Transcriptional regulation of SARS-CoV-2 receptor ACE2 by SP1
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Curated by eLife
eLife assessment
This is a valuable report that describes that ACE2 expression is upregulated by SARS-CoV-2 infection via activation of transcription factor Sp1 and inhibition of HNF4α through the PI3K/AKT pathway. Inhibition of Sp1 reduces SARS-CoV-2 infection in vitro and in an animal model. This work is solid and will be of interest to those interested in ACE2 biology and its impact in COVID-19.
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Abstract
Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation—a marker of its activity—and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters ( Mesocricetus auratus ) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.
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Author Response
Joint Public Review:
- For the in vitro work, only one cell line is used in this article: HPAEpiC cells, an immortalized human cell line derived from alveolar epithelial type II cells. This limits the generalizability of the results obtained in this study, as SARS-CoV-2 is known to infect several kinds of cells.
We appreciate the concerns of the reviewing editor. To test whether our findings were applicable to other cells, we performed similar experiments in human hepatoma cells (Huh-7) and renal tubular cells (HK-2), which are highly susceptible to SARS-CoV-2 (Yeung et al., 2021). We found that infection by SARS-CoV-2 upregulated the protein levels of ACE2, while colchicine treatment significantly inhibited the expression of ACE2 in HK-2 cells and Huh-7 cells (Revised Figure 3-figure supplement 2A-D). In addition, we …
Author Response
Joint Public Review:
- For the in vitro work, only one cell line is used in this article: HPAEpiC cells, an immortalized human cell line derived from alveolar epithelial type II cells. This limits the generalizability of the results obtained in this study, as SARS-CoV-2 is known to infect several kinds of cells.
We appreciate the concerns of the reviewing editor. To test whether our findings were applicable to other cells, we performed similar experiments in human hepatoma cells (Huh-7) and renal tubular cells (HK-2), which are highly susceptible to SARS-CoV-2 (Yeung et al., 2021). We found that infection by SARS-CoV-2 upregulated the protein levels of ACE2, while colchicine treatment significantly inhibited the expression of ACE2 in HK-2 cells and Huh-7 cells (Revised Figure 3-figure supplement 2A-D). In addition, we found that colchicine treatment also reduced the viral load of SARS-CoV-2 in HK-2 cells and Huh-7 cells (Revised Figure 3-figure supplement 2E and F).
- From the results of two separate experiments (colchicine leading to reduced ACE2-expression in HPAEpiC cells & colchicine leading to reduced SARS-CoV-2 replication in HPAEpiC cells), the authors infer that inhibition of ACE2 expression by colchicine suppresses SARS-CoV-2 infection. However, their experiments do not explicitly prove this hypothesis and do not give weight to the importance of this reduced ACE2 expression in the colchicine antiviral effect they observed, as other mechanisms may play a (bigger) role in producing this effect.
It has been well-established that the infection of SARS-CoV-2 and the Spike-RBD binding are dependent on ACE2 expression in different cell lines. ACE2 knockdown dramatically reduces SARS-CoV-2 infection in Caco2 cells (Shen et al., 2022), Spike-RBD binding, and SARS-CoV-2 replication in Calu-3 cells (Samelson et al., 2022). In contrast, overexpression of ACE2 greatly enhances SARS-CoV-2 virus infection in both A549 and H1299 cells (Chen et al., 2021). Meanwhile, two recent studies have demonstrated that androgen receptor positively regulates the expression of ACE2 at a transcriptional level (Qiao et al., 2021; Samuel et al., 2020). Importantly, inhibition of ACE2 expression by reducing the AR signaling attenuates SARS-CoV-2 infectivity (Qiao et al., 2021). A very recent study has demonstrated that ursodeoxycholic acid (UDCA), an inhibitor of the farnesoid X receptor (FXR), reduces ACE2 expression in human lung, intestinal, and liver organoids, thereby inhibiting SARS-CoV-2 infection (Brevini et al., 2022). These results clearly demonstrate that ACE2 expression levels determine the efficiency of SARS-CoV-2 infection to host cells.
- The authors refer to colchicine as a drug leading to mortality benefit when used as treatment for COVID-19 (line 101-105). However, whether colchicine is beneficial in COVID-19 is unclear. For instance, the randomized controlled trial by the RECOVERY Collaborative Group (Lancet Respir Med 2021), which included more than 11,000 patients, did not find benefit from colchicine in patients admitted to hospital with COVID-19. The authors refer to the review of Drosos et al to infer benefit of colchicine in COVID-19, however this review ignores the numerous trials contradicting this (as also stated in a letter from Finsterer in response to this review). The meta-analysis by Elshafei to which the authors refer was published before the largest RCT by the RECOVERY Group was published.
We agree with the assessment made by the reviewing editor. Our goal is to discover a new mechanism of regulating ACE2 expression. Using colchicine, we have- identified that SP1 is a crucial transcription factor that regulates ACE2 expression. In response to the reviewer’s comments, we added the sentences “This study has several limitations. Firstly, although SP1 was identified as a pivotal transcription factor in modulating ACE2 expression via the action of colchicine and MithA, neither of these compounds currently qualify as a candidate for the treatment of COVID-19.…Additionally, the efficacy of colchicine as a treatment for COVID-19 remains inconclusive. While some studies suggest benefits (Chiu et al., 2021; Drosos et al., 2022; Elshafei et al., 2021), others indicate negligible impact on mortality or disease progression (Group, 2021; Mikolajewska et al., 2021).” in Discussion of revised manuscript (Lines 329-342).
- The authors did not let a pathologist blinded to the infection/treatment state of the animals score the samples obtained in the animal experiments, which could have introduced bias in these results.
We appreciate the concerns of the reviewing editor. Actually, histological observations were made by one of authors, Dr. Li-Qiong Wang, who is a pathologist, blinded to group identity. In response to the reviewer’s suggestion, we have now added a sentence “Tissue sections were evaluated by a trained pathologist (L.-Q. W.) blinded to group identity” in the section of Material and Methods (Lines 516 and 517).
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eLife assessment
This is a valuable report that describes that ACE2 expression is upregulated by SARS-CoV-2 infection via activation of transcription factor Sp1 and inhibition of HNF4α through the PI3K/AKT pathway. Inhibition of Sp1 reduces SARS-CoV-2 infection in vitro and in an animal model. This work is solid and will be of interest to those interested in ACE2 biology and its impact in COVID-19.
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Joint Public Review:
The authors clearly state the current mystery surrounding transcriptional regulation of ACE2-expression, and how SARS-CoV-2 infection might impact this regulation. Several medications have been identified impacting the gene expression of ACE2, such as colchicine. However, the mechanism behind this regulation of ACE2 gene expression is currently unknown, yet worth investigating. Indeed, getting to know the mechanism behind the transcriptional regulation of ACE2 might lead to development of therapies targeting this expression in order to attenuate COVID-19 severity.
In order to achieve insight in the regulation of ACE2 expression by SARS-CoV-2, the authors used a luciferase reporter based assay to investigate a range of signaling pathways. The authors found that ACE2 expression is upregulated by SARS-CoV-2 infection …Joint Public Review:
The authors clearly state the current mystery surrounding transcriptional regulation of ACE2-expression, and how SARS-CoV-2 infection might impact this regulation. Several medications have been identified impacting the gene expression of ACE2, such as colchicine. However, the mechanism behind this regulation of ACE2 gene expression is currently unknown, yet worth investigating. Indeed, getting to know the mechanism behind the transcriptional regulation of ACE2 might lead to development of therapies targeting this expression in order to attenuate COVID-19 severity.
In order to achieve insight in the regulation of ACE2 expression by SARS-CoV-2, the authors used a luciferase reporter based assay to investigate a range of signaling pathways. The authors found that ACE2 expression is upregulated by SARS-CoV-2 infection via activation of transcription factor Sp1 and inhibition of HNF4α through the PI3K/AKT pathway. This led to the discovery that inhibition of Sp1 using mithramycin A reduces SARS-CoV-2 infection in vitro and in an animal model.Strengths
- The authors used an elegant design for their investigation. Based on a broad luciferase based assay, and keeping in mind the opposite effects of SARS-CoV-2 infection and colchicine administration on the expression of ACE2, they identified transcription factors as potential candidates for regulating ACE2 expression.
- Throughout the several experiments performed, the antagonizing effects of SARS-CoV-2 infection and colchicine on the identified transcription factors (Sp1 and HNF4α) are consistent and therefore strengthen the conclusions.Weaknesses
- For the in vitro work, only one cell line is used in this article: HPAEpiC cells, an immortalized human cell line derived from alveolar epithelial type II cells. This limits the generalizability of the results obtained in this study, as SARS-CoV-2 is known to infect several kinds of cells.
- From the results of two separate experiments (colchicine leading to reduced ACE2-expression in HPAEpiC cells & colchicine leading to reduced SARS-CoV-2 replication in HPAEpiC cells), the authors infer that inhibition of ACE2 expression by colchicine suppresses SARS-CoV-2 infection. However, their experiments do not explicitly prove this hypothesis and do not give weight to the importance of this reduced ACE2 expression in the colchicine antiviral effect they observed, as other mechanisms may play a (bigger) role in producing this effect.
- The authors refer to colchicine as a drug leading to mortality benefit when used as treatment for COVID-19 (line 101-105). However, whether colchicine is beneficial in COVID-19 is unclear. For instance, the randomized controlled trial by the RECOVERY Collaborative Group (Lancet Respir Med 2021), which included more than 11,000 patients, did not find benefit from colchicine in patients admitted to hospital with COVID-19. The authors refer to the review of Drosos et al to infer benefit of colchicine in COVID-19, however this review ignores the numerous trials contradicting this (as also stated in a letter from Finsterer in response to this review). The meta-analysis by Elshafei to which the authors refer was published before the largest RCT by the RECOVERY Group was published.
- The authors did not let a pathologist blinded to the infection/treatment state of the animals score the samples obtained in the animal experiments, which could have introduced bias in these results.These results add to the existing knowledge that the characteristics of ACE2 (its functionality and abundance) in the respiratory tract are pivotal to understand infection by SARS-CoV-2. The author conclusions are supported by the results. The identification of the two transcription factors influenced by SARS-CoV-2 infection is valuable, but needs further research to assess whether their effect on ACE2 expression is also seen in other cell types than the one assessed by the authors. More in-depth research will have to follow to assess if and how targeting the identified transcription factors could ultimately benefit patients with COVID-19.
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