IL-6 receptor expression and IL-6 effects change during osteoblast differentiation

Studies of the effects of interleukin-6 on osteoblasts have yielded conflicting results. In several earlier in vitro studies it has been stated that IL-6 has no effects on osteoblasts unless soluble IL-6 receptor is added. These results are contradictory to the fact that IL-6 receptors are expressed in osteoblasts in vivo. In this study, MC3T3 preosteoblast cells and rat bone marrow stromal cells were cultured in bone inducing medium containing ascorbic acid, β-glycerophosphate or dexamethasone. We found that IL-6 receptor expression increased in both types of cells during in vitro differentiation. Furthermore in MC3T3 cells IL-6 decreased proliferation and enhanced expression of two osteoblast-specific differentiation markers, Runx2 and osteocalcin, in proper sequential order. Interestingly, in both cell types IL-6-induced apoptosis only in later culture stages. We also found in MC3T3 cells that IL-6 induced STAT3 activation was significantly higher in later culture stages, i.e. when IL-6 receptor expression was high. The present study shows that IL-6 receptor expression increases during in vitro osteoblast differentiation and that IL-6 functions as a differentiation regulator of preosteoblast cells and an apoptosis initiator in more mature cells.     

CCAAT/enhancer-binding protein-beta has a role in osteoblast proliferation and differentiation

CCAAT/enhancer binding protein beta (C/EBPbeta) is known to play an important role in the expression of several genes necessary for bone development and homeostasis including osteocalcin, IGF-1, and IL-6. In this study, we show that C/EBPbeta protein levels and, consequently, DNA-binding activity are temporally regulated, dramatically decreasing upon differentiation of MC3T3-E1 mouse osteoblasts. Corresponding with these results, the constitutive expression of C/EBPbeta LAP in MC3T3-E1 osteoblasts increased proliferation and suppressed osteogenic differentiation. Thus, C/EBPbeta LAP not only appears to participate in the regulation of genes associated with mature bone physiology, but is also a critical regulator of osteoblast growth and differentiation.     

Role of Endoplasmic Reticulum Stress in Aberrant Activation of Fluoride-Treated Osteoblasts

The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis. The endoplasmic reticulum (ER) stresses and unfolded protein response (UPR) are initiated to alleviate the accumulation of unfolded proteins against cell injury. The previous researches had demonstrated that fluoride induced ER stress in other cells or tissues. In this study, we determined the ER stress and UPR to investigate their roles in aberrant activation of fluoride-treated osteoblasts. The gene expression of bone markers and UPR factors in MC3T3-E1 cells treated with varying doses of fluoride administration was analyzed. Meantime, levels of glutathione and glutathione disulfide were tested by the ultraperformance liquid chromatography–tandem mass spectrometry applications. Our results indicated that a certain dose and period of fluoride administration induced cell proliferation and differentiation, and Runx2 was involved in the regulation of osteoblastic differentiation of MC3T3-E1 cells. Increase trend of Runx2 expression was consistent with change of marker of ER stress. Fluoride caused ER stress and stimulated UPR during the process of osteoblast maturation, while oxidative stress was also active in the occurrence of ER stress. These data indicated that ER stress and UPR were possibly involved in the action of fluoride on osteoblasts.     

Bone morphogenetic protein-3b (BMP-3b) gene expression is correlated with differentiation in rat calvarial osteoblasts

BMP-3b (also called GDF-10) is a novel BMP-3-related protein recently discovered in rat femur tissue. Gene expression of BMP-3b in osteoblastic cells and its regulation by prolonged culture, BMP-2 and transforming growth factor beta1 (TGF-beta1) were examined. The BMP-3b gene was highly expressed in rat osteoblasts obtained from calvarial bones but not in the osteoblastic cell lines (MC3T3-E1 and U2-OS). BMP-3b mRNA increased during osteoblastic differentiation in prolonged culture and was associated with increased alkaline phosphatase (ALPase) activity. When BMP-2, an enhancer of ALPase activity, was added to the primary osteoblast culture, BMP-3b mRNA increased 6.9-fold after 24 h. In contrast, TGF-beta1 treatment, which suppresses ALPase activity, rapidly and completely inhibited gene expression of BMP-3b. The regulation of BMP-3 mRNA differed from that of BMP-3b, even though both proteins share 81% identity. These findings indicate that BMP-3b gene expression is regulated by osteoblastic differentiation and BMP-3b functions in highly differentiated osteoblasts.