The growth hormone receptor associates with Jak1, Jak2 and Tyk2 in human liver.

Studies in cell lines have shown that Jak2 is the primary tyrosine kinase involved in signal transduction by the growth hormone receptor (GHR). In addition, growth hormone (GH) stimulates tyrosine phosphorylation of Jak1 and Jak3 in certain cell lines, while the effect on Tyk2 has not been analysed.We have investigated the expression of Jak proteins in human liver and analysed their interactions with the GHR. Using Western blot analysis and immunohistochemistry, we demonstrate that Jak1, Jak2, Jak3 and Tyk2 are present in human liver. Immunoprecipitation by an antibody against the GHR (Mab 263) followed by immunoblotting with specific antibodies against Jak proteins showed that Jak1, Jak2 and Tyk2 were associated with the GHR in this tissue. We conclude that the GHR associates with Jak1, Jak2 and Tyk2 in human liver. Although experiments in vitro indicate that Jak2 mediates GH signalling, our results open the possibility that other Jak proteins may influence GHR signalling in human liver.     

Allelic variation in the Tyk2 and EGF genes as potential genetic determinants of CNS repair

The potential for endogenous remyelination and axonal protection can be an important factor in determining disease outcome in demyelinating diseases like multiple sclerosis. In many multiple sclerosis (MS) patients CNS repair fails or is incomplete whereas in others the disease is accompanied by extensive repair of demyelinated lesions. We have described significant differences in the ability of two strains of mice to repair CNS damage following Theiler’s virus-induced demyelination: FVB/NJ (FVB) mice repair damaged myelin spontaneously and completely, whereas B10.D1-H2^q/SgJ (B10.Q) mice are deficient in the repair process. A QTL analysis was performed to identify genetic loci that differentially regulate CNS repair following chronic demyelination in these strains and two QTL were detected: one on chromosome 3 with a LOD score of 9.3 and a second on chromosome 9 with a LOD score of 14.0. The mouse genes for epidermal growth factor (EGF) and Tyk2 are encoded within the QTL on chromosomes 3 and 9, respectively. Sequence polymorphisms between the FVB and B10.Q strains at both the EGF and Tyk2 loci define functional variations consistent with roles for these genes in regulating myelin repair. EGF is a key regulator of cell growth and development and we show a sevenfold increase in EGF expression in FVB compared to B10.Q mice. Tyk2 is a Janus kinase that plays a central role in controlling the T_H1 immune response and we show that attenuation of Tyk2 function correlates with enhanced CNS repair.     

A dual role for the kinase-like domain of the tyrosine kinase Tyk2 in interferon-alpha signaling

Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for alpha-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-alpha/beta signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-alpha binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed. Interferon-alpha induced phosphorylation, and gene expression could be detected in V584D- or G596V-expressing cells, but not in H669P- or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-alpha receptor complex.     

Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice

Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-gamma (IFN-gamma) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-kappaB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-gamma production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-gamma was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways in cytokine signaling.     

Expression of Jak3, STAT1, STAT4, and STAT6 in inflammatory arthritis: unique Jak3 and STAT4 expression in dendritic cells in seropositive rheumatoid arthritis

BACKGROUND: Modulation of Jak-STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis. OBJECTIVE: To document Jak-STAT expression in a cohort of patients with active rheumatoid arthritis (RA), spondyloarthritis (SpA), and osteoarthritis (OA) and compare these subsets with normal synovial tissue. METHODS: Synovial tissue biopsy specimens from patients with RA, OA, and SpA and histologically normal tissue (n = 10 in each arthritis group) were examined for the presence of Jak3, STAT1, STAT4, and STAT6 expression using immunohistochemistry. Phenotyping was performed using immunohistochemistry and immunofluorescence. Clinical and serological characteristics of patients with RA expressing Jak3-STAT4 were assessed. RESULTS: STAT1, STAT4, and Jak3 protein expression was generally increased in inflammatory arthritis. In contrast, STAT6 expression was relatively heterogeneous. A subpopulation of CD1a positive dendritic cells unique to seropositive patients with RA was detected. These cells showed intense protein expression for Jak3, STAT4, and STAT6. CONCLUSION: CD1a positive dendritic cells intensely express Jak3, STAT4, and STAT6 in seropositive RA tissue and may be an alternative marker for dendritic cells in their early stages of activation as well as providing a tool for identifying RA at the level of the synovium. Jak3 inhibition may be a potential therapeutic target to prevent dendritic cell maturation in RA. STAT1 expression is increased in inflammatory arthritis, suggesting that its pro-apoptotic and anti-inflammatory effects cannot effectively counteract inflammation. STAT6 expression is heterogeneous in synovium, suggesting a possible homoeostatic role in addition to any anti-inflammatory effects.     

Expression of Tyk2 in dendritic cells is required for IL-12, IL-23, and IFN-gamma production and the induction of Th1 cell differentiation

It is well documented that dendritic cells (DCs), representative antigen-presenting cells, are important sources of Th1-promoting cytokines and are actively involved in the regulation of T-helper-cell differentiation. However, the intracellular event that regulates this process is still largely unknown. In this study, we examined the role of Tyk2, a JAK kinase that is involved in the signaling pathway under IL-12 and IL-23, in DC functions. While the differentiation and maturation of DCs was normal in Tyk2-deficient (Tyk2(-/-)) mice, IL-12-induced Stat4 phosphorylation was diminished in Tyk2(-/-) DCs. IL-12-induced IFN-gamma production was also significantly diminished in Tyk2(-/-) DCs to levels similar to those in Stat4(-/-) DCs. Interestingly, Tyk2(-/-) DCs were defective in IL-12 and IL-23 production upon stimulation with CpG ODN. Furthermore, Tyk2(-/-) DCs were impaired in their ability to induce Th1-cell differentiation but not Th2-cell differentiation. Taken together, these results indicate that the expression of Tyk2 in DCs is crucial for the production of Th1-promoting cytokines such as IL-12 and IFN-gamma from DCs and thereby for the induction of antigen-specific Th1-cell differentiation.     

STAT2 deficiency and susceptibility to viral illness in humans

Severe infectious disease in children may be a manifestation of primary immunodeficiency. These genetic disorders represent important experiments of nature with the capacity to elucidate nonredundant mechanisms of human immunity. We hypothesized that a primary defect of innate antiviral immunity was responsible for unusually severe viral illness in two siblings; the proband developed disseminated vaccine strain measles following routine immunization, whereas an infant brother died after a 2-d febrile illness from an unknown viral infection. Patient fibroblasts were indeed abnormally permissive for viral replication in vitro, associated with profound failure of type I IFN signaling and absence of STAT2 protein. Sequencing of genomic DNA and RNA revealed a homozygous mutation in intron 4 of STAT2 that prevented correct splicing in patient cells. Subsequently, other family members were identified with the same genetic lesion. Despite documented infection by known viral pathogens, some of which have been more severe than normal, surviving STAT2-deficient individuals have remained generally healthy, with no obvious defects in their adaptive immunity or developmental abnormalities. These findings imply that type I IFN signaling [through interferon-stimulated gene factor 3 (ISGF3)] is surprisingly not essential for host defense against the majority of common childhood viral infections.