Retinoic acid and BMP4 cooperate to induce the expression of a subset of extracellular matrix genes during preosteoblast differentiation

Extracellular matrix (ECM) proteins include structural and nonstructural proteins, both of which serve as biological modulators of cell function by interacting directly with cells or by controlling the activity of growth factors and proteases. Recent studies indicate that retinoic acid (RA) and Bone Morphogenetic Protein 4 (BMP4) have concerted effect on bone formation. However, its underlying mechanism, especially with respect to their effect on the dynamic of ECM gene expression, remains poorly defined. To address this question, the changes in the gene expression of ECM associated protein in C2C12 preosteoblasts upon stimulation by BMP4 and/or RA were analyzed. Following stimulation with each or both reagents for 2, 12 and 48 hours, the expression of ECM genes were analyzed by mouse MAC array and the results were validated by RT-PCR. With the exception of Hapln2 whose expression was increased only during early stages of differentiation, BMP4 and RA had a synergistic effect on the expression of many genes for ECM proteins and ECM structural proteins such as Egfl9, Ltbp2, Ogn and Matn2 during 48 hour window. Among 8 ECM associated genes analyzed, Egfl9 and Matn2 appeared to be induced primarily by BMP4 and to a lesser extent by RA. Our study demonstrates that BMP4 and RA can synergistically induce the differentiation of C2C12 preosteoblasts and modulate the gene expression of a subset of ECM associated proteins.