Inhibition of protein kinase II (CK2) prevents induced signal transducer and activator of transcription (STAT) 1/3 and constitutive STAT3 activation.

The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT pathway.

Important scaffold function of the Janus kinase 2 uncovered by a novel mouse model harboring a Jak2 activation-loop mutation.

Janus kinases (Jak) play essential roles in cytokine and growth factor signaling. Conventional gene targeting of Jak2, creating a null allele, leads to a block in definitive erythropoiesis as a result of failing signal transduction at the homomeric erythropoietin receptor (EpoR) and at the heteromeric interferon γ receptor (IFNGR). To investigate the in vivo relevance of the activation loop of Jak2, a Jak2-YY1007/1008FF knockin mutation was introduced into the germline of mice. The phenotype of the Jak2(FF/FF) mouse line reveals that tyrosine residues 1007/1008 are absolutely essential for kinase function and signal transduction at the homomeric EpoR. Detailed studies using the Jak2 activation loop mutant uncover an essential scaffolding function of Jak2 within the IFNGR receptor complex and reveal that Jak1 can mediate a semi-redundant function for IFNGR signal transduction. These studies are highly important for the molecular understanding of cytokine and growth factor signaling and provide new insights for future strategies in the design of pharmacological blockers of Jak2.

Regulation of IDO activity by oxygen supply: inhibitory effects on antimicrobial and immunoregulatory functions.

Tryptophan is an essential amino acid for human beings as well as for some microorganisms. In human cells the interferon-γ (IFN-γ) inducible enzyme indoleamine 2,3-dioxygenase (IDO) reduces local tryptophan levels and is therefore able to mediate broad-spectrum effector functions: IDO activity restricts the growth of various clinically relevant pathogens such as bacteria, parasites and viruses. On the other hand, it has been observed that IDO has immunoregulatory functions as it efficiently controls the activation and survival of T-cells. Although these important effects have been analysed in much detail, they have been observed in vitro using cells cultured in the presence of 20% O2 (normoxia). Such high oxygen concentrations are not present in vivo especially within infected and inflamed tissues. We therefore analysed IDO-mediated effects under lower oxygen concentrations in vitro and observed that the function of IDO is substantially impaired in tumour cells as well as in native cells. Hypoxia led to reduced IDO expression and as a result to reduced production of kynurenine, the downstream product of tryptophan degradation. Consequently, effector functions of IDO were abrogated under hypoxic conditions: in different human cell lines such as tumour cells (glioblastoma, HeLa) but also in native cells (human foreskin fibroblasts; HFF) IDO lost the capacity to inhibit the growth of bacteria (Staphylococcus aureus), parasites (Toxoplasma gondii) or viruses (herpes simplex virus type 1). Additionally, IDO could no longer efficiently control the proliferation of T-cells that have been co-cultured with IDO expressing HFF cells in vitro. In conclusion, the potent antimicrobial as well as immunoregulatory functions of IDO were substantially impaired under hypoxic conditions that pathophysiologically occurs in vivo.

Up-regulation of c-FLIP short by NFAT contributes to apoptosis resistance of short-term activated T cells.

Upon encounter with pathogens, T cells activate several defense mechanisms, one of which is the up-regulation of CD95 ligand (CD95L/FasL) which induces apoptosis in sensitive target cells. Despite expression of the CD95 receptor, however, recently activated T cells are resistant to CD95L, presumably due to an increased expression of antiapoptotic molecules. We show here that, in contrast to naive or long-term activated T cells, short-term activated T cells strongly up-regulate the caspase-8 inhibitor, cellular FLICE-inhibitory protein (c-FLIP). Intriguingly, upon activation, T cells highly induced the short splice variant c-FLIP(short), whereas expression of c-FLIP(long) was only marginally modulated. In contrast to the general view that c-FLIP transcription is controlled predominantly by nuclear factor-kappaB (NF-kappaB), induction of c-FLIP(short) in T cells was primarily mediated by the calcineurin-nuclear factor of activated T cells (NFAT) pathway. Importantly, blockage of NFAT-mediated c-FLIP expression by RNA interference or inhibition of calcineurin rendered T cells sensitive toward CD95L, as well as activation-induced apoptosis. Thus, the resistance of recently activated T cells depends crucially on induction of c-FLIP expression by the calcineurin/NFAT pathway. Our findings imply that preventing autocrine CD95L signaling by c-FLIP facilitates T-cell effector function and an efficient immune response.

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis.

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.