Abstract Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by β1 integrins. The biochemical pathways that control CD3-mediated increases in β1 integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of β1 integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase β1 integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the β1 integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interestingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phosphorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in β1 integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of β1 integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of β1 integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation.
We previously have reported that SRG3 is required for glucocorticoid (GC)-induced apoptosis in the S49.1 thymoma cell line. Activation of Notch1 was shown to induce GC resistance in thymocytes. However, the specific downstream target of Notch1 that confers GC resistance on thymocytes is currently unknown. We found that the expression level of SRG3 was critical in determining GC sensitivity in developing thymocytes. The expression of SRG3 also was down-regulated by the activated form of Notch1 (NotchIC). The promoter activity of the SRG3 gene also was down-regulated by NotchIC. Expression of transgenic SRG3 resulted in the restoration of GC sensitivity in thymocytes expressing transgenic Notch1. These results suggest that SRG3 is the downstream target of Notch1 in regulating GC sensitivity of thymocytes.
SRG3 (SWI3-related gene) is a core subunit of mouse SWI/SNF complex and is known to play a critical role in stabilizing the SWI/SNF complex by attenuating its proteasomal degradation. SWI/SNF chromatin remodeling complex is reported to act as an important endogenous regulator in the proliferation and differentiation of mammalian neural stem cells. Because limited expression of SRG3 occurs in the brain and thymus during mouse embryogenesis, it was hypothesized that the altered SRG3 expression level might affect the process of adult hippocampal neurogenesis. Due to the embryonic lethality of homozygous knockout mice, this study focuses on dissecting the effect of overexpressed SRG3 on adult hippocampal neurogenesis. The BrdU incorporation assay, immunostaing with neuronal markers for each differentiation stage, and imunoblotting analysis with intracellular molecules involved in survival in adult hippocampal neurogenesis found no alteration, suggesting that the overexpression of SRG3 protein in mature neurons had no effect on the entire process of adult hippocampal neurogenesis including proliferation, differentiation, and survival.
447 The need for development of an effective therapeutic agent to estrogen receptor (ER)-negative breast cancer cells has been recognized because they are more aggressive and resistant to conventional hormonal therapy. Decursin ((S)-8,8-dimethyl-2-oxo-7,8-dihydro-2H,6H-pyrano(3,2-g)chromen-7-yl 3-methyl-2-butenoic acid), a naturally existing coumarin compound originated from the root of Korean angelica, has been shown to possess therapeutic potential in various cancers including prostate cancer and leukemia. Thus, we tested the anti-tumor effect of decursin in ER-negative breast cancer cell lines, MDA-MB-231 and SK-BR-3, which overexpress epidermal growth factor receptor (EGFR) and c-ErbB2, respectively. Cell growth was attenuated by decursin (50-200μM) in a time- and dose-dependent manner. Under these conditions, apoptosis was induced as evidenced by increased sub G1 population, enhanced nuclear fragmentation, up-regulation of Bax and cleaved Bid proteins, down-regulation of Bcl-2 protein. Decursin also affected signaling pathways involved in cell growth and proliferation. The expression of membrane receptors, EGFR and c-ErbB2, were reduced and their downstream effectors, Akt and Erk, were also downregulated as evidenced by reduced phosphorylated proteins. Suppressed Akt activity was seen by reduced phosphorylation of its substates, Bad, p27Kip1 and GSK-3β. Total expression of Bad was also enhanced and such changes in Bad may have contributed to augmented apopotosis by decursin. On the other hand, increased nuclear localization of p27Kip1 was observed, which was consist with reduced phosphorylation at T157 and may have provided for attenuated cell proliferation. Total β-catenin protein as well as Cyclin D1 protein and mRNA were reduced. Moreover, cytoplasmic localization of β-catenin was increased. Therefore, these results strongly suggest the suppression Akt-mediated Wnt/β-catenin signaling pathway by decursin. Finally, the in vivo anti-tumor effect of decursin was seen in the mouse xenograft model. When MDA-MB-231 or SK-BR-3 cells were engrafted into nude mice by subcutaneous injection, tumor growth was significantly reduced in decursin-administered mice orally for 28 days. By showing the anti-cancer effect of decursin both in vitro and in vivo and suggest cell cycle arrest and apoptosis as a molecular mechanism, our novel findings provide decursin as a potential preventive or therapeutic agent against the more aggressive ER-negative breast cancers through reduced Akt activity and downregulation of EGFR and c-ErbB2.
Autism or autism spectrum disorder (ASD) is a behavioral syndrome characterized by persistent deficits in social interaction, and repetitive patterns of behavior, interests, or activities. The gene encoding Methyl-CpG binding protein 2 (MeCP2) is one of a few exceptional genes of established causal effect in ASD. Although genetically engineered mice studies may shed light on how MeCP2 loss affects synaptic activity patterns across the whole brain, such studies are not considered practical in ASD patients due to the overall level of impairment, and are technically challenging in mice. For the first time, we show that hippocampal MeCP2 knockdown produces behavioral abnormalities associated with autism-like traits in rats, providing a new strategy to investigate the efficacy of therapeutics in ASD. Ketamine, an N-Methyl-D-aspartate (NMDA) blocker, has been proposed as a possible treatment for autism. Using the MeCP2 knockdown rats in conjunction with a rat model of valproic acid (VPA)-induced ASD, we examined gene expression and ASD behaviors upon ketamine treatment. We report that the core symptoms of autism in MeCP2 knockdown rats with social impairment recovered dramatically following a single treatment with ketamine. [BMB Reports 2022; 55(5): 238-243].
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