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Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.

Furuse M, Furuse K, Sasaki H, Tsukita S - J. Cell Biol. (2001)

Bottom Line: Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease).Similar results were obtained in mouse epithelial cells, Eph4.These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

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Establishment of MDCK I transfectant clones stably expressing claudin-2. Dog claudin-2 and neomycin-resistance genes were introduced into MDCK I cells, and eight independent clones for each were established (dCL2-MDCK I and neo-MDCK I, respectively). (A) Immunoblotting. Total lysates of neo-MDCK I and dCL2-MDCK I clones (and also MDCK II cells) were subjected to SDS-PAGE in the same amount of total proteins, followed by immunoblotting. There were no significant differences in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the dCL2-MDCK I–specific expression of claudin-2 between neo-MDCK I and dCL2-MDCK I clones. The amounts of endogenous claudin-2 in MDCK II cells as well as exogenous claudin-2 in dCL2-MDCK I cells were quantified as described in Materials and Methods, and their dCL2-MDCK I/MDCK II ratios (relative amounts of claudin-2) were calculated. (B) Immunofluorescence microscopy. In clone 2 of dCL2-MDCK I cells, together with endogenous claudin-1, claudin-4, occludin, and ZO-1, exogenously expressed claudin-2 was co-concentrated at TJs. Bar, 10 μm.
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Figure 3: Establishment of MDCK I transfectant clones stably expressing claudin-2. Dog claudin-2 and neomycin-resistance genes were introduced into MDCK I cells, and eight independent clones for each were established (dCL2-MDCK I and neo-MDCK I, respectively). (A) Immunoblotting. Total lysates of neo-MDCK I and dCL2-MDCK I clones (and also MDCK II cells) were subjected to SDS-PAGE in the same amount of total proteins, followed by immunoblotting. There were no significant differences in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the dCL2-MDCK I–specific expression of claudin-2 between neo-MDCK I and dCL2-MDCK I clones. The amounts of endogenous claudin-2 in MDCK II cells as well as exogenous claudin-2 in dCL2-MDCK I cells were quantified as described in Materials and Methods, and their dCL2-MDCK I/MDCK II ratios (relative amounts of claudin-2) were calculated. (B) Immunofluorescence microscopy. In clone 2 of dCL2-MDCK I cells, together with endogenous claudin-1, claudin-4, occludin, and ZO-1, exogenously expressed claudin-2 was co-concentrated at TJs. Bar, 10 μm.

Mentions: The question has naturally arisen of whether the MDCK I transfectants expressing exogenous claudin-2 can mimic MDCK II cells in terms of the barrier property of TJs. If we used mouse claudin-2 cDNA in this transfection experiment, it would be possible that it exhibits some dominant-negative effect in dog epithelial cells such as MDCK cells due to its sequence diversity between mouse and dog. We then cloned dog claudin-2 cDNA using mouse cDNA as a probe. Sequencing identified some substitutions of amino acid residues between mouse and dog claudin-2 (Fig. 2). Then, this dog claudin-2 cDNA was introduced into MDCK I cells. As control experiments, only the neomycin-resistance gene was transfected. Cells were screened by immunofluorescence microscopy as well as immunoblotting, and finally eight independent stable clones were obtained each for control (neo-MDCK I) and claudin-2–expressing MDCK I cells (dCL2-MDCK I) (Fig. 3 A). As compared with neo-MDCK I cells (clone 1–8), in dCL2-MDCK I cells no significant differences were detected by immunoblotting in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the exogenous expression of claudin-2. When the expression levels of claudin-2 in dCL2-MDCK I cells were quantitatively compared with that in MDCK II cells, the ratios of dCL2-MDCK I/MDCKII were distributed from 0.7 to 3.8 (Fig. 3 A). Immunofluorescence microscopy revealed that in these dCL2-MDCK I cells, the exogenously expressed claudin-2 was concentrated at TJs together with endogenous claudin-1 and -4 as well as occludin and ZO-1 (Fig. 3 B), although the degree of concentration of exogenous claudin-2 at TJs varied depending on clones.


Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.

Furuse M, Furuse K, Sasaki H, Tsukita S - J. Cell Biol. (2001)

Establishment of MDCK I transfectant clones stably expressing claudin-2. Dog claudin-2 and neomycin-resistance genes were introduced into MDCK I cells, and eight independent clones for each were established (dCL2-MDCK I and neo-MDCK I, respectively). (A) Immunoblotting. Total lysates of neo-MDCK I and dCL2-MDCK I clones (and also MDCK II cells) were subjected to SDS-PAGE in the same amount of total proteins, followed by immunoblotting. There were no significant differences in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the dCL2-MDCK I–specific expression of claudin-2 between neo-MDCK I and dCL2-MDCK I clones. The amounts of endogenous claudin-2 in MDCK II cells as well as exogenous claudin-2 in dCL2-MDCK I cells were quantified as described in Materials and Methods, and their dCL2-MDCK I/MDCK II ratios (relative amounts of claudin-2) were calculated. (B) Immunofluorescence microscopy. In clone 2 of dCL2-MDCK I cells, together with endogenous claudin-1, claudin-4, occludin, and ZO-1, exogenously expressed claudin-2 was co-concentrated at TJs. Bar, 10 μm.
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Figure 3: Establishment of MDCK I transfectant clones stably expressing claudin-2. Dog claudin-2 and neomycin-resistance genes were introduced into MDCK I cells, and eight independent clones for each were established (dCL2-MDCK I and neo-MDCK I, respectively). (A) Immunoblotting. Total lysates of neo-MDCK I and dCL2-MDCK I clones (and also MDCK II cells) were subjected to SDS-PAGE in the same amount of total proteins, followed by immunoblotting. There were no significant differences in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the dCL2-MDCK I–specific expression of claudin-2 between neo-MDCK I and dCL2-MDCK I clones. The amounts of endogenous claudin-2 in MDCK II cells as well as exogenous claudin-2 in dCL2-MDCK I cells were quantified as described in Materials and Methods, and their dCL2-MDCK I/MDCK II ratios (relative amounts of claudin-2) were calculated. (B) Immunofluorescence microscopy. In clone 2 of dCL2-MDCK I cells, together with endogenous claudin-1, claudin-4, occludin, and ZO-1, exogenously expressed claudin-2 was co-concentrated at TJs. Bar, 10 μm.
Mentions: The question has naturally arisen of whether the MDCK I transfectants expressing exogenous claudin-2 can mimic MDCK II cells in terms of the barrier property of TJs. If we used mouse claudin-2 cDNA in this transfection experiment, it would be possible that it exhibits some dominant-negative effect in dog epithelial cells such as MDCK cells due to its sequence diversity between mouse and dog. We then cloned dog claudin-2 cDNA using mouse cDNA as a probe. Sequencing identified some substitutions of amino acid residues between mouse and dog claudin-2 (Fig. 2). Then, this dog claudin-2 cDNA was introduced into MDCK I cells. As control experiments, only the neomycin-resistance gene was transfected. Cells were screened by immunofluorescence microscopy as well as immunoblotting, and finally eight independent stable clones were obtained each for control (neo-MDCK I) and claudin-2–expressing MDCK I cells (dCL2-MDCK I) (Fig. 3 A). As compared with neo-MDCK I cells (clone 1–8), in dCL2-MDCK I cells no significant differences were detected by immunoblotting in the expression levels of claudin-1, claudin-4, occludin, or ZO-1, except for the exogenous expression of claudin-2. When the expression levels of claudin-2 in dCL2-MDCK I cells were quantitatively compared with that in MDCK II cells, the ratios of dCL2-MDCK I/MDCKII were distributed from 0.7 to 3.8 (Fig. 3 A). Immunofluorescence microscopy revealed that in these dCL2-MDCK I cells, the exogenously expressed claudin-2 was concentrated at TJs together with endogenous claudin-1 and -4 as well as occludin and ZO-1 (Fig. 3 B), although the degree of concentration of exogenous claudin-2 at TJs varied depending on clones.

Bottom Line: Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease).Similar results were obtained in mouse epithelial cells, Eph4.These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

ABSTRACT
There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

Show MeSH