Gene expression patterns in bone following lipopolysaccharide stimulation

Bone displays suppressed osteogenesis in inflammatory diseases such as sepsis and rheumatoid arthritis. However, the underlying mechanisms have not yet been clearly explained. To identify the gene expression patterns in the bone, we performed Affymetrix Mouse Genome 430 2.0 Array with RNA isolated from mouse femurs 4 h after lipopolysaccharide (LPS) administration. The gene expressions were confirmed with real-time PCR. The serum concentration of the N-terminal propeptide of type I collagen (PINP), a bone-formation marker, was determined using ELISA. A total of 1003 transcripts were upregulated and 159 transcripts were downregulated (more than twofold upregulation or downregulation). Increased expression levels of the inflammation-related genes interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were confirmed from in the period 4 h to 72 h after LPS administration using real-time PCR. Gene ontogene analysis found four bone-related categories involved in four biological processes: system development, osteoclast differentiation, ossification and bone development. These processes involved 25 upregulated genes. In the KEGG database, we further analyzed the transforming growth factor β (TGF-β) pathway, which is strongly related to osteogenesis. The upregulated bone morphogenetic protein 2 (BMP2) and downregulated inhibitor of DNA binding 4 (Id4) expressions were further confirmed by real-time PCR after LPS stimulation. The osteoblast function was determined through examination of the expression levels of core binding factor 1 (Cbfa1) and osteocalcin (OC) in bone tissues and serum PINP from 4 h to 72 h after LPS administration. The expressions of OC and Cbfa1 decreased 6 h after administration (p < 0.05). Significantly suppressed PINP levels were observed in the later stage (from 8 h to 72 h, p < 0.05) but not in the early stage (4 h or 6 h, p > 0.05) of LPS stimulation. The results of this study suggest that LPS induces elevated expressions of skeletal system development- and osteoclast differentiation-related genes and inflammation genes at an early stage in the bone. The perturbed functions of these two groups of genes may lead to a faint change in osteogenesis at an early stage of LPS stimulation. Suppressed bone formation was found at later stages in response to LPS stimulation.