Inhibition of FOXO1/3 Promotes Vascular Calcification

Abstract

Objective Vascular calcification is a characteristic feature of atherosclerosis, diabetes and end-stage renal disease. We have demonstrated that activation of AKT upregulates Runx2, a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of PTEN, a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO-mediated Runx2 upregulation and VSMC calcification. Approaches and 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.

Dietary potassium regulates vascular calcification and arterial stiffness

“…Heart and aortic tissues were collected, processed, and analyzed as previously reported (12,13,32,76). Descending aorta was used for total calcium measurement by the Arsenazo III method (Stanbio Laboratory), or protein expression and phosphorylation by Western blot analysis.…”

“…Aortic rings were obtained from descending aortas of wild-type C57BL/6 mice (The Jackson Laboratory) as we previously described (32). Calcification of aortic rings was performed as previously reported (32), using 3-mm mouse aortic rings cultured in osteogenic media containing 1% FBS with low (3.7 mM), normal (5.4 mM), or high (6.0 mM) potassium for up to 3 weeks.…”

“…The amount of calcium was normalized to the dry weight of the tissues and expressed as millimolar/gram dry weight. In separate experiments, aortic rings were fixed in 4% paraformaldehyde, paraffin embedded, and consecutive 7-μm sections were used for H&E and Alizarin red staining as we previously reported (12,13,32).…”

“…Primary VSMCs were isolated from descending aortas from wild-type C57BL/6 mice and cultured in growth media as described previously (13,32,38,76). All experiments were performed with VSMCs at passages 3 to 5.…”

“…To determine if there was a direct effect of extracellular potassium level on calcification of the arteries and VSMCs in their natural milieu, we employed an ex vivo ring culture model that we and others have recently developed for histological and quantitative analysis of arterial calcification (32,33). Based on normal physiological levels of serum potassium in adult C57BL/6 mice (34-36), we determined the effects of potassium at the lower (3.7 mM, low K + ), middle (5.4 mM, control), and higher (6.0 mM, high K + ) end of the physiological range on aortic calcification.…”