The onset and progression of osteoblast differentiation is functionally related to cellular proliferation.

The relationship of proliferation to the developmental sequence associated with bone cell differentiation was examined in primary osteoblast cultures derived from fetal rat and embryonic chick calvaria. A reciprocal and functional relationship exists between the decline in proliferative activity which occurs during the initial stages of the developmental sequence and the induction of genes encoding osteoblast phenotype proteins associated with matrix maturation and mineralization. This relationship is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase (AP) and osteopontin (OP) genes immediately following the proliferative period and expression of osteocalcin with the onset of mineralization, and 2) increases in AP and OP when DNA synthesis is inhibited. By determining cellular mRNA levels and rates of mRNA synthesis in isolated nuclei, we found that the down-regulation of cell growth-related genes is modified at both the levels of transcription and mRNA stability. For a histone gene where down-regulation is transcriptionally mediated, we have observed that the shutdown of osteoblast proliferation is associated with the selective loss of the interaction of a promoter binding factor (HiNF-D) with a proximal regulatory element (Site II). A relationship between Site II occupancy by HiNF-D and the onset of osteoblast differentiation is supported by the persistence of Site II-HiNF-D interactions when proliferating rat osteoblasts are growth arrested under conditions that do not induce differentiation; and additionally, by the loss of Site II-HiNF-D interactions during the shut-down of proliferation when HL60 promyelocytic leukemia cells are induced to differentiate into monocytes. Our results are consistent with a requirement of proliferation for expression of genes involved with production, deposition and possibly organization of the osteoblast extracellular matrix. It is also reasonable to postulate that properties of the mineralizing matrix are related to the shut-down of proliferation.