We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.
The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAPlbinding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAPl-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1-and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.Transcription of the Saccharomyces cerevisiae HIS4 gene is regulated by two separate systems. One system, general amino acid control, increases HIS4 transcription upon starvation for any amino acid(s) (17). In this system, amino acid starvation causes an increase in the levels of the GCN4 protein (for a review, see reference 13). GCN4 stimulates transcription by binding directly to the promoters of genes that are under general control (1, 15). In vitro, GCN4 binds to five 5'-TGACTC-3' sites located within the HIS4 promoter but has a much higher affinity for one of these sites, site C, than for the other four sites (1). The GCN4 protein is also expressed at low levels under nonstarvation conditions and is responsible for some of the H1S4 transcription under these conditions.The second system that regulates HIS4 transcription is basal control. Basal control activates HIS4 transcription in the absence of amino acid starvation but further stimulates HIS4 transcription under conditions of phosphate or adenine limitation (2, 28). Basal-control regulation of HIS4 requires two trans-acting proteins, BAS1 and BAS2 (PH02). BAS1 has a DNA-binding region that is similar to Myb proteins of higher organisms (28), and BAS2 contains a region that is similar to the homeo box (7). Neither BAS1 nor BAS2 alone can activate HIS4 transcription. BAS1 and BAS2 bind adjacently on the HIS4 promoter at a site upstream of the high-affinity GCN4 site C (28). BAS1 and BAS2 can activate HlS4 transcription independently of GCN4. Likewise, GCN4 can activate HIS4 transcription independently of both BAS1 and BAS2.A yeast strain that does not contain GCN4, BAS1, and BAS2 (gcn4-2 basl-2 bas2-2) is His-as a result of lack of transcription of the HIS4 gene (3). Therefore, normal activation of HIS4 transcrip...
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(−8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(−8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
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