Molecular mechanisms of thalidomide effectiveness on COVID-19 patients explained: ACE2 is a new ΔNp63α target gene

Abstract

COVID-19 pandemic is caused by the SARS-CoV-2 virus, whose internalization and infection are mediated by the angiotensin-converting enzyme 2 (ACE2). The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2-associated cytokine storm, the transcription factor ΔNp63α and ACE2 are stabilized, and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the ACE2 gene. We previously showed that ΔNp63α is degraded upon thalidomide treatment and now found that treatment with this drug-or with its analogue lenalidomide-downregulates ACE2 in a p63-dependent manner. Finally, we found that thalidomide treatment reduces in vitro infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19. KEY MESSAGES: Thalidomide treatment results in p63-dependent ACE2 downregulation. ACE2 is a p63 transcriptional target. Thalidomide reduces the "cytokine storm" associated to COVID-19. Thalidomide prevents viral re-entry of SARS-CoV-2 by p63-dependent ACE2 downregulation. Thalidomide is a modulator of SARS-CoV-2 or other ACE2-dependent infections. ACE2 is modulated by a pharmacological substance.

Keywords: ACE2; COVID 19; SARS-CoV-2; Thalidomide; ΔNp63α.

Fig. 1

Thal and Len at clinically relevant concentrations reduce ACE2 expression. A HaCaT, A431, and A549 cells were treated for 24 h with Len (1 or 5 µM) or Thal (10 or 100 µM). Cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. B p63-null U-2 OS cells were treated for 24 h with increasing concentrations of Thal (10, 50, 100 µM) or Len (1, 5, 15, 50, 80, 100 µM). Cell extracts were analyzed by WB with anti-ACE2 Ab. Actin was used as the loading control, and one representative experiment is shown. C HaCaT, A431, A549, and H1299 cells were treated with Thal 100 µM or Len 5 µM for 8 h; DMSO was used as the control (NT). Lower panel: cell extracts were prepared and analyzed by WB with anti-p63 Ab; GAPDH was used as the loading control, and one representative experiment is shown. Upper panel: compared to the relative Ctrl, ACE2 mRNA levels decreased in p63-proficient HaCaT, and A431 and A459 cells were treated with Thal or Len but not in p63-deficient H1299 cells. ACE2 mRNA was evaluated by qRT-PCR, mRNA levels were normalized using GAPDH, and the means ± standard errors (SE) of three replicates are shown

Fig. 2

ΔNp63α and ACE2 parallel modulation at both protein and mRNA levels. A HaCaT and A431 cells were transiently transfected with 100 and 250 ng of CRBN encoding plasmid. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2, anti-p63, and anti-CRBN Abs. Actin was used as the loading control; one representative experiment is shown. Two bands of the CRBN protein are evident: °stays for endogenous CRBN, *for transfected CRBN that has higher molecular weight due to an HA tag. B p63-null U-2 OS cells were transiently transfected with 10 and 25 ng of ΔNp63α expression vector. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 or anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. C ACE2 mRNA levels were evaluated by qRT-PCR, and mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates are shown. D HaCaT and A431 cells were transiently transfected with 100 ng of small hairpin RNA (shRNA) plasmids targeting p63 mRNA; four different p63 shRNA vectors with sequence homology to four different regions of p63 mRNA were used (#1, #2, #3, #4); the cells were also transfected with a control (100 ng), shRNA-SCR, that encodes for an RNA that is not complementary to any mRNA sequence. After 24 h from transfection, cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs. Actin was used as the loading control; one representative experiment is shown. E A431 cells were stably transfected with both p63shRNA#3 and p63shRNA#4, and polyclonal populations were isolated by puromycin selection. As the control, cells were also transfected with a shRNA-SCRB vector. Cell extracts were prepared and analyzed by WB with anti-ACE2 and anti-p63 Abs, and parallel samples were used for ACE2 mRNA level evaluation by qRT-PCR; mRNA levels were normalized using GAPDH as housekeeping gene. Means ± SE of three replicates of three cultures are shown

Fig. 3

ACE2 is a new ΔNp63α target gene. A HaCaT cells were treated with 5 ng/mL of TNF-α or with 0.5 µg/mL of LPS for the indicated times. Cell extracts were prepared and analyzed by WB with an anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. The cell supernatants were collected, and IL-8 levels were quantified by the ELISA assay. B The schematic representation and sequence of the p53/p63RE were identified in the first intron of ACE2. C A431 cells were treated or not with LPS 0.5 μg/mL for 2 h. Cell extracts were prepared and analyzed by WB using anti-ACE2 and anti-p63 Abs. Actin was used as the loading control, and one representative experiment is shown. D, E A431 cells were treated with LPS for 2 h and then collected for chromatin immunoprecipitation (ChIP) analysis

Fig. 4

Thal impairs in vitro infection by GFP-expressing pseudo-SARS-CoV-2. A The schematic representation of the experiment is shown in B. B Immunofluorescent staining of A431 cells treated with Thal 100 µM; after 24-h treatment, 50 µL of pseudo-SARS-CoV-2 suspensions was added to the cells following the manufacturer’s instructions; the viral titer was 2 × 10¹⁰ viral genes (VG) per mL. After 24 h post-infection, cells were washed with PBS and fixed with 4% formaldehyde in 1X PBS for 10 min at room temperature. Nuclei were stained with DAPI. Images were acquired using a Nikon CSU-W1 microscope in widefield mode, using a × 20 objective and analyzed with FIJI [24], and representative images are shown. Scale bar 150 μm. C Quantification of GFP reduction in Thal-pretreated samples. D FACS analysis was performed 24 h after pseudo-SARS-CoV-2 infection of shp63#3/shp63#4 and shSCRB stable transfectants