RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells

Abstract

Edited by Dennis VoelkerTranscription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/ chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain-and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-B activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2. Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.The intervertebral disc is well suited to fulfill its mechanical role in the human spine, where it permits flexion and rotation, and absorbs compressive loads (1). The matrix-rich nucleus pulposus (NP) 2 at the center of the disc gives the tissue its ability to resist compression through high osmotic swelling pressure (2-4), loss of which correlates with degeneration and back pain (5). Although the high fixed charge density of the aggrecan-rich matrix allows the nucleus pulposus its water-imbibing properties, it also results in a hypertonic environment for NP cells. Importantly, tonicity of the extracellular environment fluctuates widely with diurnal cycle: water is forced out of the disc during the day when the spine is loaded and imbibed during the unloaded phase at night (6).In mammalian cells, a key transcription factor TonEBP (NFAT5) is activated by elevated hypertonicity and promotes transcription of genes that produce or transport organic osmolytes (7). In addition, TonEBP controls transcription of several genes that are important for cell survival under hypertonic conditions independ...

Bicarbonate Recycling by HIF-1–Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells

“…Analysis of our previous RNA-Seq studies indicated that NP cells express various plasma membrane bicarbonate transporters of the SLC4 and SLC26 families (GEO Accession #GSE86552) (25). Of the bicarbonate transporters expressed, SLC4A2 (AE2), SLC4A4 (NBCe1), SLC4A7 (NBCn1), and SLC26A4 (Pendrin) had the highest levels.…”

“…In fact, contrary to the general understanding, rates of CO 2 hydration by CAs are substantial enough to contribute up to 100% of total extracellular proton production, a metric often erroneously associated solely with glycolytic lactate/H + production (24). Interestingly, our published RNA-Seq (GEO Accession #GSE86552) and microarray data (#GSE42611) along with other expression studies (GSE49560, GSE24612) show that levels of CA9, CA12 and CA3 expression are the highest in rat NP cells (25–28). These studies also show that NP cells express CA2 (cytosolic) and CA5b (mitochondrial) at lower levels while the remaining CA isoforms were undetectable.…”

Differential Effect of Long-Term Systemic Exposure of TNFα on Health of the Annulus Fibrosus and Nucleus Pulposus of the Intervertebral Disc

“…TNFα may be involved in maintaining cell viability and matrix homeostasis in the physiologically challenging disc niche that is hypoxic, hyperosmolar, and under mechanical stress . Earlier work from our group found that TNFα expression is maintained in the postnatal NP cells by tonicity‐responsive binding‐protein (TonEBP); this transcription factor controls cell adaptive responses in the hyperosmolar disc environment likely in part by regulating the expression of pro‐survival and anabolic matrix genes . Additionally, complete removal of IL‐1β, another cytokine closely related to disc degeneration, renders mice more susceptible to age‐related disc degeneration, further reinforcing the importance of the physiological role that certain cytokines play in the preservation of disc health .…”

“…This alteration results is deregulated overexpression of hTNFα . Organ culture was performed using previously described methods . Briefly, dissected vertebra‐disc‐vertebra motion segments from WT (C57BL6) mice were cultured with or without hTNFα (10 ng/mL) in DMEM (1 g/L glucose) supplemented with 10% FBS for 24 hours ( n = 3 mice/group, six lumbar and nine caudal discs/animal).…”

“…(19) Organ culture was performed using previously described methods. (20) Briefly, dissected vertebradisc-vertebra motion segments from WT (C57BL6) mice were cultured with or without hTNFα (10 ng/mL) in DMEM (1 g/L glucose) supplemented with 10% FBS for 24 hours (n = 3 mice/group, six lumbar and nine caudal discs/animal). Discs were dissected under stereo microscope, Stemi 503 (Zeiss, Inc., Thornwood, NY, USA) and RNA was extracted as described in RNA isolation and microarray analysis section.…”