MC3T3-El 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 timedependent expression of alkaline phosphatase (ALP)-a marker of osteoblastic maturation-in MC3T3-El cells grown in the presence of fetal bovine serum (FBS) or resinkharcoal-stripped (AXC) serum. ALP was assessed by measuring enzyme activity, immunoblotting, and Northern analysis. Growth of MC3T3-EI cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) postproliferatively during osteoblast differentiation. In the presence of coniplcte 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-EI cells were refractory to forskdin (FSK) stimulation of ALP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-El cells grown in AXC serum displayed limited growth and failed to show a time-dependent increase in alkaline phosphatase. Neither the addition of IGF-l to AXC serum to augment cell number or plating at high density restored thc 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.