The Intracellular Mechanism Researching For S5a Inducing THP-1Cells Differentiation

Angiocidin is a protein which was isolated from lung cancer cells, it can induce THP-1cells to differentiate toward macrophages, previous research indicated that MAPK,PI3K andNF-κB pathways mediate this effect. S5a is a subunit of26S proteasome enzyme complex,its protein sequence is consistent with angiocidin. Our previous research indicated that S5aalso could promote THP-1cells to differentiate towards macrophages. S5a can bind withdeath receptor6(DR6), then activate the NF-κ B pathway, thereby promoting thedifferentiation of THP-1cells. However, the intracellular mechanism of S5a inducing THP-1differentiation remains unknown. It is valuable to investigate the key hub genes duringTHP-1differentiation. Which molecule further transmitted the signal from activation of NF-κB pathway to the downstream change was also unknown. This article will demonstrate theintracellular mechanism of S5a inducing THP-1differentiation from the following twoaspects.Part Ⅰ: the key hub genes which mediate THP-1differentiation induced by S5a.In order to investigate which genes are the key hub genes during THP-1differentiation induced by S5a, firstly we used recominbant MBP-S5a to induce THP-1cells for12h, and cells treated with only MBP was a control. Subsequently, we usedaffymetrix microarray to detect the global gene expression after S5a treatment for12h, wetotally found994differential expressed genes. We performed pathway analysis with thesedifferential expressed genes, and found20pathways were significant during the THP-1cells differentiation. The20pathways contained174differential expressed genes. Weconstructed gene-gene interaction network with the174genes, and deleted some geneswhich lacked interaction with other genes. At last we obtained a network which contained102differential expressed genes, and we thought the network reflected the mechanism ofS5a inducing THP-1cells differentiation. Finally we obtained10key hub genes byselecting the genes whose degree value was on the top10.Part Ⅱ the research about how S5a further promoted THP-1differentiation afteractivation of NF-κB pathway.We found that the phosphorylation level of P65protein in THP-1cells significantlyincreased after S5a treatment, this phenomenon suggested that S5a activated NF-κBpathway. Our commercial biotin-S5a could also activate the NF-κB pathway. Our previous research indicated that the phagocytic capacity of THP-1nolonger significantly increasedafter NF-κB pathway inhibitor pretreated, even if S5a treated. We also found that cellsgrowth inhibition and high expression of inflammatory mediators decreased after NF-κBpathway was inhibited. Overall, we speculated S5a could activate NF-κB pathway andfurther mediated THP-1cells differentiation. We proved that the expression of transcriptionfactor wt1significantly increased at mRNA and protein level after S5a treatment. Thisup-regulation was blocked after NF-κB pathway was inhibited. After the up-regulation ofwt1was successfully blocked by wt1siRNA, cell growth inhibition and enhancedphagocytic capacity were significantly weakened even if S5a treated. It suggested that S5aincreased the expression of wt1by activation of NF-κB pathway, further mediated THP-1differentiation. We also found that the expression of c-myb significantly decreased duringS5a inducing THP-1differentiation. Previous research indicated high expression of c-mybblocked the differentiation of monocytes, and its down-regulation was a necessary conditionfor many kinds of cell differentiation, our results were consistent with theory expectations.The expression of c-myb was nolonger significantly down-regulated after NF-κB pathwayinhibitor treatment. The down-regulation of c-myb expression was also inhibited after wt1expression up-regulation was successfully blocked by wt1siRNA. ChIP-PCR showed thatwt1bound to the “cgcccccgc” sequence on the c-myb promoter after S5a treatment.Previous research indicated that wt1could suppress c-myb transcription by binding on thesequence. Overall, we concluded that S5a activated NF-κB pathway and increased wt1expression, further suppressed the transcription of c-myb and overcame the block of THP-1cells differentiation.This research revealed the intracellular mechanism of S5a inducing THP-1differentiation more comprehensively based on the previous study. We have found some keyhub genes which are very important during THP-1differentiation, and showed how thesignal further transmitted after activation of NF-κB pathway, finally caused THP-1cellsdifferentiation. Our study will provide new drug targets for inducing differentiation therapyand anti-inflammatory treatment. Our understanding about the mechanism for monocytesdifferentiation has further deepened.