No abstract

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization. (J Bone Miner Res 1999;14:893-903)

The bone-specific transcription factor, Cbfa1, regulates expression of the osteocalcin (OCN) gene and is essential for bone formation. However, little is known about the mechanisms regulating Cbfa1 activity. This work examines the role of the MAPK pathway in regulating Cbfa1-dependent transcription. Stimulation of MAPK by transfecting a constitutively active form of MEK1, MEK(SP), into MC3T3-E1 preosteoblast cells increased endogenous OCN mRNA, while a dominant negative mutant, MEK(DN), was inhibitory. MEK(SP) also stimulated activity of a 147-base pair minimal OCN promoter, and this stimulation required an intact copy of OSE2, the DNA binding site for Cbfa1. Effects of MEK(SP) were specific to Cbfa1-positive osteoblast-like cells. A purified His-tagged Cbfa1 fusion protein was directly phosphorylated by activated recombinant MAPK in vitro. Furthermore, (32)P metabolic labeling studies demonstrated that MEK(SP) clearly enhanced phosphorylation of Cbfa1 in intact cells, while MEK(DN) decreased phosphorylation. The specific MEK1/MEK2 inhibitor, PD98059, inhibited extracellular matrix-dependent up-regulation of the OCN promoter, indicating that the MAPK pathway and, presumably, Cbfa1 phosphorylation are also required for responsiveness of osteoblasts to extracellular matrix signals. This study is the first demonstration that Cbfa1 is controlled by MAPKs and suggests that this pathway has an important role in the control of osteoblast-specific gene expression.

The Cbfa1/Runx2 is an important transcription factor necessary for osteoblast differentiation and bone formation. However, the signaling pathways regulating Runx2 activity are just beginning to be understood. Inconsistencies between Runx2 mRNA or protein levels and its transcriptional activity suggests that posttranslational modification and/or protein-protein interactions may regulate this factor. Runx2 can be phosphorylated and activated by the mitogen-activated protein kinase (MAPK) pathway. This pathway can be stimulated by a variety of signals including those initiated by extracellular matrix (ECM), osteogenic growth factors like bone morphogenic proteins (BMPs) and fibroblast growth factor-2 (FGF-2), mechanical loading and hormones such as parathyroid hormone (PTH). Protein kinase A (PKA) may also phosphorylate/activate Runx2 under certain conditions. In addition, Runx2 activity is enhanced by protein-protein interactions as are seen with PTH-induced Runx2/AP-1 and BMP-mediated Runx2/Smads interactions. Mechanisms for interaction with Runx2 are complex including binding of distinct components such as AP-1 factors and Smads proteins to separate DNA regions in target gene promoters and direct physical interactions between Runx2 and AP-1/Smad factors. Post-translational modifications such as phosphorylation may influence interactions between Runx2 and other nuclear factors. These findings suggest that Runx2 plays a central role in coordinating multiple signals involved in osteoblast differentiation.

Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast-specific gene expression requires ascorbic acid (AA)-dependent assembly of a collagenous ECM. Matrix responsiveness requires an ␣ 2 ␤ 1 integrin-collagen interaction and mitogen-activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast-specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK-mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3-E1 cells were shown to constitutively express BMP-2, BMP-4, and BMP-7. Noggin, a specific BMP inhibitor, reversibly blocked AA-induced gene expression, indicating that BMP production by MC3T3-E1 cells was necessary for differentiation. The ability of exogenously added BMP-2, BMP-4, or BMP-7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP-7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal-regulated kinase (MEK), blocked AA-or BMP-7/AA-dependent gene expression in a time-and dose-dependent manner that was closely correlated with inhibition of extracellular signalregulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin-mediated cell-collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity. (J Bone Miner Res 2002;17:101-110)

Fibroblast growth factor 2 (FGF-2) is an important regulator of bone formation and osteoblast activity. However, its mechanism of action on bone cells is largely unknown. A major route for FGF signaling is through the mitogen-activated protein kinase (MAPK) pathway. We showed recently that this pathway is important for activation and phosphorylation of Cbfa1/ Runx2, an osteoblast-related transcription factor (Xiao, G

Extracellular matrix molecules such as type I collagen are required for the adhesion, migration, proliferation, and differentiation of a number of cell types including osteoblasts. Matrix components often affect cell function by interacting with members of the integrin family of cell surface receptors. Previous work showed that collagen matrix synthesis, induced by addition of ascorbic acid to cells, precedes and is essential for the expression of osteoblast markers and induction of the osteocalcin promoter in murine MC3T3-E1 cells. This later response requires OSE2, the promoter element recognized by Osf2 (also called Cbfa1/AML3/PEBP2␣A), a recently identified osteoblast-specific transcription factor. Osteoblasts express several integrins including ␣2␤1 which is a major receptor for type I collagen. This paper examines the role of the ␣ 2 -integrin subunit in osteocalcin promoter activation and osteoblast differentiation. Disruption of ␣ 2 -integrin-ECM interactions with a blocking antibody or DGEA peptide containing the cell-binding domain of type I collagen blocked activation of the mouse osteocalcin gene 2 promoter by ascorbic acid as well as induction of endogenous osteocalcin mRNA and mineralization. Furthermore, anti-␣ 2 -integrin blocking antibody or peptide reduced ascorbic aciddependent binding of Osf2 to OSE2 without affecting levels of transcription factor mRNA. Time course studies revealed that ascorbic acid-dependent binding of Osf2 to OSE2 preceded increases in osteocalcin and bone sialoprotein expression and this increase in Osf2 binding was not accompanied by comparable changes in levels of transcription factor mRNA or protein. Taken together, these studies demonstrate that an ␣ 2 -integrincollagen interaction is required for activation of Osf2 and induction of osteoblast-specific gene expression. Furthermore, matrix signals may regulate Osf2 through a post-translational pathway or via an accessory factor.As a cell primarily devoted to matrix production, the osteoblast must have the ability to monitor the composition of the extracellular matrix (ECM) 1 it is secreting as well as adapt matrix composition to the changing mechanical needs of bone. Consistent with the concept that there is a dialogue between the osteoblast and its ECM, osteoblast precursors must secrete a collagenous matrix before they will differentiate. Inhibition of collagen synthesis by growing cells in the absence of ascorbic acid (AA) or through the use of specific inhibitors totally blocks osteoblast differentiation (1-6). In vivo, both bone formation and osteoblast differentiation, as assessed by expression of osteocalcin (OCN) and alkaline phosphatase mRNAs, are also severely reduced in vitamin C-deficient animals (7, 8). Thus, the ECM is an important, but poorly understood regulator of the osteoblast differentiation pathway.Integrins are the principle mediators of the molecular dialogue between a cell and its ECM environment (for reviews, see Refs 9 and 10). These transmembrane receptors convey information from the ECM to ...

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.