Construction of Subtracted Osteoblast cDNA Library with Oxidative Stress-modulated Transcription

Objectives: It is known that bone formation steadily declines with age resulting in significant loss of bone mass. Reactive oxygen species (ROS) are produced by various environmental agents and are considered to be responsible for the aging process and a number of pathological conditions. However, the mechanisms underlying the aging process and its effect on bone metabolism are not well understood. To identify the genes altered those expression by ROS, a suppression subtractive hybridization procedure was carried out between cDNAs from H(2)O(2)-treated and untreated mouse osteoblastic cells, MC3T3-E1. Methods: Tester cDNA was synthesized from total RNA of MC3T3-E1 cells after 6h of H(2)O(2) treatment (0.4mM) using template-switching oligonucleotide (SMART PCR cDNA Synthesis Kit, CLONTECH) and driver cDNA was from total RNA of untreated cells, then both cDNAs were selectively amplified by PCR. After two successive hybridizations with tester and driver cDNAs, the resulting mixtures were diluted and amplified by PCR to produce subtracted and unsubtracted PCR fragment libraries. Cloned cDNA inserts were evaluated for differential expression by DNA dot-blot analysis and then sequenced. Sequenced data were performed BLASTN homology search with NCBI DNA database. Results: Among the clones of reduced expression, F0F1-ATPase, alpha 1-tubulin, fibronectin 1 and calnexin were found. The clones of increased expression were contained interleukin 1 receptor-like 1gene. Conclusion: Reduced gene expressions of F0F1-ATPase and fibronectin 1 by ROS in osteoblasts may lead to reduce cellular ATP turnover, proliferation and differentiation through FAK signals, and increased expression of interleukin 1 receptor may diminish bone forming activity of MC3T3-E1 cells.