DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

Lysophosphatidic acid (LPA) is a bioactive lipid with functional properties that overlap those of growth factors and cytokines. LPA production in vivo is linked to platelet degranulation and the biological activities of this lipid are associated with wound healing. Osteoblasts and their progenitor cells are exposed to high levels of this lipid factor in regions adjacent to bone fractures and we postulate a role for LPA in skeletal healing. The regeneration of bone injuries requires a complex array of changes in gene expression, but the effects of LPA on mRNA levels in bone cells have not been investigated. We performed a genome-wide expression analysis in LPA-treated MC3T3-E1 pre-osteoblastic cells using Affymetrix GeneChip arrays. Cells exposed to LPA for 6 h exhibited 513 regulated genes, whereas changes in the levels of 54 transcripts were detected after a 24-h LPA treatment. Gene ontology analysis linked LPA-regulated gene products to biological processes that are known to govern bone healing, including cell proliferation, response to stress, organ development, chemotaxis/motility, and response to stimuli. Among the gene products most highly up-regulated by LPA were transcripts encoding the anti-inflammatory proteins sST2, ST2L, and heat-shock protein 25 (HSP25). RT-PCR analysis confirmed that these mRNAs were increased significantly in MC3T3-E1 cells and primary osteoblasts exposed to LPA. The response of cells to LPA is mediated by G-protein-coupled receptors, and the stimulation of anti-inflammatory gene expression in MC3T3-E1 cells was blocked by Ki16425, an inhibitor of LPA1 and LPA3 receptor forms. Pertussis toxin impaired only the LPA-induced expression of sST2. LPA-stimulated levels of sST2, ST2L and HSP25 mRNAs persisted if the cytosolic Ca2+ elevations elicited by this lipid were blocked with BAPTA. In contrast to the stimulatory effect of LPA, exposure of MC3T3-E1 cells to fluid shear reduced the transcript levels of all three anti-inflammatory genes. The induction of sST2, ST2L and HSP25 expression by LPA suggests a role for this lipid factor in the regulation of osteoblastic cell function during periods of inflammation.