Joanna Wegrzyn

Function of Mitochondrial Stat3 in Cellular Respiration

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3–/– cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the proteins function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Tyk2 Tyrosine Kinase Expression Is Required for the Maintenance of Mitochondrial Respiration in Primary Pro-B Lymphocytes

ABSTRACT Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-α/β). Although Tyk2−/− mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2−/− mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-β. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-β. Finally, Tyk2−/− mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.

Activation of Tyk2 and Stat3 Is Required for the Apoptotic Actions of Interferon-β in Primary Pro-B Cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNα/β) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNβ, but not IFNα, induces programmed cell death (PCD). IFNβ-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1–/– mice. However, in pro-B cells from Tyk2–/– mice, where there is normal activation of Stat1 and Stat2, IFNβ-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNα/β (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2–/– mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNα/β-mediated PCD. In contrast to IFNβ-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2–/– pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNβ. Furthermore, expression of a constitutively active form of Stat3 in Tyk2–/– B cells partially restores IFNβ-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNβ to stimulate apoptosis of primary pro-B cells.

HIF-1 attenuates Ref-1 expression in endothelial cells: Reversal by siRNA and inhibition of geranylgeranylation

Redox factor-1 (Ref-1), a multifunctional protein with DNA repairing activities, plays a cytoprotective function by post-translational redox modification of numerous transcription factors, including hypoxia inducible factor-1 (HIF-1). In the present study, activation of HIF-1 by hypoxia and dimethyloxaloylglycine (DMOG), a hypoxia mimic, diminished Ref-1 mRNA and protein expression in human microvascular endothelial cells (HMEC-1). Similarly, adenoviral delivery of the stabilized form of HIF-1alpha decreased Ref-1 mRNA and protein levels. Accordingly, HIF-1alpha siRNA abolished the hypoxia-induced inhibition of Ref-1 expression, indicating the role of HIF-1 in down-regulation of Ref-1. Also, translocation of Ref-1 from nucleus to cytoplasm after HIF-1 activation was noted. Interestingly, we observed the restoration of Ref-1 expression in hypoxia by pharmacologically relevant doses of atorvastatin. This effect was dependent on the inhibition of protein geranylgeranylation, but not farnesylation, as only the inhibitor of the former but not the latter prenylation step restored the Ref-1 expression. The regulation of Ref-1 by statins may be considered as a novel mechanism of their beneficial effects on endothelium.