Slug / Snail 2 is involved in the repression of proliferation 6 genes by TGF-in bronchial epithelial progenitor cells and is 7 deregulated in abnormal epithelium

Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors that are involved in development and diseases. Slug is also highly expressed in normal adult stem/progenitor cells of several epitheliums, and in such is unique among these transcription factors. By comparing primary bronchial basal cells from normal subjects to those from subjects with Chronic Obstructive Pulmonary Disease (COPD), a respiratory disease in which subjects present many anomalies of their bronchial epithelium and higher levels of Transforming Growth Factor (TGF)-{beta} in their lungs, in an air-liquid interface culture system that allows regenerating a bronchial epithelium similar to the one in vivo, we reveal that Slug has higher expression levels in basal/progenitor cells from COPD when in presence of TGF-{beta} but that it does not repress the epithelial marker E-cadherin either in normal or in COPD cells, even when treated with TGF-{beta}. To investigate Slug role in human primary bronchial basal/progenitor cells we performed loss of function experiments to determine Slug downstream genes and we characterized the impact of TGF-{beta} on these genes. We show that Slug downstream genes are different in normal and COPD subjects. In particular, we identified a set of proliferation-related genes whose expression is decreased by TGF-{beta} when cells are induced to differentiate, and that are among the genes repressed downstream of Slug in normal but not in COPD cells. In COPD the levels of expression of these genes are higher than in normal cells in presence of TGF-{beta}, and they positively correlate with the effect of TGF-{beta} on Slug. Our findings show that Slug is involved in the repression of proliferation genes by TGF-{beta} in normal basal/progenitor cells, but that in contrast, in subjects that present many anomalies in their bronchial epithelium this function of Slug is lost and Slug and proliferation genes are simultaneously but independently regulated by TGF-{beta}.


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We used COPD subjects to compare with normal subjects as they present many anomalies in their 89 bronchial epithelium that can be reproduced in the ALI cell culture system. Moreover, features of 90 EMT have been reported in COPD airways (9, 16) and Transforming Growth Factor (TGF)- is 91 found at higher levels in COPD lung tissues (17). We also wanted to characterize the effects of 92 TGF-β, since it has been shown to regulate Slug expression (18), to be, in certain conditions, an 93 EMT-inducing factor (19), and to play a role in stem/progenitor cell fate (20), we also wanted to 94 characterize its effects. We thus determines Slug expression and characterized its downstream 95 genes in bronchial epithelial progenitor cells, comparing cells from normal and COPD subjects at 96 the onset of differentiation in presence or absence of TGF-β.

98 Materials and methods
99 Study subjects and cell isolation 100 Human lung tissues, non-COPD (non-smokers (n=6) and smokers (n=6)) and COPD Image capture and analysis 229 Images of cell layers stained by immunofluorescence were captured using a SP8 Leica

250
We confirmed by immunocytochemistry that primary basal cells grown at confluence and 251 maintained undifferentiated are all progenitor cells as shown by the expression of the marker p63,

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and that they all express Slug that co-localizes with p63 in their nuclei (Fig 1A). Similar with this property. While Slug is highly expressed, the expression of Snail1 and Zeb1 is close to 256 background and Twist1 does not exceed 10% of Slug levels, and this in both normal and COPD 257 cells (Fig 1B). To determine the epithelial status of these progenitor cells, we studied the 258 expression of epithelial and mesenchymal EMT-related markers. We found that among the genes 259 coding for junction proteins, the epithelial marker E-cadherin (E-cad/CDH1) is expressed while 260 N-cadherin (N-cad/CDH2), an EMT-related mesenchymal marker, is not expressed (Fig 1C). We 261 also observed a high level of expression of the genes coding for the cytoskeletal proteins 262 cytokeratin 5 (KRT5) and vimentin (Vim), respectively an epithelial and a mesenchymal marker, as well as expression of ACTA2, the gene coding for the mesenchymal marker -smooth muscle 264 actin (a-sma). (Fig 1D). In conclusion, both COPD and normal epithelial progenitors co-express 265 epithelial and mesenchymal markers.

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GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH.

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Slug/Snail2 expression is increased in COPD cells when compared to 281 normal cells at the onset of differentiation in presence of TGF-. 282 We then determined the expression of Slug and the EMT-related markers in cells induced 283 to differentiate using the ALI cell culture system that allows basal/progenitor cells to differentiate 284 into a characteristic bronchial pseudostratified epithelium. The complete differentiation takes 3 to 285 4 weeks in this system. However, we were interested in the onset of the genetic differentiation 286 program and therefore studied the early timepoints. TGF-β can play a role in stem/progenitor cell 287 fate (20); it also regulates Slug expression, is a potential EMT-inducing factor and is expressed at higher levels in COPD airways compared to normal (17, 18). We thus compared the expression of 289 Slug and the EMT-related markers at the onset of differentiation in absence or presence of TGF-β 290 to determine if progenitor cells from normal and COPD responded differently to TGF-β. We used 291 low concentration of TGF-β (1ng/ml) to be in conditions similar to the physiological range.

304
Primary bronchial epithelial basal cells, normal and COPD, were grown on filters and at confluence changed to ALI 305 culture to induce differentiation, without TGF-β or in presence of 1ng/ml of TGF-β. Cells were analyzed at day 6 of 306 ALI culture for mRNA expression. RNA were extracted from normal and COPD cells and analyzed by RT-qPCR.

307
GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH.

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In response to TGF-β, the resulting levels of Slug mRNA are similar in normal and COPD 316 cells, and this is also the case for E-cad/CDH1 and Vim mRNA (Fig 3A). However, when we 317 determined the expression at the protein level by Reverse Phase Protein Array (RPPA), which 318 allows studying all the samples simultaneously, thus, reducing variability due to technical bias 319 (31), we found that TGF-β leads to significantly higher levels of Slug in COPD cells. E-cad 320 protein levels are also higher in COPD in presence of TGF-β, while Vim levels do not differ 321 significantly between normal and COPD cells (Fig 3B). Both in normal and COPD cells, and in 322 absence or presence of TGF-β, a strong positive correlation between Slug and E-cad protein 323 levels is observed (Fig 3C). These results indicate that Slug is deregulated in COPD 324 basal/progenitor cells at the onset of differentiation in presence of TGF-β. and COPD cells using shRNA from Mission RNAi that were validated and selected the most 351 efficient shRNA that was within the coding strand in order to limit non-specific downstream 352 genes (32). To determine the genes whose expression was modified by Slug knockdown 353 compared to a control siRNA, we performed a total RNA microarray analysis. We used an we also performed a microarray analysis on RNA from basal cells at the onset of differentiation,

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in presence or absence of TGF-β, using undifferentiated cells as control. S1 Table is  that 45 out of these 68 genes are involved in processes related to proliferation or cell cycle. Table   387 1 shows that all these 45 genes, except for 3, are much less or not repressed downstream of Slug

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in COPD compared to normal cells.

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We further studied the expression of 5 of these proliferation-related genes and first

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Knockdown with shRNA specific of Slug were performed on primary bronchial epithelial basal cells, normal and 412 COPD, Knockdown cells were grown on filters and, at confluence, changed to ALI culture to induce differentiation.

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Cells were analyzed at day 2 of ALI culture for mRNA expression. RNA were extracted from normal and COPD  429 We found a set of proliferation-related genes that have an increased expression following 430 Slug knockdown in normal but not in COPD cells; this suggests that these genes are directly or 431 indirectly repressed by Slug in normal cells but that this regulation does not take place in COPD.

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This is supported by the strong and statistically significant inverse correlation between Slug and 433 the mRNA levels of these genes that is observed in undifferentiated normal but not COPD cells 434 (Table 2). Moreover, in normal cells, in addition to Ki67 mRNA, a strong inverse correlation also 21 435 exists between Slug and Ki67 protein, a widely used marker strictly related to proliferation (35) 436 (Fig 5A). These correlations are found with Slug mRNA and not with the protein.

Table 2. Correlation between proliferation genes and Slug in normal and COPD
438 undifferentiated basal cells

439
Pearson correlation coefficients and associated P value between expression levels.

440
Computation was performed on log2 of values.

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Statistical significance was determined with a two-sided t-test. Correlations are significant at P value < show only a tendency in the same direction (Fig 5B). However, in cells at the onset of 447 differentiation in presence of TGF-β, significant lower levels are found in normal cells including 448 for Ki67 protein, with the exception of PCNA that shows only a weak not statistically significant 449 tendency (Fig 5C). TGF-β decreases the expression of these proliferation-related genes and its 450 mean effect is in accordance with the mean difference of their expression levels between normal 451 and COPD cells (Fig 5D).

452
To determine the implication of Slug in the repression of the proliferation-related gene 453 expression by TGF-β in normal cells, we analyzed the correlation between Slug and these genes.
454  Pearson correlation coefficients and associated P value between the effect of differentiation and TGF-.

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Statistical significance was determined with a two-sided t-test. Correlations are significant at P value < TGF-β (Fig 3B and Fig 5C) and, in contrast to normal cells, positive correlations, significant or 491 with a good tendency, are observed between the combined effects of differentiation and TGF-β 492 on Slug protein levels and that on the proliferation-related gene levels, with the exception of 493 PCNA. A strong positive correlation is also found between Slug and Ki67 protein ( Table 3 and 494 Fig 5F). To understand the link between the higher expression of Slug protein and of the the levels of total Smad3 and β-cat also only in COPD (Fig 6 A and B).

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Slug protein levels correlate positively with both total Smad3 and β-cat levels, but do not 504 correlate with the phosphorylation levels of Smad3 or β-cat (Fig 6C and D). In contrast, a 505 positive correlation, significant or as a strong tendency, is found between the activation of β-cat 24 506 (i.e. decrease of β-cat phosphorylation) and the levels of Ki67 protein and of the proliferation-507 related genes, with the exception of PCNA (Table 4, Fig 6E). These results show that, in COPD 508 cells, the higher levels of Slug protein and of the proliferation-related gene expression found in 509 presence of TGF-β, are induced by TGF-β through different downstream pathways.
510 Table 4. Correlation between proliferation genes and Smad3 or -cat in COPD basal/progenitor 511 cells in presence of TGF-.

512
Pearson correlation coefficients and associated P value between protein levels (phosphorylated/Total or 513 Total). Computation was performed on log2 of values.

514
Statistical significance was determined with a two-sided t-test. Correlations are significant at P value <

542
We also found a negative correlation between the effect of TGF- on their expression and that on  showing only a slight lower expression of E-cad in COPD. Also theses studies concern cells from 564 ex-vivo epithelium or fully differentiated epithelium in vitro ALI culture, and suggest that COPD 565 cells may be rather imprinted for EMT, and in such, have a higher potential to enter EMT in 566 permissive conditions (9, 17, 40).

567
In search of Slug function in basal/progenitor cells, we identified genes involved in 568 proliferation that are repressed downstream of Slug in normal but not COPD cells. Among the 569 genes studied, PCNA is apart: It is the gene with the least difference between normal and COPD 570 of Slug KD effect, and this is coherent with the fact that there is no significant difference of 571 expression between normal and COPD and no correlation with Slug for the effect of TGF-. In 572 addition to its role in DNA replication, PCNA has also a role in DNA repair, making it a poor 573 proliferation marker. In normal cells, Slug mRNA, but not protein, correlates with these 574 proliferation-related genes. We cannot exclude that, by an unknown mechanism, Slug mRNA is 575 involved in the repression of these genes; alternatively, Slug being regulated at the post- (33). We can also speculate that such deregulations of the progenitor cells could ultimately lead to