MicroRNA-199b targets the nuclear kinase Dyrk1a in an auto-amplification loop promoting calcineurin/NFAT signalling

MicroRNAs (miRs) are a class of single-stranded, non-coding RNAs of about 22 nucleotides in length. Increasing evidence implicates miRs in myocardial disease processes. Here we show that miR-199b is a direct calcineurin/NFAT target gene that increases in expression in mouse and human heart failure, and targets the nuclear NFAT kinase dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1a (Dyrk1a), constituting a pathogenic feed forward mechanism that affects calcineurin-responsive gene expression. Mutant mice overexpressing miR-199b, or haploinsufficient for Dyrk1a, are sensitized to calcineurin/NFAT signalling or pressure overload and show stress-induced cardiomegaly through reduced Dyrk1a expression. In vivo inhibition of miR-199b by a specific antagomir normalized Dyrk1a expression, reduced nuclear NFAT activity and caused marked inhibition and even reversal of hypertrophy and fibrosis in mouse models of heart failure. Our results reveal that microRNAs affect cardiac cellular signalling and gene expression, and implicate miR-199b as a therapeutic target in heart failure.     

Synthesis and biological activities of aminopyrimidyl-indoles structurally related to meridianins

The synthesis of new meridianin derivatives substituted at the C-5 position of the 2-aminopyrimidine ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described. These compounds were tested for their kinase inhibitory potencies toward five kinases (CDK5/p25, CK1δ/ε, GSK-3α/β, Dyrk1A and Erk2) as well as their in vitro antiproliferative activities toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).     

Small-molecule pyrimidine inhibitors of the cdc2-like (Clk) and dual specificity tyrosine phosphorylation-regulated (Dyrk) kinases: Development of chemical probe ML315

Substituted pyrimidine inhibitors of the Clk and Dyrk kinases have been developed, exploring structure–activity relationships around four different chemotypes. The most potent compounds have low-nanomolar inhibitory activity against Clk1, Clk2, Clk4, Dyrk1A and Dyrk1B. Kinome scans with 442 kinases using agents representing three of the chemotypes show these inhibitors to be highly selective for the Clk and Dyrk families. Further off-target pharmacological evaluation with ML315, the most selective agent, supports this conclusion.     

Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment

Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca²⁺ signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca²⁺ was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca²⁺] and environmental temperatures. Therefore, Ca²⁺ signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.     

Regulation of Dyrk1A kinase activity by 14-3-3

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.     

Dyrk1 inhibition improves Alzheimer's disease‐like pathology

There is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual‐specificity tyrosine phosphorylation‐regulated kinase‐1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD‐like pathology developed by 3xTg‐AD mice, a widely used animal model of AD. We dosed 10‐month‐old 3xTg‐AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1‐inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg‐AD mice. These effects were associated with a reduction in amyloid‐β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.     

QSAR analysis of pyrazolidine-3,5-diones derivatives as Dyrk1A inhibitors

Individuals with Down syndrome (DS) suffer from mental retardation. Overexpression and the resulting increased specific activity of Dyrk1A kinase located on chromosome 21 cause a learning and memory deficit in Dyrk1A transgenic mice. To search for therapeutic agents with Dyrk1A inhibition activity, previously we obtained HCD160 as a new hit compound for Dyrk1A inhibition. In the present study, we synthesized 34 HCD160 derivatives to investigate the quantitative structure–activity relationship (QSAR). This analysis could provide important information for novel drug discovery for treatment of DS related learning and memory deficits.     

Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer

Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.