Download osmolarity regulates gene expression in intervertebral disc cells

OSMOLARITY REGULATES GENE EXPRESSION IN INTERVERTEBRAL DISC CELLS QUANTIFIED
WITH HIGH DENSITY OLIGONUCLEOTIDE ARRAY TECHNOLOGY
*Boyd L M; *Chen J; ^Richardson W J; ^^Kraus VB; +*^Setton LA
+*Department of Biomedical Engineering, Duke University, Durham, 919-660-5376, Fax:919-660-5362, setton@duke.edu
^Department of Surgery, Division of Orthopaedic Surgery and ^^Department of Medicine, Division of Rheumatology, Duke University Medical Center
INTRODUCTION: Cells of the intervertebral disc respond to
mechanical and biophysical stimuli in their environment, including
deformations, hydrostatic and osmotic pressures [1]. Previous studies
have shown that fibrochondrocytes of the disc respond to altered
osmotic pressure with changes in post-translational biosynthesis of
proteoglycans (i.e., 35S-incorporation) [2, 3], as well as changes in gene
expression for type II collagen and select proteoglycans [4]. In other
cell types, a wide range of genes are transcriptionally activated
following exposure to hypo- or hyper-osmotic conditions [5]. We
hypothesize that fibrochondrocytes of the intervertebral disc
differentially respond to hypo- and hyper-osmotic conditions with
changes in gene expression for a large number of proteins. In this study,
the gene expression profile of human intervertebral disc cells was
quantified with gene array technology following exposure to varying
osmolarity in alginate culture in vitro. The results of the study
demonstrate that genes encoding a broad functional range of proteins are
regulated by osmotic conditions in cells of the intervertebral disc.
METHODS: Cell culture. Human intervertebral disc tissue was
obtained from patients (average age 51 yrs) undergoing surgery for
interbody fusion (n=3) or herniation (n=1). Cells were isolated,
passaged for two subcultures, and embedded in 1.2% crosslinked
alginate beads (2×106 cells/ml) [4]. Cell-alginate beads were cultured
for 4 hours in either iso-osmotic (F-12 medium; 293 mOsm/kg H2O),
hypo-osmotic (medium diluted with distilled water; 255 mOsm/kg H2O)
or hyper-osmotic media (medium with sucrose added; 450 mOsm/kg
H2O). Immediately after osmotic treatment, cells were released from
alginate, lysed and stored at –80°C. Gene Array. The Human Genome
U133A array (Affymetrix) was used to study the expression of 22,283
gene sequences, transcript variants and expressed sequence tags. Total
RNA was isolated from each cell sample and 10µg used for synthesis of
cDNA.
Biotin-labelled cRNA targets were made by in vitro
transcription, fragmented and hybridized to the U133A array. After
hybridization, arrays were stained with phycoerythrin and scanned for
intensity values. Statistical determinations of the presence or absence of
a given target were performed based on information from 10 - 20 sets of
oligos for each gene (Microarray v5.0, Affymetrix). Data Analysis.
The iso-osmotic condition was used as a control for assessment of genes
differentially expressed under or hypo- (n=3) or hyper- osmotic (n=4)
conditions. A global scaling algorithm was applied across all arrays.
Pairwise comparisons were made between control and experimental
arrays for each target gene based on intensity information from all oligo
sets. A significant difference was noted where a Wilcoxon’s signed rank
test detected a difference between “perfect match” and “mismatch”
probe pair intensities (typically <0.003) and the change in intensity was
2-fold or greater relative to control values. Results are presented here
for targets for which a significant difference was detected between
control and experimental arrays in a majority of samples (2/3 for hypoand 3/4 for hyper-osmotic conditions). Genes that were significantly
regulated by osmotic environment were classified by their biological
functions using information provided by the array manufacturer
(www.netaffx.com) and terminology defined by the Gene Ontology
Consortium (www.geneontology.org).
bolded categories discussed in text
Classification
Signal transduction/transcription
Cell-cell interaction/adhesion
Cytoskeleton
Ion/small molecule transport
Cell cycle/apoptosis
Protease
Growth factor/cytokine
HYPO
Inc
0
0
0
0
0
0
0
Dec
5
3
1
1
0
0
1
HYPER
Inc
5
0
0
3
3
1
1
Dec
3
1
0
1
5
0
2
RESULTS & DISCUSSION: A total of 8,704 transcripts were common
to all samples cultured under iso-osmotic conditions (n=4). This number
represents 81% of all transcripts found to be present in the cRNA target
pool, averaged across all 4 human samples. For transcripts falling
within the top 2% of highest average intensity values (n=174), a
significant number related to extracellular matrix (21, 28%) and
cytoskeleton proteins (12, 16%). Many of these proteins were
recognized as important products of fibrochondrocyte biosynthesis,
including types I, III and VI collagen, decorin, and fibronectin. Thus,
the human intervertebral disc cells appear to retain important
characteristics of the fibrochondrocytic phenotype in in vitro culture.
Hypo-osmotic conditions. 18 transcripts were identified as significantly
increased or decreased following exposure to hypo-osmotic conditions.
Of these, 7 were related to either translation (i.e., ribosomal proteins),
lipid or carbohydrate metabolism, or of unknown or poorly understood
function. The remaining genes were categorized by function (Table).
Genes encoding proteins involved in cytoskeletal-mediated signaling
were downregulated, including a protein phosphatase (PTPG1/
PTPN12) known to dephosphorylate cytoskeletal and cell adhesion
molecules, and an actin-binding protein known to inhibit GTPase
activity (IQGAP-1). In articular chondrocytes and other cell types,
hypo-osmotic shock induces changes in cell volume that are regulated
by the breakdown and reorganization of the actin cytoskeleton [5,6].
Thus, the observed modification of gene expression for cytoskeletalmediated signaling molecules following hypo-osmotic shock is
consistent with the idea that cytoskeletal changes occur in the disc cells
studied here. Cell volume changes also regulate cell metabolism and
growth [7], which may be linked to the finding that gene expression for
the growth factor, TGF-β2, was down-regulated by hypo-osmotic
conditions. The implication of this change is unknown as TGF-β has
numerous functions in the cell, but may be important in regulating both
biosynthesis and cell proliferation following hypo-osmotic shock.
Hyper-osmotic conditions: 42 transcripts were significantly changed
following exposure to hyper-osmotic conditions. As for hypo-osmotic
conditions, several genes were involved in cytoskeletal-mediated signal
transduction (UP: ephrin-B2 ligand (EFNB2), muskelin 1(MKLN1);
DOWN: guanylate binding protein 1 (GBP1)). Furthermore, a number
of genes were involved in ion/small molecule transport (UP: organic ion
and inositol transporters, (SLC21A12 and SLC5A3); DOWN:
monocarboxylic acid transporter, (SLC16A6)). Cells subjected to
hyper-osmotic shock may experience regulatory volume changes that
involve an influx of osmolytes via transport across the c ell membrane [5,
7, 8], which suggests why inositol transporter gene expression may be
upregulated under hyper-osmotic conditions. Hyper-osmotic stimuli
also resulted in increased gene expression for ADAMTS1, a disintegrintype metalloproteinase shown to be active in degrading aggrecan, as well
as decreases in IL-6, a known inflammatory mediator in cartilage. We
also found that a brain-derived neurotrophic factor (BDNF), a central
pain modulator of unknown function in the intervertebral disc, was upregulated under hyper-osmotic conditions.
While the majority of the genes regulated by osmotic stimuli have
poorly understood functions in the intervertebral disc, a significant
number of genes were related to cytoskeleton and osmolyte transport.
This pattern of gene expression may be expected in light of the cell
volume changes that occur following altered osmotic conditions.
Studies are ongoing to evaluate transient regulation and independent
confirmation of the specific genes identified here.
ACKNOWLEDGMENTS: Supported by funds from the NIH
(1R01AR47442, 5T32GM08555).
We acknowledge Dr. Holly
Dressman for helpful discussions.
REFERENCES: 1) Urban JPG, In: Musculo Soft-tissue Aging, 1993,
AAOS;391-412. 2) Ishihara H et al., Am J Physiol 1997; C1499-C1506.
3) Bayliss MT et al., JOR 1986; 10-17. 4) Chen J et al., BBRC 2002;
932-38. 5) Waldegger S et al., J Mem Biol, 1998; 95-100. 6) Erickson
G and Guilak F, Trans ORS, 2001; 180. 7) O'Neill WC, Am J Physiol Cell, 1999; C995-C1011. 8) Lang F et al., Physiol Rev, 1998; 247-306.
49th Annual Meeting of the Orthopaedic Research Society
Poster #1133