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.

ABSTRACT

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)

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 ...

s recently introduced general theory of crime has received considerable empirical support. Researchers have found that low self-control, the general theory's core concept, is related to lawbreaking and to deviant behaviors considered by Gottfredson and Hirschi to be "analogous" to crime. In this article, we extend this research by assessing the effects of low self-control on crime and analogous behaviors and by using two distinct measures of self-control, an attitudinal measure and the analogous/behavior scale. Thus, following Gottfredson and Hirschi, we use analogous imprudent behaviors as outcomes of low self-control and as indicators of low self-control's effects on crime. We also examine an important but thus far neglected part of the theory: the claim that low self-control has effects not only on crime but also on life chances, life quality, and other social consequences. Consistent with the general theory, we found that both measures of self-control, attitudinal and behavioral, have effects on crime, even when controlling for a range of social factors. Further, the analysis revealed general support for the theory's prediction of negative relationships between low self-control and social consequences other than crime-life outcomes and quality of life. University of Cincinnati. We thank several anonymous reviewers for their valuable comments. CRIMINOLOGY VOLUME 35 NUMBER 3 1997 475 476 EVANS ET AL.Starting with its publication in 1990 and continuing to the present, Gottfredson and Hirschi's A General Theory of Crime has generated considerable interest and much controversy (see, e.g.