TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro, preventing development of the mature bone phenotype

Abstract

This study examines the mechanism by which TGF-beta 1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-beta 1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-beta 1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-beta 1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and alpha 1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-beta 1 response throughout the course of osteoblast differentiation. The initiation of a TGF-beta 1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-beta 1. These in vitro results indicate that TGF-beta 1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml.

Human Adipose Tissue Is a Source of Multipotent Stem Cells

“…This pattern, similar to AP activity, may be the response of PLA cell subpopulations at distinct developmental stages to osteogenic induction. In support of this, several other induction agents have been shown to stage-specific effects on osteogenesis, including TGF␤ (Breen et al, 1994). Finally, OC expression by induced PLA cells was dependent upon osteogenic agent as OC expression was inhibited upon dexamethasone exposure, an effect not observed in MSC controls.…”

Erk pathway and activator protein 1 play crucial roles in FGF2‐stimulated premature cranial suture closure

“…The resulting dimers can bind to DNA at a consensus AP1 binding site termed the TPA response element (TRE), thereby transactivating the expression of AP1-responsive genes (St-Arnaud and Quelo, 1998). Experiments where fos and jun expression in the bone was modulated by steroid hormone treatment or by overexpression or ablation of fos or jun genes suggest that the Fos and Jun proteins are important in the regulation of bone formation (Clohisy et al, 1992;Breen et al, 1994). This suggestion is supported by a study by McCabe et al (1996), who showed that the developmental expression and activity of particular Fos and Jun proteins are functionally related to osteoblast maturation.…”

Transforming growth factor‐β1 downregulates dexamethasone‐indunced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV‐HFO

“…It is becoming clear that the TGF-fl supergene family plays an instrumental role for the osteoblastic cell lineage to display its full catalogue of phenotypic characteristics during osteogenesis. In various experimental systems for studying bone formation, contradictory results have been obtained on whether TGF-fll enhances or inhibits bone matrix formation, mineralization and the expression of bone marker proteins [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. This has in part been explained by differences in the source and type of cells, stage of osteoblastic maturation, culture conditions and presence of other cytokines and their receptors.…”

“…Based on all these in vitro and in vivo studies, several authors suggested that, while TGF-fll initiates the proliferation, differentiation and extracellular matrix synthesis of bone cells, TGF-fll may be less involved in or actually inhibit the later phases of osteogenesis, i.e. mineralization [17,20,21]. The biological responses to TGF-fl may be mediated by the retinoblastoma gene product [38], which binds to the SV-40 T antigen, and therefore the effect of T antigen expression by the SV-HFO cells on the TGF-fll signal transduction pathway will require further analysis.…”

“…Bone is an abundant source of TGFofll [1,2] and in vivo studies have shown that TGF-fll is a local regulator of bone formation. In osteoblast cell culture systems, TGF-fll regulates the mineralization process and the expression of osteoblastic markers, including alkaline phosphatase, c~1 (I) procollagen, osteopontin, osteonectin and osteocalcin [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22].…”