[EO-30] Comparative analysis of extracellular matrix proteins and their gene expression profile during osteoblast differentiation
[Abstract]
[Objective] Alveolar bone atrophy has been a factor challenging for successful prosthodontic treatment in broad aspects. However, current bone augmentation procedures do not necessarily result in satisfactory outcomes due to an insufficient understanding of bone biology. The key mechanism in this regard could be the reciprocal relationship between cells and extracellular matrix (ECM); while the cells metabolize ECM, the ECM provides an extracellular environment to the cells. To better understand these mutual regulatory mechanism, we aimed to analyze the changes in ECM that occur during osteoblast differentiation, which partly mimics the process of bone regeneration. Therefore, in this study, we analyze the compositional changes of ECM protein in conjunction with a comprehensive gene expression profile.
[Materials and Methods] Mouse osteoblast cell line (MC3T3-E1) was cultured and ECM-rich cell layer was harvested at 1,2,3, and 4W. Samples were decellularized, solubilized by hydroxylamine/guanidine hydrochloride, and analyzed by liquid chromatography with tandem mass spectrometry. Total RNA was isolated from the cultured cells at the same time point for proteomic analysis and comprehensive gene expression analysis was performed using RNA-seq. Changes in protein composition and gene expression profile were analyzed using integrative bioinformatic approaches.
[Results and Discussion] Proteomic analysis revealed that the composition of collagen increased while that of non-collagenous ECM decreased as time progressed. Principal component analysis revealed that the 1W sample has a distinctive proteomic profile compared to 2,3, and 4W. The profile of the top 2000 statistically significant genes indicates that the genes highly expressed at 1W were associated with cell cycle, while those at 3-4W were associated with ECM. The major fibrillar collagens (Col1a1, Col1a2, and Col3a1) and key transcriptional factors in osteoblasts (Runx2 and Sp7) showed a similar gene expression pattern, which gradually decreased overtime. Gene encoding elastic fibers (Eln and Fbn1) showed a strong time-dependent increasing pattern, indicating their important roles in ECM maturation. The increased level of Fbn1 protein was further confirmed at the protein level. Our integrated analysis of proteomic and gene expression profiles provides vital insights into the regulation of osteoblast-produced ECM, which has multiple roles in the bone generation process.
[Objective] Alveolar bone atrophy has been a factor challenging for successful prosthodontic treatment in broad aspects. However, current bone augmentation procedures do not necessarily result in satisfactory outcomes due to an insufficient understanding of bone biology. The key mechanism in this regard could be the reciprocal relationship between cells and extracellular matrix (ECM); while the cells metabolize ECM, the ECM provides an extracellular environment to the cells. To better understand these mutual regulatory mechanism, we aimed to analyze the changes in ECM that occur during osteoblast differentiation, which partly mimics the process of bone regeneration. Therefore, in this study, we analyze the compositional changes of ECM protein in conjunction with a comprehensive gene expression profile.
[Materials and Methods] Mouse osteoblast cell line (MC3T3-E1) was cultured and ECM-rich cell layer was harvested at 1,2,3, and 4W. Samples were decellularized, solubilized by hydroxylamine/guanidine hydrochloride, and analyzed by liquid chromatography with tandem mass spectrometry. Total RNA was isolated from the cultured cells at the same time point for proteomic analysis and comprehensive gene expression analysis was performed using RNA-seq. Changes in protein composition and gene expression profile were analyzed using integrative bioinformatic approaches.
[Results and Discussion] Proteomic analysis revealed that the composition of collagen increased while that of non-collagenous ECM decreased as time progressed. Principal component analysis revealed that the 1W sample has a distinctive proteomic profile compared to 2,3, and 4W. The profile of the top 2000 statistically significant genes indicates that the genes highly expressed at 1W were associated with cell cycle, while those at 3-4W were associated with ECM. The major fibrillar collagens (Col1a1, Col1a2, and Col3a1) and key transcriptional factors in osteoblasts (Runx2 and Sp7) showed a similar gene expression pattern, which gradually decreased overtime. Gene encoding elastic fibers (Eln and Fbn1) showed a strong time-dependent increasing pattern, indicating their important roles in ECM maturation. The increased level of Fbn1 protein was further confirmed at the protein level. Our integrated analysis of proteomic and gene expression profiles provides vital insights into the regulation of osteoblast-produced ECM, which has multiple roles in the bone generation process.