ANALYSIS OF DIFFERENTIAL GENE-EXPRESSION IN RAT TIBIA AFTER AN OSTEOGENIC STIMULUS IN-VIVO - MECHANICAL LOADING REGULATES OSTEOPONTIN AND MYELOPEROXIDASE

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse t ranscriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Spr ague-Dawley rats, approximately 8 months old, received either a sham o r bending load using a four-point loading apparatus on the right tibia . RNA was collected at 1 h and 24 h after load was applied, reverse-tr anscribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulat ed bands. Further analysis of seven of these bands allowed us to isola te two genes that are regulated in response to a loading stimulus. Nuc leotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollage nous bone matrix protein. Northern blot analysis confirms that OPN mRN A expression is increased by nearly 4-fold, at 6 h and 24 h after load ing. The second band shares 90% homology with mouse myeloperoxidase (M PO), a bactericidal enzyme found primarily in neutrophils and monocyte s. Semiquantitative PCR confirms that MPO expression is decreased 4-to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocyte s. These findings reinforce the association of OPN expression to bone turnover and describes for the first lime, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function. (C) 1998 Wiley-Liss , Inc.