Molecular pathology for down-regulation of erythroid-specific genes in prion diseases

AHSP是一种红细胞特异性分子伴侣，稳定新合成的a-球蛋白。先前的研究表明，在朊病毒感染的动物造血组织中，AHSP mRNA水平特异性降低。本研究的目的是阐明AHSP转录下调的机制。对MELhide8细胞中AHSP基因上游2.5 kb 5′区进行了截断和突变分析，并进行了电泳迁移迁移实验，结果表明，最小的5′-启动子区位于328-286处，位于翻译起始位点，包含一个GATA结合基序。两个上游GATA元件at-403和381中的任何一个仅在上述最小GATA元件存在时增强报告基因转录。这些发现表明，红细胞特异性转录因子GATA-1对AHSP基因表达至关重要，并提示AHSP的下调涉及GATA-1转录激活的变化。当MELhide8细胞与瘙痒病感染小鼠的脑匀浆孵育120小时后，细胞中AHSP、a-球蛋白、b-球蛋白、GATA-1、EKLF和NF-E2的基因表达无显著变化。此外，即使经过16代，MELhide8细胞也没有出现PrPsc的积累。这些数据表明，Prrc对红细胞AHSP基因表达无直接影响。相反，IL-6和IL-1β以剂量依赖性方式显著降低MELhide8细胞中AHSP mRNA表达水平和AHSP启动子报告基因表达。在STAT3通路抑制剂存在的情况下，这种减少得以恢复，这表明炎症细胞因子IL-6通过STAT3通路的信号转导会调节GATA-1/AHSP启动子相互作用，从而导致AHSP基因的下调。

AHSP is an erythroid-specific molecular chaperone that stabilizes newly synthesised a-globin. Previous studies demonstrated that mRNA levels of AHSP were specifically reduced in hematopoietic tissues of prion-infected animals. The purpose of the present study is to clarify the mechanism for down-regulation of AHSP transcription. A series of truncation and mutation analyses on 2.5-kb 5' upstream region of the AHSP gene in MELhide8 cells and electrophoretic mobility shift assay showed that the minimal 5'-promoter region located at-328-286 to the translation initiation site including a GATA binding motif. Either of two upstream GATA elements at-403 and-381 enhanced reporter gene transcription only in the presence of the minimal GATA element described above. These findings indicate that an erythroid-specific transcription factor GATA-1 is essential to AHSP gene expression and suggest that the down-regulation of AHSP involves changes in GATA-1 transcriptional activation. There was no significant change in gene expression of AHSP, a-globin, b-globin, GATA-1, EKLF, and NF-E2 in MELhide8 cells when the cells were incubated with brain homogenates from scrapie-infected mice for up to 120 hours. Moreover, MELhide8 cells exhibited no accumulation of PrPsc even after 16 passages. These data demonstrated that Prrc has no direct effect on AHSP gene expression in erythroid cells. Instead, IL-6 significantly and IL-1β weakly reduced the expression of AHSP mRNA levels and the AHSP promoter-reporter gene expression in MELhide8 cells in a dose-dependent manner. The reduction was recovered in the presence of the inhibitor of the STAT3 pathway, suggesting that the signal transduction of an inflammatory cytokine IL-6 through STAT3 pathway would modulate GATA-1/AHSP promoter interaction and subsequently causes down-regulation of the AHSP gene.