Occludin OCEL-domain interactions are required for maintenance and regulation of the tight junction barrier to macromolecular flux

Abstract

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.

FIGURE 1:

Occludin knockdown affects expression of other tight junction proteins. (A) Protein expression assessed by Western blot in two independent occludin-knockdown (ocln KD) or control clones. Claudin-4 and claudin-15 expression consistently increased, whereas claudin-1 and claudin-8 expression decreased. (B) Densitometric analysis of immunoblots (as in A). Average of three separate experiments, each with n = 3, of two independent occludin-knockdown clones (black and gray bars), normalized to the paired control lines. (C) Immuno­fluorescence microscopy demonstrates normal tight junction localization of ZO-1, ZO-3, claudin-2, and MarvelD3 in occludin-knockdown lines. Expression of claudin-1, claudin-4, and claudin-15 increased, but localization was not affected. In contrast, tricellulin was redistributed from tricellular tight junctions (arrowheads) to bicellular tight junctions (arrows) after occludin knockdown. Data are representative of two independent control and knockdown clones. Bars, 10 μm. (D) Transmission electron microscopy shows that occludin knockdown did not cause significant ultrastructural alterations of the brush border or apical junctional complex. Representative images from two independent occludin-knockdown clones and corresponding controls. AJ, adherens junction; D, desmosome; Mv, microvilli; TJ, tight junction. Bar, 200 nm. *p <0.05, **p < 0.001.

FIGURE 2:

Occludin knockdown increases leak pathway permeability. (A) TER of occludin-knockdown monolayers (gray bars) was reduced relative to control monolayers (white bars). Data are the averages of three experiments, each with n = 4, from two independent control or occludin-knockdown clones. (B) Charge selectivity, measured as PNa⁺/PCl⁻, was reduced in occludin-knockdown monolayers (gray bars) relative to control monolayers (white bars). Data are from a representative experiment with n = 7. (C) Occludin knockdown increased paracellular flux of cations with radii from 0.95 to 3.65 Å (gray circles) relative to control monolayers (white circles). EA, ethylamine; MA, methylamine; NMDG, N-methyl-d-glucamine. TEA, tetraethylammonium; TMA, tetramethylammonium. Data are the averages of three experiments, each with n = 4. (D) Occludin knockdown (gray circles) increased paracellular flux of larger macromolecules (FITC and 3-, 10-, and 40-kDa FITC-dextran) relative to shRNA control monolayers (white circles). Data shown are the averages of two experiments, each with n = 4. (E) The net increase in flux induced by occludin knockdown, that is, the difference between the lines in D, is indicated by the white circles and overlaid with solutions of the Renkin sieving equation using size cutoffs of 45, 55, 62.5, 70, or 80 Å. The occludin-dependent component of paracellular macromolecular flux fits the curve modeling a 62.5 Å pore (solid line). *p < 0.05, **p < 0.001.

FIGURE 3:

Occludin is required for TNF-induced barrier loss. (A) TNF reduced the TER of shRNA control Caco-2_BBe (white circles) but not occludin-knockdown (ocln KD; gray circles) Caco-2_BBe monolayers. Data are the average of three independent experiments, each with n = 10. (B) Protection from TNF-induced barrier loss was independent of initial TER. Each data point represents a separate occludin-knockdown (ocln KD, gray circles, eight clones) or shRNA control (white circles, four clones) clone. Data are from a representative experiment with n = 4 monolayers (for each point). (C) Transient siRNA-mediated occludin knockdown (gray bar) reduced occludin expression by 46 ± 2% relative to control siRNA (white bar) in T84 monolayers. Data are from a representative experiment with n = 6. (D) TNF (hatched bars) reduced TER of siRNA control (white bars) but not occludin-knockdown (gray bars) T84 monolayers. Data are representative of three independent experiments, each with n = 3. (E) TNF (hatched bars) did not significantly alter the charge selectivity (PNa⁺/PCl⁻) of occludin-knockdown (ocln KD) monolayers (gray bars). Data are representative of three independent experiments, each with n = 3. (F). TNF (dashed lines) increased paracellular permeability of cations with radii from 0.95 to 3.65 Å in control (white circles) but not occludin-knockdown (gray circles) monolayers. Data are representative of four independent experiments, each with n = 4. *p < 0.05, **p < 0.001.

FIGURE 4:

Occludin is not required for TNF-induced MLC phosphorylation or TNF-independent, MLCK-mediated tight junction regulation. (A) TNF induced occludin internalization and ZO-1 profile undulations in shRNA control monolayers. Although occludin internalization was not detected in occludin-knockdown monolayers, TNF-induced ZO-1 profile undulations were present. Arrows show tricellulin at the tricellular junction of TNF-treated shRNA control monolayers. Bar, 10 μm. Results are typical of three independent experiments. (B) TNF treatment did not affect expression of other tight junction proteins in shRNA control or occludin-knockdown clones. β-Actin was used as a loading control. Results are representative of three experiments, each with n = 3. (C) TNF induced similar increases in phosphorylated MLC (pMLC) in shRNA control or occludin-knockdown clones. Total MLC is shown as a loading control. Results are representative of three experiments, each with n = 3, p < 0.05. (D) PIK (250 μM) caused similar TER increases in shRNA control (white bars) or occludin-knockdown (gray bars) monolayers with active Na⁺-glucose cotransport. Results are representative of three experiments, each with n = 4. **p < 0.001.

FIGURE 5:

The occludin OCEL domain is required for TNF-induced barrier loss. (A) Doxycycline-inducible (tet-on) EGFP-occludin (EGFP-ocln), EGFP-occludin^ΔOCEL (EGFP-ocln^ΔOCEL), and free EGFP were expressed in occludin-knockdown (ocln KD) and shRNA control (control) monolayers. EGFP-occludin was expressed at levels similar to endogenous occludin at 10 ng/ml doxycycline. Results are representative of four experiments, each with n = 4. (B) Three-dimensional reconstructions (top; bar, 15 μm), and an xy plane image at the level of the tight junction (bar, 10 μm), along with corresponding xz and yz sections. Expression of EGFP-occludin or EGFP-occludin^ΔOCEL in occludin-knockdown monolayers did not alter ZO-1 distribution at the tight junction. EGFP-occludin was localized at the junction and along lateral membranes, but EGFP-occludin^ΔOCEL was found only at lateral membranes. Results are representative of five experiments. (C) Expression of EGFP-occludin, but not EGFP-occludin^ΔOCEL or free EGFP, in occludin KD monolayers restored TER. At 10 and 20 ng/ml doxycycline, EGFP-occludin expression significantly increased TER of occludin-knockdown monolayers. Results are representative of four experiments, each with n = 4. (D) Expression of EGFP-occludin, but not EGFP-occludin^ΔOCEL or EGFP, in occludin KD monolayers restored tight junction size selectivity. Results are representative of three experiments, each with n ≥ 3. (E) Expression of EGFP-occludin, but not EGFP-occludin^ΔOCEL or EGFP, in occludin KD monolayers reduced claudin-4 expression. β-Actin is shown as a loading control. Data are representative of three independent experiments, each with n = 3. **p < 0.001.

FIGURE 6:

The OCEL domain is required for TNF-dependent regulation of occludin stability at the tight junction. (A) TNF treatment induced EGFP-occludin, but not EGFP-occludin^ΔOCEL, internalization. EGFP-occludin–containing vesicles (green) were readily detected after TNF treatment (arrows). EGFP-occludin^ΔOCEL–containing vesicles (green) were not seen. ZO-1 (red) was detected by immunostaining. Bar, 10 μm. Results are representative of four independent experiments. (B) Occludin-knockdown monolayers were resensitized to TNF-induced barrier loss after EGFP-occludin expression. In contrast, occludin-knockdown monolayers expressing EGFP or EGFP-occludin^ΔOCEL were resistant to TNF. Results are representative of four experiments, each with n = 4. (C, F) FRAP kymographs of occludin-knockdown monolayers expressing EGFP-occludin or EGFP-occludin^ΔOCEL 4 h after TNF treatment. Results are representative of n ≥ 8 recordings per condition. Bar, 5 μm. (D, G) EGFP-occludin or EGFP-occludin^ΔOCEL fluorescence recovery curves for control (blue circles) and TNF-treated (red circles) monolayers. Results are averages from two experiments with at least eight recordings per condition. (E, H) Mobile fractions and t_1/2 for control or TNF-treated monolayers expressing EGFP-occludin or EGFP-occludin^ΔOCEL. Results are averages of two experiments with at least eight recordings per condition. *p < 0.05, **p < 0.001.

FIGURE 7:

The OCEL domain acts as a dominant-negative effector to prevent TNF-mediated barrier loss. (A) EGFP and EGFP fused to the occludin OCEL domain (EGFP-OCEL) were stably expressed at similar levels. Proteins were separated by SDS–PAGE and blotted with anti-GFP or anti–β-actin antibodies. Results are representative of four experiments, each with n = 3. (B) EGFP-OCEL was expressed at levels similar to endogenous occludin. Proteins were separated by SDS–PAGE and blotted with antibodies to occludin (occludin and EGFP-OCEL) or other proteins indicated. Claudin-4, -8, and -15 expression was reduced in EGFP-OCEL–expressing cells, regardless of TNF treatment. TNF induced a modest but nonsignificant reduction in endogenous occludin expression, similar to that reported previously (Wang et al., 2005), which was not affected by EGFP-OCEL expression. Results are representative of three experiments, each with n = 3. (C) EGFP-OCEL, but not EGFP, expression prevented TNF-induced TER loss. Results are the average of five experiments, each with n = 4. (D) EGFP-OCEL expression prevented TNF-induced endocytosis of endogenous (full-length) occludin. Occludin vesicles were present in TNF-treated monolayers expressing EGFP (arrows). Endogenous occludin was detected by immunostaining. Images are maximum projections assembled from z stacks and are representative of three independent experiments. Bar, 10 μm. *p < 0.05.

FIGURE 8:

Lysine 433 regulates OCEL interactions and is critical to TNF-induced barrier loss and occludin endocytosis. (A) Expression of mCherry, mCherry-OCEL, or mCherry-OCEL mutants (indicated residues are lysines that were mutated to aspartic acids) does not affect expression of endogenous occludin or ZO-1. Fusion proteins were detected with an anti-DsRed antibody that also recognizes mCherry. Results are representative of three experiments, each with n = 3. (B) Expression of mCherry, mCherry-OCEL, or mCherry-OCEL mutants did not affect ZO-1 localization at the tight junction. Images are representative of two independent experiments. Bar, 10 μm. (C) Expression of mCherry, mCherry-OCEL, or mCherry-OCEL mutants did not affect the EGFP-occludin mobile fraction. Results are averages of two experiments with at least eight recordings per condition. (D) Expression of mCherry-OCEL or mCherry-OCEL mutants, but not mCherry, altered the EGFP-occludin t_1/2. Only mCherry-OCEL or mCherry-OCEL^485/488 blocked TNF-induced reduction of EGFP-occludin t_1/2. Results are averages of two independent experiments, with at least eight recordings per condition. (E) TNF induced accumulation of intracellular EGFP-occludin (green) vesicles (arrows) in cells that did not express mCherry or mCherry-OCEL proteins (red). Occludin vesicles were also present in cells expressing mCherry or mCherry-OCEL⁴³³ but not in cells expressing Cherry-OCEL or mCherry-OCEL^485/488. Images are representative of three independent experiments. Bar, 10 μm. *p < 0.05.