Identification of dopamine responsive mRNAs in glial cells by suppression subtractive hybridization.
Recent studies have established that glial cells are important targets of the neurotransmitter dopamine (DA), but the regulatory effects of DA on glial cells have not been extensively studied. In the present study, we have investigated the influence of DA on gene transcription in glial cells. Two-directional (forward and backward) suppression subtraction hybridization (SSH) was performed on astrocytes cultured from rat cerebral tissues in standard media or in culture media treated with DA. PCR-select differential screening was used to further verify the differentially expressed cDNA clones, positive clones were sequenced, and the mRNAs were re-examined on Northern blots. Fourteen sequences were identified of which eleven are homologous to known genes, three are homologous to expressed sequence tags (ESTs). Three novel full-length cDNAs were isolated using the EST fragments as probes to screen a cDNA library constructed from human brain. Analysis of these sequences suggested that complex intracellular signaling pathways, involving crosstalk with growth factor pathways, steroid hormone pathways, and an interferon-regulated 2-5 A pathway, are responsive to DA in astrocytes. The responsive proteins downstream from the signaling pathways were found to fall into at least three groups, including a series of metabolic enzymes, stress proteins, transfer proteins, etc. In addition, several of them have established their relationships with specific neurodegenerative diseases, showing that there is overlap in the pathogenic mechanisms of different diseases. Our results have provided a foundation for better understanding of the molecular basis of glial cell functions in dopaminergic transmission and an approach to find possible medication for the related disorders.
Shi
J
J
Department of Histology and Embryology, Third Military Medical University, Chongqing 400038, People's Republic of China.
Cai
W
W
Chen
X
X
Ying
K
K
Zhang
K
K
Xie
Y
Y
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence / Q000187:drug effects / Q000502:physiology; D015152:Blotting, Northern; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism* / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D005796:Genes / Q000187:drug effects / Q000502:physiology; D017403:In Situ Hybridization / Q000379:methods*; D008969:Molecular Sequence Data; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D018076:DNA, Complementary; D012333:RNA, Messenger; D004298:Dopamine