In our experiments, both basal as well as NECA induced LIF gene expression and release selleckchem Axitinib in cultured astrocytes were inhibited by spe cific inhibitors of p38 and ERK1 2, but not JNK MAPKs. In line with our findings, it has been shown that LIF ex pression in Schwann cells is mediated through PKC pathway induced ERK1 2 activation. Furthermore, we show here that adenosine dependent LIF expression in astrocytes is regulated through the NF ��B transcrip tion factor. This observation is in line with several stud ies showing an NF ��B dependent regulation of IL 6 gene by this transcription factor in several cell types. It has been shown that NECA induced NF ��B activation and the resultant IL 6 gene expression was abolished by inhibitors of MAPK pathways.
In our study, preliminary observations indicate that NECA induced activation of the NF ��B pathway is reduced by selective inhibitors Inhibitors,Modulators,Libraries of p38 and ERK1 2 pathways, suggesting that these pathways might play as upstream mediators in NF ��B dependent LIF expres sion in astrocytes. Recent evidence indicates that, depending on the cell type, different secretory pathways are employed Inhibitors,Modulators,Libraries for cyto kine release. For example, T cells use two different release mechanisms, IL 2 and IFN are secreted at the immunological synapse whereas CCL3 and TNF are secreted multidirectionally, suggesting different secretory pathways. In neurons or neuron like cells, secretory granules called LDCVs are the organelles used for the selective secretion of IL 6, TGF B2 and CCL21. The same organelles are also used in immune cells such as mast cells and neutrophils.
Here we show that LIF protein Inhibitors,Modulators,Libraries is transported through Golgi but its secretion by astrocytes is not mediated by secretory granules. Instead, LIF co localizes with Rab11, a known marker of recycling endosomes. Moreover, we observed a partial co localization of LIF with clathrin, which also associates with recycling endosomes where it is implicated in protein sorting. Recycling endo somes have now been shown to be responsible Inhibitors,Modulators,Libraries for cyto kine secretion in several cell types. For example, IFN and TNF secretion from natural killer cells require Rab11. Recycling endosomes are also responsible for the constitutive secretion of IL 6 and TNF in macrophages. Further studies will be needed to bet ter understand LIF sorting, trafficking and release by these vesicles.
Inhibitors,Modulators,Libraries Interestingly, our data indicate that LIF is constitu tively released from astrocytes. Indeed constant levels of LIF were present in the supernatants of untreated astro cytes when measured by ELISA. Similar data were observed in human astrocyte cultures. Whether this observation is representative of the physiological behav ior of astrocytes in vivo selleck chem or is due to the culture condi tions remains to be determined. We further show that by blocking the early secretory pathway with BFA, the LIF concentration in the culture supernatant was not increased upon NECA stimulation.