Dexamethasone and retinoic acid differentially regulate growth and differentiation in an immortalised human clonal bone marrow stromal cell line with osteoblastic characteristics

Abstract

Clonogenic immortalised human pre-osteoblastic cell lines provide useful species-specific experimental tools for the study of the regulation of osteoblastic proliferation and differentiation. Steroid hormones are major regulators of bone formation. Although much is known about the effects of dexamethasone on osteoblastic growth and differentiation in vitro, there is less information on the effects of trans-retinoic acid (RA), particularly in human cultures. We have established a clonal adult human cell line (C1) derived from a bone marrow aspirate. The cell line appeared to be bi-potential. The cells were able to differentiate into an adipocytic phenotype under appropriate culture conditions. When grown in osteogenic medium, the cells expressed alkaline phosphatase (ALP) and osteocalcin mRNA. The C1 cells also expressed several other osteoblastic markers such as collagen type 1 (COL 1), PTH/PTH-rp receptor constitutively. Transcripts for the osteoblast transcription factor Cbfa1 was also detected under basal conditions. In addition treatment with 1,25(OH) 2D 3 (10 −7 M) led to a marked increase in osteocalcin mRNA expression suggesting that this cell line represents a pre-osteoblastic population. We compared the effects of Dex and RA on osteoblastic function. For the assessment of PTH/PTH-rp receptor, osteocalcin and Cbfa1 mRNA expression and PTH-stimulated adenylate cyclase responsiveness, the cells were grown in the presence of Dex and RA and harvested on Days 1, 3, 7 and 14. RA (10 −7 M) had a mitogenic effect on the C1 cells. In contrast, Dex (10 −7 M) inhibited proliferation. A similar effect was observed with primary human bone marrow stromal cultures. Both Dex and RA inhibited COL 1 synthesis and decreased COL1 mRNA. Dex stimulated ALP activity and increased ALP mRNA expression whilst RA had an inhibitory effect. Dex treatment led to an increase in PTH/PTH-rp receptor mRNA and PTH-induced cAMP accumulation with a peak response at 24 h and this effect was sustained for up to 14 days. In contrast, long-term culture with RA resulted in a reduction in the cAMP response to PTH (Days 7 and 14) with no effect on PTH/PTH-rp receptor mRNA expression. Osteocalcin and Cbfa1 mRNA expression did not alter in the presence of Dex and RA at these time points. This study shows that Dex and RA have differential effects on the expression of the phenotypic markers and genes associated with osteoblast maturation. This homogeneous cell line can therefore be used further to elucidate the cellular and molecular mechanisms of action of Dex and RA at the different developmental stages of human osteoblastic differentiation. This cell line may thus provide a useful species-specific in vitro model for the evaluation of key genes and signalling molecules involved in osteogenesis. This would be of help in the design of ‘in vivo’ studies.