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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 G1/S-phase transition by ZO-1 and ZONAB. Wild-type (wt MDCK) and transfected MDCK cells expressing the indicated cDNAs were plated at low confluence and then synchronized in low serum. Cell cycle entry was then triggered by serum addition, and progression to S-phase was monitored by measuring incorporation of [3H]thymidine (A) or BrdU (B). Note, whereas in A total DNA synthesis was measured, B shows the fraction of cells in S-phase. Expression of full-length ZO-1 significantly inhibited entry into S-phase (t test; A, P < 0.05; B, P < 0.01). Shown are means ± 1 SD of three independent clones per construct that were analyzed in at least three different experiments performed in quadruplicate.
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fig3: Regulation of G1/S-phase transition by ZO-1 and ZONAB. Wild-type (wt MDCK) and transfected MDCK cells expressing the indicated cDNAs were plated at low confluence and then synchronized in low serum. Cell cycle entry was then triggered by serum addition, and progression to S-phase was monitored by measuring incorporation of [3H]thymidine (A) or BrdU (B). Note, whereas in A total DNA synthesis was measured, B shows the fraction of cells in S-phase. Expression of full-length ZO-1 significantly inhibited entry into S-phase (t test; A, P < 0.05; B, P < 0.01). Shown are means ± 1 SD of three independent clones per construct that were analyzed in at least three different experiments performed in quadruplicate.

Mentions: Y-box transcription factors have been proposed to regulate cell cycle entry (Bargou et al., 1997; Matsumoto and Wolffe, 1998), and ZONAB cannot be detected in the nucleus in nonproliferating high density cells. To test whether ZONAB regulates cell cycle entry, we synchronized low density cultures of MDCK cells in Go/G1 phase by serum starvation. Based on FACS analysis, around 75% of the cells were arrested in Go/G1 after the 48-h incubation in 0.1% serum (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200210020/DC1). Entry into S-phase on addition of serum was then quantified by determining the incorporation of [3H]thymidine. Fig. 3 A shows that ZO-1 overexpression indeed reduced DNA synthesis, suggesting that entry into S-phase was affected. The ZO-1 effect depended on the presence of the SH3 domain, and overexpression of ZONAB again reversed the levels of [3H]thymidine incorporation to control levels. This suggests that ZO-1 indeed regulates cell cycle entry.


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 G1/S-phase transition by ZO-1 and ZONAB. Wild-type (wt MDCK) and transfected MDCK cells expressing the indicated cDNAs were plated at low confluence and then synchronized in low serum. Cell cycle entry was then triggered by serum addition, and progression to S-phase was monitored by measuring incorporation of [3H]thymidine (A) or BrdU (B). Note, whereas in A total DNA synthesis was measured, B shows the fraction of cells in S-phase. Expression of full-length ZO-1 significantly inhibited entry into S-phase (t test; A, P < 0.05; B, P < 0.01). Shown are means ± 1 SD of three independent clones per construct that were analyzed in at least three different experiments performed in quadruplicate.
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fig3: Regulation of G1/S-phase transition by ZO-1 and ZONAB. Wild-type (wt MDCK) and transfected MDCK cells expressing the indicated cDNAs were plated at low confluence and then synchronized in low serum. Cell cycle entry was then triggered by serum addition, and progression to S-phase was monitored by measuring incorporation of [3H]thymidine (A) or BrdU (B). Note, whereas in A total DNA synthesis was measured, B shows the fraction of cells in S-phase. Expression of full-length ZO-1 significantly inhibited entry into S-phase (t test; A, P < 0.05; B, P < 0.01). Shown are means ± 1 SD of three independent clones per construct that were analyzed in at least three different experiments performed in quadruplicate.
Mentions: Y-box transcription factors have been proposed to regulate cell cycle entry (Bargou et al., 1997; Matsumoto and Wolffe, 1998), and ZONAB cannot be detected in the nucleus in nonproliferating high density cells. To test whether ZONAB regulates cell cycle entry, we synchronized low density cultures of MDCK cells in Go/G1 phase by serum starvation. Based on FACS analysis, around 75% of the cells were arrested in Go/G1 after the 48-h incubation in 0.1% serum (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200210020/DC1). Entry into S-phase on addition of serum was then quantified by determining the incorporation of [3H]thymidine. Fig. 3 A shows that ZO-1 overexpression indeed reduced DNA synthesis, suggesting that entry into S-phase was affected. The ZO-1 effect depended on the presence of the SH3 domain, and overexpression of ZONAB again reversed the levels of [3H]thymidine incorporation to control levels. This suggests that ZO-1 indeed regulates cell cycle entry.

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