Lithium inhibits tryptophan catabolism via the inflammation-induced kynurenine pathway in human microglia

SummaryActivation of the kynurenine pathway may lead to depletion of the serotonin precursor tryptophan, which has been implicated in the neurobiology of depression. This study describes a mechanism whereby lithium inhibits inflammatory tryptophan breakdown. Upon activation, immortalized human microglia showed a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3βS9 phosphorylation and reducing STAT1S727 and STAT3Y705 phosphorylation levels in activated cells. Experiments in primary human microglia and human induced pluripotent stem cell (hiPSC)-derived microglia corroborated lithium’s effects. Moreover, IDO activity was reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1S727 and P-STAT3Y705. Our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.

Inhibition of GSK-3β restores delayed gastric emptying in obesity-induced diabetic female mice

Inhibition of glycogen synthase kinase 3β (GSK-3β) with SB 216763 attenuates delayed gastric emptying through gastric nuclear factor erythroid 2-related factor 2 (Nrf2) -phase II enzymes in high-fat diet-fed female mice. SB 216763 restored impaired gastric PI3K/AKT/ β-catenin/caspase 3 expression. Inhibition of GSK-3β normalized gastric dihydrofolate reductase, neuronal nitric oxide synthase-α expression, dimerization and nitrergic relaxation. SB 216763 normalized both serum estrogen and nitrate levels in female obese/Type 2 diabetes mice. SB 216763 reduced downstream signaling of GSK-3β in enteric neuronal cells in vitro.

Newly Synthesized Indolylmalemide PDA-66 and Its Derivates Induce Antiproliferative Effects In Acute Lymphoblastic Leukemia

The PI3K/Akt pathway is dysregulated is some acute lymphoblastic leukemias (ALL) and might therefore serve as therapeutic target. Indolylmaleimides exhibit inhibitory potencies against different protein kinases -like glycogen synthase kinase 3 (GSK3β) or protein kinase c- influencing thereby several cellular processes. Recently, it was demonstrated that PDA-66, a newly synthesized indolylmalemide based on the well known GSK3β inhibitor SB-216763, hinders microtubule polymerization in human neuronal progenitor and neuroblastoma cells. GSK3β is a downstream substrate of the PI3K/Akt and Wnt pathways and is often deregulated in tumor tissues. Herein, we investigated the effects of PDA-66 and its derivates (PDA-66E und PDA-377) on B and T-lymphoblastic leukemia cells. Methods B- and T-ALL cell lines (SEM, RS4;11, REH, Jurkat, MOLT-4 and CEM) were incubated for 72 h with increasing concentrations (0.1 µM-5.0 µM) of PDA-66, PDA-66E, PDA-377 and comparatively analyzed to SB-216763. To evaluate the effect of each substance WST-1 assay, cell proliferation, cell cycle analyses as well as apoptosis rates were determined. Activities of indolylmalemides were analyzed by GSK3β kinase assay. Detection of key molecules of Wnt and PI3K/Akt signaling pathway was performed using Western blot. Results PDA-66 and derivates inhibited proliferation and metabolism of ALL cells significantly in a dose dependent manner. Interestingly, all PDA derivates showed a stronger inhibitory effect on proliferation than SB-216763 but the inhibitory effect on GSK3β kinase was lower than SB-216763. Antiproliferative effects of PDA-66 were studied in more detail. The incubation of 1 µM PDA-66 led to condensation of chromatin in the nucleus, karyorrhexis and an increasing amount of vacuoles after 48 h of treatment. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT-4 cells were arrested in G2 phase, SEM cells remained increasingly in G0/1 phase. After 48 h all PDA-66 treated cell lines showed a significant increase in apoptosis compared to control cells (SEM: 2.1 ± 0.9 % to 10.5 ± 1.3 %; RS4;11: 2.5 ± 0.7 % to 7.4 ± 1.1 %; Jurkat: 3.8 ± 0.6 % to 8.3 ± 1.9 %; MOLT-4: 3.7 ± 1.2 % to 16.3 ± 5.1 %). Apoptosis of ALL cells was initiated by cleavage of caspase 3 and 7 and Poly (ADP-ribose) polymerase (PARP). Furthermore, with increasing concentration of PDA-66 a decrease of pGSK3βSer9 was observed after 4 h in SEM cells. However, no influence on the total form of β-catenin was detectable. Nevertheless, there was an influence of PDA-66 on the expression of 4EBP-1 and p4EBP-1Ser65. SEM, RS4;11 and Jurkat cells showed a decrease of the phosphorylated as well as the total form of 4EBP-1 after an incubation of 4 and 24 h. In conclusion, our results demonstrate that the newly synthesized indolymalemides have pronounced antiproliferative effects in ALL cells. In particular, the indolymalemide PDA-66 should be further investigated concerning it’s clinical efficiency as well as well as it's intracellular ways of action. Disclosures: No relevant conflicts of interest to declare.

TNF-induced activation of pulmonary microvessel endothelial cells: a role for GSK3β

The hypothesis tested was PKCα mediates the phosphorylation of glycogen synthetase kinase 3β (GSK3β) and that the GSK3β inhibition modulates the response to tumor necrosis factor-α (TNF) in rat pulmonary microvessel endothelial cells (PMEC). PMEC were treated with TNF for 4.0 h (100 ng/ml) or vehicle. First, to assess the role of PKCα in the phosphorylation of GSK3β (i.e., an indicator of GSK3β inhibition), PMEC were pretreated with 1) nonsense-RNA-PKCα, 2) siRNA-PKCα, and 3) the PKC inhibitor Gö6983. In the nonsense RNA-PKCα+TNF and TNF groups, there was increased phosphorylated GSK3β-Ser9 that did not occur in the Gö6983+TNF group. In the TNF groups, there was a significant correlation between PKCα protein and phosphorylated GSK3β-Ser9 that did not occur in the groups without TNF. Second, to assess the role of GSK3β in β-catenin activity, PMEC were pretreated with 1) wild-type (w) GSK3β plasmid to enhance GSK3β activity, 2) kinase dead (kd)-GSK3β plasmid, and 3) the GSK3β inhibitor SB-216763. In the TNF group, there was increased unphosphorylated β-catenin-Ser37/33 compared with the control group. In the GSK3β-inhibited groups (i.e., SB-216763 and kdGSK3β) ± TNF, the unphosphorylated β-catenin-Ser37/33 was similar to the TNF group. In the GSK3β-enhanced group ± TNF, the unphosphorylated β-catenin-Ser37/33 was similar to the control. Finally, PMEC were also treated with TOPflash, a β-catenin-dependent promoter luciferase reporter, or the mutant construct FOPflash, 2 days before treatment with TNF. In the TNF group, there was an increased TOPflash/FOPflash activity ratio compared with the control group. In the GSK3β-inhibited groups (i.e., SB-216763 and kdGSK3β) ± TNF, the TOPflash/FOPflash activity ratio was similar to the TNF group. In the GSK3β-enhanced group ± TNF, the TOPflash/FOPflash activity ratio was similar to the control. The data indicate that TNF induces endothelial activation that is modulated by a PKCα-dependent inhibition of GSK3β.