High expression of the Pi-transporter SLC20A1/Pit1 in calcific aortic valve disease promotes mineralization through regulation of Akt-1

Abstract

The regulation of phosphate (Pi) handling is crucial during calcification of the aortic valve. Gene profiling of Pi transporters revealed that VIC culture expresses SLC201A1/Pit1 and SLC20A2/Pit2. On exposure to a mineralizing medium (2 mM Pi), the expression of Pi transporters in VIC culture is increased several folds, with the highest magnitude for SLC20A1. By using siRNAs, we established that silencing SLC20A1 significantly reduced Pi-induced mineralization of VICs. In human pathological specimens, we found that the expression of SCL20A1 was increased in CAVD tissues compared to control non-mineralized aortic valves. Treatment of VIC culture with Pi promoted the loss of mitochondrial membrane potential (ΔΨm) and cytochrome c release within the cytosol, leading to apoptosis. Inhibition of Pi transporters with phosphonoformic acid (PFA) prevented Pi-mediated apoptosis of VICs. Moreover, we discovered that the level of the Akt-1 transcript is diminished in CAVD tissues compared with control valves. Accordingly, treatment with Pi caused a reduction of the Akt-1 transcript in VIC culture, and treatment with PFA or siRNA against SLC20A1 restored the level of Akt-1. Overexpression of Akt-1 (pCMVAkt-1) prevented both Pi-induced apoptosis and mineralization of VIC culture. These results strongly suggest that overexpression of SLC20A1 promotes apoptosis and mineralization by altering the level of Akt-1.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Pi transporters in human valve interstitial cells (VICs).

A. In isolated VICs, only transcripts of SLC20A1 and SL20A2 were expressed. B. Following exposure to Pi, VICs have increased expression of SLC20A1 and SLC20A2 transcripts by several folds, with the highest magnitude for SLC20A1 (results expressed in function of SLC20A2 as the referent) (SLC20A1 increased by 5.3-folds when compared to SLC20A2) C. Treatment with PFA, a Pi transporter inhibitor, blocked the rise of SLC20A1 and SLC20A2 transcripts induced by the mineralizing medium (Pi). D. PFA prevented the mineralization of VIC cultures induced by Pi. E. PFA prevented the Pi-induced rise of osteopontin, osteonectin, osteocalcin, Runx2 and alkaline phosphatase transcripts. F and G. In isolated VICs, siRNA-mediated knockdown of SLC20A1 (F) resulted in a decreased Pi-induced mineralization (G). When compared with the knockdown of SLC20A2, the siRNA against SLC20A1 provided a greater reduction of mineralization of VIC cultures (G). (For in vitro experiments n = 3); PFA: Phosphonoformic acid; * p<0.0001 compared to negative control (Ctn); # p<0.005 compared to mineralizing medium (PO4).

Figure 2. Expression of SCL20A1 in human calcific aortic valve disease.

A. In human calcific aortic valve disease (CAVD) tissues, expression of SLC20A1 was increased, both in tricuspid and bicuspid aortic valves, when compared to control non-mineralized aortic valves. B, C and D. Immunostaining for SLC20A1 revealed faint expression in control aortic valves (B), whereas in CAVD tissues we observed a strong immunostaining in areas of tissue remodelling in the vicinity of calcific nodules (C) (100×) (in panel D magnification 200× of inset in C). * p<0.005 compared to control (Ctn).

Figure 3. Pi induces apoptosis of valve interstitial cells through the mitochondrial pathway.

A. The percentage of apoptotic cells, measured by TUNEL assay, increased significantly during VIC mineralization, whereas treatment with PFA blocked this response. B. The mitochondrial membrane potential (ΔΨm) decreased following treatment with Pi, but addition of PFA protected the ΔΨm. C. Epifluorescence images of VICs with the MitoPT TRME fluorescent dye in different conditions. In control cells, the mitochondrial uptake of MitoPT TRME gives a clear and distinct fluorescent pattern, indicating a normal ΔΨm. Following treatment with Pi, however, the fluorescent pattern is diffuse and accompanied by an abnormal mitochondrial morphology indicating a loss in the ΔΨm. The addition of PFA prevented Pi-mediated loss in the ΔΨm. D. This protection with PFA was also confirmed with an immunofluorescence assay measuring cytochrome c release in VICs under mineralizing condition. E. Cyclosporin A which is an inhibitor of MTP, prevented Pi-mediated apoptosis of VIC cultures as detected with TUNEL assay. F. The effect of cyclosporine A on Pi-mediated apoptosis was confirmed by the APOPercentage assay, which relies on changes in membrane asymmetry during apoptosis. G. Cyclosporin A prevented Pi-induced mineralization of VIC cultures. (For in vitro experiments n = 3); PFA: Phosphonoformic acid; MTP: mitochondrial permeability transition pore; * p<0.0001 compared to negative control (Ctn); # p<0.0001 compared to mineralizing medium (PO4).

Figure 4. Pi-mediated regulation of Akt levels.

A. In isolated VICs Akt-1 and Akt-2 were expressed. B. The levels of Akt-1 transcript were reduced significantly in CAVD tissues (n = 50) when compared to control non-mineralized aortic valves (n = 28). C and D. In isolated VICs, the levels of Akt-1 transcript were lowered following exposure to Pi, whereas PFA (C) and siRNA targeting SLC20A1 (D) prevented this response. E and F. Both Akt (E) and pAkt (F) protein levels were reduced by Pi treatment of VICs, whereas in the presence of PFA, levels were maintained. (For in vitro experiments n = 3); PFA: Phosphonoformic acid; CAVD: Calcific Aortic Valve Disease; * p<0.0001 compared to negative control (Ctn); # p<0.005 compared to mineralizing medium (PO4).

Figure 5. Akt-1 a regulator of Pi-induced mineralization.

Transfection of VICs with a pCMVAkt-1 resulted in higher expression of Akt-1 transcripts. B. The transfection of Akt-1 (pCMVAkt-1) prevented Pi-induced apoptosis of VICs (measured with the TUNEL assay). C. Transfection of Akt-1 reduced significantly Pi-induced mineralization of VIC cultures. D. Inhibition of PI3K, a kinase acting upstream of Akt, with Ly294002 increased mineralization of VIC cultures by several-folds. E and F. Similarly, the knockdown of Akt-1 (F) resulted in higher mineralization of VIC cultures (E). (For in vitro experiments n = 3); pCMV empty is the control (ctn) in panels A–C * p<0.0001 compared to negative control (Ctn); # p<0.0001 compared to mineralizing medium (PO4).