Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.     

Extracellular enzymatically synthesized glycogen promotes osteogenesis by activating osteoblast differentiation via Akt/GSK-3β signaling pathway

Glycogen is the stored form of glucose and plays a major role in energy metabolism. Recently, it has become clear that enzymatically synthesized glycogen (ESG) has biological functions, such as the macrophage-stimulating activity. This study aimed to evaluate the effect of ESG on osteogenesis. MC3T3-E1 cells were cultured with ESG, and their cell proliferative activity and osteoblast differentiation were measured. An in vivo study was conducted in which ESG pellets with BMP-2 were grafted into mouse calvarial defects and histomorphometrically analyzed for the new bone formation. To confirm the effect of ESG on bone growth in vivo, ESG was orally administered to pregnant mice and the femurs of their pups were examined. We observed that ESG stimulated cell proliferation and enhanced messenger RNA expression of osteocalcin and osteopontin in MC3T3-E1 cells. ESG was taken up by the cells associated with GLUT-1 and activated the Akt/GSK-3β pathway. In vivo, the new bone formation in the calvarial defect was significantly accelerated by ESG and the maternal administration of ESG promoted fetal bone growth. In conclusion, ESG stimulates cell proliferation and differentiation of preosteoblasts via the activation of Akt/GSK-3β signaling and promotes new bone formation in vivo, suggesting that ESG could be a useful stimulant for osteogenesis.     

PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis

The temporal sequence of PTH/PTHrP receptor mRNA, binding, biologic activity, and its dependence on matrix synthesis was determined using MC3T3-E1 preosteoblast-like cells and primary rat calvarial cells in vitro. Osteoblastic cells were induced to differentiate and form mineralized nodules with the addition of ascorbic acid and β-glycerophosphate, and samples were collected from 0–26 days of culture. DNA levels as determined by fluorometric analysis increased 12- and 17-fold during the collection period for both MC3T3-E1 and primary calvarial cells respectively. Steady state mRNA levels for the PTH/PTHrP receptor as determined by northern blot analysis, were initially low for both cell types, peaked at day 4 and 5 for MC3T3-E1 and primary calvarial cells respectively, and declined thereafter. Competition binding curves were performed during differentiation using ¹²⁵I-PTHrP. The numbers of receptors per μg DNA were greatest at days 3 and 5 for MC3T3-E1 and primary calvarial cells respectively. The biologic activity of the receptor was evaluated by stimulating the cells with 10 nM PTHrP and determining cAMP levels via a binding protein assay. The PTHrP-stimulated cAMP levels increased 5-fold to peak values at day 5 for MC3T3-E1 cells and 6-fold to peak values at day 4 for the primary calvarial cells. Ascorbic acid was required for maximal development of a PTH-dependent cAMP response since ascorbic acid-treated MC3T3-E1 cells had twice the PTH-stimulated cAMP levels as non-treated cells. When the collagen synthesis inhibitor 3,4-dehydroproline was administered to MC3T3-E1 cultures prior to differentiation, there was a subsequent diminution of the PTH/PTHrP receptor mRNA gene expression and numbers of receptors per cell; however, if administered after the initiation of matrix synthesis there was no reduction in PTH/PTHrP receptor mRNA. These findings indicate that the PTH/PTHrP receptor is associated temporally at the level of mRNA, protein, and biologic activity, with a differentiating, matrix-producing osteoblastic cell in vitro. © 1996 Wiley-Liss, Inc.     

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.     

Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism

The adipose tissue is the site of expression and secretion of a range of biologically active proteins, called adipokines, for example, leptin, adiponectin, and resistin. Leptin has previously been shown to be expressed in osteoblasts and to promote bone mineralization, whereas adiponectin expression is enhanced during osteoblast differentiation. In the present study we explored the possible role of resistin in bone metabolism. We found that resistin is expressed in murine preosteoclasts and preosteoblasts (RAW 264.7, MC3T3-E1), in primary human bone marrow stem cells and in mature human osteoblasts. The expression of resistin mRNA in RAW 264.7 was increased during differentiation and seemed to be regulated through PKC- and PKA-dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NFkappaB promoter activity, indicating a role in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3-E1 cells in a PKA and PKC-dependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3-E1 into osteoblasts. All together, our results indicate that resistin may play a role in bone remodeling.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.