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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 CDK4 accumulation in the nucleus. (A) Nuclear fractions isolated from low density wild-type and ZO-1–overexpressing MDCK cells were immunoblotted for the indicated proteins. Equal amounts of protein were loaded for all homogenates and for all nuclear fractions. The weak upper band in the CDK4 blot is a background band that was only detected by early batches of the commercial anti-CDK4 antibody, but not by later ones (see panel B for comparison). (B) Wild-type cells, clones transfected with cDNAs for either ZO-1 or the HA-tagged SH3 (HASH3), or cells expressing antisense ZONAB RNA (ZONABas) were grown, franctionated, and analyzed as in the experiment shown in A. To increase the sensitivity and accuracy of quantification, higher amounts of nuclear protein were loaded in B as compared with A to obtain bands with a similar intensity. (C) Quantification of nuclear accumulation. Immunoblots such as those shown in B were quantified by densitometric scanning, and the ratio of nuclear versus total signal was calculated. For each experiment, the ratio obtained from wild-type cells was set to 1 and the other cell lines were normalized to this value. Shown are averages ± 1 SD from at least three independent experiments. The values obtained for ZONAB, CDK4, and cyclin D1 in ZO-1, HA-SH3, and ZONABas cells were significantly different from those of wild-type cells with a confidence level of P < 0.02. (D) Reduced cell density in cells expressing a CDK4 inhibitor. Wild-type and transfected MDCK cells were grown as in Fig. 5 C, and were then harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. Two independent clones expressing VSV-tagged p16-INK4a were analyzed in two independent experiments performed in triplicate. For comparison, the values of ZONAB-overexpressing or depleted clones from Fig. 5 are also shown. Note that expression of p16-INK4a significantly reduced the final cell density (t test; P < 0.05).
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fig8: Regulation of CDK4 accumulation in the nucleus. (A) Nuclear fractions isolated from low density wild-type and ZO-1–overexpressing MDCK cells were immunoblotted for the indicated proteins. Equal amounts of protein were loaded for all homogenates and for all nuclear fractions. The weak upper band in the CDK4 blot is a background band that was only detected by early batches of the commercial anti-CDK4 antibody, but not by later ones (see panel B for comparison). (B) Wild-type cells, clones transfected with cDNAs for either ZO-1 or the HA-tagged SH3 (HASH3), or cells expressing antisense ZONAB RNA (ZONABas) were grown, franctionated, and analyzed as in the experiment shown in A. To increase the sensitivity and accuracy of quantification, higher amounts of nuclear protein were loaded in B as compared with A to obtain bands with a similar intensity. (C) Quantification of nuclear accumulation. Immunoblots such as those shown in B were quantified by densitometric scanning, and the ratio of nuclear versus total signal was calculated. For each experiment, the ratio obtained from wild-type cells was set to 1 and the other cell lines were normalized to this value. Shown are averages ± 1 SD from at least three independent experiments. The values obtained for ZONAB, CDK4, and cyclin D1 in ZO-1, HA-SH3, and ZONABas cells were significantly different from those of wild-type cells with a confidence level of P < 0.02. (D) Reduced cell density in cells expressing a CDK4 inhibitor. Wild-type and transfected MDCK cells were grown as in Fig. 5 C, and were then harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. Two independent clones expressing VSV-tagged p16-INK4a were analyzed in two independent experiments performed in triplicate. For comparison, the values of ZONAB-overexpressing or depleted clones from Fig. 5 are also shown. Note that expression of p16-INK4a significantly reduced the final cell density (t test; P < 0.05).

Mentions: Overexpression of ZO-1 in low density cells causes a redistribution of ZONAB from the nucleus to the cytoplasm (Balda and Matter, 2000) and, as shown in Fig. 2, reduced proliferation. Because ZONAB binds CDK4, the nuclear pools of the two proteins may decrease in parallel. To test this, we isolated nuclear fractions from proliferating wild-type and ZO-1–transfected cells and measured the amount of nuclear ZONAB and CDK4. Fig. 8 A shows that the nuclear pools of CDK4 were strongly reduced by overexpression of ZO-1, as were ZONAB levels. Quantification of the immunoblots revealed that the nuclear pools of both proteins were reduced by >60% compared with wild-type cells. This effect was specific for CDK4 because the nuclear accumulation of CDK6, a kinase that does not coprecipitate with ZONAB, was not affected. This indicates that ZO-1 expression levels regulate the subcellular distribution of CDK4. This is also supported by the observation that in high density MDCK cells, which express high levels of ZO-1, CDK4 is primarily cytoplasmic (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 CDK4 accumulation in the nucleus. (A) Nuclear fractions isolated from low density wild-type and ZO-1–overexpressing MDCK cells were immunoblotted for the indicated proteins. Equal amounts of protein were loaded for all homogenates and for all nuclear fractions. The weak upper band in the CDK4 blot is a background band that was only detected by early batches of the commercial anti-CDK4 antibody, but not by later ones (see panel B for comparison). (B) Wild-type cells, clones transfected with cDNAs for either ZO-1 or the HA-tagged SH3 (HASH3), or cells expressing antisense ZONAB RNA (ZONABas) were grown, franctionated, and analyzed as in the experiment shown in A. To increase the sensitivity and accuracy of quantification, higher amounts of nuclear protein were loaded in B as compared with A to obtain bands with a similar intensity. (C) Quantification of nuclear accumulation. Immunoblots such as those shown in B were quantified by densitometric scanning, and the ratio of nuclear versus total signal was calculated. For each experiment, the ratio obtained from wild-type cells was set to 1 and the other cell lines were normalized to this value. Shown are averages ± 1 SD from at least three independent experiments. The values obtained for ZONAB, CDK4, and cyclin D1 in ZO-1, HA-SH3, and ZONABas cells were significantly different from those of wild-type cells with a confidence level of P < 0.02. (D) Reduced cell density in cells expressing a CDK4 inhibitor. Wild-type and transfected MDCK cells were grown as in Fig. 5 C, and were then harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. Two independent clones expressing VSV-tagged p16-INK4a were analyzed in two independent experiments performed in triplicate. For comparison, the values of ZONAB-overexpressing or depleted clones from Fig. 5 are also shown. Note that expression of p16-INK4a significantly reduced the final cell density (t test; P < 0.05).
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fig8: Regulation of CDK4 accumulation in the nucleus. (A) Nuclear fractions isolated from low density wild-type and ZO-1–overexpressing MDCK cells were immunoblotted for the indicated proteins. Equal amounts of protein were loaded for all homogenates and for all nuclear fractions. The weak upper band in the CDK4 blot is a background band that was only detected by early batches of the commercial anti-CDK4 antibody, but not by later ones (see panel B for comparison). (B) Wild-type cells, clones transfected with cDNAs for either ZO-1 or the HA-tagged SH3 (HASH3), or cells expressing antisense ZONAB RNA (ZONABas) were grown, franctionated, and analyzed as in the experiment shown in A. To increase the sensitivity and accuracy of quantification, higher amounts of nuclear protein were loaded in B as compared with A to obtain bands with a similar intensity. (C) Quantification of nuclear accumulation. Immunoblots such as those shown in B were quantified by densitometric scanning, and the ratio of nuclear versus total signal was calculated. For each experiment, the ratio obtained from wild-type cells was set to 1 and the other cell lines were normalized to this value. Shown are averages ± 1 SD from at least three independent experiments. The values obtained for ZONAB, CDK4, and cyclin D1 in ZO-1, HA-SH3, and ZONABas cells were significantly different from those of wild-type cells with a confidence level of P < 0.02. (D) Reduced cell density in cells expressing a CDK4 inhibitor. Wild-type and transfected MDCK cells were grown as in Fig. 5 C, and were then harvested and counted. The cell number per cm2 of culture was calculated and expressed as percent changes relative to wild-type MDCK cells. Two independent clones expressing VSV-tagged p16-INK4a were analyzed in two independent experiments performed in triplicate. For comparison, the values of ZONAB-overexpressing or depleted clones from Fig. 5 are also shown. Note that expression of p16-INK4a significantly reduced the final cell density (t test; P < 0.05).
Mentions: Overexpression of ZO-1 in low density cells causes a redistribution of ZONAB from the nucleus to the cytoplasm (Balda and Matter, 2000) and, as shown in Fig. 2, reduced proliferation. Because ZONAB binds CDK4, the nuclear pools of the two proteins may decrease in parallel. To test this, we isolated nuclear fractions from proliferating wild-type and ZO-1–transfected cells and measured the amount of nuclear ZONAB and CDK4. Fig. 8 A shows that the nuclear pools of CDK4 were strongly reduced by overexpression of ZO-1, as were ZONAB levels. Quantification of the immunoblots revealed that the nuclear pools of both proteins were reduced by >60% compared with wild-type cells. This effect was specific for CDK4 because the nuclear accumulation of CDK6, a kinase that does not coprecipitate with ZONAB, was not affected. This indicates that ZO-1 expression levels regulate the subcellular distribution of CDK4. This is also supported by the observation that in high density MDCK cells, which express high levels of ZO-1, CDK4 is primarily cytoplasmic (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