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The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density.

Balda MS, Garrett MD, Matter K - J. Cell Biol. (2003)

Bottom Line: Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells.Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density.ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, UK. m.balda@ucl.ac.uk

ABSTRACT
Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1-associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction-associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1-based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4.

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Association of CDK4 with intercellular junctions. (A and B) MDCK cells were plated on coverslips and grown for 3 d. The cells were then fixed with methanol and processed for double immunofluorescence using rabbit anti-CDK4 and rat anti–ZO-1 antibodies. Shown are confocal XY-sections (A, CDK4; B, ZO-1 staining). (C and D) Confocal XY-sections of MDCK cells transiently transfected with a cDNA coding for HA-tagged CDK4. The cells were stained with rabbit anti-HA and rat anti–ZO-1 antibodies (C, HA-CDK4; D, ZO-1 staining). Bars, 10 μm.
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fig7: Association of CDK4 with intercellular junctions. (A and B) MDCK cells were plated on coverslips and grown for 3 d. The cells were then fixed with methanol and processed for double immunofluorescence using rabbit anti-CDK4 and rat anti–ZO-1 antibodies. Shown are confocal XY-sections (A, CDK4; B, ZO-1 staining). (C and D) Confocal XY-sections of MDCK cells transiently transfected with a cDNA coding for HA-tagged CDK4. The cells were stained with rabbit anti-HA and rat anti–ZO-1 antibodies (C, HA-CDK4; D, ZO-1 staining). Bars, 10 μm.

Mentions: Because CDK4 was found to coimmunoprecipitate with ZONAB, one would expect that the two proteins have a similar subcellular distribution. To test this, proliferating MDCK cells, in which ZONAB localizes to the nucleus as well as tight junctins, were fixed and processed for immunofluorescence using an antibody against CDK4. Fig. 7 A shows that endogenous CDK4 was indeed detected in the nucleus as well as at cell–cell junctions, which were visualized with anti-ZO-1 antibodies (Fig. 7 B). Similar stainings were obtained with two different CDK4 antibodies. To test CDK4 association with cell–cell junctions in an independent manner, we transiently expressed HA-tagged CDK4 in MDCK cells. Fig. 7 C shows that HA-CDK4 also localizes to intercellular junctions that were labeled with anti-ZO-1 antibody (Fig. 7 D).


The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density.

Balda MS, Garrett MD, Matter K - J. Cell Biol. (2003)

Association of CDK4 with intercellular junctions. (A and B) MDCK cells were plated on coverslips and grown for 3 d. The cells were then fixed with methanol and processed for double immunofluorescence using rabbit anti-CDK4 and rat anti–ZO-1 antibodies. Shown are confocal XY-sections (A, CDK4; B, ZO-1 staining). (C and D) Confocal XY-sections of MDCK cells transiently transfected with a cDNA coding for HA-tagged CDK4. The cells were stained with rabbit anti-HA and rat anti–ZO-1 antibodies (C, HA-CDK4; D, ZO-1 staining). Bars, 10 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172662&req=5

fig7: Association of CDK4 with intercellular junctions. (A and B) MDCK cells were plated on coverslips and grown for 3 d. The cells were then fixed with methanol and processed for double immunofluorescence using rabbit anti-CDK4 and rat anti–ZO-1 antibodies. Shown are confocal XY-sections (A, CDK4; B, ZO-1 staining). (C and D) Confocal XY-sections of MDCK cells transiently transfected with a cDNA coding for HA-tagged CDK4. The cells were stained with rabbit anti-HA and rat anti–ZO-1 antibodies (C, HA-CDK4; D, ZO-1 staining). Bars, 10 μm.
Mentions: Because CDK4 was found to coimmunoprecipitate with ZONAB, one would expect that the two proteins have a similar subcellular distribution. To test this, proliferating MDCK cells, in which ZONAB localizes to the nucleus as well as tight junctins, were fixed and processed for immunofluorescence using an antibody against CDK4. Fig. 7 A shows that endogenous CDK4 was indeed detected in the nucleus as well as at cell–cell junctions, which were visualized with anti-ZO-1 antibodies (Fig. 7 B). Similar stainings were obtained with two different CDK4 antibodies. To test CDK4 association with cell–cell junctions in an independent manner, we transiently expressed HA-tagged CDK4 in MDCK cells. Fig. 7 C shows that HA-CDK4 also localizes to intercellular junctions that were labeled with anti-ZO-1 antibody (Fig. 7 D).

Bottom Line: Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells.Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density.ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Institute of Ophthalmology, University College London, London EC1V 9EL, UK. m.balda@ucl.ac.uk

ABSTRACT
Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1-associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction-associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1-based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4.

Show MeSH
Related in: MedlinePlus