ABSTRACT
DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1alpha (HIF-1alpha) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1alpha but not with ARNT1. The binding of HIF-1alpha to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.
Subject(s)
Cell Hypoxia/genetics , Transcription Factors/metabolism , Vascular Endothelial Growth Factor A/genetics , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites/genetics , COS Cells , Cell Line, Tumor , Chlorocebus aethiops , Gene Expression Regulation , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Plasmids/genetics , Promoter Regions, Genetic , RNA, Small Interfering/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoma 180/genetics , Sarcoma 180/metabolism , Transcription Factors/genetics , TransfectionABSTRACT
The basic helix-loop-helix (bHLH) transcription factors, DEC2 and DEC1, play critical roles in the circadian rhythm of the suprachiasmatic nucleus (SCN). It is known that mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative-feedback loop, and CLOCK/BMAL1 and CLOCK/BMAL2 enhance DEC2 transcription via CACGTG E-boxes. To understand the role of arginine 57 ((57)Arg) within the basic region of DEC2, we examined the effect of substituting this residue into DEC2 on CLOCK/BMAL2-mediated transactivation. A luciferase assay showed that substituting (57)Arg for Ala or Lys in DEC2 diminished the suppressive activity of wild-type DEC2 on CLOCK/ BMAL2-mediated transactivation, while substituting (48)Pro for Ala in DEC2 did not alter it, and the same was true for wild-type DEC2. We also showed that proteins which were wild-type and substitution mutants of DEC2 were expressed at nearly equivalent levels by Western blotting. These findings demonstrate that (57)Arg in the basic region of DEC2 is essential for its activity in suppressing CLOCK/BMAL2-mediated transactivation.
