Inhibition of FOXO1/3 promotes vascular calcification.
OBJECTIVE
Vascular calcification is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease. We have demonstrated that activation of protein kinase B (AKT) upregulates runt-related transcription factor 2 (Runx2), a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of phosphatase and tensin homolog (PTEN), a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO (forkhead box O)-mediated Runx2 upregulation and VSMC calcification.


RESULTS
SMC-specific PTEN deletion mice were generated by crossing PTEN floxed mice with SM22α-Cre transgenic mice. The PTEN deletion resulted in sustained activation of AKT that upregulated Runx2 and promoted VSMC calcification in vitro and arterial calcification ex vivo. Runx2 knockdown did not affect proliferation but blocked calcification of the PTEN-deficient VSMC, suggesting that PTEN deletion promotes Runx2-depedent VSMC calcification that is independent of proliferation. At the molecular level, PTEN deficiency increased the amount of Runx2 post-transcriptionally by inhibiting Runx2 ubiquitination. AKT activation increased phosphorylation of FOXO1/3 that led to nuclear exclusion of FOXO1/3. FOXO1/3 knockdown in VSMC phenocopied the PTEN deficiency, demonstrating a novel function of FOXO1/3, as a downstream signaling of PTEN/AKT, in regulating Runx2 ubiquitination and VSMC calcification. Using heterozygous SMC-specific PTEN-deficient mice and atherogenic ApoE(-/-) mice, we further demonstrated AKT activation, FOXO phosphorylation, and Runx2 ubiquitination in vascular calcification in vivo.


CONCLUSIONS
Our studies have determined a new causative effect of SMC-specific PTEN deficiency on vascular calcification and demonstrated that FOXO1/3 plays a crucial role in PTEN/AKT-modulated Runx2 ubiquitination and VSMC calcification.
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham.
Liang
L
Deng
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham.
Lu
L
Huang
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham.
Yong
Y
Sun
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham.
Jack M
JM
Heath
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham.
Hui
H
Wu
From the Departments of Pathology (L.D., L.H., Y.S., J.M.H., Y.C.) and Pediatric Dentistry (H.W.), University of Alabama at Birmingham and Birmingham Veterans Affairs Medical Center; and Department of Research Service (Y.C.), University of Alabama at Birmingham. ybchen@uab.edu.
Yabing
Y
Chen
D000818:Animals; D001011:Aorta / Q000378:metabolism / Q000473:pathology; D001018:Aortic Diseases / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004195:Disease Models, Animal; D004789:Enzyme Activation; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D005838:Genotype; D019426:Integrases / Q000235:genetics; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000473:pathology; D051059:PTEN Phosphohydrolase / Q000172:deficiency / Q000235:genetics; D010641:Phenotype; D010766:Phosphorylation; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D054875:Ubiquitination; D061205:Vascular Calcification / Q000235:genetics / Q000378:metabolism* / Q000473:pathology
D001057:Apolipoproteins E; D050659:Core Binding Factor Alpha 1 Subunit; D000071161:Forkhead Box Protein O1; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C474033:FoxO3 protein, mouse; C467200:Foxo1 protein, mouse; D008840:Microfilament Proteins; D009124:Muscle Proteins; C493716:Runx2 protein, mouse; C058992:Tagln protein, mouse; D051057:Proto-Oncogene Proteins c-akt; C045073:Cre recombinase; D019426:Integrases; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse
AKT; FOXO1/3; PTEN; Runx2; ubiquitination; vascular calcification