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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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Interaction of ZONAB with CDK4. MDCK cells were extracted under stringent conditions (A) or in PBS-Triton X-100 (B), and cell extracts were subjected to immunoprecipitation with the indicated antibodies. CDK4 and cyclin D1 were then detected by immunoblotting using a γ-chain–specific secondary antibody. (C) In vitro interaction between recombinant His-tagged ZONAB and GST-tagged CDK4. Glutathione beads loaded with either GST or GST-CDK4 were incubated with His-tagged ZONAB fusion protein. The beads were then washed, and pull-down was monitored by immunoblotting. A His-tagged control fusion protein was not detected in the precipitates (not depicted). (D) Kinase activity of recombinant CDK4/cyclin D1 was assayed in the presence of recombinant GST-ZONAB or GST using GST-retinoblastoma (GST-Rb) as a substrate. Note that CDK4 activity was not affected by GST-ZONAB.
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fig6: Interaction of ZONAB with CDK4. MDCK cells were extracted under stringent conditions (A) or in PBS-Triton X-100 (B), and cell extracts were subjected to immunoprecipitation with the indicated antibodies. CDK4 and cyclin D1 were then detected by immunoblotting using a γ-chain–specific secondary antibody. (C) In vitro interaction between recombinant His-tagged ZONAB and GST-tagged CDK4. Glutathione beads loaded with either GST or GST-CDK4 were incubated with His-tagged ZONAB fusion protein. The beads were then washed, and pull-down was monitored by immunoblotting. A His-tagged control fusion protein was not detected in the precipitates (not depicted). (D) Kinase activity of recombinant CDK4/cyclin D1 was assayed in the presence of recombinant GST-ZONAB or GST using GST-retinoblastoma (GST-Rb) as a substrate. Note that CDK4 activity was not affected by GST-ZONAB.

Mentions: First, we immunoprecipitated extracts generated from wild-type MDCK cells with a stringent buffer (i.e., containing deoxylcholate, SDS, Empigen BB, and Triton X-100), and monitored the presence of CDKs by immunoblotting using a γ-chain–specific secondary antibody. Under stringent extraction conditions, CDK4 was present in immunoprecipitates generated with anti-ZONAB antibodies, but absent from control precipitates (Fig. 6 A). Other tested CDKs (e.g., CDK6) were not detected in ZONAB precipitates (unpublished data).


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)

Interaction of ZONAB with CDK4. MDCK cells were extracted under stringent conditions (A) or in PBS-Triton X-100 (B), and cell extracts were subjected to immunoprecipitation with the indicated antibodies. CDK4 and cyclin D1 were then detected by immunoblotting using a γ-chain–specific secondary antibody. (C) In vitro interaction between recombinant His-tagged ZONAB and GST-tagged CDK4. Glutathione beads loaded with either GST or GST-CDK4 were incubated with His-tagged ZONAB fusion protein. The beads were then washed, and pull-down was monitored by immunoblotting. A His-tagged control fusion protein was not detected in the precipitates (not depicted). (D) Kinase activity of recombinant CDK4/cyclin D1 was assayed in the presence of recombinant GST-ZONAB or GST using GST-retinoblastoma (GST-Rb) as a substrate. Note that CDK4 activity was not affected by GST-ZONAB.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172662&req=5

fig6: Interaction of ZONAB with CDK4. MDCK cells were extracted under stringent conditions (A) or in PBS-Triton X-100 (B), and cell extracts were subjected to immunoprecipitation with the indicated antibodies. CDK4 and cyclin D1 were then detected by immunoblotting using a γ-chain–specific secondary antibody. (C) In vitro interaction between recombinant His-tagged ZONAB and GST-tagged CDK4. Glutathione beads loaded with either GST or GST-CDK4 were incubated with His-tagged ZONAB fusion protein. The beads were then washed, and pull-down was monitored by immunoblotting. A His-tagged control fusion protein was not detected in the precipitates (not depicted). (D) Kinase activity of recombinant CDK4/cyclin D1 was assayed in the presence of recombinant GST-ZONAB or GST using GST-retinoblastoma (GST-Rb) as a substrate. Note that CDK4 activity was not affected by GST-ZONAB.
Mentions: First, we immunoprecipitated extracts generated from wild-type MDCK cells with a stringent buffer (i.e., containing deoxylcholate, SDS, Empigen BB, and Triton X-100), and monitored the presence of CDKs by immunoblotting using a γ-chain–specific secondary antibody. Under stringent extraction conditions, CDK4 was present in immunoprecipitates generated with anti-ZONAB antibodies, but absent from control precipitates (Fig. 6 A). Other tested CDKs (e.g., CDK6) were not detected in ZONAB precipitates (unpublished data).

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