sq-23377 and 3-3--4-5--tetrahydroxystilbene

Studies of patients with congenital immunodeficiency due to mutation of the NF-kappaB essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-kappaB activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IkappaB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-kappaB activation. NK cell IkappaB degradation corresponded to increases in nuclear NF-kappaB as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-kappaB used. NF-kappaB function was evaluated in NK92 cells transduced with a kappaB GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-kappaB dependent proteins during the initiation of cytotoxicity.

Topics: Cell Line; Green Fluorescent Proteins; Humans; Ionomycin; K562 Cells; Killer Cells, Natural; Leupeptins; Natural Cytotoxicity Triggering Receptor 3; NF-kappa B; Receptors, Immunologic; Signal Transduction; Stilbenes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

A range of selective tyrosine kinase inhibitors, piceatannol, methyl-2,5-dihydroxycinnamate (MDC), genistein, psi-tectorigenin and lavendustin A, all reduced luteinizing hormone-releasing hormone (LHRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from pro-oestrous rat hemipituitaries incubated in vitro. In general, both 'initial' release and the augmented release resulting from LHRH self-priming, were reduced in parallel in a concentration-dependent fashion. The effects of piceatannol were independent of the steroidal status of the pituitary tissue. Both piceatannol and MDC greatly reduced LH release by ionomycin and a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), suggesting that the tyrosine kinase(s)-dependent step is in the later stages of the stimulus-secretion pathway activated by the LHRH receptor. These data were supported by immunoblots for phosphotyrosine showing that in the gonadotrope-derived alpha T3-1 cell line, treatment with LHRH caused piceatannol-sensitive increases in specific tyrosine phosphorylation of several proteins (major bands at 65-75 and 120-130 kDa). Treatment of cells with PDBu mimicked the tyrosine phosphorylations evoked by LHRH whereas the PKC inhibitor, GF109203X, partially reduced both LHRH- and PDBu-induced tyrosine phosphorylations. Direct effects of MDC and piceatannol on PKC were assessed in an in vitro PKC assay; piceatannol, but not MDC, inhibited PKC activity but at considerably higher concentrations than required for inhibition of LHRH-induced gonadotropin secretion. These data support a role for tyrosine kinase activation in LHRH-induced secretion.

Topics: Animals; Cinnamates; Female; Follicle Stimulating Hormone; Genistein; Gonadotropin-Releasing Hormone; In Vitro Techniques; Ionomycin; Isoflavones; Luteinizing Hormone; Phenols; Phorbol 12,13-Dibutyrate; Pituitary Gland, Anterior; Proestrus; Protein-Tyrosine Kinases; Rats; Stilbenes