The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation can be examined in primary diploid cultures of fetal calvarial-derived osteoblasts by the combination of molecular, biochemical, histochemical, and ultrastructural approaches. Modifications in gene expression define a developmental sequence that has 1) three principal periods: proliferation, extracellular matrix maturation, and mineralization; and 2) two restriction points to which the cells can progress but cannot pass without further signals. The first restriction point is when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle and cell growth regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which an enhanced expression of alkaline phosphatase occurs immediately after the proliferative period, and later an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited; and 3) enhanced levels of expression of the osteoblast markers when collagen deposition is promoted, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and development of the osteoblast phenotype. The loss of stringent growth control in transformed osteoblasts and in osteosarcoma cells is accompanied by a deregulation of the tightly coupled relationship between proliferation and progressive expression of genes associated with bone cell differentiation.

Rat calvaria osteoblasts derived from 21-day-old fetal rat pups undergo a temporal expression of markers of the osteoblast phenotype during a 5 week culture period. Alkaline phosphatase and osteocalcin are sequentially expressed in relation to collagen accumulation and mineralization. This pattern of expression of these osteoblast parameters in cultured rat osteoblasts (ROB) is analogous to that seen in vivo in developing fetal rat calvaria tissue (Yoon et. al: Biochem. Biophis. Res. Commun. 148:1129, 1987) and is similar to that observed in cultures of subcultivated 16-day-old embryonic chick calvaria-derived osteoblasts (COB) (Gerstenfeld, et.al: Dev. Biol. 122:46, 1987). While the cellular organization of subcultivated COB and primary ROB cultures are somewhat different, the temporal expression of the parameters remains. Both the rat and chick culture systems support formation of matrix mineralization even in the absence of beta-glycerol-phosphate. A systematic examination of factors which constitute conditions supporting complete expression of the osteoblast phenotype in ROB cultures indicate requirements for specific serum lots, ascorbic acid and the ordered deposition of mineral in the extracellular matrix. The present studies suggest that formation of a collagenous matrix, dependent on ascorbic acid, is requisite for expression of the osteoblast phenotype. In ROB cultures, expression of osteocalcin synthesis occurs subsequent to initiation of alkaline phosphatase activity and accompanies the formation of mineralized nodules. Thus, extracellular matrix mineralization (deposition of hydroxyapatite) is required for complete development of the osteoblast phenotype, as reflected by a 200-fold increase in osteocalcin synthesis. These data show the temporal expression of the various osteoblast parameters during the formation and mineralization of an extracellular matrix can provide markers reflective of various stages of osteoblast differentiation/maturation in vitro.

Members of the transforming growth factor-␤ (TGF-␤)superfamily of growth and differentiation factors have been identified in a wide variety of organisms, ranging from invertebrates to mammals. Bone morphogenetic proteins (BMPs) constitute a subgroup of proteins belonging to the TGF-␤ superfamily. BMPs were initially identified by their ability to induce endochondral bone formation at ectopic sites, suggesting a critical role for this family in development and regeneration of the skeleton. They are also expressed at a variety of nonskeletal sites during development, suggesting possible extraskeletal roles for these proteins. We cloned a novel member of the BMP family that is expressed at high levels in the placenta and the prostate and that we have designated as prostate-derived factor (PDF). Based on cDNA sequence analysis, the predicted PDF protein contains two cysteines in addition to the seven conserved cysteines that are the hallmark of the members of the TGF-␤ superfamily. In addition, Northern blot hybridization to poly(A) ؉ RNA showed low levels of expression in the kidney and pancreas. We further characterized the expression of this member of the BMP family by in situ hybridization and immunohistochemistry. These results show high expression in the terminal villae of the placenta. The expression of the protein as visualized by immunohistochemistry shows an expression pattern identical to that of the message in the terminal villae of the placenta. In day 18 rat embryos, protein expression was also seen in the skin and in the cartilaginous tissue of developing skeleton. Orchidectomy and dihydrotestosterone treatment of rats revealed that PDF expression is regulated by androgens in the prostate. In addition, subcutaneous implantation of recombinant PDF induced cartilage formation and the early stages of endochondral bone formation. These data indicate that PDF has a functional relationship to the BMPs.

Normal rat osteoblasts in culture undergo a developmental sequence consisting of a proliferation period in which high levels of the histone and collagen type I genes are expressed, followed by periods of matrix maturation [high levels of alkaline phosphatase (AP)] and mineralization that signal a high level of production of osteopontin (OP) and osteocalcin (OC). Since these parameters are regulated by vitamin D, the effects of both short term and chronic treatment with 1,25-dihydroxyvitamin D3 were examined during osteoblast growth and differentiation. In acute studies, during the proliferation period, histone mRNA (reflecting DNA synthesis) was inhibited (20-60%). Matrix Gla protein (MGP) and OP mRNA were significantly elevated during proliferation (30- and 15-fold), in contrast to OC which is not expressed and was not induced by hormone treatment. OP and MGP remained stimulated throughout the developmental sequence, but to a lesser degree (from 6- to 10-fold). Collagen and AP mRNA were inhibited by hormone at their peak levels of expression, but were stimulated at their lowest basal levels in the mineralization period. OC expression, which was initiated at the onset of mineralization, was stimulated 13- to 15-fold when basal levels were low, then from 6- to 8-fold by hormone throughout its period of expression. In chronic studies a different profile of gene expression was observed. When hormone treatment was initiated during the proliferation period on day 6, type I collagen and AP expression were suppressed, mineralized nodules did not develop, and induced levels of OP and OC gene expression did not occur. When chronic treatment was initiated on day 20 after the development of a mineralized matrix, OC, but not collagen and OP, levels were stimulated by the hormone. This observation is consistent with the requirement of a competent or mineralized bone matrix for expression of OC. In contrast, MGP expression was stimulated in the chronic vitamin D-treated cultures similar to acute treatments. Taken together these studies demonstrate that vitamin D, a physiological mediator of bone formation and remodelling, can both positively and negatively regulate expression of osteoblast phenotypic markers as a function of duration of hormone treatment and basal levels of gene expression, which is a reflection of bone matrix competency and the differentiated state of the osteoblast.