Role of serum in the developmental expression of alkaline phosphatase in MC3T3‐E1 osteoblasts

Abstract

MC3T3-E1 cells in culture exhibit a temporal sequence of development similar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived factors, we compared the time-dependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation- in MC3T3-E1 cells grown in the presence of fetal bovine serum (FBS) or resin/charcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-proliferatively during osteoblast differentiation. In the presence of complete serum, actively proliferating cells during the initial culture period expressed low ALP levels consistent with their designation as pre-osteoblasts, whereas postmitotic cultures upregulated ALP protein, message, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-I to AXC serum to augment cell number or plating at high density restored the time-dependent upregulation of alkaline phosphatase. Cells incubated in AXC serum for 14 days, however, though expressing low alkaline phosphatase levels, maintained the capacity to upregulate ALP after FBS re-addition or forskolin activation of cAMP-dependent pathways. Such time-dependent acquisition of FSK responsiveness and serum stimulation of ALP expression only in mature osteoblasts indicate the possible presence of differentiation switches that impart competency for a subset of osteoblast developmental events that require complete serum for maximal expression.

Role of Cx43-Based Gap Junction in Murine Osteoblast-Like Mc3t3-E1Cells Exposed to 17-Βestradiol

“…Following growth arrest, they begin to differentiate by developing markers of mature osteoblasts, including the high-level expression of ALP and the capacity to form mineralized bone matrix [53,54]. In this study, on the first day, MC3T3-E1 cells were firmly attached within 8 h after seeding, and short dendritic protrusions were visible.…”

.BETA.-Cryptoxanthin and Bone Metabolism: The Preventive Role in Osteoporosis

“…30) Alkaline phosphatase participates in the mineralization process in osteoblastic cells. 31) β-Cryptoxanthin has a stimulatory effect on the gene expression of for various proteins involved in osteoblasic bone formation. The effect of β-cryptoxanthin in stimulating mineralization in osteoblastic cells is summarized in Fig.…”

Isolation of GnafC, a Polysaccharide Constituent ofGnaphalium affine, and Synergistic Effects of GnafC and Ascorbate on the Phenotypic Expression of Osteoblastic MC3T3-E1 Cells

“…ALPase is present early in osteoblastic development and has been shown to be required for the mineralization of ECM, 14,27,28) probably because ALPase hydrolyses organic phosphates and boosts the local phosphate concentration, leading to diŠerentiation toward the mineralizing phenotype. ECM plays many essential roles to exert various cellular functions, including osteoblastic diŠerentiation and proliferation.…”