Effects of phospholipase D during cultured osteoblast mineralization and bone formation

Abstract

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos‐2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos‐2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1‐selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high‐resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.

Cyanidin-3-glucoside Regulates Osteoblast Differentiation via the ERK1/2 Signaling Pathway

“…30 Abdallah et al evaluated the phospholipase D on the mineralization of osteoblasts. 31 The alizarin red staining method was also used to evaluate the differentiation and osteogenic ability of osteoblasts. In this study, after 7 days of induced mineralization, alizarin red staining confirmed that C3G has a positive regulatory effect on the mineralization ability of osteoblasts, and C3G treatment can increase mineralization in cell slides in a dose-dependent manner.…”

“…Both et al stained osteoblast-mineralized nodules using alizarin red dye in a study of hydroxychloroquine, inhibiting osteoblast mineralization . Abdallah et al evaluated the phospholipase D on the mineralization of osteoblasts . The alizarin red staining method was also used to evaluate the differentiation and osteogenic ability of osteoblasts.…”

Intracellular density is a novel indicator of differentiation stages of murine osteoblast lineage cells

“…Pathway enrichment analysis was performed to determine the dominant phenomenon ongoing in each fraction (Figure S1). The genes reduced in the 50/70 fraction compared with the 10/30 fraction included the pathways involved in the immature state, such as “hippo signaling pathways” and “pathways regulating pluripotency of stem cells.” 26,27 In contrast, genes upregulated in the 50/70 cell fraction compared with the 10/30 fraction were enriched in pathways involved in osteoblast differentiation, such as “osteoclasts,” “phospholipase D signaling,” and “lysosome.” 28,29 …”

“…26,27 In contrast, genes upregulated in the 50/70 cell fraction compared with the 10/30 fraction were enriched in pathways involved in osteoblast differentiation, such as "osteoclasts," "phospholipase D signaling," and "lysosome." 28,29…”

From organic and inorganic phosphates to valvular and vascular calcifications

“… 134 PLD releases lysophosphatidic acid, an important signalling molecule capable of enhancing procalcifying genes, 135 and also promotes mineralization. 136 …”