Elsevier

Bone

Volume 48, Issue 6, 1 June 2011, Pages 1328-1335
Bone

Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth

https://doi.org/10.1016/j.bone.2011.02.020Get rights and content

Abstract

Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24 h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6 h and 844 proteins at 24 h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6 h and 24 h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among the proteins with elevated abundances in dendrites were molecules that regulate cytoskeletal function, cell motility and membrane adhesion. Our combined transcriptomic/proteomic analysis of the response of MLO-Y4 osteocytic cells to LPA indicates that dendritogenesis is a membrane- and cytoskeleton-driven process with actin dynamics playing a particularly critical role.

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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