An organ culture system to model early degenerative changes of the intervertebral disc II: profiling global gene expression changes

Abstract

Introduction: Despite many advances in our understanding of the molecular basis of disc degeneration, there remains a paucity of preclinical models which can be used to study the biochemical and molecular events that drive disc degeneration, and the effects of potential therapeutic interventions. The goal of this study is to characterize global gene expression changes in a disc organ culture system that mimics early nontraumatic disc degeneration.

Methods: To mimic a degenerative insult, rat intervertebral discs were cultured in the presence of TNF-α, IL-1β and serum-limiting conditions. Gene expression analysis was performed using a microarray to identify differential gene expression between experimental and control groups. Differential pattern of gene expression was confirmed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or Western blot.

Results: Treatment resulted in significant changes in expression of more than 1,000 genes affecting many aspects of cell function including cellular movement, the cell cycle, cellular development, and cell death and proliferation. Many of the most highly upregulated and downregulated genes have known functions in disc degeneration and extracellular matrix hemostasis. Construction of gene networks based on known cellular pathways and expression data from our analysis demonstrated that the network associated with cell death, cell cycle regulation and DNA replication and repair was most heavily affected in this model of disc degeneration.

Conclusions: This rat organ culture model uses cytokine exposure to induce wide gene expression changes with the most affected genes having known reported functions in disc degeneration. We propose that this model is a valuable tool to study the etiology of disc degeneration and evaluate potential therapeutic treatments.

Figure 1

(A) Schematic showing disc organ culture setup. Culturing discs under low pO₂, in hyperosmolar, nutritionally limiting media with exposure to proinflammatory cytokines mimics the molecular changes characteristic of disc degeneration. (B) Microarray analysis of rat discs treated with TNF-α and IL-1β for 10 days. The heat map displays gene expression patterns of 1,036 genes after treatment. The relative levels of gene expression are depicted with a color scale, where blue represents the lowest and red represents the highest level of expression. (C) Volcano plot of microarray data depicting the significant differences (P < 0.05) in expression patterns between the control (Ctr.) and experimental (Exp.) groups, where the red plots represent a 1.5-fold or more difference in expression patterns between the two groups. (D) Pie chart showing differentially expressed genes based on major molecular and cellular functions. The total numbers of genes associated with each specific function are listed. Note that some genes may have multiple functions and can be classified in several categories (see Table 2), N = 4 independent experiments.

Figure 2

(A-B) Diagrams illustrating genes with the highest upregulation (A) and downregulation (B) in expression between the experimental (Exp.) and control (Ctr.) discs. (C-E) Quantitative RT-PCR confirms the changes in gene expression identified by microarray analysis between the Ctr. and Exp. discs. Quantitative PCR expression levels were normalized to hypoxanthine phosphoribosyltransferase 1 (Hprt1) and shown relative to the Ctr., N = 3 independent experiments. (F) Western blot analysis of rat nucleus pulposus (NP) tissue lysates shows an increase in expression of Syndecan-4 (SDC4) and a decrease in expression of Keratin 19 (KRT19) in the experimental group, N = 3 independent experiments.

Figure 3

Functional gene networks identified using Ingenuity Pathway Analysis (IPA) from differentially expressed genes between the control and treated discs. (A) Network-1: cell death, cell cycle, and DNA replication, recombination, and repair (score 31). (B) Network-2: cellular growth and proliferation, tissue development, and cancer (score 27). Node color indicates degree of overexpression (red) and the degree of downregulation (green). Colored nodes represent genes of focus, whereas genes in uncolored nodes were not identified as being differentially expressed and were consequently integrated into the networks based on information in the IPA database.

Figure 4

Functional gene networks identified using Ingenuity Pathway Analysis (IPA) from differentially expressed genes between the control and treated discs. (A) Network-3: cellular development, hematological system development and function, and hematopoiesis (score 27). (B) Network-4: carbohydrate metabolism and protein synthesis (score 16).

Figure 5

Functional gene networks identified using Ingenuity Pathway Analysis (IPA) from differentially expressed genes between the control and experimental discs. (A) Network-5: cellular movement, immune cell trafficking, and cell-to-cell signaling and interaction (score 24).