Molecular Characteristics And Functional Analysis Of Porcine STEAP4 Gene

STEAP4 (six transmembrane epithelial antigen of the prostate 4) is a new member of the 6 transmembrane epithelial antigen of the prostate family, which has 6 transmembrane domains and ferric reduct domain. STEAP4 is first identified as a TNFa-induced adipose-related protein, subsequent studies show it has multiple functions in glucose and lipid metabolism, anti-inflammatory effects, metabolic homeostasis, cell proliferation and apoptosis, cancer and reduction of metal ions. In this study, we reported the molecular characteristics and functional analysis of steap4 gene in pigs. The full-length coding sequence of porcine steap4 was cloned using RT-PCR, and subjected to bioinformatic analysis. The expression profile of STEAP4 was examined by semi-quantitative RT-PCR. Ectopic overexpression of steap4 inhibited the expression of inflammatory cytokines TNFa. To undertand the transcriptional regulation of porcine STEAP4, we isolated the promoter region of porcine STEAP4 and tested its functionally activity using the dual-luciferase reporter assay system. Bioinformatic analysis predicted multiple potential binding sites for various transcription factors, including C/EBPa, C/EBPβ, KLF6 and PPARy, within the promoter region of porcine STEAP4. We further investigated the regulation of these transcription factors on STEAP4. Finally, the effects of inflammatory stimuli LPS and resistin on steap4 expression was studied. In conclusion, our results demonstrate that STEAP4 plays an important role in anti-inflammation and the transcription of its gene is regulated by C/EBP(3. Simultaneously, our data provide a platform for further STEAP4 functional study in pig model. The present results are as follows:1. The sequences of porcine STEAP4 and its variant (STEAP4v) were obtained and analyzed. The full-length open reading frame of porcine steap4 covers 1413bp and encodes a 470 amino acid polypeptide, whereas the alternative splicing variant lacks the first part of third exon and encodes a polypeptide of 353 amino acids. The genomic sequence of porcine STEAP4 covers 23kb in chromosome 9q11.1, and consists of five exons and four introns. 2. STEAP4 mRNA is ubiquitous expressed in the examined porcine tissues, with the highest level in fat, kidney, heart and lung, moderate in liver, spleen and small intestine, and the lowest level in brain, stomach, large intestine and muscle. The mRNA level of STEAP4 splicing variant is lower than its normal counterpart in all the tissues examined, the highest expression was found in lung, followed by adipose tissue and heart, and nearly undetectable in other tissues.3. STEAP4 and STEAP4v were respectively overexpressed in HepG2 cells, the cellular ATP concentration was measured to reflect the cell status. The results showed that ectopic overexpression of STEAP4 does not affect the cell state, whereas overexpression of STEAP4v decreases cell activity. In addition, overexpression of STEAP4v in mouse RAW264.7 macrophage cells downregulates the expression of inflammatory cytokines TNFα, suggesting that STEAP4v plays an important role in anti-inflammation.4. The promoter region (-1402/+10 bp) of porcine STEAP4 was isolated and subjected to its activity was analysed in hepG2 and IBRS-2 cells using dual-luciferase reporter assay system. To determine the characteristics of porcine STEAP4 proximal promoter, various truncated regions (-1402/+10,-801/+10,-546/+10,-349/+10,-191/+10,-101/+10,-46/+10 bp) were produced and subjected to activity tests. The results showed that the region-801/+10bp has the maximum promoter activity.5. Putative binding sites for adipogenesis-related transcription factors, including C/EBPα, C/EBPβ, KLF6 and PPARγ, were identified within porcine STEAP4 promoter-801/+10bp region by bioinformatic analysis. We further investigated the effects of these transcription factors on the promoter activity of porcine STEAP4. The results show that C/EBPβupregulates porcine STEAP4 promoter activity, whereas C/EBPα, KLF6 and PPARγhave no significant influence. Accordingly, overexpression of C/EBPβin HepG2 cells increased the mRNA level of STEAP4 mRNA. Comparison between pig and mouse steap4 promoter (-801/+10bp) reveals that unlike porcine STEAP4 which is transcriptionally regulated solely by C/EBPβ,the transcription of mouse STEAP4 is up-regulated by both C/EBPαand C/EBPβ. 6. Transcriptional regulation of C/EBPβon STEAP4 was analyzed in detail. Our datas show that the promoter region-101/-46bp of porcine STEAP4 is critical region for C/EBPβregulation. Further experiments demonstrate that the mutation of a putative C/EBP binding site within STEAP4 promoter (-73/-59bp) eliminates the up-regulation of C/EBPP on steap4 transcription.7. Either inflammatory stimuli LPS or resistin significantly increase STEAP4 mRNA expression in porcine MSCs and HepG2 cells.