Integrative transcriptomic and proteomic analysis of osteocytic cells exposed to fluid flow reveals novel mechano-sensitive signaling pathways

Abstract

Osteocytes, positioned within bone's porous structure, are subject to interstitial 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 adaptation to mechanical load. The objective of this study was to examine the time course of flow-induced changes in osteocyte gene transcript and protein levels using high-throughput approaches. Osteocyte-like MLO-Y4 cells were subjected to 2 hours of oscillating fluid flow (1 Pa peak shear stress) and analyzed following 0, 2, 8, and 24 hours post-flow incubation. Transcriptomic microarray analysis, followed by gene ontology pathway analysis, demonstrated fluid flow regulation of genes consistent with both known and unknown metabolic and inflammatory responses in bone. Additionally, two of the more highly up-regulated gene products—chemokines Cxcl1 and Cxcl2, supported by qPCR—have not previously been reported as responsive to fluid flow. Proteomic analysis demonstrated greatest up-regulation of the ATP-producing enzyme NDK, calcium-binding Calcyclin, and G protein-coupled receptor kinase 6. Finally, an integrative pathway analysis merging fold changes in transcript and protein levels predicted signaling nodes not directly detected at the sampled time points, including transcription factors c-Myc, c-Jun, and RelA/NF-κB. These results extend our knowledge of the osteocytic response to fluid flow, most notably up-regulation of Cxcl1 and Cxcl2 as possible paracrine agents 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.

Bone Homeostasis and Repair: Forced Into Shape

“…Osteocytes subjected to mechanical stimulation respond rapidly by mobilizing a series of second messengers, including calcium [59,60], nitric oxide [61,62], and prostaglandins [63]; by activating kinase signaling cascades, including the MAP kinase and PKC pathways [64]; and by exhibiting alterations in gene expression [65]. In addition, osteocytes respond to biophysical cues by releasing soluble factors important for cellular proliferation and differentiation and for recruitment of osteoblasts and osteoclasts [66][67][68][69][70]. Leucht et al showed that Cxcl12, the gene encoding the chemotactic molecule stromal cell-derived factor-1 (SDF-1), is upregulated in osteocytes and periosteal cells using in vivo and in vitro mechanical loading models.…”

“…Systemic inhibition of CXCR4 (SDF-1 receptor) signaling attenuated in vivo loadinduced bone formation, suggesting that CXCL12 is an important paracrine regulator of osteoblast function. Using in vitro techniques, Govey et al [67] reported the altered expression of a variety of genes in osteocytes exposed to fluid flow shear stress. The greatest increases were observed in three chemokine genes (Cxcl1, 2, and 5) involved in chemotaxis and inflammation [71], suggesting that these factors may also serve as important paracrine signals.…”

“…In response to loading, osteocyte-osteocyte and paracrine signaling [66,67], via the release of soluble factors from osteocytes that travel through the canalicular space to bone surfaces, are likely key mechanisms in bone adaptation.…”

Chemokines and Bone

“…In osteoblasts isolated from patients with osteoarthritis (OA), CXCL12 and CXCL13 increased proliferation and expression of type I collagen. Analysis of osteocyte responses to fluid flow identified CXCL1 and CXCL2 as responsive genes [50].…”

A novel miniature dynamic microfluidic cell culture platform using electro-osmosis diode pumping

“…Specifically, non-uniform distribution of molecules in a static in vitro model may result in local deviation of results and potentially increase unnecessary cell movement to the most abundant source of nutrients. 3 In addition, fluid flow shears can significantly regulate the metabolism of certain types of cells, such as endothelial and smooth muscle cells in blood vessels, 4,5 fibroblasts in muscles, 6,7 bone cells, 8,9 and chondrocytes in articular cartilages. 10,11 Subsequently, fluidic flow conditions are favorable for these types of cells to live in vivo.…”