Expression and regulation of glial-cell-line-derived neurotrophic factor (GDNF) mRNA in human astrocytes in vitro.
The expression and modulation of mRNA for glial-cell-line-derived neurotrophic factor (GDNF) in human glial cells was investigated. Astrocyte cell cultures were isolated from human fetal brains, characterized by immunocytochemistry and maintained in vitro in conditions of high purity; sister cultures were exposed to protein kinase C (PKC) inhibitors for 20 min. Total RNA was extracted from the cell pellets, reverse-transcribed into cDNA and amplified by the polymerase chain reaction (PCR) with primers specific for GDNF. A reverse-transcription/PCR procedure was also performed on mRNA extracted from human fibroblast and lymphocyte cell lines. Human astrocytes grown in the absence of neurons expressed detectable amounts of mRNA for GDNF but no amplification products were observed in fibroblasts and lymphocytes, thus confirming that GDNF production was cell-type specific. After exposure to PKC inhibitors, a dramatic down-regulation of GDNF mRNA was observed in astrocyte cell cultures. Thus, human astrocytes are constitutively capable of producing GDNF, such trophic activity is restricted to neural cells, and PKC plays key roles in signal pathways that regulate the gene activation and production of GDNF.
Moretto
G
G
Sezione di Neurologia, Dipartimento di Scienze Neurologiche e della Visione, Policlinico Borgo Roma, Università di Verona, I-37134 Verona, Italy. moretto@borgoroma.univr.it
Walker
D G
DG
Lanteri
P
P
Taioli
F
F
Zaffagnini
S
S
Xu
R Y
RY
Rizzuto
N
N
D001253:Astrocytes / Q000166:cytology / Q000502:physiology*; D002478:Cells, Cultured / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D009414:Nerve Growth Factors / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D018696:Neuroprotective Agents / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D015533:Transcriptional Activation
D018076:DNA, Complementary; C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D012333:RNA, Messenger; D011493:Protein Kinase C