Discovery of novel Jak2–Stat pathway inhibitors with extended residence time on target

The discovery of the activating mutation V617F in the JH2 domain of Jak2 and the modulation of oncogenic Stat3 by Jak2 inhibitors have spurred a great interest in the inhibition of the Jak2/Stat pathway in oncology. In this Letter, we communicate the discovery of novel inhibitors of the Jak2/Stat5 axis, the N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives. The rationale, synthesis and biological evaluation of these derivatives are reported. Two lead analogs from this series, 6 and 9, displayed prolonged residence time on Jak2, at enzymatic level. Although 6 and 9 exhibited moderate selectivity in a selected kinase panel, we chose to test these inhibitors in vivo as a consequence to their long residence time. However, extended inhibition of Jak2 due to the long residence time, in the form of inhibiting phosphorylation of downstream Stat5, was not recapitulated in an in vivo setting.     

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γ_c chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.     

A non-catalytic function of the Src family tyrosine kinases controls prolactin-induced Jak2 signaling

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or Src?K, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src^?/? mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.     

The role of Jak/STAT signaling in heart tissue renin-angiotensin system

The involvement of the Renin Angiotensin System (RAS) and the role of its primary effector, angiotensin II (Ang II), in etiology of myocardial hypertrophy and ischemia is well documented. In several animal models, the RAS is activated in cardiac cell types that express the receptor AT₁, and/or AT₂, through which the Ang II mediated effects are promoted. In this article, we briefly review recent experimental evidence on the critical role of a prominent signaling pathway, the Jak/Stat pathway in activation and maintenance of the local RAS in cardiac hypertrophy and ischemia. Recent studies in our laboratory document that the promoter of the prohormone angiotensinogen (Ang) gene serves as the target site for STAT proteins, thereby linking the Jak/Stat pathway to activation of heart tissue autocrine Ang II loop. Stat5A and Stat6, are selectively activated when the heart is subjected to ischemic injury, whereas activation of Stat3 and Stat5A is involved in myocardial hypertrophy. Blockage of RAS activation by treatment with specific inhibitor promotes a remarkable recovery in functional hemodynamics of the myocardium. Thus, activation of selective sets of Stat proteins constitutes the primary signaling event in the pathogenesis of myocardial hypertrophy and ischemia.     

Sesquiterpene dimer (DSF-52) from Artemisia argyi inhibits microglia-mediated neuroinflammation via suppression of NF-κB,JNK/p38 MAPKs and Jak2/Stat3 signaling pathways

Microglia-involved neuroinflammation is thought to promote brain damage in various neurodegenerative disorders. Therefore, novel therapeutics suppressing microglia over-activation could prove useful for neuroprotection in inflammation-mediated neurodegenerative diseases. DSF-52 is a novel sesquiterpene dimer compound isolated from medical plant Artemisia argyi by our group. In this study, we investigated whether DSF-52 inhibited the neuroinflammatory responses in lipopolysaccharide (LPS)-activated microglia. Our findings showed that DSF-52 inhibited the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), as well as mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein-1α (MIP-1α) in LPS-activated BV-2 microglia. Moreover, DSF-52 markedly up-regulated mRNA levels of anti-inflammatory cytokine IL-10. Mechanism study indicated that DSF-52 suppressed Akt/IκB/NF-κB inflammation pathway against LPS treatment. Also, DSF-52 down-regulated the phosphorylation levels of JNK and p38 MAPKs, but not ERK. Furthermore, DSF-52 blocked Jak2/Stat3 dependent inflammation pathway through inhibiting Jak2 and Stat3 phosphorylation, as well as Stat3 nuclear translocation. We concluded that the inhibitory ability of DSF-52 on microglia-mediated neuroinflammation may offer a novel neuroprotective modality and could be potentially useful in inflammation-mediated neurodegenerative diseases.     

Akt regulates IL-10 mediated suppression of TNFα-induced cardiomyocyte apoptosis by upregulating Stat3 phosphorylation

Background

We have already reported that TNF-α increases cardiomyocyte apoptosis and IL-10 treatment prevented these effects of TNF-α. Present study investigates the role of Akt and Jak/Stat pathway in the IL-10 modulation of TNF-α induced cardiomyocyte apoptosis.

Methodology/Principal findings

Cardiomyocytes isolated from adult Sprague Dawley rats were exposed to TNF-α (10 ng/ml), IL-10 (10 ng/ml) and TNF-α+IL-10 (ratio 1) for 4 h. Exposure to TNF-α resulted in an increase in cardiomyocyte apoptosis as measured by flow cytometry and TUNEL assay. IL-10 by itself had no effect, but it prevented TNF-α induced apoptosis. IL-10 treatment increased Akt levels within cardiomyocytes and this change was associated with an increase in Jak1 and Stat3 phosphorylation. Pre-exposure of cells to Akt inhibitor prevented IL-10 induced Stat3 phosphorylation. Furthermore, in the presence of Akt or Stat3 inhibitor, IL-10 treatment was unable to block TNF-α induced cardiomyocyte apoptosis.

Conclusion

It is suggested that IL-10 modulation of TNF-α induced cardiomyocyte apoptosis is mediated by Akt via Stat3 activation.     

Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe

The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. Our findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions.     

Loss of Jak2 Selectively Suppresses DC-Mediated Innate Immune Response and Protects Mice from Lethal Dose of LPS-Induced Septic Shock

Given the importance of Jak2 in cell signaling, a critical role for Jak2 in immune cells especially dendritic cells (DCs) has long been proposed. The exact function for Jak2 in DCs, however, remained poorly understood as Jak2 deficiency leads to embryonic lethality. Here we established Jak2 deficiency in adult Cre^+/+Jak2^fl/fl mice by tamoxifen induction. Loss of Jak2 significantly impaired DC development as manifested by reduced BMDC yield, smaller spleen size and reduced percentage of DCs in total splenocytes. Jak2 was also crucial for the capacity of DCs to mediate innate immune response. Jak2^−/− DCs were less potent in response to inflammatory stimuli and showed reduced capacity to secrete proinflammatory cytokines such as TNFα and IL-12. As a result, Jak2^−/− mice were defective for the early clearance of Listeria after infection. However, their potency to mediate adaptive immune response was not affected. Unlike DCs, Jak2^−/− macrophages showed similar capacity secretion of proinflammatory cytokines, suggesting that Jak2 selectively modulates innate immune response in a DC-dependent manner. Consistent with these results, Jak2^−/− mice were remarkably resistant to lethal dose of LPS-induced septic shock, a deadly sepsis characterized by the excessive innate immune response, and adoptive transfer of normal DCs restored their susceptibility to LPS-induced septic shock. Mechanistic studies revealed that Jak2/SATA5 signaling is pivotal for DC development and maturation, while the capacity for DCs secretion of proinflammatory cytokines is regulated by both Jak2/STAT5 and Jak2/STAT6 signaling.