Regulation of adenine nucleotide translocator 1 expression by transforming growth factor beta 1 in astrocytes

At sites of CNS injury astrocytes undergo a reactive astrogliosis response resulting in the glial scar that is inhibitory to neuronal regeneration. Reactive astrocytes elevate the expression of many proteins, including axonal regeneration inhibitory extracellular matrix (ECM) molecules. The secreted signaling molecule transforming growth factor-beta1 (TGF-β1), which is rapidly and chronically elevated following CNS injury, regulates gene expression in cultured astrocytes and in reactive astrocytes in vivo. Despite the important role of TGF-β1 in regulating gene expression in reactive astrogliosis however, the underlying mechanism(s) are poorly understood.; The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein important for mitochondrial energy mobilization. Recently, TGF-β1 was found to specifically increase expression of the Ant1 gene in reactive astrocytes of the glial scar. Consequently, the molecular mechanisms through which TGF-β1 regulates Ant1 gene expression in primary rodent astrocytes were investigated to further understand TGF-β1-directed gene expression. Transfection of Ant1 promoter transcription reporter constructs into primary astrocyte cell cultures verified that TGF-β1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. This activity was localized to a 69 basepair TGF-β1 responsive element of the Ant1 promoter. Electrophoretic mobility shift assays (EMSAs) demonstrated that astrocyte nuclear proteins bind specifically to this response element and TGF-β1 induces additional nuclear protein binding to this element. Antibody supershift EMSA and deletion analyses demonstrated that Sp1 binding sites in the RE are important for TGF-β1 regulation of Ant1 in astrocytes. Additionally, TGF-β1 activated Smad transcription factors contribute to this regulation. Expression of Smad family members in injured and uninjured cerebral cortex and in primary astrocyte cultures was also demonstrated for the first time. Transcription reporter assays including co-transfection of dominant negative Smads 3 and 4 significantly reduced induction of Ant1 by TGF-β1 demonstrating the functional significance of astrocyte Smad expression. These results demonstrate that TGF-β1 regulates Ant1 gene expression through the coordinated action of Sp1, Smad3 and Smad4 transcription factors.