Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth
Research highlights
► LPA treatment elicits extensive alterations in osteocyte gene and protein expression. ► Dendrite outgrowth is associated with osteocyte protein redistribution. ► Computational analysis links cytoskeletal dynamics to LPA-induced dendrite outgrowth. ► Perturbation of the actin cytoskeleton blocks LPA-induced dendritogenesis in vitro.
Introduction
Bone cell functions are modulated by a wide array of chemical and physical stimuli, and we and others have found that the bioactive lipid lysophosphatidic acid (LPA) elicits potent receptor-mediated regulatory effects on cultured osteoblastic and osteocytic cells. LPA induces acute signaling events in osteoblastic cells, such as elevations in cytosolic free Ca2+ and the activation of MAP kinase [1], [2], [3], [4], [5]. This lipid factor also triggered osteoblast mitogenesis and differentiation, and prolonged the survival of these cells when they were exposed to pro-apoptotic conditions [5], [6], [7], [8], [9]. LPA recently was reported to be a key autocrine mediator of nucleotide-coupled osteogenic activity in osteoblasts [10], and this lipid also has potential roles in the regulation of osteoclast function [11], [12], [13]. The role of LPA in the control of bone tissue function in vivo is not known, but mice lacking expression of the LPA1 receptor exhibited craniofacial malformations that might reflect effects on skeletal development [14], [15].
Platelets activated during early responses to tissue damage are the major source of LPA in vivo [16], [17], and the primary physiological roles for this lipid appear to relate to the stimulation of wound healing and angiogenesis [18]. It is likely that bone cells in the vicinity of skeletal damage are exposed to high levels of LPA released from hematomas. Pre-osteoblast migration is essential for proper fracture healing [19], and LPA has robust chemotactic effects on osteoblastic cells [1], [20], [21]. LPA induced membrane blebbing in primary cultured calvarial osteoblasts and stimulated the formation of membrane extensions in MC3T3-E1 pre-osteoblastic cells and MLO-Y4 osteocytic cells [21], [22], [23]. Osteocyte dendrites are critical for intercellular communication [24], and an enhancement of osteocyte membrane outgrowth in vivo would facilitate the re-establishment of the mechanosensory network in the newly-formed bone during fracture healing.
LPA exerts its effects on target cells through G protein-coupled receptors that subsequently are linked to signaling networks [25]. However, the mechanisms by which rapid signaling events elicit broader changes in bone cell function are less clear. We previously employed DNA microarray analysis to reveal that LPA treatment was linked to the regulation of over 500 gene products in MC3T3-E1 pre-osteoblastic cells [26]. The functions of many of these LPA-regulated transcripts were associated with cellular processes that control phenomena known to be important for skeletal healing, such as proliferation and migration. Thus, transcriptional profiling provided new insights into the mechanisms by which osteoblastic cells alter their function in response to lipid growth factors. We postulated that LPA would have similar effects on gene expression in osteocytic cells, particularly with respect to the ability of the lipid to stimulate dendrite outgrowth, and here we report the results of transcriptomic and proteomic profiling of LPA-treated MLO-Y4 cells.
Section snippets
Materials
The bovine serum albumin (BSA) used in this study was essentially fatty acid-free (MP Biomedicals, Solon, OH). Ammonium bicarbonate and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ), sequencing grade modified trypsin was purchased from Promega (Madison, WI), bicinchoninic acid (BCA) assay reagents and standards were purchased from Pierce (Rockford, IL). Unless otherwise noted, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Cell culture
MLO-Y4 osteocyte-like cells
Transcriptomic analysis of LPA-treated MLO-Y4 cells
We postulated that the effects of LPA on osteocytic cells would include the regulation of gene expression, including the modulation of transcripts encoding proteins involved in cellular processes that control LPA-induced membrane outgrowth. MLO-Y4 cells were cultured for 6 and 24 h with 1.0 μM LPA, a dose that we previously found to induce maximal dendrite outgrowth in vitro [22], after which global transcriptional profiling was performed using DNA microarrays. LPA induced statistically
Discussion
The exposure of osteocytic cells to a physiological concentration of LPA led to statistically significant changes in the levels of many gene transcripts. GO analysis revealed distinct biological processes to which the functions of the encoded proteins are associated. As expected for the response of cells to a pleiotropic growth factor, a variety of diverse biological processes appeared to be modulated in LPA-treated MLO-Y4 cells. For example, LPA treatment led to elevated levels of transcripts
Acknowledgments
This work was supported by National Institutes of Health grant AR055192 (N.J.K.) and the Laboratory-Directed Research and Development Program at the Pacific Northwest National Laboratory, operated by Battelle for the U.S. Department of Energy under contract DE-AC06-76RLO1830. 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
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