Regulation of Collagenase-3 and Osteocalcin Gene Expression by Collagen and Osteopontin in Differentiating MC3T3-E1 Cells

Abstract

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the ␣ v integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain ␣ v integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and ␣ v integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.When primary or clonal osteoblastic cells, such as MC3T3-E1 cells, are cultured with ␤-glycerophosphate (␤-gly) 1 and ascorbic acid (AA), the cells undergo a differentiation process and eventually produce bone nodules resembling woven bone in vitro (1-4). Mineral deposition occurs in the final stages of osteoblast differentiation and is associated with maximal osteoblastic expression of osteocalcin and collagenase-3 (5-7). The mechanisms controlling osteoblast differentiation are unclear, but research suggests that a reciprocal interaction exists between the osteoblast and the surrounding extracellular matrix. For example, when osteoblasts or osteoblast-like cells are grown on plates pre-coated with collagen, they exhibit early signs of osteoblast maturation and osteoblastic gene expression (8 -12). Further accumulating data suggest that the extracellular matrix mediates many effects through activation of integrin signaling pathways in osteoblasts (12-14). Osteoblasts and osteoblast-like cells have been reported to express a wide variety of integrin receptor subunits, including ␣ 1 , ␣ 2 , ␣ 3 , ␣ 4 , ␣ 5 , ␣ v , ␤ 1 , and ␤ 3 (15-17).Osteocalcin (OC), or bone gla protein, is a calcium-binding protein found in bone extracellular matrix and expressed by osteoblasts, odontoblasts, and hypertrophic chondrocytes (reviewed in Ref. 18). It has an affinity for hydroxyapatite, suggesting a role in the regulation of mineralization. In fact, OCnull mutant mice exhibit increased bone density (19), although defects in mineralization have been detected (20). Recent evidence suggests that the up-regulation of OC late in osteoblast diff...

Parathyroid Hormone-responsive Smad3-related Factor, Tmem119, Promotes Osteoblast Differentiation and Interacts with the Bone Morphogenetic Protein-Runx2 Pathway

“…Although Runx2 is critical for the commitment of mesenchymal stem cells into the osteoblast lineage (16,(31)(32)(33), transgenic mice overexpressing Runx2 under the control of the osteoblast-specific Col1a1 promoter exhibit decreased bone mineralization and increased bone resorption (34). Although in the Runx2 osteoblast-overexpressing transgenic mice, the osteoblast number is increased at an early differentiation stage, osteoblast functions (matrix production and mineralization) were impaired in these cells (34).…”

Cooperative Interactions between Activating Transcription Factor 4 and Runx2/Cbfa1 Stimulate Osteoblast-specific Osteocalcin Gene Expression

“…For example, bone morphogenetic proteins induce osteoblast differentiation and bone formation through SMADs-Runx2 interactions (19,45). Likewise, PTH increases the expression of rat collagenase 3 in osteoblasts via cooperative interactions between AP-1 and Runx2 (17,18). Our recently published work demonstrates that the OSE1 in the mOG2 promoter is necessary and sufficient for PTH induction of the Ocn gene (35).…”

Parathyroid Hormone Induction of the Osteocalcin Gene

“…Studies on the collagenase-3 gene promoter showed that both a RUNX2 binding site and a conserved AP-1 binding site are necessary for PTH stimulation (38,39,53,54). Mutations in either site that abrogated the binding of RUNX2 or c-Fos/c-Jun, respectively, abolished PTH stimulation of promoter activity.…”

“…Whether Osf1 is regulated by this mechanism needs to be determined. Finally, PTH may alter interactions between Osf1 and other proteins much as it modifies interactions between RUNX2 and AP-1 factors on the collagenase gene (39,53). Determining the precise mechanisms through which PTH regulates Osf1 transcriptional activity must await the availability of molecular reagents for Osf1.…”

“…The ability of PTH to regulate gene expression is dependent on the activation of specific transcription factors such as cAMPresponse element-binding protein (CREB) (36, 37), AP-1 family members (38,39), pituitary-specific transcription factor 1 (40), and RUNX2 (38). Although it is well known that PTH induces OCN gene expression, the mechanism mediating this regulation is not known.…”