Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection

Abstract

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.

Keywords: ACE2; Akt; COVID-19; SARS-CoV2; triciribine.

Figure 1

Treatment with TCBN suppresses ACE2 expression in A549 cells. (a)–(c) Western blot images and bar graphs of band densitometry analysis of MK2206 (MK) and TCBN‐treated A549 cell lysates, indicating changes in the expressions of ACE2, phosphorylated (Ser‐473) Akt, and total Akt normalized to β‐tubulin expression, respectively. (d) and (e) Bar graphs showing changes in the mRNA expression of ACE2 and TMPRSS2 normalized to the β‐actin expression on MK2206 and TCBN‐treated A549 cells compared to the vehicle‐treated (PBS) control. Data are shown as mean ± SEM. ACE2, angiotensin‐converting enzyme 2; mRNA, messenger RNA; PBS, phosphate‐buffered saline; SEM, standard error of mean; TCBN, triciribine

Figure 2

TCBN suppresses HMGB1‐induced ACE2 expression in A549 cells. (a) and (b) Western blot images and bar graphs of band densitometry analysis of ACE2 expression induced by various doses of HMGB1 treatment in A549 cells. (c)–(e) Western blot images and bar graphs of band densitometry analysis of 10 nM HMGB1‐induced ACE2 expression and Akt phosphorylation, and the effect of TCBN in A549 cells, respectively. Data are shown as mean ± SEM. ACE2, angiotensin‐converting enzyme 2; SEM, standard error of mean; TCBN, triciribine

Figure 3

TCBN suppresses hyperglycemia‐induced ACE2 expression in A549 cells. Western blot images (a) and bar graph of band densitometry analysis (b) of hyperglycemia (50 mM or 100 mM glucose) induced expression changes in ACE2 and phosphorylated Akt expression in A549 cells (c) in the presence and absence of TCBN, respectively. Data are shown as mean ± SEM. ACE2, angiotensin‐converting enzyme 2; HG, hyperglycemia; SEM, standard error of mean; TCBN, triciribine

Figure 4

Akt inhibition blunts hyperglycemia‐induced increase in ACE2 expression in H441 cells. (a) and (b) Western blot images and bar graphs of band densitometry analysis of hyperglycemia (HG) (50 mM or 100 mM glucose) induced expression changes in ACE2 in H441 cells. (c)–(e) Western blot images and bar graphs of band densitometry analysis of 50 mM glucose‐induced changes in ACE2 phosphorylated Akt expression in H441 cells, respectively. Data are shown as mean ± SEM. ACE2, angiotensin‐converting enzyme 2; SEM, standard error of mean; TCBN, triciribine

Figure 5

Schematic representation of the potential effects of HMGB1, hyperglycemia, and TCBN on ACE2 expression and SARS‐CoV2 binding to lung epithelial cells. (a) HMGB1 or high glucose‐induced AGE (advanced glycation end‐products) promotes the expression of ACE2 receptors, thereby increasing the risk of SARS‐CoV‐2 interaction with the lung epithelial cell surface ACE2 leading to their internalization and infection. (b) Whereas Akt inhibitor MK‐2206 does not affect the expression of ACE2 receptors, treatment with TCBN blunts HMGB1, and hyperglycemia‐induced ACE2 expression in lung epithelial cells potentially in an Akt‐independent but adenosine‐receptor dependent mechanism. ACE2, angiotensin‐converting enzyme 2; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; TCBN, triciribine