DEVELOPMENTAL STAGE-SPECIFIC CELLULAR-RESPONSES TO VITAMIN-D AND GLUCOCORTICOIDS DURING DIFFERENTIATION OF THE OSTEOBLAST PHENOTYPE - INTERRELATIONSHIP OF MORPHOLOGY AND GENE-EXPRESSION BY IN-SITU HYBRIDIZATION

Fetal rat calvarial-derived osteoblasts, in vitro, undergo a developme ntal sequence of events leading to bone tissue-like organization and o steoblast differentiation. Previous studies have documented temporal e xpression of genes reflecting stages of osteoblast phenotype developme nt in relation to tissue organization. Two steroid hormones are known to modify the developmental sequence; 1,25(OH)(2)D-3 can block differe ntiation when added to proliferating cells, while glucocorticoid addit ion to proliferating cultures increases the population of cells compet ent to produce a bone-like matrix and accelerates the differentiation time course. We have addressed the mechanisms contributing to these ob servations at the single cell level by analysis of a growth-related ge ne (H4 histone which is coupled with DNA synthesis) and matrix-associa ted genes (collagen, osteopontin, and osteocalcin) in hormone-treated cells. Our results demonstrate (1) a window of responsiveness for modi fications in phenotype development; (2) distinct morphological changes and selective modifications in gene expression in response to both ho rmones as a function of whether the cell is proliferating or different iated; and (3) location of the cell with respect to the mineralized no dule was a contributing factor to the levels of gene expression and ho rmonal responses. In response to vitamin D, surface osteoblasts associ ated with the nodules became flattened, elongated, and aligned, remini scent of a bone lining cell. In glucocorticoid-treated cultures, proli ferating cells became cuboidal and nodule-associated differentiated ce lls were approximately one-third the size of control osteoblasts. We a lso find subsets of hormone-responsive cells in the proliferating cult ures in response to glucocorticoid but not vitamin D. In postprolifera tive cultures, both hormones increased osteocalcin mRNA in the more di fferentiated osteoblasts associated with the mineralized matrix but no induction occurred in monolayer internodular cells. Osteopontin was i nduced by glucocorticoid in a larger population of cells. Thus, our st udies at the single cell level show selective morphological changes an d changes in the level of gene expression supporting the hypothesis th at hormones have differential effects on osteoblasts in relation to th eir stage of phenotype development. (C) 1995 Academic Press, Inc.