Identification of a novel GDNF mRNA induced by LPS in immune cell lines.
Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types and has wide ranging effects within the central nervous system. To investigate the expression of the GDNF gene in immune cell lines under inflammatory conditions, we pharmacologically estimated the induction of GDNF mRNA in RAW264.7 cells. RT-PCR analysis revealed that LPS-induced GDNF mRNA in RAW264.7 cells does not include exon 3, which encodes the translational start site of this gene. A novel type of GDNF mRNA cloned by 5'-RACE consisted of the previous exon 4 and its flanking 5' upstream region, akin to a single exon gene. A similar type of human GDNF mRNA was also detected in a human neuroblastoma cell line, SH-SY5Y, without any stimulation. This novel (Ex4) GDNF mRNA was also upregulated by LPS in primary cultured rat macrophages, microglia and astrocytes and was found to exist in mouse brain. Ex4 GDNF protein produced by transfected HEK293 cells was mainly detected in cell lysates, but in conditioned medium only after PMA stimulation. Ex4 GDNF protein was found to exist as an unglycosylated form in both the transfected cells and the conditioned medium while full-type GDNF protein is glycosylated. PMA-stimulated augmentation of unglycosylated Ex4 GDNF protein was demonstrably regulated at the post-translational level.
Tanaka
Tatsuhide
T
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, Japan.
Oh-hashi
Kentaro
K
Ito
Masatoshi
M
Shitara
Hironobu
H
Hirata
Yoko
Y
Kiuchi
Kazutoshi
K
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005091:Exons / Q000235:genetics; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000096:biosynthesis* / Q000235:genetics; D005966:Glucuronidase / Q000494:pharmacology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000099175:Heparanase
D018076:DNA, Complementary; D051100:Glial Cell Line-Derived Neurotrophic Factor; D005966:Glucuronidase; D008070:Lipopolysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012333:RNA, Messenger; D000099175:Heparanase