NF-κB Orchestrates a Dual Transcriptional Program in Hypoxia with Predominant Control over Gene Repression

Hypoxia triggers conserved adaptive cellular programmes that restore oxygen homeostasis or mitigate its deficit. Central to the oxygen-sensing machinery are the hypoxia-inducible factors (HIFs), whose stability and activity are governed by prolyl hydroxylase domain enzymes (PHDs) and the von Hippel–Lindau ubiquitin ligase complex (Kaelin and Ratcliffe, 2008; Kenneth and Rocha, 2008). In parallel, hypoxia elicits activation of the NF-κB transcription factor family, a pathway traditionally associated with inflammation but increasingly recognised for its response to diverse physiological stresses (Perkins and Gilmore, 2006; Cummins and Taylor, 2005).

The NF-κB family comprises five subunits—RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52)—that form homo- and heterodimers with distinct regulatory roles. Canonical activation proceeds through IκB kinase (IKK)-mediated phosphorylation and degradation of IκB inhibitors, whereas non-canonical pathways involve processing of p100 to p52. Hypoxia-induced NF-κB activation is IKK-dependent across species and cell types, requires calcium signalling and TAK1 in many systems, and is modulated by PHD activity, yet proceeds without classical IκB degradation in certain contexts (Culver et al., 2010; Bandarra et al., 2014; Wilson et al., 2020).

Although hypoxia-repressed genes are well documented, the transcriptional mechanisms underlying their downregulation remain poorly defined (Batie et al., 2018; Cavadas et al., 2017). Here we systematically evaluated the contribution of NF-κB to the global transcriptional response to prolonged (24 h) severe hypoxia (1% O₂) using integrative transcriptomics across multiple human cell lines.

NF-κB Is Essential for a Large Fraction of the Hypoxic Transcriptome, Especially Repressed Genes

RNA sequencing of HeLa cells subjected to normoxia or 24 h hypoxia following siRNA-mediated depletion of RelA, RelB, or c-Rel revealed that NF-κB governs approximately one-third of hypoxia-induced genes and nearly two-thirds of hypoxia-repressed genes. Of 1,568 genes upregulated by hypoxia in control cells, 556 (35.5%) failed to be induced upon depletion of any NF-κB subunit. Strikingly, of 655 genes repressed by hypoxia, 410 (62.6%) escaped repression when NF-κB signalling was impaired, indicating a dominant repressive role.

Individual subunit contributions differed markedly. RelB exerted the broadest influence, followed by c-Rel and RelA. Only 41 genes required all three subunits for induction, whereas the majority displayed subunit-specific dependence. Hallmark pathway analysis showed RelA-linked induced genes enriched in hypoxia, glycolysis, and TNF-α/NF-κB signatures; RelB-linked genes in epithelial–mesenchymal transition and myogenesis; and c-Rel-linked genes in inflammation. Repressed gene sets converged on oxidative phosphorylation and reactive oxygen species pathways irrespective of subunit.

Motif and public ChIP-seq analyses confirmed co-enrichment of HIF-1α/β and HIF-2α binding sites predominantly at NF-κB-dependent induced genes, consistent with functional crosstalk. In contrast, repressed genes lacked HIF enrichment but displayed diverse NF-κB-associated motifs, supporting direct or indirect repressive mechanisms.

Validation by RT-qPCR across a panel of candidates (e.g., VIM, USP28, EGLN3, SAP30, TGFA, JUNB, GCLM, IDH1) confirmed the RNA-seq findings, with RelB again showing the most pervasive effects. Protein-level analysis of oxidative phosphorylation components (ATP5A, UQCRC2, NDUFB8, COX2, IDH1) and superoxide dismutase 1 (SOD1) corroborated repression in hypoxia and its reversal upon NF-κB depletion.

Conservation Across Cell Types and Physiological Relevance

Comparative analysis of public hypoxia RNA-seq datasets from lung (A549), glioblastoma (U87), colorectal (HCT116), breast (MCF-7), melanoma (501-mel), and neuroblastoma (SKNAS), and non-transformed HUVEC cells demonstrated substantial overlap with the HeLa NF-κB-dependent signature. The highest concordance occurred in U87, HCT116, and MCF-7 cells, with RelB-dependent genes again most conserved. Orthogonal inhibition of canonical and non-canonical NF-κB signalling via IKKα/β double-knockout HCT116 cells recapitulated failure to repress oxidative phosphorylation proteins, ruling out off-target siRNA effects.

Notably, NF-κB-dependent regulation was hypoxia-specific: the same genes showed no altered basal expression under normoxia despite subunit depletion.

NF-κB Sustains Hypoxic ROS Signalling and Metabolic Reprogramming

Hypoxia elevates mitochondrial-derived reactive oxygen species (ROS), which function as signalling intermediates. Depletion of RelA, and to a lesser extent RelB and c-Rel, significantly blunted hypoxia-induced ROS accumulation measured by CellROX fluorescence. Mechanistically, hypoxia repressed SOD1 protein (but not always mRNA), an effect partially relieved by RelB or c-Rel knockdown, implicating NF-κB-mediated SOD1 downregulation in sustained superoxide signalling. NRF2 protein and canonical target induction were minimal, indicating ROS levels remained sub-threshold for antioxidant response activation.

Treatment with the broad-spectrum HDAC inhibitor SAHA reversed hypoxia-mediated repression of several oxidative phosphorylation components (ATP5A, UQCRC2, COX2) but not SOD1, suggesting NF-κB recruits HDAC-containing co-repressor complexes to a subset of targets while employing additional mechanisms for others.

Model and Implications

Under hypoxic stress, NF-κB functions bidirectionally: it co-operates with HIF at a subset of promoters to drive adaptive induction while actively recruiting co-repressors—partly HDAC-dependent—to downregulate oxidative phosphorylation and ROS-detoxification machinery (Fig. 8). This dual programme conserves energy, sustains ROS-based signalling required for full hypoxic adaptation, and contrasts with the predominantly activating role of NF-κB during inflammatory stimuli. The prominence of RelB and the extensive repressive signature distinguish the hypoxic NF-κB programme from canonical cytokine responses and highlight context-dependent deployment of this versatile transcription factor.

In conclusion, NF-κB emerges as a master regulator of the hypoxic transcriptome whose previously underappreciated repressive activity shapes metabolic reprogramming and redox signalling, with broad conservation across transformed and non-transformed human cells. These findings redefine the hierarchy of transcriptional control in oxygen deprivation and nominate NF-κB as a therapeutic target in pathologies driven by chronic hypoxia.