yes / AMP-activated protein kinase (AMPK) is a pivotal regulator of metabolism at the cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the SH2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect which required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPKmediated inhibition of JAK-STAT signaling stimulated either by the sIL-6Rα/IL-6 complex or by expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

Critical care has undergone several developments in the recent years leading to improved survival. However, acquired muscle weakness in the intensive care unit (ICU) is an important complication that affects severely ill patients and can prolong their ICU stay. Critical illness myopathy (CIM) is the progressive decline in the function and mass of the limb muscles in response to exposure to the ICU condition, while ventilator-induced diaphragm dysfunction (VIDD) is the time dependent decrease in the diaphragm function after the initiation of mechanical ventilation. Since the complete underlying mechanisms for CIM and VIDD are not completely understood, there is a compelling need for research on the mechanisms of CIM and VIDD to develop intervention strategies targeting these mechanisms. The aim of this thesis was to investigate the effects of several intervention strategies and rehabilitation programs on muscle wasting associated with ICU condition. Moreover, muscle specific differences in response to exposure to the ICU condition and different interventions was investigated. Hence, a rodent ICU model was used to address the mechanistic and therapeutic aspects of CIM and VIDD. The effects of heat shock protein 72 co-inducer (HSP72), BGP-15, on diaphragm and soleus for rats exposed to different durations of ICU condition was investigated. We showed that 5 and 10 days treatment with BGP-15 improved diaphragm fiber and myosin function, protected myosin from posttranslational modification, induced HSP72 and improved mitochondrial function. Moreover, BGP-15 treatment for 5 days improved soleus muscle fibers function, improved mitochondrial structure and reduced the levels of some ubiquitin ligases. In addition to BGP-15 treatment, passive mechanical loading of the limb muscles was investigated during exposure to the ICU condition. We showed that mitochondrial dynamics and mitophagy gene expression was affected by Mechanical silencing while mechanical loading counteracted these effects. Our investigation for other pathways that can be involved in muscle wasting associated with ICU condition showed that the Janus kinase 2/ Signal transducer and activator of transcription 3 (JAK2/STAT3) pathway is differentially activated in plantaris, intercostals and diaphragm. However, further studies are required with JAK2/STAT3 inhibitors to fully examine the role of this pathway in the pathogenesis of CIM and VIDD prior to translation to clinical research.

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

Janus kinase 3 (JAK3) est un membre de la famille JAK de protéines tyrosine kinase impliquées dans la transduction du signal intracellulaire médiée par les récepteurs de cytokines via la voie de signalisation JAK/STAT. JAK3 s'est avéré exprimé de manière différentielle dans les cellules de la granulosa (GC) des follicules pré-ovulatoires bovins et régulé à la baisse par l'hormone lutéinisante. Ces observations suggèrent que la régulation de JAK3 pourrait moduler la prolifération des GC, l'activité stéroïdienne et l'activation/l'inhibition des cibles en aval. Pour étudier les mécanismes des actions de JAK3 dans GC, nous avons utilisé JANEX-1, un inhibiteur pharmacologique de JAK3, et des traitements FSH et analysé des marqueurs de prolifération, des enzymes stéroïdogènes et la phosphorylation de protéines cibles, y compris STAT3 et les partenaires JAK3 précédemment identifiés CDKN1B/p27Kip1 et MAPK8IP3/JIP3. Les GC en culture ont été traités avec ou sans FSH en présence ou non de JANEX-1. L'ARN total et les protéines ont été extraits et analysés par RT-qPCR, western blot et UHPLC-MS/MS. L'expression de l'enzyme stéroïdogène CYP11A1, mais pas du CYP19A1, était significativement régulée à la hausse dans les GC traités avec la FSH et les deux étaient significativement diminuées lorsque JAK3 était inhibé par rapport au contrôle. Les marqueurs de prolifération CCND2 et PCNA ont été significativement réduits dans les GC traités au JANEX-1 et régulés positivement par la FSH. Les analyses Western blots ont montré que le traitement JANEX-1 réduisait de manière significative les quantités de pSTAT3 tandis que la surexpression de JAK3 augmentait pSTAT3. De même, le traitement à la FSH a augmenté pSTAT3 même dans les GC traités au JANEX-1. Les analyses UHPLC-MS/MS ont montré une phosphorylation et des modifications supplémentaires de résidus d'acides aminés spécifiques dans JAK3 ainsi que ses partenaires de liaison CDKN1B et MAPK8IP3 révélant une activation ou une inhibition possible de JAK3 après des traitements FSH ou JANEX-1, respectivement. L'abondance de la protéine totale JAK3 a augmenté après le traitement par FSH et a diminué de manière significative, avec MAPK8IP3, dans le GC traité par JANEX-1, tandis que l'abondance totale de CDKN1B a été modifiée après FSH et augmentée après JANEX-1. Nous montrons que JAK3 influence l'activité GC par la phosphorylation de protéines cibles en réponse à des stimulations telles que la FSH, ce qui conduit à l'activation de JAK/STAT et module probablement d'autres voies de signalisation impliquant CDKN1B et MAPK8IP3. / Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine preovulatory follicles and downregulated by the luteinizing hormone. These observations suggested JAK3 regulation could modulate GC proliferation, steroidogenic activity and activation/inhibition of downstream targets. To investigate the mechanisms of JAK3 actions in GC, we used JANEX-1, a pharmacological JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3 and previously identified JAK3 partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GCs were treated with or without FSH in the presence or not of JANEX-1. Total RNA and proteins were extracted and analyzed by RT-qPCR, western blotting and UHPLC-MS/MS. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was significantly upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited as compared to control. Proliferation markers CCND2 and PCNA were significantly reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment significantly reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses showed phosphorylation and additional modifications of specific amino acid residues within JAK3 as well as its binding partners CDKN1B and MAPK8IP3 revealing possible activation or inhibition of JAK3 following FSH or JANEX-1 treatments, respectively. Abundance of JAK3 total protein was increased post-FSH treatment and significantly decreased, along with MAPK8IP3, in JANEX-1-treated GC while CDKN1B total abundance was altered post-FSH and increased post-JANEX-1. We show that JAK3 influences GC activity through phosphorylation of target proteins in response to stimulations such as FSH, which leads to the activation of JAK/STAT and likely modulating other signaling pathways involving CDKN1B and MAPK8IP3.

Neointimal hyperplasia (NIH) is a cardiovascular disease characterised by increased smooth muscle cell (SMC) inflammation and proliferation. Suppressor of cytokine signalling 3 (SOCS3) limits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in vascular remodelling but is limited by its short biological half-life. Therefore, mutation of all 9 Lys residues that are potential sites of ubiquitylation to Arg should produce a mutated SOCS3 resistant to ubiquitin-mediated proteasomal degradation (“Lys-less” SOCS3). This study hypothesise that enhancing SOCS3 stability and limiting JAK/STAT signalling may provide sustained inhibition of the vascular remodelling in NIH. Lentiviral transduction of WT and Lys-less SOCS3 in human saphenous vein (HSVSMCs) was highly efficient after 48 hours (>97%) and was sustained over 2 weeks. Lys-less SOCS3 was resistant to ubiquitylation contrary to WT-transduced HSVECs, and Lys-less SOCS3 was more stable (t1/2=4h) than WT (t1/2<4h) (n=6, P<0.001) in HSVSMCs. In HSVSMCs, both Lys-less SOCS3 and WT inhibited sIL-6Rα/IL-6 mediated STAT3 activation but not extracellular signal regulated protein kinase 1/2 (ERK1/2) by 80±7% (Lys-lessSOCS3/pSTAT3) and 74±6% (WT/pSTAT3) (n=3, P<0.05) and similarly inhibited PDGF-mediated STAT3 activation but not ERK1/2 by 67±17% (Lys-less SOCS3/pSTAT3) and 72±18% (WT/pSTAT3) (n=3, P<0.05). Functionally, Lys-less SOCS3 and WT were equivalent in inhibiting sIL-6Rα/IL-6 and PDGF-induced proliferation, whilst having no effects on PDGF-induced migration in HSVSMCs. Lys-less SOCS3 can be successfully transduced into primary HSVSMCs. It is more stable than WT yet retains its functional ability to ameliorate pro-inflammatory signalling and SMC proliferation, making it an attractive option for developing treatment of NIH. / University of Botswana

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