Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells
Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams, Makoto Miyazaki
Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams, Makoto Miyazaki
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Research Article Vascular biology

Activating transcription factor-4 promotes mineralization in vascular smooth muscle cells

Abstract

Emerging evidence indicates that upregulation of the ER stress–induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global Atf4 KO, smooth muscle cell–specific (SMC-specific) Atf4 KO, and transgenic (TG) mouse models. Reduced expression of ATF4 in global ATF4-haplodeficient and SMC-specific Atf4 KO mice reduced medial and atherosclerotic calcification under normal kidney and CKD conditions. In contrast, increased expression of ATF4 in SMC-specific Atf4 TG mice caused severe medial and atherosclerotic calcification. We further demonstrated that ATF4 transcriptionally upregulates the expression of type III sodium-dependent phosphate cotransporters (PiT1 and PiT2) by interacting with C/EBPβ. These results demonstrate that the ER stress effector ATF4 plays a critical role in the pathogenesis of vascular calcification through increased phosphate uptake in vascular SMCs.

Authors

Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams, Makoto Miyazaki

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Figure 6

The transcriptional regulation of PiT1 by the ATF4-C/EBPβ complex.

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The transcriptional regulation of PiT1 by the ATF4-C/EBPβ complex. (A) D...
(A) Deletion and mutational analysis of the PiT1 gene using a luciferase (Luc) reporter gene assay. The schematic illustrations represent the serially deleted PiT/Luc reporter constructs. Results are expressed as the relative Luc/β-galactosidase units of induction (n-fold) over the control value for each construct. (B) Yeast 2-hybrid screen. Y2HGold yeasts expressing ATF4 and/or C/EBPβ were grown onto an SD agar plate placing 4 amino acids (Leu, Trp, Ade, and His) in the presence of X-α-Gal and Aureobasidin A. Quadrant 1 of the plate contained a bait vector, pGBKT7-Atf4, and a prey vector, pGADT7-C/ebpβ; quadrant 2 contained a bait vector, pGBKT7-Atf4, and a prey vector, pGADT7-Empty; quadrant 3 contained a bait vector, pGBKT7-C/ebpβ, and a prey vector, pGADT7-Empty; and quadrant 4 contained a bait vector, pGBKT7-C/ebpβ, and a prey vector, pGADT7-Atf4. (C) The ATF4-C/EBPβ complex induces PiT1 transcription in mammalian cells. NIH 3T3 cells were cotransfected with pGL3 Luc plasmid containing the –617/+825 PiT1 gene, ATF4-pcDNA3, and/or C/EBPβ-pcDNA3 expression plasmid. (D) The ATF4-C/EBPβ complex but not ATF4 or C/EBPβ alone specifically binds to the PiT1 ATF4RE. EMSA was performed using 32P-radiolabeled double-stranded oligonucleotides corresponding to the ATF4RE of the PiT1 gene. Competitive assays were performed using a 50-fold molar excess of unlabeled oligonucleotides corresponding to the WT ATF4RE or the mutant ATF4 of the PiT1 gene as indicated. Supershift assay was performed using C/EBPβ and ATF4 antibody. Black arrow: binding ATF4-C/EBPbeta complex; white arrow: supershift. (E) ATF4 and C/EBPβ belong to the same protein complex, which binds to the PiT1 ATF4RE. Double ChIP assays were performed using VSMCs incubated for 6 hours with 500 μM stearic acid that were first immunoprecipitated with ATF4 antibody, eluted, and then subjected to a second immunoprecipitation with C/EBPβ antibody or normal rabbit IgG antibody. The enrichment of ATF4-C/EBPβ protein was analyzed by real-time quantitative PCR using primer sets specific for the PiT1 ATF4RE or for the 5′ distal promoter region of PiT1 as a negative control (data not shown). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin (% of input). Two-tailed unpaired Student’s t test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.