Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells

Abstract

Uncarboxylated osteocalcin, an osteoblast-derived protein, plays an important role in the regulation of glucose metabolism. It has previously been demonstrated that high glucose levels inhibit osteoblast proliferation and differentiation. However, the mechanisms through which uncarboxylated osteocalcin regulates osteoblast proliferation and differentiation under high glucose conditions remain unclear. Thus, in the present study, we aimed to examine the effects of uncarboxylated osteocalcin on the proliferation and differentiation of MC3T3-E1 cells under high glucose conditions. We demonstrated that high glucose levels induced the production of reactive oxygen species (ROS) in MC3T3-E1 cells, and this production was inhibited by treatment with uncarboxylated osteocalcin and N-acetyl-L-cysteine (NAC), a ROS scavenger. In addition, we found that uncarboxylated osteocalcin reduced high glucose‑induced oxidative stress and increased the mRNA expression of the osteogenic markers, runt-related transcription factor 2 (Runx2), osterix and osteocalcin, as well as the formation of mineralized nodules; it also inhibited adipogenic differentiation, as shown by a decrease in the mRNA expression of the adipogenic markers, peroxisome proliferator‑activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (adipocyte protein 2; aP2) and fatty acid synthase (FAS), and reduced lipid drop accumulation. Furthermore, we found that uncarboxylated osteocalcin inhibited PI3K/Akt signaling which was induced by ROS and facilitated the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. Taken together and to the best of ou knowledge, our results demonstrate for the first time that uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by inhibiting the activation of PI3K/Akt in MC3T3-E1 cells. Therefore, we suggest that uncarboxylated osteocalcin may be a potential therapeutic agent for diabetes-related osteoporosis.

Uncarboxylated osteocalcin promotes osteogenic differentiation of mouse bone marrow–derived mesenchymal stem cells by activating the Erk‐Smad/β‐catenin signalling pathways

“…In addition, increasing evidence indicates that Gprc6a can respond to osteocalcin in the pancreas, muscle, and testis . Previous studies in our laboratory showed that unOc not only upregulates Gprc6a expression but also simulates differentiation of MC3T3‐E1 cells into mature osteoblasts under high‐glucose conditions . Furthermore, osteocalcin gene knockdown in human MSCs decreased mineral species maturation and the total hydroxyapatite content .…”

“…The vector containing His‐unOc was transformed into Escherichia coli , and the expression and purification of the unOc protein were performed as previously described . In short, His‐unOc protein expressed in E coli was purified on a Ni Sepharose™ 6 Fast Flow column (GE, 17531801) according to the manufacturer's instructions . Coomassie blue staining results show that the purity of the His‐unOc obtained by purification was greater than 95%.…”

Salidroside promotes human periodontal ligament cell proliferation and osteocalcin secretion via ERK1/2 and PI3K/Akt signaling pathways

“…In addition, the osteocalcin-associated cell proliferation is associated with PI3K/Akt and ERK1/2 signaling pathways (38,39). Osteocalcin is an osteogenic marker (40) and activation of the ERK1/2 signaling pathway induces osteocalcin expression (39,40). Accordingly, in the present study, it was speculated that salidroside treatment induces the secretion of osteocalcin via the PI3K/Akt and ERK1/2 pathways.…”

“…A previous study reported that salidroside enhanced the expression of osteocalcin and reversed H 2 O 2 -induced bone loss in overiectomized rats (1). In addition, the osteocalcin-associated cell proliferation is associated with PI3K/Akt and ERK1/2 signaling pathways (38,39). Osteocalcin is an osteogenic marker (40) and activation of the ERK1/2 signaling pathway induces osteocalcin expression (39,40).…”

Rankl Expression and Metabolic Changes in Ovariectomized Rats and the Possible Protection by Vegetable Formula.

“…Alterations in bone and mineral metabolism were reported in some studies as a result of elevated blood sugar [43] which is accompanied with oxidative stress and partly contributing to the development of osteoporosis [44]. Supplementation with the vegetable formula, in OVXT group, showed hypoglycemic effect.…”