Bone adaptation to mechanical loading is regulated via signal transduction by mechano-sensing osteocytes. Mineral-embedded osteocytes experience strain-induced interstitial fluid flow and fluid shear stress, and broad shifts in gene expression are key components in the signaling pathways that regulate bone turnover. RNA sequencing analysis, or RNA-Seq, enables more complete characterization of mechano-responsive transcriptome regulation than previously possible.
Researchers at Penn State College of Medicine hypothesized that RNA-Seq of osteocytic cells would reveal both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays. MLO-Y4 cells were flowed for 2 h with 1 Pa oscillating fluid shear stress and post-incubated 2 h. RNA-Seq of original samples detected 55 fluid flow-regulated gene transcripts, the same number previously detected by microarray. However, RNA-Seq demonstrated greater dynamic range, with all 55 transcripts increased >1.5-fold or decreased <0.67-fold whereas 10 of 55 met this cut-off by microarray.
As part of a broad inflammatory response inferred by gene ontology analyses, the researchers again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1α and AMPK signaling pathways. Importantly, they detected both expected fluid flow-sensitive transcripts and transcripts not previously identified as flow-sensitive. They found RNA-Seq advantageous over microarrays because of its greater dynamic range and ability to analyze unbiased estimation of gene expression, informing our understanding of osteocyte signaling.
(A) Experimental timeline for fluid flow treatment of osteocytic MLO-Y4 cells. (B) Workflow for RNA-Seq analysis using the Tuxedo Suite tools Tophat, Cufflinks, and Cuffdiff. (C) Representative bar graphs of reads vs. base pair from Control and Flow samples illustrate mapping to corresponding regions of the mouse genome encoding Cxcl1 and Cxcl2, indicated by thicker bars (reference exons) below reads. Expression was visualized with the UCSC Genome Browser (http://genome.ucsc.edu).