 nM EGF. H₂O₂ (20 μM) was administered 10 min after EGF. Control cells without treatments, as indicated, are also shown. Occludin was immunoprecipitated (IP) from detergent-insoluble fractions of 4-day-old or 14-day-old old Caco-2 cells and immunoblotted for PKCζ and PP2A-Cα. The molecular mass in kDa is indicated. Densitometric analysis of (D) PP2A-Cα and (E) PKCζ bands in experiments described in (C). Values are means ± S.E.M. (n = 3–5). *P < 0.05 compared with control values, #P < 0.05 compared with the corresponding H₂O₂ values, @P < 0.05 compared with the corresponding EGF+H₂O₂ values.

Figure 7. Differential effects of EGF on redistribution of PKCζ in under-differentiated and differentiated cell monolayers

Under-differentiated 3-day-old and differentiated 14-day-old cell monolayers were pretreated with 10 μM U0126 for 50 min prior to administration of 30 nM EGF. H₂O₂ (20 μM) was administered 10 min after EGF. Control cells without treatments, as indicated, are also shown. Cell monolayers were fixed and immunostained for PKCζ.

Figure 8. Differential distribution of active ERK in EGF-treated cell monolayers

(A and B) Extracts from under-differentiated (UD) 4-day-old and differentiated (D) 14-day-old Caco-2 cell monolayers were immunoblotted for total ERK and p-ERK (active), and the bands were quantified by densitometric analysis. Values are means ± S.E.M. (n = 6). *P < 0.05 compared with corresponding 3-day-old values. (C) Caco-2 cell monolayers were incubated with or without EGF for various times and the cell extracts were immunoblotted for total and p-ERK. The molecular mass in kDa is indicated. (D) Caco-2 cell monolayers were incubated with EGF for varying times and the cell monolayers fixed and immunostained for p-ERK.