Deoxyhypusine synthase haploinsufficiency attenuates acute cytokine signaling

Abstract

Deoxyhypusine synthase (DHS) catalyzes the post-translational formation of the amino acid hypusine. Hypusine is unique to the eukaryotic translational initiation factor 5A (eIF5A), and is required for its functions in mRNA shuttling, translational elongation, and stress granule formation. In recent studies, we showed that DHS promotes cytokine and ER stress signaling in the islet β cell and thereby contributes to its dysfunction in the setting of diabetes mellitus. Here, we review the evidence supporting a role for DHS (and hypusinated eIF5A) in cellular stress responses, and provide new data on the phenotype of DHS knockout mice. We show that homozygous knockout mice are embryonic lethal, but heterozygous knockout mice appear normal with no evidence of growth or metabolic deficiencies. Mouse embryonic fibroblasts from heterozygous knockout mice attenuate acute cytokine signaling, as evidenced by reduced production of inducible nitric oxide synthase, but show no statistically significant defects in proliferation or cell cycle progression. Our data are discussed with respect to the utility of sub-maximal inhibition of DHS in the setting of inflammatory states, such as diabetes mellitus.

Figures

Figure 1

Dhps heterozygosity in mice does not alter growth or metabolic homeostasis. (A) Schematic diagram of the Dhps gene targeting vector (KO) and the wild-type mouse locus (WT). Targeting construct and mice were generated by a contract to InGenious Targeting Labs (Stony Brook, NY). Neo, neomycin selection cassette. Dotted lines indicate homologous recombination regions; (B) serial body weights of Dhps+/+ and Dhps+/− mice between 5–25 weeks of age; (C) results of glucose tolerance tests in mice at 8 weeks of age. Glucose (1 g/kg body weight) was injected intraperitoneally following an overnight fast; (D) results of glucose tolerance tests in mice at 20 weeks of age. Glucose (1 g/kg body weight) was injected intraperitoneally following a 6 h fast.

Figure 2

Dhps heterozygosity attenuates acute cytokine signaling in mouse embryonic fibroblasts. (A) Top: immunoblot analysis for DHS and actin from whole-cell extracts of Dhps+/+ and Dhps+/− MEFs. Immunoblots were visualized using a LiCor Odysseyr fluorescence system following electrophoresis on a 4–20% SDS-polyacrylamide gel. Bottom: quantitation of DHS protein levels (normalized to actin levels) from three independent experiments. (B) Top: MEFs were incubated with 1 µCi/ml 3H-spermidine for 4 h, then whole cell extracts were subjected to immunoblot analysis (for actin and eIF5A) or to fluorography (for 3H-eIF5A) following electrophoresis on a 4–20% SDS-polyacrylamide gel. Bottom: quantitation of 3H-eIF5A levels (normalized to actin levels) expressed as the mean ± SEM from 5 independent experiments. (C) MEFs were incubated in the absence or presence of a cytokine mixture (5 ng/ml IL-1β, 10 ng/ml TNFα and 100 ng/ml IFNγ) for 4 h, then total RNA was isolated and subjected to real-time RT-PCR analysis for Nos2 mRNA encoding iNOS. Data were normalized to Actb mRNA levels (encoding actin), and are presented as fold-induction relative to non-cytokine treatment. (D) Top: immunoblot analysis for iNOS and actin from whole cell extracts of MEFs treated without and with a cytokine mixture for 4 h. Bottom: quantitation of iNOS protein levels (normalized to actin levels) following incubation with cytokines. Data represent the mean ± SEM from three independent experiments. In all parts, * indicates that the value for Dhps+/− MEFs is statistically different from corresponding value for Dhps+/+ MEFs (p < 0.05 by a Student's t-test).

Figure 3

Dhps heterozygosity does not lead to significant inhibition of proliferation or cell cycle progression. (A) MEFs were incubated with 1 µCi/ml of 3H-methyl thymidine for 4 h, then washed and lysed. 3H-thymidine incorporation was measured and normalized to protein content. (B) MEFs were incubated with Guava cell cycle reagent (Millipore) for 30 min. Intercalation of propidium iodide into cellular DNA was quantitated using a FACS Calibur instrument, and the data were analyzed for cell cycle status using Modfit software. (C) same as in (A), except cells were concurrently treated with a cytokine mixture (5 ng/ml IL-1β, 10 ng/ml TNFα and 100 ng/ml IFNγ) during the 4 h 3H-methyl thymidine incubation period. (D) Same as in (B), except cells were treated with a cytokine mixture for 4 h prior to incubation with Guava reagent.