Thalidomide and Lenalidomide at clinically relevant concentrations reduce ACE2 expression. We first investigated whether Thal or Len could modulate the expression of ACE2 receptor, as a consequence of ΔNp63α degradation in HaCaT human keratinocytes and A431 human epidermoid cells, which express both ΔNp63α and ACE2 proteins. Cells were treated for 24 hrs with 10 or 100 µM Thal or 1 or 5 µM Len. As expected (17), ΔNp63α protein was degraded by the pharmacological treatments, with the ACE2 levels decreasing in parallel with the reduction of ΔNp63α levels in both cell lines (Fig. 1A). We then verified whether Thal or Len could modulate ACE2 expression in p63-null cells in a dose-dependent manner, since it has been reported that Len at high concentrations induces ACE2 downmodulation by a posttranslational mechanism (16). At the highest concentrations of Thal, we did not observe ACE2 downmodulation in the p63-null U-2 OS human osteosarcoma cells. In contrast, the treatment with Len at the high concentrations reported to modulate ACE2 expression (16) (i.e., 80–100 µM) resulted to be toxic, as it can be inferred by the concomitant reduction of the actin levels (Fig. 1B). Next, to investigate the modulation of ACE2 by ΔNp63α at the transcriptional level in response to Thal or Len treatment, we treated for 24 hrs with 100 µM Thal or 5 µM Len the following cells: A431, HaCaT and A549 cells, all expressing ΔNp63α and ACE2, and H1299 cells, not expressing any of the p63 isoforms. In ΔNp63α proficient cells, ΔNp63α protein was degraded by both treatments with a concomitant decrease of ACE2 mRNA levels suggesting that ACE2 might be a p63 transcriptional target, whereas, we did not observe ACE2 mRNA decrease in the p63-null cells (Fig. 1C).
Taken together, these results suggest that ACE2 could be a ΔNp63α target gene with Thal or Len leading to CRBN-mediated ΔNp63α degradation (CRBN is part of the E3 ubiquitin ligase complex that targets ΔNp63α for degradation upon Thal treatment (17, 25) that in turn would give rise to reduced ACE2 transcription.
Thalidomide reduce ACE2 expression through CRBN-mediated ΔNp63α degradation. To verify this hypothesis, we transiently transfected the p63-null U-2 OS cells with a ΔNp63α encoding plasmid and observed a positive correlation between expression levels of ACE2 and ΔNp63α (Fig. 2A). Moreover, evaluation of ACE2 mRNA by qRT-PCR in parallel samples showed increased ACE2 mRNA levels in the samples transfected with the ΔNp63α plasmid, thus indicating that ACE2 might be a ΔNp63α transcriptional target (Fig. 2B). This point was further reinforced by p63 silencing in HaCaT and A431 cells transfected with small hairpin RNA (shRNA) plasmids targeting the p63 mRNA. For this type of experiment, we used four different p63 shRNA vectors (OriGene) with sequence homology to four different regions of the p63 mRNA, with the sh-4 vector resulting in the strongest effect on ΔNp63α silencing and a concomitant decrease of ACE2 protein levels, in both cell lines (Fig. 2C and 2D). Finally, we reduced ΔNp63α protein levels by transfecting HaCaT and A431 cells with the CRBN encoding plasmid. As expected (17), CRBN overexpression led to a dose-dependent ΔNp63α degradation in both cell lines (Fig. 2E) that was paralleled, also in this case, by ACE2 downmodulation, supporting the idea that ACE2 levels are directly correlated with ΔNp63α levels.
ACE2 is a new ΔNp63α target gene. One of the main problems with SARS-CoV-2 infection is the triggering of a “cytokine storm” (26) a hyper-inflammatory state characterized by production of extremely high levels of proinflammatory cytokines that eventually leads to patient death. In order to mimic the hyper-inflammatory state in vitro, HaCaT cells were treated with LPS or the proinflammatory cytokine TNF-α, both known to stabilize ΔNp63α protein levels and to stimulate cytokine production, possibly by stabilized ΔNp63α acting on the promoter of several cytokine genes (27–29). Upon TNF-α and LPS treatments, both ΔNp63α and ACE2 expression levels were induced in dose-dependent and time-dependent manner (Fig. 3A, lower panel). The levels of IL-8, known to be overproduced in COVID-19 patients and expressed in HaCaT cells (30–31) were also increased by our treatments (Fig. 3A, upper panel), likely by stabilized ΔNp63α.
We then verified whether p53-family Responsive Elements (RE) were present in the regulatory regions of the ACE2 gene. For this purpose, we queried the p53Fam-Tag database (32) and identified one strong putative p53/p63-RE composed by three decamers in the first intron of the ACE2 gene (Fig. 3B). To evaluate the in vivo recruitment of ΔNp63α on the identified p53/p63-RE, a Chromatin ImmunoPrecipitation assay (ChIP) was performed. Cross-linked chromatin from A431 cells treated with LPS 0.5 µg/ml for 2 hrs was immunoprecipitated with anti-acetylated H4-histone or anti-p63α Abs. In the presence of LPS (i.e., 2 hrs treatment), but not in the untreated control cells, ΔNp63α was consistently recruited on the p53/p63-RE of the ACE2 gene (Fig. 3D). The increased p63 occupancy was accompanied by an increase in histone H4 acetylation (Fig. 3D) and, consistently, in ACE2 protein levels (Fig. 3C). As negative control, the exon 8 of the ACE2 gene, not containing any p53/p63-RE, was not amplified in the same samples (Fig. 3E). Taken together, these results clearly indicate that ACE2 is a new target gene of ΔNp63α.
Thalidomide weakens in vitro infection by pseudo-SARS-CoV-2. It has been reported that COVID-19 patients treated with Thal had a faster recovery in respect to untreated patients (15). From the data obtained, we hypothesized that the observed protection might be due to diminished viral re-entry due to ACE2 downmodulation upon Thal treatment as a consequence of ΔNp63α degradation. To verify this hypothesis, we pretreated A431 cells with 100 µM Thal for 24 hrs before adding, for additional 24 hrs, the pseudo-SARS-CoV-2, a GFP-expressing baculovirus pseudotyped with the SARS-CoV-2 spike protein (Fig. 4A). We found that Thal pretreatment impairs pseudoviral infection in vitro, as evidenced by reduced GFP signals in the Thal pretreated samples compared with the controls (Fig. 4B and 4C).
Altogether, these data offer a mechanistic explanation of the protective effect from severe COVID-19 observed in patients treated with Thal (15).