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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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Regulation of final cell density by ZONAB. (A) Expression of ZONAB in high density wild-type and transfected MDCK cells. Wild-type (wt MDCK) and ZONAB-A or -B overexpressing cells were grown for 7 d. Cells cultured for 7 d had reached maximal cell density. Cultures were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. (B) Cell cycle arrest of high density MDCK cells. Wild-type or transfected MDCK cells overexpressing ZO-1 or ZONAB were grown to full density, and cell cycle arrest was determined by BrdU incorporation. Shown are representative images of propidium iodide and BrdU labelings, and the indicated numbers are the percentage of BrdU-positive cells (averages of two independent experiments are shown). Note the different cell densities in the propidium iodide staining. (C) Cell density of mature monolayers. Cells grown as those in B were harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. The value shown for control transfections (control T) represents cell densities obtained from cell lines expressing three different control cDNAs generated with the same expression vector. Shown are averages ± 1 SD of independent clones that were analyzed twice independently using triplicate cultures. Analyzed were three different clones expressing ZONAB-A, three ZONAB-RD-I clones, and two independent clones each for ZONAB-B, ZO-1, ZONAB-RD-II, and Control-RD. All cell lines with the exception of the two types of control transfection were significantly different from wild-type cells (t test; P < 0.05).
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fig5: Regulation of final cell density by ZONAB. (A) Expression of ZONAB in high density wild-type and transfected MDCK cells. Wild-type (wt MDCK) and ZONAB-A or -B overexpressing cells were grown for 7 d. Cells cultured for 7 d had reached maximal cell density. Cultures were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. (B) Cell cycle arrest of high density MDCK cells. Wild-type or transfected MDCK cells overexpressing ZO-1 or ZONAB were grown to full density, and cell cycle arrest was determined by BrdU incorporation. Shown are representative images of propidium iodide and BrdU labelings, and the indicated numbers are the percentage of BrdU-positive cells (averages of two independent experiments are shown). Note the different cell densities in the propidium iodide staining. (C) Cell density of mature monolayers. Cells grown as those in B were harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. The value shown for control transfections (control T) represents cell densities obtained from cell lines expressing three different control cDNAs generated with the same expression vector. Shown are averages ± 1 SD of independent clones that were analyzed twice independently using triplicate cultures. Analyzed were three different clones expressing ZONAB-A, three ZONAB-RD-I clones, and two independent clones each for ZONAB-B, ZO-1, ZONAB-RD-II, and Control-RD. All cell lines with the exception of the two types of control transfection were significantly different from wild-type cells (t test; P < 0.05).

Mentions: Because reduced nuclear expression of ZONAB in low density cells reduces proliferation and ZONAB becomes down-regulated with increasing cell density (Fig. 1 A; Balda and Matter, 2000), we used cell lines overexpressing either one of the ZONAB isoforms to test whether overexpression of ZONAB affects either cell cycle arrest in high density cells or the final cell density (Fig. 5 A). First, we labeled high density cells with BrdU to determine whether overexpression of ZONAB affects cell cycle arrest in mature monolayers.


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)

Regulation of final cell density by ZONAB. (A) Expression of ZONAB in high density wild-type and transfected MDCK cells. Wild-type (wt MDCK) and ZONAB-A or -B overexpressing cells were grown for 7 d. Cells cultured for 7 d had reached maximal cell density. Cultures were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. (B) Cell cycle arrest of high density MDCK cells. Wild-type or transfected MDCK cells overexpressing ZO-1 or ZONAB were grown to full density, and cell cycle arrest was determined by BrdU incorporation. Shown are representative images of propidium iodide and BrdU labelings, and the indicated numbers are the percentage of BrdU-positive cells (averages of two independent experiments are shown). Note the different cell densities in the propidium iodide staining. (C) Cell density of mature monolayers. Cells grown as those in B were harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. The value shown for control transfections (control T) represents cell densities obtained from cell lines expressing three different control cDNAs generated with the same expression vector. Shown are averages ± 1 SD of independent clones that were analyzed twice independently using triplicate cultures. Analyzed were three different clones expressing ZONAB-A, three ZONAB-RD-I clones, and two independent clones each for ZONAB-B, ZO-1, ZONAB-RD-II, and Control-RD. All cell lines with the exception of the two types of control transfection were significantly different from wild-type cells (t test; P < 0.05).
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Related In: Results  -  Collection

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fig5: Regulation of final cell density by ZONAB. (A) Expression of ZONAB in high density wild-type and transfected MDCK cells. Wild-type (wt MDCK) and ZONAB-A or -B overexpressing cells were grown for 7 d. Cells cultured for 7 d had reached maximal cell density. Cultures were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. (B) Cell cycle arrest of high density MDCK cells. Wild-type or transfected MDCK cells overexpressing ZO-1 or ZONAB were grown to full density, and cell cycle arrest was determined by BrdU incorporation. Shown are representative images of propidium iodide and BrdU labelings, and the indicated numbers are the percentage of BrdU-positive cells (averages of two independent experiments are shown). Note the different cell densities in the propidium iodide staining. (C) Cell density of mature monolayers. Cells grown as those in B were harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. The value shown for control transfections (control T) represents cell densities obtained from cell lines expressing three different control cDNAs generated with the same expression vector. Shown are averages ± 1 SD of independent clones that were analyzed twice independently using triplicate cultures. Analyzed were three different clones expressing ZONAB-A, three ZONAB-RD-I clones, and two independent clones each for ZONAB-B, ZO-1, ZONAB-RD-II, and Control-RD. All cell lines with the exception of the two types of control transfection were significantly different from wild-type cells (t test; P < 0.05).
Mentions: Because reduced nuclear expression of ZONAB in low density cells reduces proliferation and ZONAB becomes down-regulated with increasing cell density (Fig. 1 A; Balda and Matter, 2000), we used cell lines overexpressing either one of the ZONAB isoforms to test whether overexpression of ZONAB affects either cell cycle arrest in high density cells or the final cell density (Fig. 5 A). First, we labeled high density cells with BrdU to determine whether overexpression of ZONAB affects cell cycle arrest in mature monolayers.

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