Prostaglandin D2 pathway and peroxisome proliferator‐activated receptor γ‐1 expression are induced by mechanical loading in an osteoblastic cell line

Abstract

The hypothesis underlying the current study was that the arachidonic acid cascade, specifically activation of the prostaglandin (PG) D(2) pathway in osteoblasts, is an anabolic signal induced by mechanical loading. Previous studies have shown that mechanical loading of osteoblasts triggers cyclooxygenase (COX)-2, PGE(2) and prostacyclin (PGI(2)) synthesis. Since modest mechanical loading of osteoblasts promotes bone formation, we sought to determine whether mechanical stress activates the osteoblastic PGD(2) pathway resulting in the synthesis of osteogenic cyclopentenones, including Delta(12)PGJ(2). Osteoblast monolayers were stretched using a Bioflex apparatus at a frequency of 1 Hz with 1% elongation. Cells and cell media were collected at various time points: 5, 10, 15, 30 min; and 1, 4, 16, 24 h. RNA was extracted for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In certain experiments, cells were pre-labeled with (14)C arachidonic acid prior to stretching. Radiolabeled metabolites in cell media were identified by reverse-phase high performance liquid chromatography (RP-HPLC). Osteoblasts were evaluated for an induction in bone nodule formation by stretching. Mechanical strain significantly increased mRNA expression of COX-1, COX-2, PGD(2) synthase and peroxisome proliferator-activated receptor (PPAR) gamma-1, but not of PPARgamma-2 as compared to control unstretched cells (p < 0.05). Mechanical loading stimulated the release of PGE(2), PGD(2) and the PGD(2) metabolite Delta(12)PGJ(2). Mechanical strain resulted in the induction of bone nodules. This report indicates that mechanical loading of osteoblasts results in activation of PGD(2) and the concomitant expression of transcription factor PPARgamma-1 mRNA. The coordinated synthesis of Delta(12)PGJ(2), a natural ligand for PPARgamma-1, with the increased expression of PPARgamma-1, suggests that biomechanical transduction pathways that initially involve the activation of cyclooxygenases may also involve the activation of the Delta(12)PGJ(2)-PPAR pathway.