GRP78-binding protein regulates cAMP-induced glial fibrillary acidic protein expression in rat C6 glioblastoma cells

AbstractWe previously reported that a novel GRP78-binding protein (GBP) is predominantly expressed in rat brain and its expression declines through the aging process. To characterize its biological function, we established C6 glioblastoma cells that stably overexpressed GBP. Stable overexpression of GBP attenuated cAMP-induced expression of the glial fibrillary acidic protein (GFAP) gene, which was accompanied by a decrease in cAMP-induced signal transducer and activators of transcription 3 (STAT3) phosphorylation. Other distinct cAMP-induced events, including a transient reduction in extracellular signal-regulated protein kinase phosphorylation and a slowdown in cell proliferation, were hardly affected by GBP overexpression. Most importantly, treatment with siRNA against endogenous GBP markedly downregulated GBP expression in C6 glioblastoma cells, and dramatically augmented cAMP-induced GFAP mRNA expression in parallel with hyper-phosphorylation of STAT3. These results suggest a novel function of GBP in regulating GFAP gene expression via STAT3 phosphorylation

A Comparative Analysis of the Molecular Features of MANF and CDNF.

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF