Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways
Introduction
Mounting evidence suggests that osteocytes, positioned within bone mineral׳s interstitial space, coordinate cellular remodeling leading to functional adaptation in response to mechanically-induced stimuli (Bonewald, 2011, Schaffler et al., 2014). Interstitial fluid flow is one such stimulus (Kufahl and Saha, 1990, Weinbaum et al., 1994) observed upon cyclic whole bone loading (Knothe Tate and Knothe, 2000, Price et al., 2011). Fluid flow exposes osteocytes to enhanced solute transport (Price et al., 2011), streaming potentials (Cowin et al., 1995), and cyclic fluid shear stress transduced via cellular adhesion molecules, the cell membrane, the actin cytoskeleton, and possibly primary cilium (Bonewald and Johnson, 2008).
In vitro, specific responses of osteocytic cells to steady, pulsating, and oscillating fluid flow (OFF) have been widely studied. However, these studies are generally constrained to a limited set of candidate genes or proteins as part of known or suspected signaling pathways. Osteocytic cells elastically deform when subject to physiological peak fluid shear stresses up to 5 Pa (Kwon and Jacobs, 2007, Price et al., 2011), initiating the rapid release of adenosine triphosphate (ATP) and prostaglandin E2 via gap junctions or hemichannels (Batra et al., 2012, Cherian et al., 2005, Genetos et al., 2007, Klein-Nulend et al., 1995). In turn, these factors contribute to cell network propagation of intracellular calcium waves in proportion to flow-induced shear stress in vitro (Huo et al., 2008, Lu et al., 2012) as well as during in situ dynamic bone loading (Jing et al., 2013). Subsequent to OFF, osteocytic cells demonstrate stress amplitude-, frequency-, and duration-dependent shifts in mRNA levels with increased prostaglandin E2-synthesizing Ptgs2 (Cox-2) and a decreased Rankl/Opg mRNA ratio associated with reduced recruitment of bone-resorbing osteoclasts (Kim et al., 2006, Li et al., 2012, Xiong and O׳Brien, 2012). Fluid flow also regulates molecules involved with Wnt/β-catenin pathway activation (Kamel et al., 2010, Santos et al., 2009) and protects against osteocyte apoptosis (Cheung et al., 2011, Kitase et al., 2010).
Related studies have broadened our knowledge of skeletal mechano-sensing through transcriptomic (Mantila Roosa et al., 2011, McKenzie et al., 2011, Reijnders et al., 2013, Rolfe et al., 2014, Xing et al., 2005) or proteomic investigations (Li et al., 2011, Zhang and Wang, 2009) of heterogeneous cell populations from whole bones subject to in vivo mechanical loading. Others have analyzed global gene expression specifically in osteocytes isolated from loaded rat trabeculae (Wasserman et al., 2013) and osteocyte-like MLO-Y4 cells subjected to cyclic compressive force stimulation (Chen et al., 2010). However, no single study has taken an integrated transcriptomic and proteomic approach.
In this study we evaluated the utility of two unbiased high-throughput approaches, gene transcript microarrays and protein mass spectrometry, to investigate the response of osteocytes exposed to fluid flow. We mapped a time course of flow-induced fluctuations in both gene transcript levels and protein abundances at corresponding time points. Additionally, in spite of various post-transcriptional modifications, we computationally predicted sequences of critical signaling nodes using an integrative bioinformatics approach. We examined the hypothesis that this broadened inquiry will reveal a mechano-sensitive shift in gene transcript and protein abundances in response to fluid flow, reflecting regulation of known mechano-sensitive signaling pathways as well as novel signaling networks. Our results demonstrate both individual and global shifts in signaling molecules consistent with known regulation of bone metabolism. More importantly, we identified signaling molecules and pathways not previously implicated in mechanotransduction in bone, most notably, up-regulation of Cxcl1 and Cxcl2.
Section snippets
Cell culture
MLO-Y4 osteocyte-like cells (Kato et al., 1997), courtesy of Dr. Lynda Bonewald (University of Missouri—Kansas City) were maintained in normal growth medium (α-MEM [Invitrogen, Grand Island, NY] with 2.5% CS [Hyclone, Logan, UT], 2.5% FBS [Lonza, Walkersville, MD], 1% Penicillin/Streptomycin) throughout all portions of the experiment. Cells were seeded 48 h prior to fluid flow on 75×38×1 mm glass slides coated with 300 μg/ml Type I Collagen (BD Biosciences, Bedford, MA) for 1 h and washed. Cell
Transcriptome regulation by fluid flow
We detected approximately 14,000 unique gene products using microarray analysis of whole cell lysates from flowed and non-flowed MLO-Y4 cells. Among these, 171 gene products increased or decreased abundance relative to non-flow controls at one or more of the given time points of 0, 2, 8, or 24 h post-flow at a significance level of p<0.05 (Supplementary Table 1). Similarly, 423 gene products were regulated at p<0.1 (Supplementary Table 2). As we have done previously (Waters et al., 2011), we
Discussion
The goal of this study was to establish the utility of high-throughput transcriptomic and proteomic analyses to unravel the mechano-sensitive response of MLO-Y4 cells subjected to OFF. We hypothesized that this global analysis would reflect regulation of genes and proteins previously known to be responsive to fluid flow while also identifying novel mechano-sensitive signaling molecules and pathways. Gene transcription and protein levels demonstrated a shift attributable to two variables: fluid
Conflicts of interest statement
The authors have no conflicts to disclose.
Acknowledgments
A portion of this research was performed using the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy׳s Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory. Pacific Northwest National Laboratory is operated by Battelle Memorial Institute for the U.S. Department of Energy under contract DE-AC05-76RLO-1830. This work was supported by R01 AG13087-15 from the National
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