Cloning And Functional Analysis Of The Promoter Of MANF Gene

MANF (Mesencephalic astrocyte derived neurotrophie factor) gene is also knownas ARP (argininerich protein) or Aremt (Arginine Rich, Mutated in Early stage ofTumors). MANF was initially identified as highly mutated genes in the different tumortissues and its encoding protein is located in the endoplasmic reticulum (ER) and golgiapparatus. In2003, MANF protein was isolated from an astrocyte cell line of rats andfound it promotes neural cells growth. Therefore, it was named as MANF. MANF is asecreted protein with20kD molecular weight. MANF exerted a protective effect onneurons in brain ischemic injury and Parkinson’s diseas. It also protected cells from ERstress-induced apoptosis. In a recent study, we found MANF gene highly expressed inrheumatoid arthritis and SLE. However, the transcriptional regulation mechanism ofMANF is not clear. The purpose of this study is to explore the upstream transcriptionregulatory mechanism of human MANF and analyze MANF gene expression level inER stress and inflammatory pathways in the mechanism.In this study, we cloned the human MANF promoter, a1415-bp fragment (-1240to +176) comprising of the5’-flanking sequence of the human MANF gene, intopGL3-Basic vector to generate pGL3(-1240/+176)(5’F1), which could drive fireflyluciferase. Analysis of the promoter sequence revealed the potential binding sites fortranscription factor XBP1, including ERSE (CCACT-N9-CCACG, located at-853to-835) and ERSE-like element (CCTGG-A-CCAAT, located at-94to-84). Transienttransfection of pGL3(-1240/+176) in Neu2A cells and treatment with ER stress inducertunicamycin activated MANF promoter. Moreover, over-expression of XBP1, especiallyXBP1s, enhanced MANF-luciferase activity. The results suggest that MANF expressionis up-regulated at the transcriptional level during ER stress.To identify ER stress responsive cis-acting element in the upstream region of MANF,we constructed several truncated forms of the5’-flanking sequence of MANFand testedtheir activity in Neu2A cells. Measurements of luciferase activity revealed that theactivity of three shorter truncates significantly increased, compared with5’F1(-1240/+176). However, if compared with truncate5’F3(-880/+83), the activity oftruncate5’F4(-785/+83) remarkably decreased. This region (5’F4) did not include anERSE sequence that was known to be a binding site for XBP1s, suggesting that ERSEmay be crucial for the response of MANF to ER stress. Further comparison of5’F4(-785/+83) with the5’F3(-880/+83) indicated that the luciferase activity of5’F4wasobviously lower with or without XBP1transfection. We also found that theresponsiveness of MANF to tunicamycin (TM) was remarkably repressed when wemutated ERSE.To compare the importance of ERSE and ERSE Ⅱlike element in the responsivenessof MANF to ER stress, we mutated ERSE Ⅱl ike element.Unlike ERSE, mutagenesis ofERSE Ⅱlike element only slightly repressed the responsiveness of MANF promoter to tunicamycin, which was not statistically significant. These results indicate that ERSEplays an important role in the responsiveness of MANF to ER stress.To analyze whether the ERSE of the MANF promoter could interact withtranscription factor XBP1in vivo, ChIP assay was carried out with anti-XBP1antibodyin293T cells. The immunoprecipitated DNA was amplified with a couple of primers ofERSE region. The PCR results showed a DNA band that was bound to XBP1in293Tcells stimulated with tunicamycin. This finding indicates that transcription factor XBP1can bind to the ERSE site in the promoter region of MANF. Collectively, ER stress canactivate MANF promoter through transcription factor XBP1.We also preliminarily demonstrated that, SP1and AP1play a regulation functionrole in MANF gene promoter region. Therefore, MANF gene plays an important roleER stress and inflammation.