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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 proliferation by ZONAB. (A) MDCK cells were grown for the indicated amounts of days. Cultures were harvested and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. The bands representing the ZONAB-A and -B isoforms are indicated. Parallel cultures were trypsinized, and the cells were counted to determine the cell density. (B) Reduction of ZONAB expression in low density cells by ZONAB antisense RNA. Proliferating wild-type MDCK cells (wt MDCK) and cells expressing ZONAB antisense RNA (ZONABas) were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. Based on densitometric scanning of the immunoblots, expression of ZONAB was reduced by >50% in cells transfected with the antisense construct. (C) Proliferation of low density MDCK cells expressing ZONAB antisense RNA (ZONABas), a control missense RNA (msRNA), overexpressing either one of the ZONAB isoforms, or transfected with a control cDNA were analyzed by measuring incorporation of [3H]thymidine. Data were normalized to wild-type cells (shown are means ± 1 SD of at least three independent clones per construct that were analyzed in three independent experiments with quadruplicate cultures). Note that incorporation of [3H]thymidine by ZONABas cells was reduced by >70% (t test; P < 0.01). (D) Overexpression of ZONAB isoforms in low density MDCK cells. Proliferating wild-type MDCK cells (wt MDCK) and cells overexpressing ZONAB-A or ZONAB-B were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting.
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fig1: Regulation of proliferation by ZONAB. (A) MDCK cells were grown for the indicated amounts of days. Cultures were harvested and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. The bands representing the ZONAB-A and -B isoforms are indicated. Parallel cultures were trypsinized, and the cells were counted to determine the cell density. (B) Reduction of ZONAB expression in low density cells by ZONAB antisense RNA. Proliferating wild-type MDCK cells (wt MDCK) and cells expressing ZONAB antisense RNA (ZONABas) were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. Based on densitometric scanning of the immunoblots, expression of ZONAB was reduced by >50% in cells transfected with the antisense construct. (C) Proliferation of low density MDCK cells expressing ZONAB antisense RNA (ZONABas), a control missense RNA (msRNA), overexpressing either one of the ZONAB isoforms, or transfected with a control cDNA were analyzed by measuring incorporation of [3H]thymidine. Data were normalized to wild-type cells (shown are means ± 1 SD of at least three independent clones per construct that were analyzed in three independent experiments with quadruplicate cultures). Note that incorporation of [3H]thymidine by ZONABas cells was reduced by >70% (t test; P < 0.01). (D) Overexpression of ZONAB isoforms in low density MDCK cells. Proliferating wild-type MDCK cells (wt MDCK) and cells overexpressing ZONAB-A or ZONAB-B were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting.

Mentions: ZONAB expression is high in proliferating but low in growth-arrested MDCK cells (Fig. 1 A), suggesting that high expression levels might be required for cell proliferation (Balda and Matter, 2000). To test this, we used an antisense RNA approach to reduce ZONAB expression. Stable expression of ZONAB antisense RNA resulted in a reduction of cellular ZONAB levels by about >50% (Fig. 1 B), whereas expression of a 50% homologous Y-box factor, YB-1, was not affected (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)

Regulation of proliferation by ZONAB. (A) MDCK cells were grown for the indicated amounts of days. Cultures were harvested and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. The bands representing the ZONAB-A and -B isoforms are indicated. Parallel cultures were trypsinized, and the cells were counted to determine the cell density. (B) Reduction of ZONAB expression in low density cells by ZONAB antisense RNA. Proliferating wild-type MDCK cells (wt MDCK) and cells expressing ZONAB antisense RNA (ZONABas) were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. Based on densitometric scanning of the immunoblots, expression of ZONAB was reduced by >50% in cells transfected with the antisense construct. (C) Proliferation of low density MDCK cells expressing ZONAB antisense RNA (ZONABas), a control missense RNA (msRNA), overexpressing either one of the ZONAB isoforms, or transfected with a control cDNA were analyzed by measuring incorporation of [3H]thymidine. Data were normalized to wild-type cells (shown are means ± 1 SD of at least three independent clones per construct that were analyzed in three independent experiments with quadruplicate cultures). Note that incorporation of [3H]thymidine by ZONABas cells was reduced by >70% (t test; P < 0.01). (D) Overexpression of ZONAB isoforms in low density MDCK cells. Proliferating wild-type MDCK cells (wt MDCK) and cells overexpressing ZONAB-A or ZONAB-B were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting.
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fig1: Regulation of proliferation by ZONAB. (A) MDCK cells were grown for the indicated amounts of days. Cultures were harvested and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. The bands representing the ZONAB-A and -B isoforms are indicated. Parallel cultures were trypsinized, and the cells were counted to determine the cell density. (B) Reduction of ZONAB expression in low density cells by ZONAB antisense RNA. Proliferating wild-type MDCK cells (wt MDCK) and cells expressing ZONAB antisense RNA (ZONABas) were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting. Based on densitometric scanning of the immunoblots, expression of ZONAB was reduced by >50% in cells transfected with the antisense construct. (C) Proliferation of low density MDCK cells expressing ZONAB antisense RNA (ZONABas), a control missense RNA (msRNA), overexpressing either one of the ZONAB isoforms, or transfected with a control cDNA were analyzed by measuring incorporation of [3H]thymidine. Data were normalized to wild-type cells (shown are means ± 1 SD of at least three independent clones per construct that were analyzed in three independent experiments with quadruplicate cultures). Note that incorporation of [3H]thymidine by ZONABas cells was reduced by >70% (t test; P < 0.01). (D) Overexpression of ZONAB isoforms in low density MDCK cells. Proliferating wild-type MDCK cells (wt MDCK) and cells overexpressing ZONAB-A or ZONAB-B were harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZONAB expression by immunoblotting.
Mentions: ZONAB expression is high in proliferating but low in growth-arrested MDCK cells (Fig. 1 A), suggesting that high expression levels might be required for cell proliferation (Balda and Matter, 2000). To test this, we used an antisense RNA approach to reduce ZONAB expression. Stable expression of ZONAB antisense RNA resulted in a reduction of cellular ZONAB levels by about >50% (Fig. 1 B), whereas expression of a 50% homologous Y-box factor, YB-1, was not affected (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