The Role Of Transcription Factor AKNA In The Growth Inhibition Mediated By T-2 Toxin

T-2 toxin,the most toxic member of the trichothecenes,is commonly found in cereals and feedstuffs.It is characterized by strong toxicity and detoxification difficulties.In animals,chronic exposure to low doses of T-2 toxin causes growth retardation and reduced body weight,resulting in economic losses during livestock production.Excessive inflammatory cytokines and GH deficiency are important mechanisms that contribute to growth inhibition.In our previous genomics study after the GH3 cells were exposed to 10 and 40 nM of T-2 toxin,respectively,the gene expressions of AKNA were very significantly decreased in a dose-dependent manner(25.85 and 52.15 folds),while inflammatory cytokines were significantly upregulated,and GH was significantly downregulated.However,the molecular mechanism of GH downregulation caused by T-2 toxin was still unclear.The transcription factor AKNA,belonging to AT-hook protein family,has been associated with the expression of inflammatory cytokines and has fostered interest in recent years.Knockout of AKNA in mice results in the expression of neutrophil inflammatory cytokines(IL-1? and IFN-?)and neonatal death.However,it is unclear how AKNA regulates the expression of inflammatory cytokines.We hypothesized that AKNA might play important roles in T-2 toxin-induced GH deficiency and inflammation.We used the transcription factor AKNA as the research target to further clarify the role of AKNA in T-2 toxin-mediated inflammatory response and GH deficiency and the signal transduction pathway of AKNA.This will provid novel insights into the significant role of AKNA in T-2 toxin-induced growth inhibition and be valuable for further development of safe new antagonists,as well as lay the foundation for a comprehensive assessment of the risks of T-2 toxin.GH3 cells were treated with T-2 toxin concentrations of 5 nM,10 nM,20 nM,40 nM and 80 nM,respectively,for 0.5,1,2,4,8 and 12 h,and the relationship between AKNA expression level and T-2 toxin was measured by qRT-PCR.The results showed that T-2 toxin could downregulate the expression of AKNA in a time-and dose-dependent manner.In order to identify signaling pathway involved in the regulation of AKNA,GH3 cells were pretreated with different signaling pathway inhibitors for 45 min,followed by addition of T-2 toxin.The results showed that PKA/CREB,NF-?B and MAPK/p38 signaling inhibitors significantly reversed the downregulation of AKNA expression induced by T-2 toxin.The bioinformatics analysis revealed that AKNA promoter region contained the binding sites of key proteins CREB and p65 in the PKA/CREB and NF-?B signaling pathways.The transfection of CREB and p65 overexpressing vector into GH3 cells showed that the expression of AKNA was significantly decreased after overexpression of CREB and p65,suggesting that CREB and p65 could negatively regulate the expression of AKNA,PKA/CREB and NF-?B signaling pathway might be the key signaling pathway to regulate AKNA expression.The effects of the T-2 toxin on phospho-CREB and phospho-p65 shuttling into the nucleus and the site of AKNA in GH3 cells were observed by indirect immunofluorescence double standard assays after incubation with 40 nM T-2 toxins for 0.5,1,2,4,8 and 12 h,respectively.We detected a significant increase in fluorescent intensities in the phospho-CREB translocation after incubation for 1 h.The fluorescence intensity of phospho-p65 transferred to the nucleus increased significantly after 8 h of incubation with the T-2 toxin.The transcription factor AKNA was mainly located in the cytoplasm in rat GH3 cells,and the fluorescent intensities of AKAN transferred to the nucleus increased significantly after 8 h of incubation with the T-2 toxin.These results clearly demonstrated that T-2 toxin was able to activate the PKA/CREB and NF-?B/p65 signaling pathways via phosphorylating CREB and p65 proteins and promoting phospho-CREB and phospho-p65 into the nucleus and that AKNA was a likely downstream target of p65 and CREB.In addition,we showed that AKNA was relocated from the cytoplasm to the nucleus after T-2 toxin treatment,thus regulating the expression of downstream target genes.In order to determine the manner of the interaction between CREB and p65 and AKNA,it was found that CREB and p65 could significantly downregulate the transcriptional activity of the AKNA promoter region by double luciferase reporter assay.ChIP combined with qRT-PCR analysis showed that the CREB and p65 proteins could bind directly to the proximal promoter of the AKNA gene,respectively,and T-2 toxin significantly increased the activity of CREB and p65 and AKNA promoter.These results fully demonstrated the phospho-CREB and phospho-p65 protein could directly bind to AKNA promoter to inhibit the expression of AKNA.T-2 toxin significantly downregulated AKNA expression by promoting the binding of phospho-CREB and phospho-p65 protein to the AKNA promoter.The expression level of AKNA was inhibited by transfection of pSicoR-AKNA interference vector into GH3 cells,which explored the downstream-regulatory factors of AKNA and its role in growth inhibition.The expressions of IL-1?,IL-6,IL-11,TNF-? and MMP-9 were decreased after silencing of AKNA,which suggested that AKNA could positively regulate the expression of inflammatory cytokines and MMP-9.Therefore,we speculated that,in addition to AKNA regulation of the expression of inflammatory cytokines,there were other signaling pathways involved in the upregulation of inflammatory cytokines induced by T-2 toxin.The expression of GH was also significantly downregulated after silencing of AKNA,indicating that AKNA was an important regulator and played an important role in T-2 toxin-induced GH deficiency.The transcription factor AKNA was likely to be an important new target for T-2 toxin-induced growth inhibition.In summary,here we demonstrated that AKNA was a downstream molecular target in T-2 toxin-induced GH downregulation and inflammatory cytokines expression.T-2 toxin increased the phosphorylation of CREB and p65 to activate the PKA/CREB and NF-?B/p65 signaling pathways and promoted the translocation of phospho-p65 and phospho-CREB into the nucleus.Phospho-CREB and phospho-p65 inhibited the transcriptional activity of AKNA by direct binding to the AKNA promoter.We found that AKNA was mainly cytosolic in rat GH3 cells but entered the nucleus to exert a regulatory effect after treatment with the T-2 toxin.GH expression was significantly downregulated after silencing of AKNA,suggesting that AKNA was a key regulator of T-2 toxin-mediated GH deficiency.Thus,we report the signal transduction pathway of AKNA,which will be of major significance for the future study of the regulatory mechanism of AKNA.These findings provide vital insights into the essential role of AKNA in the inflammatory response and T-2 toxin-induced growth inhibition.