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. 2006 Feb;147(2):977-89.
doi: 10.1210/en.2005-0916. Epub 2005 Oct 20.

Changes in tight junctional resistance of the cervical epithelium are associated with modulation of content and phosphorylation of occludin 65-kilodalton and 50-kilodalton forms

Ling Zhu  1 , Xin Li, Robin Zeng, George I Gorodeski
Affiliations

Changes in tight junctional resistance of the cervical epithelium are associated with modulation of content and phosphorylation of occludin 65-kilodalton and 50-kilodalton forms

Ling Zhu et al. Endocrinology. 2006 Feb.

Abstract

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (R(TJ)). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in R(TJ) induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of R(TJ) correlated with the 65-kDa form, whereas low levels of R(TJ) correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the R(TJ), 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

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Figures

FIG. 1
FIG. 1
CaSki cells on filters were treated with 10 μM ATP (A) or with 10 μM diC8 (B), added to both the luminal and subluminal solutions, in the absence (solid lines) or presence of 10 μM staurosporine (broken lines). Changes in transepithelial permeability were determined in terms of RTE. The effects of ATP include an immediate decrease in RTE (phase-I), followed by a transient increase in RTE (phase-II), and a late decrease in RTE (phase-III). Experiments were repeated four to seven times with similar trends, and data are summarized in Table 2. C, Effects of ATP on occludin expression. CaSki cells on filters were treated with 10 μM ATP for 45 min, and cells attached on filters were fixated and immunostained with rabbit antioccludin antibody (×20).
FIG. 2
FIG. 2
Effects of ATP (A–C) and diC8 (D and E) on the cellular densities of occludin. At designated times after treatments, cells were lysed and total homogenates were immunoprecipitated and immunoblotted with antioccludin antibodies as described in the text. Shown are data of changes in occludin 65- and 50-kDa forms. Membranes were deprobed and immunoblotted with antitubulin antibody. Experiments were repeated two to four times with similar trends.
FIG. 3
FIG. 3
Effects of ATP (A) and diC8 (B) on the cellular densities of occludin 65- and 50-kDa forms: densitometry analysis of the data shown in Fig. 2, C and E. A.U., Arbitrary units.
FIG. 4
FIG. 4
Effects of ATP (50 μM) (A and B) and diC8 (10 μM) (C and D) on the phosphorylation of occludin 65- and 50-kDa forms at threonine (PT) (A and C) and tyrosine (PY) (B and D) residues. At designated times after treatments, cells were lysed and total homogenates were immunoprecipitated with antioccludin antibody and immunoblotted with either antiphosphothreonine or antiphosphotyrosine antibodies. Experiments were repeated twice with similar trends.
FIG. 5
FIG. 5
Densitometry analysis of the effects of ATP on the phosphorylation of occludin 65- and 50-kDa forms at threonine (PT, B) and tyrosine (PY, C) residues. Data are from Fig. 4, and results are normalized to the densities of total occludin 65- and 50-kDa isoforms (A).
FIG. 6
FIG. 6
Densitometry analysis of the effects of diC8 on the phosphorylation of occludin 65- and 50-kDa forms at threonine (PT) (A) and tyrosine (PY) (B) residues. Data are from Fig. 4, and results are normalized to the densities of total occludin 65- and 50-kDa isoforms.
FIG. 7
FIG. 7
Staurosporine modulation of ATP changes in the densities and phosphorylation of occludin 65- and 50-kDa forms. Cells were pretreated for 15 min with 12 nM staurosporine, followed by 50 μM ATP. At times 0–5 min after treatments, cells were lysed and total homogenates were immunoprecipitated with antioccludin antibody. Immunoblots used either antioccludin antibody (WB, Western blots) (A), or antiphosphothreonine (PT) (B) or antiphosphotyrosine antibodies (PY) (C).
FIG. 8
FIG. 8
Densitometry analysis of staurosporine modulation of ATP changes in the densities of occludin 65- and 50-kDa forms (A) and occludin phosphorylation at threonine ((PT) (B) and tyrosine (PY) (C) residues. Data are from Fig. 7, and results are normalized to the densities of total occludin 65- and 50-kDa isoforms.
FIG. 9
FIG. 9
ALLN modulation of ATP (A) and diC8 (B) effects on the densities of occludin 65- and 50-kDa forms. A, Cells were treated for 30 min with 50 μM ALLN or the vehicle and then cotreated with 50 μM ATP (or the vehicle) for an additional 30 min. B, Treatments were as follows: vehicles (lane 1), 50 μM ALLN (lane 2), and 10 μM diC8 for 30 min (lanes 3 and 5); 50 μM ALLN plus 10 μM diC8 for 30 min (lane 4) (Sim, simultaneous); and 50 μM ALLN for 30 min followed by 10 μM diC8 for an additional 30 min (lane 6). At the completion of treatments, cells were lysed, and total homogenates were immunoprecipitated and immunoblotted with antioccludin antibody.
FIG. 10
FIG. 10
ALLN modulation of ATP (A) and diC8 (B) effects on the densities of occludin 65- and 50-kDa forms: densitometry analysis of the data shown in Fig. 9. In A, lanes 1 and 2, densitometry assessed the 48-kDa forms and in lanes 3 and 4 the 50-kDa forms.
FIG. 11
FIG. 11
CaSki cells were treated with the V, with CLO, or with ASO. A, RNA data; B, cell lysates were immunoblotted with the antihuman occludin antibody, and membranes were reprobed with anti-GAPDH antibody.
FIG. 12
FIG. 12
Correlation between RTE and the densities of occludin 65-kDa form (A), occludin 50-kDa form (B), and the ratio of 65-kDa/50-kDa (C). Densitometry data of occludin were obtained from the experiments described in Figs. 2 and 9 and was correlated with data of RTE obtained in Fig. 1 and Table 2. In A–C, correlations for RTE levels of 10–50 Ω-cm2 were significant (P < 0.05–0.03). In C, the correlation for RTE levels of 70–90 Ω-cm2 was significant (P < 0.02).
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References

    1. Gorodeski GI. The cervical cycle. In: Adashi EY, Rock JA, Rosenwaks Z, editors. Reproductive endocrinology, surgery, and technology. Philadelphia: Lippincott-Raven; 1996. pp. 301–324.
    1. Gorodeski GI. The cultured human cervical epithelium: a new model for studying transepithelial paracellular transport. J Soc Gynecol Invest. 1996;3:267–280. - PubMed
    1. Gorodeski GI, Merlin D, De Santis BJ, Frieden KA, Hopfer U, Eckert RL, Romero MF. Characterization of paracellular permeability in cultured human cervical epithelium: regulation by extracellular ATP. J Soc Gynecol Invest. 1994;1:225–233. - PubMed
    1. Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol. 1998;142:117–127. - PMC - PubMed
    1. Furuse M, Sasaki H, Fujimoto K, Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol. 1998;143:391–401. - PMC - PubMed

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