TY - JOUR T1 - Menaquinone-4 accelerates calcification of human aortic valve interstitial cells in high-phosphate medium through PXR JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.119.263160 SP - jpet.119.263160 AU - wei Yang AU - Zaiqiang Yu AU - Mari Chiyoya AU - Xu Liu AU - Kazuyuki Daitoku AU - Shigeru Motomura AU - Tadaatsu Imaizumi AU - Ikuo Fukuda AU - Ken-Ichi Furukawa AU - Motonori Tsuji AU - Kazuhiko Seya Y1 - 2019/01/01 UR - http://jpet.aspetjournals.org/content/early/2019/12/23/jpet.119.263160.abstract N2 - Recently, we confirmed that in human aortic valve interstitial cells (HAVICs) isolated from patients with aortic valve stenosis (AVS), calcification is induced in high inorganic phosphate (high-Pi) medium by warfarin (WFN). Because WFN is known as a vitamin K antagonist, reducing the formation of blood clots by vitamin K cycle, we hypothesized that vitamin K regulates WFN-induced HAVIC calcification. Here, we sought to determine whether WFN-induced HAVIC calcification in high-Pi medium is inhibited by menaquinone-4 (MK-4), the most common form of vitamin K2 in animals. HAVICs obtained from AVS patients were cultured in α-Modified Eagle's Medium containing 10% FBS, and when the cells reached 80%–90% confluency, they were further cultured in the presence or absence of MK-4 and WFN for 7 days in high-Pi medium (3.2 mM Pi). Intriguingly, in high-Pi medium, MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated the calcification when used alone (at 10 nM). Furthermore, MK-4 enhanced alkaline phosphatase (ALP) activity in HAVICs, and 7 days of MK-4 treatment markedly upregulated the gene expression of the calcification marker bone morphogenetic protein 2 (BMP2). Notably, MK-4-induced calcification was potently suppressed by two pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol, and, conversely, PXR activity was increased weakly, but in a statistically significant and dose-dependent manner, by MK-4. Lastly, in physiological-Pi medium, MK-4 increased BMP2 gene expression and induced HAVIC calcification in the presence of excess BMP2 (30 ng/mL). These results suggest that MK-4, namely vitamin K2 accelerates calcification of HAVICs from AVS patients like WFN via PXR-BMP2-ALP pathway.SIGNIFICANCE STATEMENT For AVS induced by irreversible valve calcification, the most effective treatment is surgical aortic or transcatheter aortic valve replacement, but ~20% of patients are deemed unsuitable due to its invasiveness. For effective drug treatment strategies for AVS, the mechanisms underlying aortic valve calcification must be elucidated. Here, we show that MK-4 accelerates WFN-induced calcification of AVS-patient HAVICs in high-Pi medium; this effect is mediated by PXR-BMP2-ALP signaling, which could be targeted for novel drug development. ER -