Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the 2019 novel coronavirus (COVID-19) epidemic. Recent studies have highlighted the important role of the human angiotensin-converting enzyme 2 (ACE2) molecule in mediating the cellular entry of SARS-CoV-2. Binding to the ACE2 receptor is a critical initial step for SARS-CoV-2 to infect target cells and is critical for human infection, and the expression and distribution of the ACE2 receptor may be related to the progression and prognosis of COVID-19.

Telomeres are regions at the ends of linear chromosomes. Telomeres in vertebrates are composed of a large number of typical TTAGGG repeat sequences. The shelterin protein complex binds to telomeric DNA and protects telomeric DNA from being recognized as DNA damage (DD), thereby preventing DDR (DNA damage response). DNA replication causes the telomeres of chromosomes to gradually shorten. When telomeres become very short, they are sensed as DNA double-strand breaks (DSBs) and activate the DDR pathway. Continuous inbreeding of mice leads to progressive telomere shortening and DDR activation at telomeres, and the accumulation of characteristics of aging and age-related diseases. Furthermore, since damage at telomeres is not easily repaired, this results in persistent DDR activation. In this article, Professor Fabrizio d'Adda di Fagagna and his team from the Institute of Molecular Genetics of the National Research Council in Pavia, Italy, aimed to better describe the regulation of ACE2 expression during aging after telomere shortening and DDR activation. .

In order to understand the regulation of ACE2 expression during aging, Professor Fabrizio d'Adda di Fagagna and his team from Italy studied The expression of ACE2 in the lungs of mice and humans of different ages was studied. By RT-qPCR detection and immunohistochemistry (IHC), increased ACE2 mRNA and protein levels were observed in the lungs of old mice (22-24 months) compared with young mice (2-3 months). Similarly, it was also found in human lungs that the ACE2 protein expression level in elderly subjects was higher than that in young subjects. To confirm that ACE2 was increased in ATII cells, the team performed double-labeled immunoassays for ACE2 in lung samples of different ages. Fluorescence detection found that the expression of ACE2 in ATII lung cells increases with age, both in mice and humans. The team further investigated the differential expression of ACE2 transcripts and observed that ACE2 increased with age in ATII lung cells, while the housekeeping gene GAPDH was widely expressed in nearly all cell types and did not change across the ages analyzed. In summary, it can be proven that the expression of ACE2 increases when cells undergo senescence, and the increased expression of ACE2 is mainly in ATII lung cells.

Next, in order to reveal the molecular mechanism controlling ACE2 upregulation during aging, the team conducted in vitro and in vivo experiments. To test whether telomere shortening is sufficient to regulate ACE2 expression, the team measured ACE2 mRNA levels in human fibroblasts (BJ) and human bronchial epithelial cells (HBECs) in different populations. Because both cell types lack telomere maintenance mechanisms and undergo progressive telomere shortening as they proliferate. It was found that compared with early passage cells, the ACE2 mRNA levels of late passage BJ and HBEC increased. Next, the team expanded their research into a mouse model lacking telomerase RNA components. Analysis of the third generation of mice lacking telomerase RNA found that compared with age- and sex-matched wild-type animals, the team lacked telomerase RNA. ACE2 expression was consistently increased in mice treated with granzyme RNA, and immunofluorescence imaging showed that ACE2 was primarily increased in ACE2-expressing ATII lung cells. And the same phenomenon was also observed in pro-SP-C cells. These results clearly demonstrate a role for telomere shortening in regulating ACE2 levels in human cells and mouse tissues.

Because when telomeres become very short, they activate the DNA damage response (DDR) pathway. So next the team tested whether telomeric DDR was sufficient to increase ACE2 mRNA levels. The team used two mammalian cell systems that allow specific activation of DDR at telomeres in the absence of telomere shortening. After the team knocked out TRF2, they found increased levels of H2AX lesions to demonstrate activation of the DDR pathway. Experiments have shown that the increase in DDR lesions is accompanied by an increase in ACE2 mRNA levels, and the same results were also observed after ionizing radiation (IR) in the same cell line, proving that the DDR signaling pathway plays a role in increasing ACE2 mRNA levels. In order to verify the role of telomeric DDR in vivo, his team administered tamoxifen in mice to induce loss of TRF2 expression, activate the DDR pathway at telomeres, and observed an increase in ACE2 mRNA levels in the livers of mice. This also proves the above results. Taken together, these results in human and mouse cell lines and mouse tissues suggest that the activated DDR pathway has a conserved role in regulating ACE2 levels. Since telomere DDR accumulates during aging, this may lead to increased ACE2 levels.

The team believes that the increase in ACE2 mRNA levels is caused by enhanced activity of its transcriptional promoter. To determine whether the ACE2 promoter responds to DDR activation, the team performed in silico analysis to identify transcription factors whose DNA-binding motifs were significantly enriched in the ACE2 promoter region. The results showed that among these transcription factors, there are pathways related to DNA damage response. The team transfected HeLa shTRF2 cells with a plasmid carrying a luciferase reporter gene under the control of the human ACE2 promoter, induced telomeric DDR by knocking out TRF2, and found that the luciferase signal increased. Similar transcriptional activation was also observed upon DDR activation in irradiation-uninduced HeLa cells. This suggests that the DDR pathway increases ACE2 mRNA levels by controlling ACE2 promoter activity.

To further demonstrate that key components of the DDR pathway are responsible for the observed increase in ACE2 mRNA levels. The team used the ATM kinase inhibitor KU-60019 (ATMi) to prevent the formation of DDR lesions. A significantly reduced increase in ACE2 mRNA levels was found, suggesting that ATM kinase activity is involved in the regulation of ACE2 transcription levels. In order to study whether the inhibition of telomeric DDR affects the expression of ACE2 in vivo, the team used telomeric antisense oligonucleotides (tASOs) to achieve specific inhibition of telomeric DDR. When the mice were sacrificed and their tissues analyzed, both tASO treatments were found to be highly effective and ACE2 mRNA levels were downregulated in the body. Next, the team extended the study to the third generation of mice with shortened telomeres and found through double-label immunostaining that ACE2 expression was significantly less after treatment with tASOs or tASOs. These results support that ACE2 regulation results from subsequent DDR activation rather than telomere shortening.

Taken together, these results indicate that the expression of the SARS-CoV-2 receptor ACE2 is directly regulated by DDR at the transcriptional level Regulation of pathway activation, telomere dysfunction is a physiological event that can participate in the DDR pathway to regulate ACE2 levels.