TonEBP/OREBP Is a Regulator of Nucleus Pulposus Cell Function and Survival in the Intervertebral Disc

Abstract

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmoregulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/ kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.The intervertebral disc is a specialized biomechanical structure that permits movement between vertebrae and protects the vertebral bone from mechanical forces. It consists of an outer ligament, the annulus fibrosus that encloses a gel-like tissue, the nucleus pulposus. While sparse, cells in the nucleus pulposus secrete a complex extracellular matrix that contains fibrillar collagens and the proteoglycan, aggrecan. The numerous glycosaminoglycan side chains of the aggrecan molecule bind cations thereby raising the osmolarity of the disc tissues (1-4). While the hydration properties of the nucleus pulposus permits dynamic loading and unloading, the mechanism by which these cells adapt to elevated osmotic forces is poorly understood.In a number of tissues, cellular adaptation to hyperosmotic stress is mediated by the tonicity enhancer-binding protein (TonEBP), 2 also called OREBP (5) or NFAT5, (5, 6). Upon activation, TonEBP binds to the tonicity-responsive enhancer element (TonE) of genes required for osmotolerance and cell survival. These genes include the betaine/␥-aminobutyric acid transporter, sodium myo-inositol co-transporter (7-9), taurine transporter (10, 11), and aldose reductase (6). By regulating levels of betaine, myo-inositol, taurine, and sorbitol, these genes control the osmotic properties ...

Activation of TonEBP by Calcium Controls β1,3-Glucuronosyltransferase-I Expression, a Key Regulator of Glycosaminoglycan Synthesis in Cells of the Intervertebral Disc

“…Null MEFs displayed decreased basal GlcAT-I promoter activity and failure to induce reporter activity even when treated with ionomycin. Together, the results of these functional studies indicated that, in addition to regulating tauT, other osmolytes, and HSP-70 (4,14,21), by controlling the expression of GlcAT-I, TonEBP adapts nucleus pulposus cells to the mechanically and osmotically restrained conditions of the disc (4,22).…”

“…In an earlier study, we showed that nucleus pulposus cells responded to changes in osmotic pressure by up-regulating the transcription factor, TonEBP (tonicity enhancer-binding protein) (4). When expressed, this protein modulated aggrecan expression; whether it can regulate chondroitin chain synthesis has not been determined.…”

Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells to growth factors and the activation of the ERK and Akt pathways

“…22 Interestingly, hypo-osmotic stress also stimulates ERK in hepatocytes and intestine cells, an effect that seems to be mediated by the Ras-Raf-MEK pathway, [31][32][33] indicating that the activation of these pathways is a primary response to cell volume changes. Several lines of evidence suggest that the activation of these pathways may serve different functions in disc cells' homeostasis beyond cell proliferation, such as regulation of metabolism and aggrecan production, 22,34 as well as adaptation to unfavorable conditions and overexpression of proteins like ASIC3, which promote cell survival and lower the activity of caspases. 35 In addition, ERK and Akt pathways are classical transduction pathways, activated by mechanical forces, 36 the latter leading also to changes in cell volume, similarly to variations in extracellular osmolality.…”

Recent advances in annular pathobiology provide insights into rim-lesion mediated intervertebral disc degeneration and potential new approaches to annular repair strategies

“…Several proteins have been investigated which convey functional adaptations to NP cells allowing them to survive under hostile low oxygen tension and high osmotic pressure environments. Tonicity enhancer binding protein (TonEBP) [139] and hypoxia inducible factor-1 alpha (HIF-1a) [4,115,119] are two candidate proteins which allow NP cells to withstand hyperosmotic stress and an avascular hypoxic environment. TonEBP binds to the aggrecan promoter, regulates its transcriptional activity and the hydration status of the IVD.…”