title: Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells.
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 alpha2beta1 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 signal-regulated 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.
authors.affiliation: Department of Periodontics, Prevention, and Geriatrics, School of Dentistry, University of Michigan, Ann Arbor 48109-1078, USA.
authors.forename: Guozhi
authors.initials: G
authors.lastname: Xiao
authors.forename: Rajaram
authors.initials: R
authors.lastname: Gopalakrishnan
authors.forename: Di
authors.initials: D
authors.lastname: Jiang
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Reith
authors.forename: M Douglas
authors.initials: MD
authors.lastname: Benson
authors.forename: Renny T
authors.initials: RT
authors.lastname: Franceschi
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000235:genetics* / Q000494:pharmacology; D002454:Cell Differentiation; D005109:Extracellular Matrix / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D058574:Integrin-Binding Sialoprotein; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000235:genetics; D015398:Signal Transduction; D016212:Transforming Growth Factor beta
chemical_list: C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; C546872:Ibsp protein, mouse; D058574:Integrin-Binding Sialoprotein; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D020928:Mitogen-Activated Protein Kinases; D001205:Ascorbic Acid