Abstract
BackgroundThe 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-β) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-β1 regulates Ant1 gene expression in cultured primary rodent astrocytes.ResultsTranscription reporter analysis verified that TGF-β1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-β1 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-β1 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-β1 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-β1 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-β1.ConclusionThe specific regulation of Ant1 by TGF-β1 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-β1 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-β1.
Highlights
The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation
Using an in vivo filter implant model of the glial scar, we have recently shown that the expression of Ant1, a gene involved in energy mobilization, is elevated in reactive astrocytes and that astrocytic Ant1 expression is regulated by TGF-β1 both in vivo and in vitro [6]
This upstream 6.5 kb of promoter sequence showed a lack of induction in response to treatment with this cytokine, but transcriptional activity from this fragment was reduced by 43% in the presence of TGF-β1 compared to untreated controls (Fig 1b)
Summary
The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant is upregulated by transforming growth factor-beta (TGF-β) in reactive astrocytes following CNS injury. Using an in vivo filter implant model of the glial scar, we have recently shown that the expression of Ant, a gene involved in energy mobilization, is elevated in reactive astrocytes and that astrocytic Ant expression is regulated by TGF-β1 both in vivo and in vitro [6]. Ant is a major mitochondrial inner membrane protein that exchanges mitochondrial ATP for cytosolic ADP and is thereby an important component of oxidative phosphorylation (OXPHOS) energy production. Mice deficient in Ant demonstrate characteristics of cardiac myopathy, including severe exercise intolerance and mitochondrial proliferation in the heart [10]
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