Title

Fluid shear stress induction of COX-2 expression in osteoblastic cells

Date of Completion

January 2002

Keywords

Health Sciences, Dentistry

Degree

Ph.D.

Abstract

Forces applied to bone drive interstitial fluid flow within the mineralized bone matrix, which exerts shear stress at surfaces of osteocytes and osteoblasts lining the interstices. Shear stresses along cell surfaces can stimulate new gene transcription. One of the genes whose transcription can be stimulated by fluid shear stress (FSS) is inducible cyclooxygenase-2 (COX-2). The goals of this thesis were to characterize and examine the signaling pathways involved in the FSS induction of COX-2 expression in osteoblastic cells. ^ We examined the FSS induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells, stably transfected with −371/+70 bp of the COX-2 5-flanking DNA (Pluc371), and in primary osteoblasts from calvaria (POBs) of mice transgenic for Pluc371. FSS (10 dynes/cm 2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at 8 h or greater in POBs. An inhibitor of new protein synthesis, puromycin (10 μg/ml), did not affect the small FSS induction of COX-2 mRNA at 1 h but inhibited the large peak induction at 4 h by 90%. ^ We next examined the signaling pathways involved in the peak induction of COX-2 mRNA by fluid shear stress in osteoblastic cells. Inhibiting ERK phosphorylation with specific inhibitors, inhibited fluid shear stress induction of COX-2 mRNA by 55–70% and luciferase activity by ≥80% in both MC3T3-E1 and POBs cells. Inhibitors of the protein kinase A (PKA) signaling pathway, reduced FSS stimulated COX-2 mRNA expression and luciferase activity at 4–4.5 h by 70% and 68%, respectively. In contrast, specific inhibitors of the protein kinase C (PKC) pathway had no effect on the FSS induction of COX-2 mRNA and luciferase activity at 4–4.5 h. Similar experiments were performed in order to evaluate the signaling pathways involved in the early induction of COX-2 by FSS in MC3T3-E1 osteoblastic cells. Inhibitors of the ERK and PKC signaling pathways decreased fluid shear stress induction of COX-2 mRNA at 1 h by 79% and 66%, respectively. We therefore conclude that the FSS early and late induction of COX-2 expression occurs by separate signaling pathways. ^