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. 2024 Apr 15;16(8):1177.
doi: 10.3390/nu16081177.

Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin

M Teresa Cabezuelo  1   2 , Luis Torres  3 , Elena Ortiz-Zapater  3 , Gerardo López-Rodas  3 , M Pilar Marín  4 , Joaquín Timoneda  3 , Juan R Viña  3 , Rosa Zaragozá  5 , Teresa Barber  3
Affiliations

Vitamin A Status Modulates Epithelial Mesenchymal Transition in the Lung: The Role of Furin

M Teresa Cabezuelo et al. Nutrients. .

Abstract

Vitamin A deficiency (VAD) induced TGF-β hyperactivation and reduced expression of cell adhesion proteins in the lung, suggesting that the disruption of retinoic acid (RA) signaling leads to epithelial-mesenchymal transition (EMT). To elucidate the role of lung vitamin A status in EMT, several EMT markers and the expression of the proprotein convertase furin, which activates TGF-β, were analyzed in two experimental models. Our in vivo model included control rats, VAD rats, and both control rats and VAD rats, treated with RA. For the in vitro studies, human bronchoalveolar epithelial cells treated with RA were used. Our data show that EMT and furin are induced in VAD rats. Furthermore, furin expression continues to increase much more markedly after treatment of VAD rats with RA. In control rats and cell lines, an acute RA treatment induced a significant increase in furin expression, concomitant with changes in EMT markers. A ChIP assay demonstrated that RA directly regulates furin transcription. These results emphasize the importance of maintaining vitamin A levels within the physiological range since both levels below and above this range can cause adverse effects that, paradoxically, could be similar. The role of furin in EMT is discussed.

Keywords: E-cadherin; N-cadherin; epithelial–mesenchymal transition; extracellular matrix; furin; lung; pulmonary disease; retinoic acid; retinol; vitamin A deficiency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Vitamin A status and EMT markers in the lung. (A) RT-qPCR was performed to analyze the mRNA expression of EMT markers Cdh1, Cdh2, and β-catenin in control rats, vitamin A-deficient rats (VAD), and vitamin A-deficient rats treated with retinoic acid (RA). The data used in the statistical analyses and histograms are the mean ± SD (n = 4–5 for each group). (B) Protein levels of different EMT markers (E-Cadherin, N-Cadherin, and β-catenin) were studied by Western blot in lungs from control (C), vitamin A-deficient (VAD), and vitamin A-deficient rats treated with RA (VAD + RA). (C) Graphs showing the Western blot quantification. Data (n ≥ 3) were quantified, normalized with β-actin, and plotted as mean fold ± SD vs. control. (A,C) significant results are shown * p ≤ 0.05; ** p ≤ 0.01 *** p ≤ 0.001 or **** p ≤ 0.0001.
Figure 2
Figure 2
Vitamin A status and furin expression in the lung. (A) RT-qPCR showing mRNA levels of proconvertase furin in control rats, vitamin A-deficient rats, or vitamin A-deficient rats treated with retinoic acid (RA) (n = 4–5 per group). (B) Western blot analysis of furin in lungs from control (C), vitamin A-deficient (VAD), and vitamin A-deficient rats treated with RA (VAD + RA) (n = 3). GAPDH was used for normalization and the graph on the right represents quantification of the blots of three independent cultures. For both panels, histograms represent the mean ± SD, and results that were significantly different were * p ≤ 0.05; ** p ≤ 0.01.
Figure 3
Figure 3
Effect of RA addition in vitro in human lung cell lines. (A) Western blot analysis showed the expression of different EMT markers and furin in two cell lines (A549 and BEAs) after incubation with 5 μM RA at 24 h and 48 h. Hsc70 was used as a loading control. A representative image is shown (n = 3 independent cultures). (B) Localization of E-cadherin (green), detected by immunofluorescence staining in A549 and BEA cells after 5 μM RA treatment for 24 h. Zoom has been made around the dotted lines and magnified images are shown on the right side. (C) Representative IF analysis of furin (green) in lung cell lines (A549 and BEAs) after 24 h of RA incubation (5 μM). In both (B,C), nuclei were stained with DAPI and F-actin with phalloidin (cyan blue). Scale bars 20 μM and 60 μM for zoom images in (B).
Figure 4
Figure 4
Effect of RA addition in vivo on EMT markers. (A) Western blot analysis to study the levels of different EMT markers in vivo in control rats after RA treatment (+RA group). GAPDH was used as a loading control. (B) Quantification of the Western blots is shown in panel (A). Data (n ≥ 3 different animals) were quantified, normalized with GAPDH, and plotted as mean fold ± SD vs. control. (C) Western blot analysis of furin in control rats after RA treatment (+RA group) (n = 3); GAPDH was used for normalization and quantification (mean fold ± S.D. vs. control). For all quantifications, asterisks indicate significant differences: * p ≤ 0.05; ** p ≤ 0.01, or *** p ≤ 0.001.
Figure 5
Figure 5
RARα ChIP assay on furin promoter. (A) Potential binding sites for RAR and/or RXR nuclear receptors in the furine promoter region. CTF1, CTF2, and Enhancer (ENH) indicate 3 regions where most transcriptional factor binding sites that modulate gene transcription are concentrated. (B) ChIP data analysis of RARα binding to furin and MMP-9 genes in control rats (RA−) and control rats injected intraperitoneally with retinoic acid (RA+) (left panel, CONTROL group). The same analysis was carried out in vitamin A-deficient rats (RA−) and vitamin A-deficient rats injected intraperitoneally with retinoic acid (RA+) (Right panel, VAD). Chromatin was immunoprecipitated with an anti-RARα antibody (black bars) or a nonrelated antibody anti-IgG as a negative control (grey bars). Data are the result of three independent experiments and bars represent mean ± SD.
Figure 6
Figure 6
Vitamin A modulates Furin expression and EMT in the lung. Schematic representation of RA signaling via the RAR–RXR pathway when RA is added. This induces a transcriptional change within the nucleus, increasing furin expression that proteolyzes target proteins, triggering EMT. CRAPB: cellular retinoic acid binding protein.
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