TGF-beta1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors.
BACKGROUND
The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-beta) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-beta1 regulates Ant1 gene expression in cultured primary rodent astrocytes.


RESULTS
Transcription reporter analysis verified that TGF-beta1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-beta1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-beta1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-beta1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-beta1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN)-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-beta1.


CONCLUSIONS
The specific regulation of Ant1 by TGF-beta1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-beta1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may represent a general mechanism that underlies the neuroprotective effects of TGF-beta1.
Law
Alick K T
AK
Department of Physiology, Emory University, Atlanta, USA. alaw@us.ibm.com
Gupta
Deepak
D
Levy
Shawn
S
Wallace
Douglas C
DC
McKeon
Robert J
RJ
Buck
Charles R
CR
D033741:Adenine Nucleotide Translocator 1 / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000473:pathology; D003101:Collodion / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D020007:Implants, Experimental; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000235:genetics; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D033741:Adenine Nucleotide Translocator 1; D004268:DNA-Binding Proteins; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D016329:Sp1 Transcription Factor; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003101:Collodion