The impact of C-reactive protein levels on the effectiveness of upadacitinib in patients with rheumatoid arthritis: a 12-month prospective, non-interventional German study.

OBJECTIVES: We investigated whether the effectiveness of upadacitinib in rheumatoid arthritis (RA) treatment is affected by baseline CRP levels in a real-world setting. METHODS: UPwArds was a prospective, non-interventional study. Patients had moderate-to-severe RA and an inadequate response or intolerance to ≥1 disease-modifying anti-rheumatic drug (DMARD). The primary endpoint was clinical remission (Clinical Disease Activity Index [CDAI] ≤2.8) at 6 months. Secondary endpoints at 12 months included clinical remission and low disease activity assessed by CDAI and Simple Disease Activity Index criteria, DAS28-CRP <2.6/≤3.2, and patient-reported outcomes. The impact of baseline CRP levels (normal vs. above the upper limit of normal [ULN]) on primary and secondary endpoints was evaluated. The effect of concomitant MTX and prior inadequate response to biologic or targeted synthetic DMARDs (b/tsDMARD-IR) on the effectiveness of upadacitinib was also assessed. Safety was evaluated through 12 months. RESULTS: 518 patients were included in the effectiveness analyses. At 6 months, 24.4% of patients achieved the primary endpoint (CDAI ≤2.8). At 12 months, similar proportions of patients with normal CRP and CRP above the ULN at baseline achieved CDAI ≤2.8 (27.3% and 29.1%) and other key secondary endpoints. The effectiveness of upadacitinib was comparable with and without concomitant MTX and in b/tsDMARD-naive and b/tsDMARD-IR patients. The safety results were consistent with the known safety profile of upadacitinib; no new safety signals were identified. CONCLUSIONS: Upadacitinib therapy was effective for RA in a real-world setting. Baseline CRP levels had no significant impact on the effectiveness of upadacitinib.

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK).

BACKGROUND: Homeodomain interacting protein kinases (HIPKs) function as modulators of cellular stress responses and regulate cell differentiation, proliferation and apoptosis. The HIPK family includes HIPK1, HIPK2 and HIPK3, which share a similar domain structure, and the more distantly related HIPK4. Although HIPKs phosphorylate their substrates on serine or threonine residues, it was recently reported that HIPK2 depends on the autophosphorylation of a conserved tyrosine in the activation loop to acquire full catalytic activity and correct subcellular localization. In this study we addressed the question whether tyrosine autophosphorylation in the activation loop has a similar function in the other members of the HIPK family. RESULTS: All HIPKs contained phosphotyrosine when expressed in HeLa cells. Catalytically inactive point mutants were not tyrosine-phosphorylated, indicating that HIPKs are dual-specificity protein kinases that autophosphorylate on tyrosine residues. HIPK point mutants lacking the conserved tyrosine residue in the activation loop showed reduced catalytic activity towards peptide and protein substrates. Analysis of these mutants revealed that HIPK1, HIPK2 and HIPK3 but not HIPK4 are capable of autophosphorylating on other tyrosines. Inhibition of tyrosine phosphatase activity by treatment with vanadate enhanced global phosphotyrosine content of HIPK1, HIPK2 and HIPK3 but did not affect tyrosine phosphorylation in the activation loop. Mutation of the activation-loop tyrosines resulted in a redistribution of HIPK1 and HIPK2 from a speckle-like subnuclear compartment to the cytoplasm, whereas catalytically inactive point mutants showed the same pattern of cellular distribution as the wild type proteins. In contrast, mutation of the activating tyrosine did not increase the low percentage of cells with extranuclear HIPK3. HIPK4 was excluded from the nucleus with no difference between the wild type kinase and the point mutants. CONCLUSIONS: These results show that HIPKs share the mechanism of activation by tyrosine autophosphorylation with the closely related DYRK family (dual-specificity tyrosine phosphorylation regulated kinase). However, members of the HIPK family differ regarding the subcellular localization and its dependence on tyrosine autophosphorylation.

Oral administration of methysticin improves cognitive deficits in a mouse model of Alzheimer's disease.

INTRODUCTION: There is increasing evidence for the involvement of chronic inflammation and oxidative stress in the pathogenesis of Alzheimer's disease (AD). Nuclear factor erythroid 2-related factor 2 (Nrf2) is an anti-inflammatory transcription factor that regulates the oxidative stress defense. Our previous experiments demonstrated that kavalactones protect neuronal cells against Amyloid ß (Aß)-induced oxidative stress in vitro by Nrf2 pathway activation. Here, we tested an in vivo kavalactone treatment in a mouse model of AD. METHODS: The kavalactone methysticin was administered once a week for a period of 6 months to 6 month old transgenic APP/Psen1 mice by oral gavage. Nrf2 pathway activation was measured by methysticin treatment of ARE-luciferase mice, by qPCR of Nrf2-target genes and immunohistochemical detection of Nrf2. Aß burden was analyzed by CongoRed staining, immunofluorescent detection and ELISA. Neuroinflammation was assessed by immunohistochemical stainings for microglia and astrocytes. Pro-inflammatory cytokines in the hippocampus was determined by Luminex multi-plex assays. The hippocampal oxidative damage was detected by oxyblot technique and immunohistochemical staining against DT3 and 4-HNE. The cognitive ability of mice was evaluated using Morris water maze. RESULTS: Methysticin treatment activated the Nrf2 pathway in the hippocampus and cortex of mice. The Aß deposition in brains of methysticin-treated APP/Psen1 mice was not altered compared to untreated mice. However, methysticin treatment significantly reduced microgliosis, astrogliosis and secretion of the pro-inflammatory cytokines TNF-α and IL-17A. In addition, the oxidative damage of hippocampi from APP/Psen1 mice was reduced by methysticin treatment. Most importantly, methysticin treatment significantly attenuated the long-term memory decline of APP/Psen1 mice. CONCLUSION: In summary, these findings show that methysticin administration activates the Nrf2 pathway and reduces neuroinflammation, hippocampal oxidative damage and memory loss in a mouse model of AD. Therefore, kavalactones might be suitable candidates to serve as lead compounds for the development of a new class of neuroprotective drugs.

DYRK protein kinases.

Soppa and Becker introduce the DYRK family of dual specificity protein kinases and their diverse functions and associations with genetic diseases.

DYRK1A: a potential drug target for multiple Down syndrome neuropathologies.

Down syndrome (DS), the most common genetic cause of intellectual disability, is caused by the trisomy of chromosome 21. MNB/DYRK1A (Minibrain/dual specificity tyrosine phosphorylation-regulated kinase 1A) has possibly been the most extensively studied chromosome 21 gene during the last decade due to the remarkable correlation of its functions in the brain with important DS neuropathologies, such as neuronal deficits, dendrite atrophy, spine dysgenesis, precocious Alzheimer's-like neurodegeneration, and cognitive deficits. MNB/DYRK1A has become an attractive drug target because increasing evidence suggests that its overexpression may induce DS-like neurobiological alterations, and several small-molecule inhibitors of its protein kinase activity are available. Here, we summarize the functional complexity of MNB/DYRK1A from a DS-research perspective, paying particular attention to the capacity of different MNB/DYRK1A inhibitors to reverse the neurobiological alterations caused by the increased activity of MNB/DYRK1A in experimental models. Finally, we discuss the advantages and drawbacks of possible MNB/DYRK1A-based therapeutic strategies that result from the functional, molecular, and pharmacological complexity of MNB/DYRK1A.

The Down syndrome-related protein kinase DYRK1A phosphorylates p27(Kip1) and Cyclin D1 and induces cell cycle exit and neuronal differentiation.

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G1 phase. Sustained overexpression of DYRK1A induced G0 cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27(Kip1) on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27(Kip1) Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.

NGF upregulates the plasminogen activation inhibitor-1 in neurons via the calcineurin/NFAT pathway and the Down syndrome-related proteins DYRK1A and RCAN1 attenuate this effect.

BACKGROUND: Plasminogen activator inhibitor 1 (PAI-1) is a key regulator of the plasminogen activation system. Although several lines of evidence support a significant role of PAI-1 in the brain, the regulation of its expression in neurons is poorly understood. In the present study we tested the hypothesis that NGF induces the upregulation of PAI-1 via the calcineurin/nuclear factor of activated T cells (NFAT) pathway and analysed whether the overexpression of the Down syndrome-related proteins DYRK1A and RCAN1 modulated the effect of NGF on PAI-1 expression. RESULTS: NGF upregulated PAI-1 mRNA levels in primary mouse hippocampal neurons cultured for 3 days in vitro and in the rat pheochromocytoma cell line PC12. Reporter gene assays revealed that NGF activated the calcineurin/NFAT pathway in PC12 cells. Induction of PAI-1 by NGF was sensitive to the calcineurin inhibitor FK506 and the specific inhibition of NFAT activation by the cell permeable VIVIT peptide. Activation of calcineurin/NFAT signalling through other stimuli resulted in a much weaker induction of PAI-1 expression, suggesting that other NGF-induced pathways are involved in PAI-1 upregulation. Overexpression of either DYRK1A or RCAN1 negatively regulated NFAT-dependent transcriptional activity and reduced the upregulation of PAI-1 levels by NGF. CONCLUSION: The present results show that the calcineurin/NFAT pathway mediates the upregulation of PAI-1 by NGF. The negative effect of DYRK1A and RCAN1 overexpression on NGF signal transduction in neural cells may contribute to the altered neurodevelopment and brain function in Down syndrome.

Sulforaphane has opposing effects on TNF-alpha stimulated and unstimulated synoviocytes.

INTRODUCTION: Rheumatoid arthritis (RA) is characterized by progressive inflammation associated with rampantly proliferating synoviocytes and joint destruction due to oxidative stress. Recently, we described nuclear factor erythroid 2-related factor 2 (Nrf2) as a major requirement for limiting cartilage destruction. NF-κB and AP-1 are the main transcription factors triggering the inflammatory progression in RA. We used sulforaphane, an isothiocyanate, which is both an Nrf2 inducer and a NF-κB and AP-1 inhibitor. METHODS: Cultured synoviocytes were stimulated with sulforaphane (SFN) with or without TNF-α pre-treatment. NF-κB, AP-1, and Nrf2 activation was investigated via dual luciferase reporter gene assays. Matrix metalloproteinases (MMPs) were measured via zymography and luminex technique. Cytokine levels were detected using ELISA. Cell viability, apoptosis and caspase activity were studied. Cell proliferation was analysed by real-time cell analysis. RESULTS: SFN treatment decreased inflammation and proliferation dose-dependently in TNF-α-stimulated synoviocytes. SFN did not reduce MMP-3 and MMP-9 activity or expression significantly. Interestingly, we demonstrated that SFN has opposing effects on naïve and TNF-α-stimulated synoviocytes. In naïve cells, SFN activated the cytoprotective transcription factor Nrf2. In marked contrast to this, SFN induced apoptosis in TNF-α-pre-stimulated synoviocytes. CONCLUSIONS: We were able to show that SFN treatment acts contrary on naïve and inflammatory synoviocytes. SFN induces the cytoprotective transcription factor Nrf2 in naïve synoviocytes, whereas it induces apoptosis in inflamed synoviocytes. These findings indicate that the use of sulforaphane might be considered as an adjunctive therapeutic strategy to combat inflammation, pannus formation, and cartilage destruction in RA.

Harmine specifically inhibits protein kinase DYRK1A and interferes with neurite formation.

DYRK1A is a dual-specificity protein kinase that autophosphorylates a conserved tyrosine residue in the activation loop but phosphorylates exogenous substrates only at serine or threonine residues. Tyrosine autophosphorylation of DYRKs is a one-off event that takes place during translation and induces the activation of the kinase. Here we characterize the beta-carboline alkaloid harmine as a potent and specific inhibitor of DYRK1A both in vitro and in cultured cells. Comparative in vitro assays of four kinases of the DYRK family showed that harmine inhibited substrate phosphorylation by DYRK1A more potently than it inhibited substrate phosphorylation by the closely related kinase DYRK1B [half maximal inhibitory concentrations (IC(50)) of 33 nm versus 166 nm, respectively] and by the more distant members of the family, DYRK2 and DYRK4 (1.9 microm and 80 microm, respectively). Much higher concentrations of harmine were required to suppress tyrosine autophosphorylation of the translational intermediate of DYRK1A in a bacterial in vitro translation system (IC(50) = 1.9 microm). Importantly, harmine inhibited the phosphorylation of a specific substrate by DYRK1A in cultured cells with a potency similar to that observed in vitro (IC(50) = 48 nm), without negative effects on the viability of the cells. Overexpression of the DYRK1A gene on chromosome 21 has been implicated in the altered neuronal development observed in Down syndrome. Here, we show that harmine interferes with neuritogenesis in cultured hippocampal neurons. In summary, our data show that harmine inhibits DYRK1A substrate phosphorylation more potently than it inhibits tyrosine autophosphorylation, and provide evidence for a role of DYRK1A in the regulation of neurite formation.