Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts.
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.
Yohay
D A
DA
Duke University Medical Center, Durham, North Carolina 27710.
Zhang
J
J
Thrailkill
K M
KM
Arthur
J M
JM
Quarles
L D
LD
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000502:physiology; D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation; D002460:Cell Line; D002606:Charcoal; D005576:Colforsin / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology*; D010641:Phenotype; D012116:Resins, Plant; D012710:Serum Albumin, Bovine / Q000502:physiology*; D013997:Time Factors; D015854:Up-Regulation
D012116:Resins, Plant; D002606:Charcoal; D005576:Colforsin; D012710:Serum Albumin, Bovine; D000469:Alkaline Phosphatase