TMPRSS2 Expression and Activity Modulation by Sex-Related Hormones in Lung Calu-3 Cells: Impact on Gender-Specific SARS-CoV-2 Infection

Abstract

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although males and females are at equivalent risk of infection, males are more prone to develop a higher severity disease, regardless of age. The factors that mediate susceptibility to SARS-CoV-2 and transmission are still under investigation. A potential role has been attributed to differences in the immune systems response to viral antigens between males and females as well as to different regulatory actions played by sex-related hormones on the two crucial molecular effectors for SARS-CoV-2 infection, TMPRSS2 and ACE2. While few and controversial data about TMPRSS2 transcript regulation in lung cells are emerging, no data on protein expression and activity of TMPRSS2 have been reported. Aim of the present study was to search for possible modulatory actions played by sex-related hormones on TMPRSS2 and ACE2 expression in Calu-3 cells, to test the effects of sex-steroids on the expression of the 32kDa C-term fragment derived from autocatalitic cleavage of TMPRSS2 and its impact on priming of transiently transfected spike protein. Cells were stimulated with different concentrations of methyltrienolone (R1881) or estradiol for 30 h. No difference in mRNA and protein expression levels of full length TMPRSS2 was observed. However, the 32 kDa cleaved serine protease domain was increased after 100 nM R1881 (+2.36 ± 1.13 fold-increase vs control untreated cells, p < 0.05) and 10 nM estradiol (+1.90 ± 0.64, fold-increase vs control untreated cells, p < 0.05) treatment. Both R1881 and estradiol significantly increased the activating proteolytic cleavage of SARS-CoV-2 Spike (S) transfected in Calu-3 cells (+1.76 ± 0.18 and +1.99±,0.76 increase in S cleavage products at R1881 100nM and 10 nM estradiol treatment, respectively, p < 0.001 and p < 0.05 vs control untreated cells, respectively). Finally, no significant differences in ACE2 expression were observed between hormones-stimulated cells and untreated control cells. Altogether, these data suggest that both male and female sex-related hormones are able to induce a proteolityc activation of TMPRSS2, thus promoting viral infection, in agreement with the observation that males and females are equally infected by SARS-CoV-2.

Apalutamide Prevents SARS-CoV-2 Infection in Lung Epithelial Cells and in Human Nasal Epithelial Cells

“…However, a strong reduction in cleaved-TMPRSS2 quantity was observed during apalutamide treatment, in a dose-dependent manner ( Figure 6 b). A recent study raised the possibility that androgen signaling may play a role in modulating TMPRSS2 catalytic cleavage, which is necessary for its protease activity [ 30 ]. We therefore aimed to confirm the effect of Apalutamide treatment on the mechanism of TMPRSS2 proteolytic activation in HNECs and lung cells.…”

“…AR and TMPRSS2 proteins have been detected in lung cells, airway cells and lung tissues [ 17 , 19 , 21 , 22 , 30 , 33 ]. In our study, we have first evaluated the expression of these two genes at the mRNA and protein levels in two different lung cell lines: A549 cells used to analyze androgen regulation in lung cells [ 33 ] and Calu-3 cells, described as a good model for SARS-CoV-2 infection via the TMPRSS2-dependent pathway [ 11 , 34 ].…”

“…In our study, we have first evaluated the expression of these two genes at the mRNA and protein levels in two different lung cell lines: A549 cells used to analyze androgen regulation in lung cells [ 33 ] and Calu-3 cells, described as a good model for SARS-CoV-2 infection via the TMPRSS2-dependent pathway [ 11 , 34 ]. Although AR expression has been reported in both A549 [ 19 , 33 , 35 ] and Calu-3 cell lines [ 30 ], two separate studies were unable to detect AR in Calu-3 cells [ 17 , 22 ]. Our results showed lower but still detectable expressions of both AR and TMPRSS2 at the mRNA and protein levels in lung cells compared to PCa cells.…”

“…Thus, Calu-3 cells, which express AR and TMPRSS-2 and thereby mimic natural infection, were used for further experiments. TMPRSS2 is known to be regulated by androgens in PCa cells [ 15 , 16 ], but controversial observations have been reported in lung and airway cells [ 17 , 19 , 20 , 22 , 30 , 33 ]. Indeed, the diversity of lung cells and models that have been used adds complexity to such analyses.…”

“…While Calu-3 cells cultured under androgen-deprived conditions with FBS-charcoal stripped serum for one week exhibited a reduction in TMPRSS2 expression, no modification of TMPRSS2 expression was observed with DHT stimulation [ 30 ]. In our study, we have shown an increase in TMPRSS2 expression after DHT stimulation in Calu-3 cells.…”