The impact of COVID-19 on populations living at high altitude: Role of hypoxia-inducible factors (HIFs) signaling pathway in SARS-CoV-2 infection and replication

Abstract

Cases of coronavirus disease 2019 (COVID-19) have been reported worldwide. However, one epidemiological report has claimed a lower incidence of the disease in people living at high altitude (>2,500 m), proposing the hypothesis that adaptation to hypoxia may prove to be advantageous with respect to SARS-CoV-2 infection. This publication was initially greeted with skepticism, because social, genetic, or environmental parametric variables could underlie a difference in susceptibility to the virus for people living in chronic hypobaric hypoxia atmospheres. Moreover, in some patients positive for SARS-CoV-2, early post-infection 'happy hypoxia" requires immediate ventilation, since it is associated with poor clinical outcome. If, however, we accept to consider the hypothesis according to which the adaptation to hypoxia may prove to be advantageous with respect to SARS-CoV-2 infection, identification of the molecular rational behind it is needed. Among several possibilities, HIF-1 regulation appears to be a molecular hub from which different signaling pathways linking hypoxia and COVID-19 are controlled. Interestingly, HIF-1α was reported to inhibit the infection of lung cells by SARS-CoV-2 by reducing ACE2 viral receptor expression. Moreover, an association of the rs11549465 variant of HIF-1α with COVID-19 susceptibility was recently discovered. Here, we review the evidence for a link between HIF-1α, ACE2 and AT1R expression, and the incidence/severity of COVID-19. We highlight the central role played by the HIF-1α signaling pathway in the pathophysiology of COVID-19.

Keywords: COVID-19; Nrf2; TRPA1; curcumin; hypoxia; hypoxia-inducible factors.

FIGURE 1

Structure of hypoxia-inducible factor, HIF-1α. (A) Schematic representation of the HIF-1α protein (left panel). The numbers in black indicate the first and last amino acid residues of each domain of the molecule. From the NH2-terminal to the COOH-terminal region of the protein, the main domains are: i) a N-terminal region with the basic domain (aa 17-30), a basic helix-loop-helix domain (bHLH; aa 31-71) composed of two amphipatic α-helices connected by a loop, and a nuclear localisation signal (NLS; aa 17-74); ii) a Per-AHR-ARNT-Sim homology domains (PAS including the PAS-A aa 85-158, and PAS-B aa 228-298) involved in heterodimerisation with HIF-1β; iii) an oxygen-dependent degradation domain (ODD; aa 401-603) which contains two PEST-like motifs (aa 499-518 and 581-600) rich in proline (P), glutamic acid (E), serine (S) and threonine (T) (not shown); iv) a transactivation domains (TADs also termed NAD aa 531-575 and CAD aa 786-826) with the conserved residues 792-796 making contacts with the surface zinc-binding TAZ1 domain of CBP; and v) a C-terminal NLS (aa 718-721). Prolines P-402 and P-564 within the ODD and asparagine N-803 within CAD are requires for the full activation of HIF-1α. Under normoxic conditions the N-803 is hydoxylated by the FIH-1 hydroxylase and P-402 and P-564 within the ODD are hydroxylated by the prolyl hydroxylase PHD and the hydroxyproline residues become buried within the hydrophobic core of pVHL leading to ubiquitinylation of HIF-1α. The HIF-1α protein stability is increased by acetylation on lysine K-709 as well as by proline P-582 substitution (P582S) and alanine A-588 substitution (A588T). Acetylation of lysine K-532 promotes interaction with and ubiquitination by pVHL. (B) Amino acid sequence of HIF-1α from Homo sapiens (NCBI reference sequence NP8001521.1). The amino acids P-402, P-564 and N-803 are highlighted.

FIGURE 2

Schematic diagram illustrating the dual regulation of HIF. At high O2 concentration, HIF-1α is hydroxylated on proline by the prolyl hydroxylase PHD. The ubiquitin ligase VHL targets HIV-1α-OH for polyubiquitinylation and proteosomal degradation. Similarly, hydroxylation of TRP by PHD and asparaginyl hydroxylase FIH targets TRP-OH for ubiquitinylation and proteosomal degradation contributing to homeostasis. Under hypoxia, HIF-1α translocates to the cell nucleus where it forms heterodimers with the HIF-β subunit and binds to the HRE element (the HRE core binding site sequence is 5'-RCGTG-3' with R being a purine) in the promoter of hypoxia-inducible genes and recruits histone acetyltransferases CREB Binding Protein (CBP)/p300. Hypoxia up-regulates ACE1 which contributes to Ang II production through the RAS pathway. Ang II can contribute to hypoxia through binding to AT1R which initiates signaling events including activation of PKC and c-Src that is required for superoxide production by NADPH oxidases (NOX1 and NOX2). NOX2 also stimulates the production of reactive oxygen species (ROS) by mitochondria. HIF-1α nuclear translocation also activates the transient receptor potential ankyrin 1 (TRPA1) gene expression which leads to increase in intracellular Ca2+ and cell injury. In parallel (not shown), Ang II triggers an increase in cytoplasmic Ca2+ that induces NOX5 to generate H2O2. The Kelch-like erythroid cell-derived protein (KEAP1) is a negative regulator of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Under hypoxia, KEAP1 is oxidated by ROS. This leads to the nuclear translocation of phosphorylated Nrf2 which maintains oxidative homeostasis in regulating PHD thereby promoting HIF-1α proteasomal degradation. The Nrf2/MAF (MAF BZIP transcription factor) heterodimers also induce transcription of anti-oxidant-response genes through binding to the antioxidant response element (ARE sequence: 5'-TGACNNNGC-3') and reduce both the concentration of ROS and pro-inflammatory cytokines. In addition, chronic hypoxia triggers HIF-dependent down regulation of the ACE2 gene and activation of ADAM17 which leads to cleavage of the ACE2 protein (not shown).

FIGURE 3

Schematic representation of the functioning of the renin-angiotensin system under normal conditions and during SARS-CoV-2 infection. The left panel illustrates the fact that ACE2 converts Ang II into Ang(1-7). Ang-(1-7) exhibits vasodilatory, anti-proliferative, and anti-inflammatory effects via the G protein-coupled receptor called Mas-1. The right panel illustrates the possible dysfunction of signals when SARS-CoV-2 is attached to its ACE2 receptor. Under this condition Ang(1-7) is no longer synthetised, Ang II accumulates and binds AT1R, leading to HIF-1α induction.

FIGURE 4

Genes upregulated by HIF “master regulator”. The consensus DNA sequence for HIF-1α/HIF-1β binding is common for many genes upregulated during hypoxia. Representative list of genes upregulated by HIF, however this list is not exhaustive and grows continuously. It is worth noting that several genes involved in the renin-angiotensin system, in the development and functioning of the vascular system (which modulate vascular tone or promote angiogenesis) and in erythropoiesis, belong to this list. HIF-1α is also known to upregulate TRP-A1 and p35srj. The p35srj protein is an alternatively spliced isoform of MRG1 which inhibits HIF-1 transactivation by blocking the HIF-1α/p300 interaction.

FIGURE 5

Schematic diagram illustrating six of the potential curcumin targets during SARS-CoV-2 infection of susceptible cells with a specific focus on: Left: viral entry pathway (right panel, targets 1–3) and, Right: Ang II induced hypoxia pathway (left panel, targets 4–6). Regarding the viral entry pathway, curcumin prevents the binding of the SARS-CoV-2 S protein to the ACE2 receptor by binding to both proteins (1,2). In addition, curcumin prevents endosomal acidification required for the initiation of viral replication (3). Regarding the Ang II pathway, curcumin inhibit ACE1 (4), thus preventing the accumulation of Ang II. Moreover, curcumin prevents hypoxia-induced HIF1-α upregulation (5) and p300 acetyl transferase activity within the HIF1-α/β/CBP-p300 complex (6). Upper left, chemical structure of curcumin (diferuloylmethane). Effects of curcumin on Trp ion channels such as TrpA1 and TrpV1, have also been reported (not shown). ACE: Angiotensin converting enzyme; Ang: Angiotensin; AT1R: Ang II type I receptor; CBP/p300: Histone acetyltransferases CREB Binding Protein (CBP)/p300; IRF: Interferon regulatory factor; MDA: Melanoma differentiation-associated gene; MVAs: Mitochondrial antiviral-signal protein; RIG: Retinoic acid inducible gene; TMPRSS2: Transmembrane serine protease two; TLR: Toll like receptor; TRAF: Tumour necrosis factor receptor-associated factor; TRIF: TIR-domain-containing adapter-inducing interferon.