RELATIONSHIP BETWEEN ALKALINE-PHOSPHATASE LEVELS, OSTEOPONTIN EXPRESSION, AND MINERALIZATION IN DIFFERENTIATING MC3T3-E1 OSTEOBLASTS

We are using viral oncogene probes to study the pathways by which oste oblast-specific gene expression is induced in ascorbic acid-treated MC 3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directl y to key cellular regulators and, as a result, represses tissue specif ic gene expression and blocks differentiation in a wide variety of cel l types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targ ets p300/CBP, but not the pRB family. Using these constructs to dissec t osteoblast differentiation, we found that targeting of p300/CBP appe ars to be sufficient to repress alkaline phosphatase expression, altho ugh a low bur functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-sta ge markers such as osteocalcin and osteopontin. Surprisingly, cell lin es exhibiting severe repression of alkaline phosphatase activity diffe rentiate to a mineral-secreting phenotype much like normal MC3T3-E1 ce lls. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induct ion of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in cond itions in which endogenous alkaline phosphatase is severely repressed. (C) 1998 Wiley-Liss, Inc.