Osteopontin transcription in aortic vascular smooth muscle cells is controlled by glucose-regulated upstream stimulatory factor and activator protein-1 activities.
The expression of the matrix cytokine osteopontin (OPN) is up-regulated in aortic vascular smooth muscle cells (VSMCs) by diabetes. OPN expression in cultured VSMCs is reciprocally regulated by glucose and 2-deoxyglucose (2-DG; inhibitor of cellular glucose metabolism). Systematic analyses of OPN promoter-luciferase reporter constructs identify a CCTCATGAC motif at nucleotides -80 to -72 relative to the initiation site that supports OPN transcription in VSMCs. The region -83 to -45 encompassing this motif confers basal and glucose- and 2-DG-dependent transcription on an unresponsive promoter. Competition and gel mobility supershift assays identify upstream stimulatory factor (USF; USF1:USF2) and activator protein-1 (AP1; c-Fos:c-Jun) in complexes binding the composite CCTCATGAC element. Glucose up-regulates both AP1 and USF binding activities 2-fold in A7r5 cells and selectively up-regulates USF1 protein levels. By contrast, USF (but not AP1) binding activity is suppressed by 2-DG and restored by glucose treatment. Expression of either USF or AP1 activates the proximal OPN promoter in A7r5 VSMCs in part via the CCTCATGAC element. Moreover, glucose stimulates the transactivation functions of c-Fos and USF1, but not c-Jun, in one-hybrid assays. Mannitol does not regulate binding, transactivation functions, USF1 protein accumulation, or OPN transcription. Thus, OPN gene transcription is regulated by USF and AP1 in aortic VSMCs, entrained to changes in cellular glucose metabolism.
Division of Bone and Mineral Diseases, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Miri
M
Bidder
Jian-Su
JS
Shao
Nichole
N
Charlton-Kachigian
Arleen P
AP
Loewy
Clay F
CF
Semenkovich
Dwight A
DA
Towler
D020816:Amino Acid Motifs; D000818:Animals; D001011:Aorta / Q000473:pathology; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005947:Glucose / Q000378:metabolism; D007150:Immunohistochemistry; D008353:Mannitol / Q000494:pharmacology; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D053495:Osteopontin; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D020798:Two-Hybrid System Techniques; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D016760:Proto-Oncogene Proteins c-fos; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; C506897:Spp1 protein, rat; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497182:Usf1 protein, rat; C497186:Usf2 protein, mouse; C497187:Usf2 protein, rat; D053495:Osteopontin; D008353:Mannitol; D005947:Glucose