| Dendritic cells(DCs)are one of the most important unique antigen presenting cells(APCs),which trigger CD8+T cells that are response for anti-tumor and defense against microbe infection.Our preliminary experiments suggested that nicotine,a N-acetylcholine receptor antagonist,promotes CD8+DCs mediated cross-presentation and thus strengthen latter anti-tumor effects.As an essential pattern recognition receptors on DC surface,mannose receptors(MRs)mediated antigen endocytosisis a key step of CD8+DCs mediated cross-presentation.It is reported that OVA stimulation can promote the K48 polyubiquitination of MR and promote the localization of the antigen to the endosome.Our further investigation revealed that nicotine stimulation not only up-regulated the expression of MR but also promoted the translocation of endocytosis to the endosome.Up to now,however,the potential ubiquitination sites or critical E3 ligase during K48 poly-ubiquitination of MR still remains to be elucidated.To address this goal,Real-time PCR and Western Blotting were employed to screen the probable E3 ligase targeting MR K48 polyubiquitination.Firstly,we demonstrate that MR undergoes Cbl-dependent ubiquitinylation under conditions of nicotine stimuli.Nextly,the location of Cbl and its interaction with MR were assessed via Co-Immunoprecipitation.And the effects of Cbl gene silencing on K48 polyubiquitination of MR were detected via Co-immunoprecipitation.At last,we analysed the effect of cross-presentation product SIINFEKL-H2Kb formation by Flow Cytometry,confocal assay under conditions of Cbl deleption.Our results showed that low dose nicotine up-regulates C-Cbl and Cbl-c expression,and efficiently activation of Cbl was observed.Most importantly,MR interaction and endosome location of Cbl were efficiently promoted by nicotine stimuli.Cbl deleption reverses nicotine-enhanced MR K48 polyubiquitination,and reduces cross-presentation of CD8+DCs.In conclusion,we report that the key E3 ligase in K48 polyubiquitination of MR.Thus,we illustrated the underlying mechnism that nicotine promotes K48 poly-ubiquitination of MR and strengthens cross-presentation of CD8+DC. |
