ROLE OF SERUM IN THE DEVELOPMENTAL EXPRESSION OF ALKALINE-PHOSPHATASEIN MC3T3-E1 OSTEOBLASTS

MC3T3-E1 cells in culture exhibit a temporal sequence of development s imilar to in vivo bone formation. To examine whether the developmental expression of the osteoblast phenotype depends on serum derived facto rs, 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 (A XC) serum. ALP was assessed by measuring enzyme activity, immunoblotti ng, and Northern analysis. Growth of MC3T3-E1 cells in FBS resulted in the programmed upregulation of alkaline phosphatase (ALP) post-prolif eratively during osteoblast differentiation. In the presence of comple te serum, actively proliferating cells during the initial culture peri od expressed low ALP levels consistent with their designation as pre-o steoblasts, whereas postmitotic cultures upregulated ALP protein, mess age, and enzyme activity. In addition, undifferentiated early cultures of MC3T3-E1 cells were refractory to forskolin (FSK) stimulation of A LP, but became forskolin responsive following prolonged culture in FBS containing media. In contrast, MC3T3-E1 cells grown in AXC serum disp layed limited growth and failed to show a time-dependent increase in a lkaline phosphatase. Neither the addition of IGF-I to AXC serum to aug ment cell number or plating at high density restored the time-dependen t upregulation of alkaline phosphatase. Cells incubated in AXC serum f or 14 days, however, though expressing low alkaline phosphatase levels , maintained the capacity to upregulate ALP after FBS re-addition or f orskolin activation of cAMP-dependent pathways. Such time-dependent ac quisition of FSK responsiveness and serum stimulation of ALP expressio n only in mature osteoblasts indicate the possible presence of differe ntiation switches that impart competency for a subset of osteoblast de velopmental events that require complete serum for maximal expression. (C) 1994 Wiley-Liss, Inc.