The POZ-ZF Transcription Factor Kaiso (ZBTB33) Induces Inflammation and Progenitor Cell Differentiation in the Murine Intestine

<div><p>Since its discovery, several studies have implicated the POZ-ZF protein Kaiso in both developmental and tumorigenic processes. However, most of the information regarding Kaiso’s function to date has been gleaned from studies in <i>Xenopus laevis</i> embryos and mammalian cultured cells. To examine Kaiso’s role in a relevant, mammalian organ-specific context, we generated and characterized a Kaiso transgenic mouse expressing a murine Kaiso transgene under the control of the intestine-specific <i>villin</i> promoter. Kaiso transgenic mice were viable and fertile but pathological examination of the small intestine revealed distinct morphological changes. Kaiso transgenics (<i>Kaiso^Tg/+</i>) exhibited a crypt expansion phenotype that was accompanied by increased differentiation of epithelial progenitor cells into secretory cell lineages; this was evidenced by increased cell populations expressing Goblet, Paneth and enteroendocrine markers. Paradoxically however, enhanced differentiation in <i>Kaiso^Tg/+</i> was accompanied by reduced proliferation, a phenotype reminiscent of Notch inhibition. Indeed, expression of the Notch signalling target HES-1 was decreased in <i>Kaiso^Tg/+</i> animals. Finally, our Kaiso transgenics exhibited several hallmarks of inflammation, including increased neutrophil infiltration and activation, villi fusion and crypt hyperplasia. Interestingly, the Kaiso binding partner and emerging anti-inflammatory mediator p120^ctn is recruited to the nucleus in <i>Kaiso^Tg/+</i> mice intestinal cells suggesting that Kaiso may elicit inflammation by antagonizing p120^ctn function.</p></div

Generation of transgenic mouse lines ectopically expressing <i>villin</i>-Kaiso.

<p>(<b>A</b>) Myc-tagged murine <i>Kaiso</i> cDNA was cloned downstream of the 9 kb v<i>illin</i> promoter sequence. (<b>B</b>) The transgene copy number in each transgenic line was evaluated via PCR. Line A transgenic animals have the greatest copy number. (<b>C</b>) RT-PCR confirmed expression of the Kaiso transgene in <i>villin</i>-expressing tissues of transgenic mice, <i>i.e.</i> the small intestine, large intestine, and kidneys. (<b>D</b>) Immunoblot analysis shows increased Kaiso expression in both small and large intestines in Kaiso transgenic (<i>Kaiso^Tg</i>^/+) Line A mice compared to non-transgenic (Non-Tg) siblings.</p

<i>Kaiso^Tg</i>^/+ mice display decreased HES-1 expression in the small intestine.

<p>Both Non-Tg and <i>Kaiso^Tg</i>^/+ tissues displayed nuclear HES-1 expression in the crypts of the small intestine, however <i>Kaiso^Tg</i>^/+ tissue displays significantly decreased HES-1 expression in the villi. Quantitative RT-PCR showed a significant decrease in HES-1 expression in <i>Kaiso^Tg</i>^/+ mice. Values were first normalized to the GAPDH housekeeping gene, followed by normalizing to non-Tg HES-1 expression (** represents p<0.05).</p

Kaiso transgenic mice exhibit inflammation of the intestinal mucosa.

<p>(<b>A</b>) <b><u>H</u></b>ematoxylin and <b><u>e</u></b>osin (H&E) stained sections were used to measure villi length (red bracket; ∼80 villi/mouse) and crypt depth (black bracket; ∼800 open crypts/mouse). <i>Kaiso^Tg</i>^/+ display increased crypt depth compared to their Non-Tg siblings, p = 0.001. (<b>B</b>) <i>Kaiso^Tg</i>^/+ mice exhibit increased immune cell infiltration of the lamina propria (yellow demarcated area) accompanied by increased MPO activity compared to their Non-Tg siblings, p = 0.014. (<b>C</b>) Line B mice do not exhibit immune cell infiltration or enhanced MPO activity compared to Non-Tg siblings. ** represents significance.</p

Subcellular localization and expression of ectopic Kaiso in Line A <i>Kaiso^Tg</i>^/+ small intestines.

<p><i>Kaiso^Tg</i>^/+ mice display strong nuclear Kaiso in the villi and crypt cells, compared to non-transgenic mice (Non-Tg), which mainly display weak Kaiso staining in the cytoplasm. Additionally, <i>Kaiso^Tg/+</i> mice display strong nuclear c-Myc staining corresponding to ectopic myc-tagged Kaiso expression, while Non-Tg mice display cytoplasmic c-Myc expression.</p

Secretory cell lineages are expanded in the intestines of <i>Kaiso^Tg</i>^/+ mice.

<p>(<b>A</b>) PAS stain for Goblet cells (black arrowheads) revealed increased numbers of Goblet cells in both the villi and crypts of <i>Kaiso^Tg</i>^/+ intestines, p = 0.011 & 0.002. (<b>B</b>) Lysozyme staining revealed increased Paneth cell numbers in <i>Kaiso^Tg</i>^/+ mice, p = 0.017. (<b>C</b>) Synaptophysin positive enteroendocrine cells (arrowheads) are increased in <i>Kaiso^Tg</i>^/+ mice, p = 0.031. n = 3 mice/genotype; measurements performed by two independent blind observers; T-test used for p-value. ** represents significance.</p

CD157 overexpression protects OV-90 cells from anoikis and enhances motility.

<p>(A) sqRT-PCR (left) and western blot analysis (right) of CD157 in OV-90/CD157 and OV-90/mock cells. GAPDH and β-actin were used as internal controls, respectively. (B) Morphology of colonies formed by OV-90/mock and OV-90/CD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 µM. (C) sqRT-PCR analysis of E-cadherin and N-cadherin in OV-90/mock and OV-90/CD157 cells. Densitometry quantifies the levels of mRNA expression of the indicated molecules relative to GAPDH. (D) Effect of CD157 overexpression on anoikis. After 48, 72. 96 and 192 h of anchorage-independent growth, cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Anoikis in OV-90/mock and OV-90/CD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean ± SEM of three independent experiments. *P<0.05; **P<0.01, two-tailed t test. (E) Anchorage-independent growth of OV-90/CD157 and mock cells was analyzed by soft agar colony formation assay. Graph represents average number of colonies formed from three independent experiments ± SEM after 3 weeks incubation of cells in soft agar. ***P<0.001, two-tailed t test. (F) Effect of CD157 expression on cell migration in a scratch-wound assay in OV-90/CD157 and mock cells. Cells were grown as monolayers, wounded, and photographed at time 0 and at 24 hr. Wound edges are indicated by black dashed lines (scale bar: 200 µM). (G) The ability of cells to close the wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 hr. Results represent the percentage reduction of the average wound width and are expressed as the mean ± SEM of three independent experiments. *P<0.05; two-tailed t test.</p

Morphological and functional modifications induced by CD157 knockdown in OV-90 cells.

<p>(A) sqRT-PCR (left) and western blot analysis (right) showing OV-90 cells retrovirally transduced with a shRNA that targets the human CD157 mRNA, resulting in efficient knockdown of CD157 expression. GAPDH and β-actin were used as internal controls, respectively. (B) Morphology of colonies formed by OV-90/scramble and OV-90/shCD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 µM. (C) sqRT-PCR for E-cadherin, N-cadherin and Snail, Twist1 and Slug transcriptional repressors in OV-90/scramble and OV-90/shCD157 cells. Densitometry quantifies the levels of mRNA expression of the indicated molecules relative to GAPDH. (D) Anoikis assay. After 48, 72, 96 and 192 h under anchorage-independent growth, cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Data analysis was performed with ModFit LT™ cell cycle analysis software. Anoikis in OV-90/scramble and OV-90/shCD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean ± SEM of three independent experiments. *P<0.05; **P<0.01, two-tailed t test. (E) Anchorage-independent growth of OV-90/scramble and OV-90/shCD157 cells was analyzed by soft agar colony formation assay. Graph represents the average number of colonies/field formed from three independent experiments ± SEM after 3 weeks incubation of cells in soft agar. *P<0.05, two-tailed t test. (F) Effect of CD157 knockdown on OV-90 cell migration in a scratch-wound assay. Cells were grown as monolayers, wounded, and photographed at time 0 and at 24 h (scale bar: 200 µM). Wound edges are indicated by black dashed lines. (G) The ability of OV-90/scramble and OV-90/shCD157 cells to close the wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 h. Results represent the percentage reduction of the average wound width and are expressed as the mean ± SEM of three independent experiments. **P<0.01, two-tailed t test.</p

High CD157 expression influences spheroid formation and disaggregation in OV-90 cells.

<p>(A) Representative morphology of spheroids formed by OV-90/mock (top) and OV-90/CD157 (bottom) (scale bar: 100 µM). (B) Phase contrast microscopy images of spheroid disaggregation on fibronectin. A representative spheroid of OV-90/mock and OV-90/CD157 cells at time 0 and at 12 h are shown (scale bar: 200 µM). (C) Spheroids were photographed at time 0 and at 12 h, and the increase in the size of disaggregation area calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043649#s2" target="_blank">Materials and Methods</a> is reported. The means ± SD of the fold change in area of 20 spheroids per condition of a representative experiment, repeated (n = 3), are shown. ***P<0.001, two-tailed t test.</p

Gene ontology analysis of genes modulated by CD157 overexpression.

<p>Go analysis of differentially induced or repressed genes shared by OVCAR-3/CD157 and OV-90/CD157 cells with respect to enrichment of genes with assignments to specific biological processes. The number of genes in a specific biological process is indicated in brackets.</p