Calu-3

ACE2 Expression Levels in Calu-3 Cells Grown in High D-Glucose Medium

Angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that negatively regulates the renin-angiotensin system, which can induce vasodilation by cleaving a single residue from the inactive decapeptide angiotensin I to generate angiotensin and degrade angiotensin II. The time dependency of ACE2 mRNA expression was investigated in Calu-3 cells; the cells were treated with normal D-glucose (NG) or high D-glucose (HG) for 24, 48, and 72 h. Real-time quantitative polymerase chain reaction (qPCR) was used to determine ACE2 mRNA expression levels. ACE2 mRNA expression levels in HG-treated cells were not significantly different from those in NG-treated cells at 24 h. However, ACE2 mRNA expression in HG-treated cells was significantly elevated at 48 and 72 h (p < 0.05, Fig. 2a). Further, dose-titration of D-glucose was performed in Calu-3 cells at 72 h. The cells were treated with different D-glucose concentrations [100 (NG), 325, 550, 1000 (HG), and 5000 mg/dL]. Interestingly, ACE2 mRNA expression was significantly enhanced with 550, 1000, and 5000 mg/dL D-glucose treatments, but not with NG treatment (p < 0.05). Additionally, lower D-glucose concentrations did not enhance ACE2 mRNA expression (Fig. 2b). To determine ACE2 protein expression levels after NG or HG treatment, western blotting of cell lysates was performed after 24, 48, and 72 h of treatment. Protein expression levels in HG-treated cells were elevated compared with those in NG-treated cells at 24, 48, and 72 h (Fig. 3a and b).

Fig. 2 Real-time quantitative polymerase chain reaction analysis of angiotensin-converting enzyme 2 (ACE2) mRNA expression. (Wakabayashi Y, et al., 2022)

Fig. 3 Western blot analysis of angiotensin-converting enzyme 2 (ACE2) expression in Calu-3 cells. (Wakabayashi Y, et al., 2022)

SARS-CoV-2 Infection Induces MUC1-C Expression in Calu-3 Cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lineage B betacoronavirus that is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. Many COVID-19 patients have difficulty breathing partially due to excessive mucus formation. As MUC1 is a major constituent of the mucus layer in the respiratory system, the impact of SARS-CoV-2 infection on MUC1 expression was investigated in Calu-3 cells using an antibody that recognizes the intracellular C-terminal region of MUC1. As controls, Calu-3 cells were cultured without infection (mock infection). MUC1-C expression was slightly elevated during culture in mock-infected cells and was markedly induced by SARS-CoV-2 (Fig. 4A). Specifically, SARS-CoV-2 infection promptly induced MUC1-C expression at 12 h. Moreover, SARS-CoV-2-induced MUC1-C expression continued to rise to 24 h and was maintained at peak levels until 72 h. As β-catenin is one of the known downstream targets activated by MUC1, expression levels of β-catenin. However, no differences were observed between SARS-CoV-2 and mock infections. In addition to its role in the cell membrane, MUC1-C can function as an oncogene by translocating to the nucleus to activate transcription. To determine if MUC1-C was imported to the nucleus after SARS-CoV-2 infection, Calu-3 cells were treated with Leptomycin B to inhibit nuclear export. A small proportion of MUC1-C was present in nuclei after SARS-CoV-2 infection (Fig. 4B). In contrast, no MUC1-C signal was observed in the nuclei of uninfected control cells. These results suggest that MUC1-C signaling activity is substantially altered in Calu-3 cells after SARS-CoV-2 infection.

SARS-CoV-2 infection induces STAT3 phosphorylation in Calu-3 cells, and others have demonstrated that STAT3 influences MUC1 expression in lung cancer cells. Therefore, the effect of STAT3 inhibitors, including AG490, JAK inhibitor I, and S3I-201, on MUC1-C expression. In mock-infected Calu-3 cells, basal expression of MUC1-C was reduced by treatment with JAK inhibitor I but not by treatment with AG490 or S3I-201. Importantly, JAK inhibitor I also markedly reduced MUC1-C expression in SARS-CoV-2-infected Calu-3 cells (Fig. 4C). These results support that increased MUC1 expression after SARS-CoV-2 infection is dependent on JAK1/3 activation and subsequent STAT3 activation.

Fig. 4 MUC1-C expression in SARS-CoV-2-infected Calu-3 cells. (Kim D, et al., 2021)