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Gene expression analysis in response to osmotic stimuli in the intervertebral disc with DNA microarray.

ABSTRACT: BACKGROUND: Intervertebral disc (IVD) cells experience a broad range of physicochemical stimuli under physiologic conditions, including alterations in their osmotic environment. At present, the molecular mechanisms underlying osmotic regulation in IVD cells are poorly understood. This study aims to screen genes affected by changes in osmotic pressure in cells of subjects aged 29 to 63 years old, with top-scoring pair (TSP) method. METHODS: Gene expression data set GSE1648 was downloaded from Gene Expression Omnibus database, including four hyper-osmotic stimuli samples, four iso-osmotic stimuli samples, and three hypo-osmotic stimuli samples. A novel, simple method, referred to as the TSP, was used in this study. Through this method, there was no need to perform data normalization and transformation before data analysis. RESULTS: A total of five pairs of genes ((CYP2A6, FNTB), (PRPF8, TARDBP), (RPS5, OAZ1), (SLC25A3, NPM1) and (CBX3, SRSF9)) were selected based on the TSP method. We inferred that all these genes might play important roles in response to osmotic stimuli and age in IVD cells. Additionally, hyper-osmotic and iso-osmotic stimuli conditions were adverse factors for IVD cells. CONCLUSIONS: We anticipate that our results will provide new thoughts and methods for the study of IVD disease.

SUBMITTER: Zhang W

PROVIDER: S-EPMC3911967 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Gene expression analysis in response to osmotic stimuli in the intervertebral disc with DNA microarray.

Zhang Wenzhi W Li Xu X Shang Xifu X Zhao Qichun Q Hu Yefeng Y Xu Xiang X He Rui R Duan Liqun L Zhang Feng F

European journal of medical research 20131227

<h4>Background</h4>Intervertebral disc (IVD) cells experience a broad range of physicochemical stimuli under physiologic conditions, including alterations in their osmotic environment. At present, the molecular mechanisms underlying osmotic regulation in IVD cells are poorly understood. This study aims to screen genes affected by changes in osmotic pressure in cells of subjects aged 29 to 63 years old, with top-scoring pair (TSP) method.<h4>Methods</h4>Gene expression data set GSE1648 was downlo ...[more]

PMID: 24369767

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Project description:Human intervertebral disc tissue was obtained from patients (average age 51 yrs) undergoing surgery for lumbar interbody fusion (n=3) or lumbar disc herniation (n=1). Cells were isolated by sequential pronase-collagenase digestion [3]. Cells were passaged twice in monolayer and suspended at a density of 2 x 106 cells/ml in 1.2% alginate (low viscosity, Sigma Chemical, St Louis, MO) dissolved in 150 mM NaCl. Alginate beads were formed by dropwise addition of the alginate from a 22 gauge needle into 102 mM CaCl2, followed by 10 minutes of curing, as described previously [13, 27]. Cell-gel beads were incubated in cell culture media consisting of Ham’s F-12 medium (Gibco BRL, Grand Island, NY), supplemented with 10% FBS (HyClone, Logan, UT), 25 μg/ml ascorbic acid (Sigma, St. Louis, MO), 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μg/ml Fungizone at 5% CO2 and 37° C. After 24 h, the cell culture medium was removed via pipette and exchanged for one of three osmotically active solutions, representing iso-osmotic, hyper-osmotic and hypo-osmotic media [12, 23, 34]. The iso-osmotic solution consisted of a defined cell culture medium (Ham’s F-12 with supplements as described above; 293 mOsm/kg H2O). The hypo-osmotic solution consisted of the same cell culture media diluted with de-ionized water to a final osmolarity of 250 mOsm/kg H2O. The hyper-osmotic solution consisted of the same cell culture media supplemented with sucrose to a final osmolarity of 450 mOsm/kg H20. The osmolarity of all solution formulations was determined using a freezing-point osmometer (Advanced Laboratory Wide Range 3W2, Advanced Instrument, Needham Heights, MA) as described previously [12]. Cell-alginate beads were cultured for a 4 hour period under one of these conditions, after which the cells were released from alginate in a dissolving buffer (55 mM Na-citrate and 150 mM NaCl), lysed and stored at –80°C. Intervertebral disc (IVD) cells experience a broad range of physical stimuli under physiologic conditions, including alterations in their osmotic environment. In this study, the gene expression profile of human IVD cells was quantified with gene array technology following exposure to varying osmolarity in order to capture the biological responses for a broad set of targets. A total of 42 genes were identified in IVD cells as significantly changed following culture under hyper-osmotic conditions, while a total of 18 genes were identified as significantly changed under hypo-osmotic conditions. Gene expression patterns were verified using RT-PCR. Genes identified in this study include those related to cytoskeleton remodeling and stabilization (ephrin-B2, sarcoglycan beta, IQGAP1), as well as membrane transport (ion transporter SLC21A12, osmolyte tranporter SLC5A3, monocarboxylic acid SLC16A6, and amino acid transporter SLC7A8). An unexpected finding was the differential regulation of the gene for the neurotrophin brain-derived neurotrophic factor by hyper-osmotic stimuli that may be indicative of a physiological response of IVD cells to physical stimuli important in regulating discogenic pain. Keywords = intervertebral disc Keywords = osmotic stimuli Keywords: other

Project description:Painful intervertebral disc (IVD) degeneration is an age-related process characterized by reduced tissue osmolarity, increased catabolism of the extracellular matrix, and elevated levels of pro-inflammatory molecules. With the aging population and constantly rising treatment costs, it is of utmost importance to identify potential therapeutic targets and new pharmacological treatment strategies for low back pain. Transient receptor potential (TRP) channels are a family of Ca2+ permeable cell membrane receptors, which can be activated by multitude of stimuli and have recently emerged as contributors to joint disease, but were not investigated closer in the IVD. Based on the gene array screening, TRPC1, TRPM7, and TRPV4 were overall the most highly expressed TRP channels in bovine IVD cells. We demonstrated that TRPV4 gene expression was down-regulated in hypo-osmotic condition, whereas its Ca2+ flux increased. No significant differences in Ca2+ flux and gene expression were observed for TRPM7 between hypo- and iso-osmotic groups. Upon hypo-osmotic stimulation, we overall identified via RNA sequencing over 3,000 up- or down-regulated targets, from which we selected aggrecan, ADAMTS9, and IL-6 and investigated whether their altered gene expression is mediated through either the TRPV4 or TRPM7 channel, using specific activators and inhibitors (GSK1016790A/GSK2193874 for TRPV4 and Naltriben/NS8593 for TRPM7). GSK1016790A induced the expression of IL-6 under iso-osmotic condition, alike to hypo-osmotic stimulation alone, indicating that this effect might be TRPV4-mediated. However, using the TRPV4 blocker GSK2193874 failed to prevent the increase of IL-6 under hypo-osmotic condition. A treatment with TRPM7-activator did not cause significant changes in the gene expression of tested targets. In conclusion, while TRPV4 and TRPM7 are likely involved in osmosensing in the IVD, neither of them mediates hypo-osmotically-induced gene expression changes of aggrecan, ADAMTS9, and IL-6.

Project description:Degeneration of the intervertebral disc (IVD), which consists of the annulus fibrosus (AF) and nucleus pulposus (NP), is a multifactorial physiological process associated with lower back pain. Despite decades of research, the knowledge of the underlying molecular mechanisms of IVD degeneration (IDD) has remained limited. The present study aimed to reveal the differential gene expression patterns in AF and NP during the process of IDD and to identify key biomarkers contributing to these differences. The microarray dataset GSE70362 containing 24 AF and 24 NP samples was retrieved from the Gene Expression Omnibus database. Of these, 8 healthy samples were discarded. GeneSpring11.5 software was employed to identify differentially expressed genes (DEGs). Metascape online tools were used to perform enrichment analyses. Finally, the DEGs were mapped with the Search Tool for the Retrieval of Interacting Genes, and a protein-protein interaction (PPI) network was constructed in Cytoscape software. A total of 87 DEGs were identified. Gene ontology enrichment revealed that these DEGs were mainly involved in the inflammatory response, the extracellular matrix and RNA polymerase II transcription factor activity. Pathway enrichment revealed that the DEGs were mainly involved in the transforming growth factor (TGF-?) and estrogen signaling pathways. Matrix metalloproteinase (MMP)1 and interleukin (IL)6 were included in the genes enriched in rheumatoid arthritis, whereas bone morphogenetic protein (BMP)2 and thrombospondin 1 (THBS1) were among the genes enriched in the TGF-? signaling pathway. In the PPI network, IL6 was identified as the central gene. In conclusion, as MMP1 has been demonstrated degrade collagen III at higher rates compared with other types of collagen (which is at a higher quantity in AF than NP), collagen types may be in different distribution patterns, which may contribute to the upregulation of MMP1 in AF. Differences in the expression of BMP2, ESR1 and THBS1 may explain for the pathological differences between AF and NP. IL6 may have a key role in different degeneration processes in AF and NP.

Dataset Information

Gene expression analysis in response to osmotic stimuli in the intervertebral disc with DNA microarray.

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Gene expression analysis in response to osmotic stimuli in the intervertebral disc with DNA microarray.

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