Abstract
Cyclosporine-A (CyA) and FK506 are potent immunosuppressive agents because of their ability to suppress the production of Th1 cytokines including interleukin (IL)-12. However, the mechanisms underlying the inhibitory effects of CyA and FK506 on the production of IL-12p40, a critical component of IL-12, remain unknown. Both CyA and FK506 are potent inhibitors of calcineurin in the calcium signaling pathway. Interestingly, calcium and phosphoinositide 3-kinase (PI3K) signaling pathways have been shown to negatively regulate lipopolysaccharide (LPS)-induced murine IL-12p40 production. Contrary to these observations, we show that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by the calcium pathway and in particular by calmodulin-(CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated PI3K. Furthermore, LPS-induced IL-12p40 production was regulated by the p110alpha catalytic subunit of PI3K. Moreover, LPS induced IL-12p40 production through the CaM/CaMK-II-activated NFkappaB and AP-1 transcription factors. LPS-induced IL-12p40 production is known to be regulated by the c-Jun N-terminal kinase (JNK) pathway. Importantly, both CyA and FK506 down-regulated LPS-induced IL-12p40 transcription by inhibiting CaM/CaMK-II-activated PI3K and their downstream transcription factors NFkappaB and AP-1 independent of the JNK pathway.
Highlights
We show that LPS-induced IL-12p40 production in human monocytic cells is positively regulated by the calcium pathway and in particular by calmodulin- (CaM) and CaM-dependent protein kinase-II (CaMK-II)-activated phosphoinositide 3-kinase (PI3K)
Since PI3K is involved in TLR-4-mediated signaling and Akt has been shown to regulate c-Raf (ERK), p38, and Jun-N-terminal kinase (JNK) mitogen-activated protein kinases [52, 53], we examined whether activation of calmodulin/calmodulin-dependent protein kinase (CaMK)-II, PI3K, and JNK by LPS constitute distinct pathways in IL-12p40 regulation
Our results show that calcium signaling through CaM and CaMK-II, as well as through PI3K positively regulated LPS-induced IL-12p40 production in human monocytic cells
Summary
Isolation of Monocytes from PBMCs, Cell Lines, Cell Culture, and Reagents—Monocytes from PBMCs of healthy blood donors were isolated as described previously [20]. Equal aliquots (5 l) of cDNA equivalent to 100 ng of RNA were subsequently amplified for IL-12p40 (373 bp) and -actin (663 bp) using their respective primers as described [20]. Cells were analyzed for Ca2ϩ levels in the presence and the absence of LPS by a FACScan flow cytometer (BD Biosciences) equipped with CellQuest software, version 3.2.1 fl. The mixture was incubated with 3 l of FuGENe 6 in 50 l of IMDM to allow formation of DNA-liposome complexes These complexes were added to the cell suspension in each well. After 24 h, cells were stimulated with LPS for another 24 h followed by analysis of luciferase and
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
