Effect of the p38 Mitogen-Activated Protein Kinase Signaling Cascade on Radiation Biodosimetry

Abstract

Radiation biodosimetry based on transcriptomic analysis of peripheral blood is a valuable tool to detect radiation exposure after a radiological/nuclear event and obtain useful biological information that could predict tissue and organismal injury. However, confounding factors, including chronic inflammation or immune suppression, can potentially obscure the predictive power of the method. Members of the p38 mitogen-activated protein kinase (MAPK) family respond to pro-inflammatory signals and environmental stresses, whereas genetic ablation of the p38 signaling pathway in mice leads to reduced susceptibility to collagen-induced arthritis and experimental autoimmune encephalomyelitis that model human rheumatoid arthritis and multiple sclerosis, respectively. p38 is normally regulated by the MAP3K-MAP2K pathway in mammalian cells. However, in T cells there is an alternative pathway for p38 activation that plays an important role in antigen-receptor-activated T cells and participates in immune and inflammatory responses. To examine the role of p38 in response to radiation, we used two mouse models expressing either a p38α dominant negative (DN) mutation that globally suppresses p38 signaling or a p38αβ double-knock-in (DKI) mutant, which inhibits specifically T-cell receptor activation. We exposed p38 wild-type (p38WT) and mutant male mice to 7 Gy X rays and 24 h later whole blood was isolated subjected to whole-genome microarray and gene ontology analysis. Irradiation of p38WT mice led to a significant overrepresentation of pathways associated with morbidity and mortality, as well as organismal cell death. In contrast, these pathways were significantly underrepresented in p38DN and p38DKI mutant mice, suggesting that p38 attenuation may protect blood cells from the deleterious effects of radiation. Furthermore, radiation exposure in p38 mutant mice resulted in an enrichment of phagocytosis-related pathways, suggesting a role for p38 signaling in restricting phagocytosis of apoptotic cells after irradiation. Finally, despite the significant changes in gene expression, it was still feasible to identify a panel of genes that could accurately distinguish between irradiated and control mice, irrespective of p38 status.

FIG. 1.

Differentially expressed genes (DEGs) between mutant (p38DKI, p38DN) vs. p38WT and between irradiated vs. unirradiated samples. Panel A: Significantly differentially expressed genes (DEGs) in mouse blood or after 7 Gy X-ray irradiation relative to unirradiated controls (P < 0.001, FDR < 0.05). Absolute numbers and percentages of upregulated and downregulated genes are shown. Panel B: Venn diagram showing overlap patterns of genes that are differentially expressed in the control and irradiated p38 mutant mice. Abbreviations, WT: p38 wild-type; DKI: p38 double knock-in mutant; DN: p38 dominant negative; up: upregulated; dn: downregulated; ui: unirradiated; ir: irradiated.

FIG. 2.

Cell death-related functions. Panel A: Significant cell death-related diseases and functions among genes differentially expressed in irradiated WT, DKI, and DN mice as identified by Ingenuity Pathway Analysis (IPA). Panel B: Significant cell-death functions among genes differentially expressed in p38DN vs. p38WT mice under basal conditions as identified by IPA. Biological functions displaying a z-score ≥ 2 or ≤ −2 and showing Benjamini-Hochberg adjusted P value < 0.05 were considered significant. Abbreviations, WT: p38 wild-type; DKI: p38 double knock-in mutant; DN: p38 dominant negative.

FIG. 3.

Phagocytosis-related biological functions are overrepresented in irradiated p38 transgenic mice. Significant phagocytosis-related diseases or functions among genes differentially expressed in irradiated WT, DKI, and DN mice as identified by Ingenuity Pathway Analysis. Biological functions displaying a z-score ≥ 2 and showing Benjamini-Hochberg adjusted P value < 0.05 were considered significant. Abbreviations, WT: p38 wild-type; DKI: p38 double knock-in mutant; DN: p38 dominant negative.

FIG. 4.

Phagocytosis-related gene expression measured by RT-qPCR. Gene expression levels of 4 phagocytosis-related genes (Axl, Gas6, Marco and Cd93) analyzed by qRT-PCR and normalized to Actb expression. Data represent the mean ± S.E.M. (n = 3). The dashed line represents the level in unirradiated controls. Abbreviations, WT: p38 wild-type; DKI: p38 double knock-in mutant; DN: p38 dominant negative.

FIG. 5.

Cancer-related functions. Panel A: Enriched cancer-related functions in irradiated p38WT, p38DKI, and p38DN mouse blood compared with control animals. Panel B: Enriched cancer-related functions in p38DN versus p38WT under basal conditions. A P value < 0.05 and a z-score ≥ 2 (marked by the red line) were considered significant. Abbreviations, DKI: p38 double knock-in mutant; DN: p38 double dominant; WT: p38 wild-type.