LAUSR

Angiotensin-converting enzyme 2 (ACE2) is the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV2 and is upregulated after infection with these viruses, which assists the entry of these viruses into the target cells. In addition, ACE2 is a human interferon-stimulated gene. In response to viral infection, host cells produce vast amounts of type I interferon (IFN) to defend against viral infection. Type I IFNs, such as IFN-α, can upregulate the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2). Thus, whether and how IFN-α could promote and whether blockade of the IFN-α-STAT pathway could inhibit the expression of ACE2 are largely unknown. Given the importance of ACE2 for SARS-CoV-2 invasion and the clinical practice of IFN treatment to combat viral infection, herein, we addressed these issues via a series of in vitro experiments. First, we assessed the expression of ACE2 in various human cell lines. Relatively high levels of ACE2 mRNA and protein expression were observed in HBE and HEK293 cells but low expression levels were observed in HeLa cells (Fig. S1a). Immunoblot analysis after IFN-α stimulation showed that the ACE2 protein level was increased in A549 and HBE cells (Fig. 1a) and in HCC-LM3, LoVo, and HEK293 cells (Fig. S1b); these findings were further confirmed by immunofluorescence staining (Figs. 1b, S1c). In addition, flow cytometric analysis showed an increase in membrane ACE2 protein expression at 12 h after IFN-α treatment in A549 and HBE cells (Fig. 1c). These data indicate that IFN-α can universally upregulate the protein expression of ACE2 in lung, kidney, liver, and intestinal cells. Next, we investigated the mechanisms by which IFN-α promotes ACE2 expression. IFN-α treatment significantly upregulated the expression of the Ace2 gene in A549 and HBE cells (Fig. 1d) and in HCC-LM3, HEK293, and LoVo cells (Fig. S1d). The increase in ACE2 protein expression in HBE cells 12 h after IFN-α treatment was abolished in the presence of the mRNA translation inhibitor cycloheximide (CHX) (Fig. 1e) but not in the presence of the proteasome inhibitor MG132 (Fig. S1e). These results suggest that the IFN-α-mediated increase in ACE2 protein expression occurs at the transcriptional level. As STAT1 is required for the transcriptional induction of IFN-α-responsive genes, we determined whether STAT1 is involved in regulating the IFN-αmediated increase in Ace2 transcription. As expected, Stat1 gene silencing or overexpression abolished or promoted, respectively, the IFN-α-induced increases in the levels of ACE2 gene and protein expression in both HBE cells (Fig. 1f–i; respectively) and HEK293 cells (Fig. S2a, b, c, d; respectively). Next, we separately cloned four sequential Ace2 promoters (each with 450 bp) into a luciferase reporter plasmid, and the resulting constructs were termed pGL3ACE2-1-Luc, pGL3-ACE2-2-Luc, pGL3-ACE2-3-Luc, and pGL3-ACE24-Luc. Overexpression of STAT1 significantly enhanced the luciferase activity of pGL3-ACE2-3-Luc (containing the Ace2 promoter region including nucleotides −1250 to −833) (Fig. 1j) but not the other three luciferase reporter plasmids (Fig. S2e). Further chromatin immunoprecipitation experiments showed that the Ace2 promoter region containing nucleotides −1232 to −1032 was enriched by precipitation with an anti-STAT1 antibody and enhanced by IFN-α stimulation (Fig. 1k); this region was within the scope of the in silico bioinformatic analysis (−1500 to −500 bp upstream of the transcription start site). These results indicate that IFN-α induces direct binding of STAT1 to the Ace2 promoter and subsequently enhances transcription of the Ace2 gene. Fludarabine is an inhibitor of STAT1 and a common chemotherapeutic drug used to treat chronic B lymphocytic leukemia in the clinic; we presumed that it could reduce ACE2 expression by inhibiting STAT1. At a low concentration (<1 μM), fludarabine did not influence the proliferation of these cells within 24 h (Fig. S3a), whereas it obviously decreased the IFN-α-induced upregulation of ACE2 gene and protein expression in HBE cells (Fig. 1l, m) and in A549, HEK293, and HCC-LM3 cells (Fig. S3b, c), accompanied by a notable reduction in both p-STAT1 and STAT1 protein levels. Furthermore, the levels of both intracellular and membrane ACE2 protein were reduced by fludarabine treatment in IFN-αstimulated HBE cells (Fig. 1n, o) and in A549 and HCC-LM3 cells (Fig. S3d, e). In contrast, these effects were abolished in HBE cells after STAT1 knockdown (Fig. 1p). These results demonstrate that fludarabine reduces IFN-α-induced ACE2 expression in a STAT1dependent manner. In conclusion, we provide direct in vitro evidence that type I interferon IFN-α promotes STAT1 expression and phosphorylation and that phosphorylated STAT1 upregulates the expression of the Ace2 gene and ACE2 protein by binding to the promoter region (positions −1232 to −1032) of Ace2, an effect that can be