NR4A proteins and neutrophil lifespan

In this issue of Blood, Prince et al provide fundamental insight into the function of proteins in the NR4A orphan nuclear receptor protein family, identifying NR4A2 and NR4A3 as critical regulators of neutrophil lifespan and homeostasis, and demonstrating that they act via a cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA)–dependent mechanism.¹ 

Neutrophils are short-lived leukocytes that are inherently programmed to undergo apoptosis within 24 hours after release into the circulation. Tight spatial and temporal control of neutrophil turnover is critical for resolution of inflammation and control of infection, and as such, defects in this process exemplify an aberrant and dysregulated inflammatory response that favors tissue destruction and disease.^2,3  Understanding of the mechanisms that control neutrophil turnover was revolutionized by the discovery that cell lifespan is regulated not only by intracellular signaling but also by global changes in gene expression that together comprise an apoptosis differentiation program.^4-6  These seminal studies overturned the notion that neutrophils have little or no capacity for transcription or translation and set the stage for subsequent studies of neutrophil lifespan in the context of specific infectious and inflammatory diseases.³ 

The NR4A family of orphan nuclear receptors is composed of 3 members: NR4A1, NR4A2, and NR4A3. All 3 proteins are transcription factors and are rapidly induced in response to stress.⁷  Although NR4A proteins are widely expressed, their effects are cell type–specific, but how specificity is determined is unknown. The current study by Prince et al provides the first definitive evidence that NR4A2 and NR4A3 are critical regulators of neutrophil homeostasis and lifespan. This was demonstrated using multiple approaches. First, global transcriptional profiling of ultrapure human neutrophils in vitro revealed that NR4A2 and NR4A3 are selectively and profoundly upregulated by PKA agonists, but not by other factors that also extend cell lifespan such as lipopolysaccharide, hypoxia, or granulocyte-macrophage colony-stimulating factor. Second, an in vivo role for NR4A proteins was confirmed by analyses of human neutrophils that migrated into skin in response to endotoxin and was validated by knockdown in genetically tractable murine neutrophil progenitors. Third, selectivity for effects on cell abundance, but not cell migration, was obtained by live imaging of neutrophils in zebrafish larvae in the presence and absence of NR4A-specific morpholinos. Of note, the data also identify prostaglandin E2 (PGE2) as an upstream signal leading to PKA activation and rapid induction of NR4A2 and NR4A3 expression. Taken together, the data confirm NR4A2 and NR4A3 as immediate early genes and identify a PGE2-cAMP-PKA-NR4A2/3-neutrophil survival signaling pathway.

Previous studies demonstrated that all 3 NR4A family members are present in the brain and central nervous system and are important for neuronal protection as well as learning and memory.⁷  These proteins also regulate liver and muscle metabolism, and murine knockout studies indicate that NR4A1 and NR4A3 protect against cardiovascular disease. With regard to inflammation and immunity, mutations in NR4A1 and/or NR4A3 have been linked to leukemia, apoptosis, and abnormal T-cell development, further highlighting the context-specific nature of NR4A protein functions. In particular, NR4A1 is rapidly induced in macrophages that have engulfed apoptotic thymocytes, with subsequent NR4A1-dependent downregulation of NF-κB apparently critical for the anti-inflammatory effects of efferocytosis.⁸  Moreover, NR4A2 can either increase or decrease expression of target genes, and both constitutive and inducible expression of NR4A2 in macrophages has been reported. Consistent with this, NR4A2 is induced by PGE2 in rheumatoid arthritis, and its anti-inflammatory effects in macrophages are associated with transrepression of NF-κB.⁷ 

The results of the current study are in agreement with the classification of NR4A family members as immediate early genes, but many important questions remain unanswered. For example, the endogenous ligands of NR4A proteins are unknown. In addition, precisely how PKA activation is linked to NR4A2/3 induction is unclear. There is evidence that NR4A proteins can be phosphorylated, but whether they are direct targets of PKA remains to be determined.⁷  Whether and how NR4A trafficking between the cytoplasm and nucleus is regulated is also unknown. Development of reliable antibodies for detection of NR4A isoforms will be required to address these questions. As NR4A2 and NR4A3 induction is rapid and their capacity for transrepression of NF-κB in other settings is established, it is also attractive to predict that NR4A proteins may be key components of feed-forward loops that regulate neutrophil apoptosis via sequential changes in gene expression. This notion is particularly attractive as NR4A2 can have positive and negative effects, as noted previously, and because NF-κB targets are known to include both pro- and antiapoptosis regulatory factors.^3,8  Finally, it is not clear how isoform and cell type–selectivity of NR4A family proteins are achieved, as all 3 have similar DNA-binding domains and regulate cAMP-response elements.⁷  Further studies of their divergent N termini may shed light on this critical question.

There is significant interest in the development of therapeutics to treat infectious and inflammatory disorders where neutrophil accumulation, prolonged cell lifespan, and necrotic tissue damage are key features of disease.³  One agent in this category is the cyclin-dependent kinase inhibitor R-roscovitine.^3,9  Pharmacological agonists and inhibitors of NR4A1 have been identified.⁷  As a link between NR4A2 and NR4A3 and neutrophil lifespan is now firmly established, it will be of interest to determine if agents that selectively target these NR4A family members in neutrophils can be identified and developed as candidate therapeutics.

In summary, NR4A family proteins are transcription factors that are induced in response to stress and have complex context-specific effects on gene expression and cell function. Recent studies establish NR4A2 and NR4A3 as critical regulators of neutrophil lifespan downstream of cAMP and PKA. Further studies of these proteins are expected to advance understanding of NR4A protein biology, both in general and in the context of neutrophil apoptosis, and may identify new targets for the development of therapeutics to treat arthritis and other disorders where neutrophils contribute to pathology rather than effective host defense.