AUTHOR=Yang Yung-Yu , Lin Chao-Ju , Wang Cheng-Chin , Chen Chieh-Min , Kao Wen-Jen , Chen Yi-Hui 

TITLE=Consecutive Hypoxia Decreases Expression of NOTCH3, HEY1, CC10, and FOXJ1 via NKX2-1 Downregulation and Intermittent Hypoxia-Reoxygenation Increases Expression of BMP4, NOTCH1, MKI67, OCT4, and MUC5AC via HIF1A Upregulation in Human Bronchial Epithelial Cells

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2020.572276

DOI=10.3389/fcell.2020.572276

ISSN=2296-634X

ABSTRACT=<p>Previous studies have shown that the experimental models of hypoxia-reoxygenation (H/R) mimics the physiological conditions of ischemia-reperfusion and induce oxidative stress and injury in various types of organs, tissues, and cells, both <italic>in vivo</italic> and <italic>in vitro</italic>, including human lung adenocarcinoma epithelial cells. Nonetheless, it had not been reported whether H/R affected proliferation, apoptosis, and expression of stem/progenitor cell markers in the bronchial epithelial cells. In this study, we investigated differential effects of consecutive hypoxia and intermittent 24/24-h cycles of H/R on human bronchial epithelial (HBE) cells derived from the same-race and age-matched healthy subjects (i.e., NHBE) and subjects with chronic obstructive pulmonary disease (COPD) (i.e., DHBE). To analyze gene/protein expression during differentiation, both the NHBE and DHBE cells at the 2nd passage were cultured at the air-liquid interface (ALI) in the differentiation medium under normoxia for 3 days, followed by either culturing under hypoxia (1% O<sub>2</sub>) for consecutively 9 days and then returning to normoxia for another 9 days, or culturing under 24/24-h cycles of H/R (i.e., 24 h of 1% O<sub>2</sub> followed by 24 h of 21% O<sub>2</sub>, repetitively) for 18 days in total, so that all differentiating HBE cells were exposed to hypoxia for a total of 9 days. In both the normal and diseased HBE cells, intermittent H/R significantly increased <italic>HIF1A</italic>, <italic>BMP4</italic>, <italic>NOTCH1</italic>, <italic>MKI67</italic>, <italic>OCT4</italic>, and <italic>MUC5AC</italic> expression, while consecutive hypoxia significantly decreased <italic>NKX2-1</italic>, <italic>NOTCH3</italic>, <italic>HEY1</italic>, <italic>CC10</italic>, and <italic>FOXJ1</italic> expression. Inhibition of <italic>HIF1A</italic> or <italic>NKX2-1</italic> expression by siRNA transfection respectively decreased <italic>BMP4</italic>/<italic>NOTCH1</italic>/<italic>MKI67</italic>/<italic>OCT4</italic>/<italic>MUC5AC</italic> and <italic>NOTCH3</italic>/<italic>HEY1/CC10/FOXJ1</italic> expression in the HBE cells cultured under intermittent H/R to the same levels under normoxia. Overexpression of <italic>NKX2-1</italic> via cDNA transfection caused more than 2.8-fold increases in <italic>NOTCH3</italic>, <italic>HEY1</italic>, and <italic>FOXJ1</italic> mRNA levels in the HBE cells cultured under consecutive hypoxia compared to the levels under normoxia. Taken together, our results show for the first time that consecutive hypoxia decreased expression of the co-regulated gene module <italic>NOTCH3/HEY1/CC10</italic> and the ciliogenesis-inducing transcription factor gene <italic>FOXJ1</italic> via <italic>NKX2-1</italic> mRNA downregulation, while intermittent H/R increased expression of the co-regulated gene module <italic>BMP4/NOTCH1/MKI67/OCT4</italic> and the predominant airway mucin gene <italic>MUC5AC</italic> via <italic>HIF1A</italic> mRNA upregulation.</p>