There is a similarity of the mechanisms of physiological and pathological mineralization. Pathological calcification of soft tissues as the case of vascular calcification is characterized by the deposition of hydroxyapaptite induced by matrix vesicles at least in the initial stage as in the case of physiological calcification in skeletal tissues. Lipid metabolism is involved in the differentiation of smooth muscle cells and bone cells suggesting that phospholipases can modulate the differentiation process. Among the phospholipases, phospholipase D (PLD) action during differentiation of smooth muscle cells and bone cells are not well established. We seek to define the role of PLD during physiological and pathological mineralization. We selected a cell line human Saos-2 cells and primary osteoblasts from murine calvarias to mimic physiological mineralization and aorta culture to mimic ex vivo vascular calcification. The cells, in the presence of osteogenic factors ascorbic acid and β-glycerophosphate became mineral competent, having a strong alkaline phosphatase (TNAP) activity, a biomarker of mineralization process. We identified the presence of two PLD isoforms: PLD1 and PLD2 in Saos-2 cells. An increase of PLD expressions and activity were observed during Saos-2 cell differentiation which reached a maximum at day-5. To confirm the influence of PLD in the mineralization process, incubation of halopemide a PLD inhibitor- in Saos-2 and primary osteoblast cells induced a 20% decrease of mineralization as probed by a calcification-marker Aliza-Red and by TNAP activity. Overexpression of PLD (by transfecting Saos-2 cells) stimulated the mineralization process and TNAP activity. The calcified aorta in presence of 5 mM phosphate stimulated its transdifferentiation, increasing PLD activity. Incubation of halopemide at 10 μM in calcified aorta decreased TNAP activity by around 40%. Our findings indicated that PLD is involved during the maturation of mineral competent cells such as osteoblasts and smooth muscle cells.
17 May 2014 - 20 May 2014