A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells - PubMed (original) (raw)
A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells
Isabelle Beau et al. J Virol. 2007 Aug.
Abstract
We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.
Figures
FIG. 1.
Rearrangement of the distribution of occludin, but not that of ZO-1 and ZO-3, in RRV-infected Caco-2 cells. Fully differentiated cells were left uninfected or were infected with RRV for 18 h. The cells were permeabilized and processed for indirect immunofluorescence labeling of occludin, ZO-1, ZO-3, and rotavirus proteins as described in Materials and Methods. All images were obtained by confocal laser scanning microscopy. Micrographs are representative of three independent experiments.
FIG. 2.
Effect of purified RRV, UV-inactivated RRV, and culture supernatants of RRV-infected Caco-2 cells on the distribution of occludin in Caco-2 cells. Fully differentiated noninfected cells are shown in panels a and b. In panels c to g, cells were infected with purified RRV (c and d) or UV-inactivated virus (e) for 18 h or exposed for 1 h to supernatants of RRV-infected cells that had been cleared of infectious virus and of proteins with molecular masses of up to 50 kDa by filtration (f). The cells were permeabilized and processed for indirect immunofluorescence labeling of occludin or rotavirus proteins as described in Materials and Methods. All images were obtained by confocal laser scanning microscopy. Micrographs are representative of three independent experiments.
FIG. 3.
Decrease in the occludin contents of Triton X-100-soluble and -insoluble fractions isolated from RRV-infected Caco-2 cells. Fully differentiated cells were left uninfected or were infected with RRV for 18 h. The cells were processed for isolation of Triton X-100-soluble and -insoluble fractions. Detergent-insoluble and -soluble fractions were resolved by SDS-PAGE and Western immunoblotted for detection of occludin, ZO-1, and ZO-3 proteins as described in Materials and Methods. Representative immunoblot assays from three independent experiments are shown. (Left side) Immunoblot analysis of occludin and ZO-1 in the Triton X-100-soluble (E) and -insoluble (R) fractions of control (C) or RRV-infected (I) Caco-2 cells. Actin was used as a loading control for immunoblotting. Arrows indicate the 65-kDa nonphosphorylated occludin band, the 220-kDa ZO-1 band, the 130-kDa ZO-3 band, and the 42-kDa actin band. (Right side) Blots were quantified with NIH Image software. Open bars, nonphosphorylated form of occludin in Triton X-100-soluble fraction. Closed bars, nonphosphorylated form of occludin in Triton X-100-insoluble fraction. Hatched bars, phosphorylated (P) form of occludin in Triton X-100-insoluble fraction. For the nonphosphorylated form of occludin in the Triton X-100-soluble and -insoluble fractions, each bar represents the mean ± the standard deviation of three independent experiments. *, P > 0.01 (Student t test) compared with appropriate control cells.
FIG. 4.
Decrease in the occludin mRNA level in RRV-infected Caco-2 cells. Fully differentiated cells were left uninfected or were infected with RRV for 18 h. Total RNA was extracted, and the occludin and ZO-1 mRNAs were quantified by Q-RT-PCR. C t values obtained for these two mRNA transcripts were normalized relative to those for GAPDH. Results are expressed as occludin/GAPDH and ZO-1/GAPDH ratios (R). Each bar represents the mean ± the standard deviation of three independent experiments. For occludin, P < 0.01 (Student t test) for the difference between RRV-infected cells and control cells. For ZO-1, the Student t test showed no significant difference between RRV-infected cells and control cells.
FIG. 5.
Rp-cAMP and PKA inhibitors, but not PKC and MSK1 inhibitors, restore the expression of occludin in RRV-infected Caco-2 cells. Detergent-insoluble and -soluble fractions were resolved by SDS-PAGE and Western immunoblotted for detection of occludin as described in Materials and Methods. Representative immunoblot assays from three independent experiments are shown. Western blot analysis of occludin in the Triton X-100-soluble (E) and Triton X-100-insoluble (R) fractions of control (C) or RRV-infected (I) Caco-2 cells which had or had not been treated with Rp-cAMP, the PKA inhibitors H89 and KT5720, the MSK1 inhibitor Ro318220, or the PKC inhibitor Gö6976. The position of the 65-kDa form of nonphosphorylated occludin is indicated. Blots were quantified with NIH Image software. Open bars, nonphosphorylated form of occludin in Triton X-100-soluble fraction; closed bars, nonphosphorylated form of occludin in Triton X-100-insoluble fraction; hatched bars, phosphorylated (P) form of occludin in Triton X-100-insoluble fraction. Each bar represents the mean ± the standard deviation of three independent experiments. *, P > 0.01 (Student t test) compared with appropriate control cells.
FIG. 6.
PKA inhibitor reversed the RRV-induced fall in the occludin mRNA level in Caco-2 cells. Fully differentiated cells were left uninfected or were infected with RRV for 18 h, with or without the PKA inhibitor H89. Total RNA was extracted, and occludin mRNAs were quantified by Q-RT-PCR. C t values obtained for occludin mRNA transcripts were normalized relative to those of GAPDH. Results are expressed as the TJs protein/GAPDH ratio (R). Each bar represents the mean ± the standard deviation of three independent experiments. *, P > 0.01 (Student t test) compared with RRV-infected or inhibitor-treated, RRV-infected cells.
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