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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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Depletion of ZONAB by RNA interference inhibits G1/S-phase transition. (A) Wild-type MDCK cells or cells stably expressing RNA duplexes corresponding to region I (ZONAB-RD-I c1 and c2) or II (ZONAB-RD-II c8 and c9) of ZONAB, or control RNA duplexes (control-RD c1 and c3) were grown for 2 d and then lysed and processed for electrophoresis. The samples were immunoblotted with anti-ZONAB and anti–α-tubulin antibodies. (B) Wild-type and transfected MDCK cells expressing the indicated RNA duplexes were plated at low confluence and synchronized in low serum. Cell cycle entry was triggered by serum addition, and progression to S-phase was monitored by determining the fraction of cell incorporation of BrdU. Shown are means ± 1 SD of at least two independent clones per type of RNA duplex that were analyzed in two independent experiments. Both ZONAB-directed RNA duplexes significantly inhibited entry into S-phase (t test; P < 0.02).
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fig4: Depletion of ZONAB by RNA interference inhibits G1/S-phase transition. (A) Wild-type MDCK cells or cells stably expressing RNA duplexes corresponding to region I (ZONAB-RD-I c1 and c2) or II (ZONAB-RD-II c8 and c9) of ZONAB, or control RNA duplexes (control-RD c1 and c3) were grown for 2 d and then lysed and processed for electrophoresis. The samples were immunoblotted with anti-ZONAB and anti–α-tubulin antibodies. (B) Wild-type and transfected MDCK cells expressing the indicated RNA duplexes were plated at low confluence and synchronized in low serum. Cell cycle entry was triggered by serum addition, and progression to S-phase was monitored by determining the fraction of cell incorporation of BrdU. Shown are means ± 1 SD of at least two independent clones per type of RNA duplex that were analyzed in two independent experiments. Both ZONAB-directed RNA duplexes significantly inhibited entry into S-phase (t test; P < 0.02).

Mentions: Next, we wanted to test whether reduction of total cellular ZONAB levels also inhibits cell cycle entry. Because expression of long antisense RNAs as the one used in the experiment described in Fig. 1 can cause nonspecific cellular responses due to activation of antiviral defense mechanisms (Williams, 1999), we used an alternative approach to reduce ZONAB expression. This second approach was based on the expression of small ZONAB-directed RNA duplexes to induce RNA interference using a recently described expression vector containing an RNA polymerase III promoter (Yu et al., 2002). Fig. 4 A shows that stable transfection of two different vectors targeting different regions of the ZONAB sequence resulted in efficient reduction of ZONAB expression, but a vector expressing a control RNA duplex with a random sequence did not affect ZONAB expression.


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)

Depletion of ZONAB by RNA interference inhibits G1/S-phase transition. (A) Wild-type MDCK cells or cells stably expressing RNA duplexes corresponding to region I (ZONAB-RD-I c1 and c2) or II (ZONAB-RD-II c8 and c9) of ZONAB, or control RNA duplexes (control-RD c1 and c3) were grown for 2 d and then lysed and processed for electrophoresis. The samples were immunoblotted with anti-ZONAB and anti–α-tubulin antibodies. (B) Wild-type and transfected MDCK cells expressing the indicated RNA duplexes were plated at low confluence and synchronized in low serum. Cell cycle entry was triggered by serum addition, and progression to S-phase was monitored by determining the fraction of cell incorporation of BrdU. Shown are means ± 1 SD of at least two independent clones per type of RNA duplex that were analyzed in two independent experiments. Both ZONAB-directed RNA duplexes significantly inhibited entry into S-phase (t test; P < 0.02).
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Related In: Results  -  Collection

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fig4: Depletion of ZONAB by RNA interference inhibits G1/S-phase transition. (A) Wild-type MDCK cells or cells stably expressing RNA duplexes corresponding to region I (ZONAB-RD-I c1 and c2) or II (ZONAB-RD-II c8 and c9) of ZONAB, or control RNA duplexes (control-RD c1 and c3) were grown for 2 d and then lysed and processed for electrophoresis. The samples were immunoblotted with anti-ZONAB and anti–α-tubulin antibodies. (B) Wild-type and transfected MDCK cells expressing the indicated RNA duplexes were plated at low confluence and synchronized in low serum. Cell cycle entry was triggered by serum addition, and progression to S-phase was monitored by determining the fraction of cell incorporation of BrdU. Shown are means ± 1 SD of at least two independent clones per type of RNA duplex that were analyzed in two independent experiments. Both ZONAB-directed RNA duplexes significantly inhibited entry into S-phase (t test; P < 0.02).
Mentions: Next, we wanted to test whether reduction of total cellular ZONAB levels also inhibits cell cycle entry. Because expression of long antisense RNAs as the one used in the experiment described in Fig. 1 can cause nonspecific cellular responses due to activation of antiviral defense mechanisms (Williams, 1999), we used an alternative approach to reduce ZONAB expression. This second approach was based on the expression of small ZONAB-directed RNA duplexes to induce RNA interference using a recently described expression vector containing an RNA polymerase III promoter (Yu et al., 2002). Fig. 4 A shows that stable transfection of two different vectors targeting different regions of the ZONAB sequence resulted in efficient reduction of ZONAB expression, but a vector expressing a control RNA duplex with a random sequence did not affect ZONAB expression.

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