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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 ZO-1. (A and B) Expression of ZO-1 in wild-type and transfected MDCK cells. Wild-type MDCK cells (A) or wild-type (wt MDCK) and ZO-1–overexpressing (ZO-1 1/21 and ZO-1 2/3) cells (B) were grown for the indicated number of days as described in Fig. 1 A. Cells were then harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZO-1 expression by immunoblotting. Note that ZO-1 was overexpressed in transfected proliferating cells to a similar extent as it was up-regulated in mature monolayers. (C) Incorporation of [3H]thymidine by low density MDCK cells stably transfected with ZO-1 or HA-tagged ZO-1 with (HA-ZO-1) or without (HA-ZO-1ΔSH3) the SH3 domain, or constructs containing specified domains. ZO-1/ZONAB indicates data obtained from double transfected cells overexpressing both proteins. 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 all cell lines expressing constructs containing the SH3 domain of ZO-1 exhibited significantly reduced [3H]thymidine incorporation (t test; P < 0.05). (D) Domain structure of ZO-1. PDZ, PSD95-DlgA-ZO-1 homology domain; SH3, src homology domain 3; GUK, guanylate kinase homology domain.
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fig2: Regulation of proliferation by ZO-1. (A and B) Expression of ZO-1 in wild-type and transfected MDCK cells. Wild-type MDCK cells (A) or wild-type (wt MDCK) and ZO-1–overexpressing (ZO-1 1/21 and ZO-1 2/3) cells (B) were grown for the indicated number of days as described in Fig. 1 A. Cells were then harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZO-1 expression by immunoblotting. Note that ZO-1 was overexpressed in transfected proliferating cells to a similar extent as it was up-regulated in mature monolayers. (C) Incorporation of [3H]thymidine by low density MDCK cells stably transfected with ZO-1 or HA-tagged ZO-1 with (HA-ZO-1) or without (HA-ZO-1ΔSH3) the SH3 domain, or constructs containing specified domains. ZO-1/ZONAB indicates data obtained from double transfected cells overexpressing both proteins. 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 all cell lines expressing constructs containing the SH3 domain of ZO-1 exhibited significantly reduced [3H]thymidine incorporation (t test; P < 0.05). (D) Domain structure of ZO-1. PDZ, PSD95-DlgA-ZO-1 homology domain; SH3, src homology domain 3; GUK, guanylate kinase homology domain.

Mentions: ZONAB localizes to the nucleus as well as tight junctions in proliferating cells, but is not detectable in the nucleus of nonproliferating high density cells (Balda and Matter, 2000), suggesting that accumulation of ZONAB in the nucleus may be required for efficient proliferation. ZO-1 levels are low in proliferating cells and increase with cell density, and overexpression of ZO-1 inhibits the nuclear accumulation of ZONAB (Balda and Matter, 2000); hence, ZO-1 may regulate proliferation by preventing ZONAB from accumulating in the nucleus. To test whether high levels of ZO-1 expression reduce proliferation, we used cell lines in which ZO-1 was three- to fivefold overexpressed in low confluent cells; an increase that is comparable to the up-regulation of the protein in wild-type cells once they reach high cell densities (Fig. 2, A and B). Next, we assessed proliferation by measuring [3H]thymidine incorporation in low density cultures. Fig. 2 C shows that exogenous expression of ZO-1 resulted in a fourfold reduction of [3H]thymidine incorporation, indicating that high levels of ZO-1 expression indeed reduced proliferation.


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 ZO-1. (A and B) Expression of ZO-1 in wild-type and transfected MDCK cells. Wild-type MDCK cells (A) or wild-type (wt MDCK) and ZO-1–overexpressing (ZO-1 1/21 and ZO-1 2/3) cells (B) were grown for the indicated number of days as described in Fig. 1 A. Cells were then harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZO-1 expression by immunoblotting. Note that ZO-1 was overexpressed in transfected proliferating cells to a similar extent as it was up-regulated in mature monolayers. (C) Incorporation of [3H]thymidine by low density MDCK cells stably transfected with ZO-1 or HA-tagged ZO-1 with (HA-ZO-1) or without (HA-ZO-1ΔSH3) the SH3 domain, or constructs containing specified domains. ZO-1/ZONAB indicates data obtained from double transfected cells overexpressing both proteins. 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 all cell lines expressing constructs containing the SH3 domain of ZO-1 exhibited significantly reduced [3H]thymidine incorporation (t test; P < 0.05). (D) Domain structure of ZO-1. PDZ, PSD95-DlgA-ZO-1 homology domain; SH3, src homology domain 3; GUK, guanylate kinase homology domain.
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fig2: Regulation of proliferation by ZO-1. (A and B) Expression of ZO-1 in wild-type and transfected MDCK cells. Wild-type MDCK cells (A) or wild-type (wt MDCK) and ZO-1–overexpressing (ZO-1 1/21 and ZO-1 2/3) cells (B) were grown for the indicated number of days as described in Fig. 1 A. Cells were then harvested, and equal amounts of protein were loaded on SDS-PAGE gels for analysis of ZO-1 expression by immunoblotting. Note that ZO-1 was overexpressed in transfected proliferating cells to a similar extent as it was up-regulated in mature monolayers. (C) Incorporation of [3H]thymidine by low density MDCK cells stably transfected with ZO-1 or HA-tagged ZO-1 with (HA-ZO-1) or without (HA-ZO-1ΔSH3) the SH3 domain, or constructs containing specified domains. ZO-1/ZONAB indicates data obtained from double transfected cells overexpressing both proteins. 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 all cell lines expressing constructs containing the SH3 domain of ZO-1 exhibited significantly reduced [3H]thymidine incorporation (t test; P < 0.05). (D) Domain structure of ZO-1. PDZ, PSD95-DlgA-ZO-1 homology domain; SH3, src homology domain 3; GUK, guanylate kinase homology domain.
Mentions: ZONAB localizes to the nucleus as well as tight junctions in proliferating cells, but is not detectable in the nucleus of nonproliferating high density cells (Balda and Matter, 2000), suggesting that accumulation of ZONAB in the nucleus may be required for efficient proliferation. ZO-1 levels are low in proliferating cells and increase with cell density, and overexpression of ZO-1 inhibits the nuclear accumulation of ZONAB (Balda and Matter, 2000); hence, ZO-1 may regulate proliferation by preventing ZONAB from accumulating in the nucleus. To test whether high levels of ZO-1 expression reduce proliferation, we used cell lines in which ZO-1 was three- to fivefold overexpressed in low confluent cells; an increase that is comparable to the up-regulation of the protein in wild-type cells once they reach high cell densities (Fig. 2, A and B). Next, we assessed proliferation by measuring [3H]thymidine incorporation in low density cultures. Fig. 2 C shows that exogenous expression of ZO-1 resulted in a fourfold reduction of [3H]thymidine incorporation, indicating that high levels of ZO-1 expression indeed reduced proliferation.

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