Effects of cortisol and bone morphogenetic protein-2 on stromal cell differentiation: correlation with CCAAT-enhancer binding protein expression

Abstract

Bone marrow stroma contain pluripotential cells with the potential to differentiate into various mesenchymal cell lineages. We compared the effect of cortisol and bone morphogenetic protein-2 (BMP-2) on the differentiation of murine ST-2 stromal cells into mature osteoblasts or adipocytes. ST-2 cells were cultured for 3–27 days in the presence of 10% fetal bovine serum, 100 μg/mL ascorbic acid, and 5 mmol/L β-glycerolphosphate in the presence or absence of cortisol at 1 μmol/L or BMP-2 at 1 nmol/L. Untreated ST-2 cells expressed high levels of alkaline phosphatase activity (APA) 15 days after confluence, and this was followed by the appearance of mineralized nodules after 24 days. BMP-2 accelerated and intensified the appearance of cells expressing APA and the presence of mineralized nodules. In contrast, cortisol decreased APA, prevented the formation of mineralized nodules, and induced a cellular phenotype characteristic of adipocytes. Untreated stromal cells expressed osteocalcin, Cbfa1, type I collagen, and alkaline phosphatase mRNA. BMP-2 increased osteocalcin and alkaline phosphatase mRNA, whereas cortisol suppressed their expression, as well as Cbfa1 and type I collagen transcripts. Cortisol enhanced, and BMP-2 downregulated, peroxisome proliferator-activated receptor gamma 2 and adipsin transcripts. The C/EBP transcription factors regulate genes critical for adipocytic and osteoblastic differentiation. Cortisol increased the expression of C/EBP α, β, δ, and γ mRNA levels, whereas BMP-2 had minor effects on C/EBP expression. In conclusion, BMP-2 accelerates the differentiation of stromal cells toward an osteoblastic phenotype, whereas glucocorticoids induce their differentiation toward an adipocytic phenotype.

Introduction

Bone marrow stroma contain pluripotential cells with the potential to differentiate into various mesenchymal cell lineages including osteoblasts, adipocytes, chondrocytes, and myoblasts. The ultimate cellular phenotype depends, to an extent, on systemic signals and influences present in the cellular microenvironment.9, 20, 38, 48

In vivo and in vitro studies indicate that glucocorticoids have the potential to induce the differentiation of bone marrow stromal cells toward the adipocytic pathway, and this may occur at the expense of osteoblastic cell differentiation.4, 34 Indeed, there is an inverse relationship between marrow fat accumulation and trabecular bone abundance in patients receiving glucocorticoid therapy.4, 33 This may play a role in the pathogenesis of glucocorticoid-induced osteoporosis.5, 6 Some in vitro studies have suggested that glucocorticoids have the capacity to induce the differentiation of preosteoblastic cells into mature osteoblasts; however, this effect is dependent on culture conditions and not consistent with the inhibitory actions of glucocorticoids on the differentiated function of the osteoblast.5, 28, 40 Furthermore, recent studies from our laboratory have demonstrated that glucocorticoids prevent the terminal differentiation of cells of the osteoblastic lineage.37 In contrast, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal cells into cells of the osteoblastic lineage and enhance the differentiated function of the osteoblast.18, 21, 47, 48 BMPs also play a stimulatory role on chondrogenesis and have an inhibitory effect on myogenesis.48

The CCAAT-enhancer binding proteins (C/EBPs) are a family of transcription factors that play a role in cell differentiation.22, 24, 49 To date, six C/EBPs have been characterized, α, β, δ, γ, ε, and ζ. The C/EBP proteins contain a highly conserved DNA-binding domain and a bZIP dimerization domain, and can form homo- and heterodimers that bind to similar sequence motifs. C/EBPs are expressed in multiple cell types, including osteoblasts and adipocytes, and C/EBP β and δ are essential for the formation of mature adipocytes.15, 27, 29, 43 Recently, we demonstrated that glucocorticoids enhance the expression of C/EBP β and δ in osteoblasts, and these two transcription factors play a role in the downregulation of insulin-like growth factor (IGF)-1 expression in these cells.16 Because C/EBP β and δ are essential for adipogenesis and IGF-1 enhances the differentiated function of the osteoblast, the results suggest that C/EBP β and δ play a role in directing mesenchymal cells away from the osteoblastic and toward an adipocytic pathway. As an initial step in establishing the role of C/EBPs in cells of the osteoblastic lineage, we examined the differentiation of the murine stromal cell line, ST-2, and correlated changes in C/EBP expression with the adoption of the adipocytic or the osteoblastic phenotype. For this purpose, ST-2 cells were treated with cortisol or BMP-2, in an attempt to induce cell differentiation toward the adipocytic or osteoblastic lineage.