The Regulation of Pulmonary ACE2 by Human Antigen R (HuR)

COVID-19 has rapidly spread through the world, and is caused by SARS-CoV-2. Patients with chronic obstructive pulmonary disease (COPD) face an increased risk for severe illness from COVID-19 because of angiotensin-converting enzyme 2 (ACE2) upregulation, an entry receptor for SARS-CoV-2. Our group recently revealed that COPD-derived lung fibroblasts express higher level of ACE2, and provide direct evidence that chronic cigarette smoke (CS) exposure significantly increases pulmonary ACE2 protein. CS is the primary risk factor for COPD. ACE2 expression may be regulated at the transcriptional, post-transcriptional and translational levels. Changes in protein expression can be controlled by RNA-binding proteins (RBPs). One of the best studied RBPs is human antigen R (HuR), a ubiquitously expressed RBP, that regulates the stability, localization and/or translation of target mRNAs. To stabilize target mRNA, HuR translocates from the nucleus to the cytoplasm. CS may increases HuR translocation to the cytoplasm, thereby indirectly augmenting cellular protein levels. Therefore, we hypothesized that HuR regulates ACE2 expression in response to CS, thereby accounting for higher ACE2 in COPD-derived cells. Aims: 1)- Evaluate HuR expression and localization in Normal, Smoker and COPD lung tissues;2) Establish that HuR induces ACE2 expression in primary human lung fibroblasts (HLF);3)- Determine if HuR controls ACE2 expression by regulating mRNA stability. Methods: 1)- Multiplex Immunohistochemistry was performed on lung tissue from Normal, Smoker and COPD subjects. The localization of HuR was assessed in HLF exposed to 2% cigarette smoke extract (CSE) for 4h by subcellular fractionation-western blot. 2)- Normal, Smoker and COPD HLF were transfected with either small interfering RNA (siRNA) against HuR or Control siRNA. ACE2 protein was evaluated by western blot. 3)- RNA-binding protein immunoprecipitation-qPCR was used to assess the association of HuR to ACE2 mRNA in Normal, Smoker and COPD HLF. The effect of HuR on the stability of ACE2 mRNA was assessed by using actinomycin D pulse-chase followed by qPCR. Results: 1)- HuR cytoplasmic localization is higher in Smoker and COPD lung tissues. There was an increase in cytoplasmic HuR in HLF exposed to 2%CSE for 4h. 2)- Knockdown of HuR slightly increases ACE2 protein. 3)- HuR associates with ACE2 mRNA in Normal and Smoker HLF. Significance: Our work is the first to highlight the association between ACE2 and HuR in COPD. The regulation of ACE2 by HuR could provide the basis to understand the upregulation of ACE2 expression in COPD HLF and the potential consequence towards COVID-19.