PNNL

Abstract

Osteocytes, positioned within bone’s interstitial space, are subject to fluid flow upon whole bone loading. Such fluid flow is widely theorized to be a mechanical signal transduced by osteocytes, initiating a poorly understood cascade of signaling events mediating bone metabolism. The objective of this study was to utilize high-throughput approaches to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels. Osteocyte-like MLO-Y4 cells were subjected to 2 hours of 10 dyn/cm2 oscillating fluid flow in parallel-plate fluid flow chambers and harvested for analysis at 0, 2, 8, and 24 hours post-flow incubation. Microarray analysis demonstrated fluid flow regulation of genes consistent with known anabolic loading responses, including Ptgs2, NF-?B inhibitors, MAP3 kinases, and Wnt/ß-catenin pathway signaling molecules. However, two of the most highly up-regulated genes—Cxcl1 and Cxcl2, confirmed by qPCR—have not previously been reported to be responsive to fluid flow. Gene ontology analysis suggested a highly significant inflammatory and immune response, with cellular functions including trafficking, cell-to-cell signaling, and tissue development. Proteomic analysis of the same samples demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. An integrative pathway analysis merging fold changes in gene and transcript levels predicted signaling nodes not directly detected at the sampled time points, including STAT3 and c-Myc. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as a possible paracrine agent for osteoblastic and osteoclastic recruitment. Moreover, these results demonstrate the utility of integrative, high-throughput approaches in place of a traditional candidate approach for identifying novel mechano-sensitive signaling molecules.