Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Abstract

Background: Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. Objective: In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Design/Methods: Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Results: Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. Conclusion(s): These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

Inhibiting the Deubiquitinase UCHL1 Reduces SARS-CoV-2 Viral Uptake by ACE2

“…Here we investigated ACE2 ubiquitination as a strategy to identify potential upstream regulators of ACE2 protein abundance. Most cellular proteins undergo posttranslational modification with ubiquitin; ACE2 is no exception ( 7 , 15 ). More recently, studies have demonstrated ACE2 ubiquitination in SARS-CoV-2 infection and identified E3 ligases Skp2 ( 16 ) and UBR4 ( 17 ) as putative regulators.…”

“…In pulmonary hypertension, ACE2 is ubiquitinated by MDM2 to regulate its abundance, which may impact pulmonary hypertension pathogenesis ( 7 ). ACE2 ubiquitination is also modified by hyperoxia in the lung ( 15 ). The E3 ligase Skp2 has also been proposed to degrade ACE2 in the setting of cigarette smoke extract ( 16 ).…”

Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

“…For example, ACE2 shows the highest levels in the kidney, small intestine and adipose tissues, the medium levels in the lung, colon and liver tissues, and the lowest levels in the brain, bone marrow and blood (Li et al, 2020). Thus, we observed ACE2 protein levels in several cell lines from different tissues, including HEK293T (kidney), A549 (lung) and Caco-2 (colon), as well as a human fibroblast cell line (2fTGH) due to the reported expression of ACE2 in lung fibroblasts (Mohamed et al, 2021), since these cell lines have been reported to be functional for SARS-CoV-2 infection. We noticed that all these cell lines can more or less express ACE2 proteins (Figure 1A).…”

Vitamin C promotes ACE2 degradation and protects against SARS‐CoV‐2 infection

“…For example, ACE2 shows the highest levels in the kidney, small intestine, and adipose tissues; medium levels in the lung, colon, and liver; and the lowest levels in the brain, bone marrow, and blood (Li et al , 2020b). Thus, we measured ACE2 protein levels in several cell lines derived from different tissues, including HEK293T (kidney), A549 (lung), and Caco‐2 (colon) cells, as well as a human fibroblast cell line (2fTGH) due to the reported expression of ACE2 in lung fibroblasts (Mohamed et al , 2021), since these cell lines have been reported to be functional for SARS‐CoV‐2 infection. We found that all these cell lines expressed the ACE2 protein to varying degrees (Fig 1A).…”