Oxysterol‐induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC‐ and PKA‐dependent pathway

Abstract

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.

Oxysterols Are Novel Activators of the Hedgehog Signaling Pathway in Pluripotent Mesenchymal Cells

“…Similar to the pluripotent marrow stromal cells, we found that C3H10T1/2 cells undergo osteoblastic differentiation in response to oxysterols, as assessed by the induction of ALP activity (Fig. 4a) and Runx2 DNA binding activity (18). Treatment with oxysterols also induced Gli-luc activity in C3H10T1/2 cells, and this activity was inhibited by cyclopamine pre-treatment (Fig.…”

“…Role of PKC and PKA in Oxysterol-induced Hh Pathway Activation-We previously reported that oxysterol-induced osteoblastic differentiation of cells is mediated via protein kinase C (PKC)-and protein kinase A (PKA)-dependent mechanisms (18). The role of these signaling pathways in regulating the different markers of osteoblastic differentiation appears to be both specific and overlapping (18).…”

“…Oxysterols can be derived from the diet or from endogenous cellular biosynthesis, and they play important roles in physiological and pathological processes. We previously reported that specific oxysterols, namely 20(S)-hydroxycholesterol in combination with 22(S)-or 22(R)-hydroxycholesterol have potent osteoinductive properties when applied to marrow stromal cells (17,18). This effect was evidenced from increased DNA binding activity of Runx2, the transcriptional regulator of osteoblastic gene expression, increased alkaline phosphatase (ALP) activity, important in mineralization of extracellular matrix and increased expression of osteocalcin (OCN), an osteoblast-specific gene.…”

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…These results reveal a prominent role for PKC␣ in osteoblast differentiation induced by MT FGFR2 in murine mesenchymal cells. A positive role of PKC in the regulation of osteoblast differentiation is supported by the recent finding that PKC activation increases osteoblast gene expression (62,63). Whether PKC␣ may up-regulate directly osteoblast gene expression is an interesting question.…”

Bioluminescence imaging in bone tissue engineering

“…Reported activities include regulation of Runx2 expression and activity [99], increasing the expression of BMP receptor IA [128], stimulating fibronectin expression [129,130] enhancing N-Cadherin expression [131], and increasing indexes of the osteoblast phenotype in MC3T3, C2C12, C3H10T1/2, M2-10B4 mouse bone marrow stromal cells [132], primary cultured rat osteoblasts and human calvaria osteoblasts.…”