title: Mapping the osteocytic cell response to fluid flow using RNA-Seq.
abstract: 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. We hypothesized that RNA-Seq of osteocytic MLO-Y4 cells reveals both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays. MLO-Y4 cells were flowed for 2h with 1Pa oscillating fluid shear stress and post-incubated 2h. RNA-Seq of original samples detected 55 fluid flow-regulated gene transcripts (p-corrected <0.05), 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. Analyses were complimentary in patterns of regulation, though only 6 transcripts were significant in both RNA-Seq and microarray analyses: Cxcl5, Cxcl1, Zc3h12a, Ereg, Slc2a1, and Egln1. As part of a broad inflammatory response inferred by gene ontology analyses, we again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1α and AMPK signaling pathways. Importantly, we detected both expected fluid flow-sensitive transcripts (e.g. Nos2 [iNOS], Ptgs2 [COX-2], Ccl7) and transcripts not previously identified as flow-sensitive, e.g. Ccl2. We 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.
authors.affiliation: Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, PA 17033, USA; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Govey
authors.affiliation: Institute for Personalized Medicine, Departments of Biochemistry and Molecular Biology and Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA.
authors.forename: Yuka Imamura
authors.initials: YI
authors.lastname: Kawasawa
authors.affiliation: Division of Musculoskeletal Sciences, Department of Orthopaedics and Rehabilitation, Penn State College of Medicine, Hershey, PA 17033, USA; Department of Biomedical Engineering, Penn State College of Engineering, University Park, PA 16802, USA; Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23284, USA. Electronic address: hjdonahue@vcu.edu.
authors.forename: Henry J
authors.initials: HJ
authors.lastname: Donahue
mesh_terms: D000818:Animals; D016723:Bone Remodeling; D002460:Cell Line; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D063990:Gene Ontology; D053263:Gene Regulatory Networks; D040542:Mechanotransduction, Cellular; D051379:Mice; D010011:Osteocytes / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D012333:RNA, Messenger
keywords: Fluid flow; Gene expression; Mechanotransduction; Osteocyte; RNA-Seq; Signaling