Extracellular osmolarity regulates matrix homeostasis in the intervertebral disc and articular cartilage: evolving role of TonEBP

Abstract

Degeneration of the intervertebral disc is characterized by changes in proteoglycan status, loss of bound water molecules, decreased tissue osmotic pressure and a resulting mechanical failure of the disc. A similar spectrum of changes is evident in osteoarthritic articular cartilage. When healthy, resident cells in these skeletal tissues respond to applied mechanical loads by regulating their own osmotic state and the hydration of the extracellular matrix. The transcription factor Tonicity-Responsive Enhancer Binding Protein (TonEBP or NFAT5) is known to mediate the osmoadaptive response in these and other tissues. While the molecular basis of how osmotic loading controls matrix homeostasis is not completely understood, TonEBP regulates the expression of aggrecan and β1,3-glucoronosyltransferase in nucleus pulposus cells, in addition to targets that allow for survival under hypertonic stress. Moreover, in chondrocytes, TonEBP controls expression of several collagen subtypes and Sox9, a master regulator of aggrecan and collagen II expression. Thus, TonEBP-mediated regulation of the matrix composition allows disc cells and chondrocytes to modify the extracellular osmotic state itself. On the other hand, TonEBP in immune cells induces expression of TNF-α, ΙL-6 and MCP-1, pro-inflammatory molecules closely linked to matrix catabolism and pathogenesis of both disc degeneration and osteoarthritis, warranting investigations of this aspect of TonEBP function in skeletal cells. In summary, the TonEBP system, through its effects on extracellular matrix and osmoregulatory genes can be viewed primarily as a protective or homeostatic response to physiological loading.

Keywords: Cartilage; Disc degeneration; Intervertebral disc; Osmoregulation; Proteoglycan-rich matrix; TonEBP.

Fig. 1

Regulation of aggrecan gene promoter activity by TonEBP. A,DNA sequence of the promoter region of rat and mouse aggrecan gene. TonE consensus sequence is marked in bold and underlined. B, promoter organization of the rat aggrecan gene. The transcription start site is marked as + 1. TonE sites are shown as ovals on either side of a conserved Sox-9 binding site. C, electromobility shift assay to examine functional binding of TonEBP to TonE motif in the rat aggrecan gene promoter. An oligonucleotide probe containing the TonE motif (−912 b) in the rat aggrecan promoter was incubated with nuclear extracts from rat nucleus pulposus cells cultured under isotonic and hypertonic (400 and 500 mosmol/kg) conditions, and binding was detected using chemiluminescence. Specificity was confirmed by inclusion of excess unlabeled wild type probe or a probe containing mutation in the TonE site (Mt probe) in the binding reaction. The binding signal is significantly diminished when either a wild type competitor probe or a mutant probe is used. D, nucleus pulposus cells were co-transfected with DN-TonEBP and aggrecan reporter plasmids. Twenty-four hours after transfection, cells were cultured in isotonic medium for 24 h and luciferase activity measured. Expression of DN-TonEBP results in decreased aggrecan promoter activity compared with control cells that receive empty backbone vector. E, aggrecan promoter construct was transiently transfected into siRNA expressing and control cells (C) and reporter activity measured in isotonic media. Compared with control cells, the silenced nucleus pulposus cells elicit a marked reduction in aggrecan reporter activity. Data represent mean ± S.D. from three independent experiments, performed in triplicate (n= 3); *, p b 0.05. This research was originally published in The Journal of Biological Chemistry. Tsai TT, Danielson KG, Guttapalli A, Oguz E, Albert TJ, Shapiro IM, Risbud MV. TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc. J Biol Chem. 2006; 1;281(35):25416-24. © the American Society for Biochemistry and Molecular Biology.

Fig. 2

Schematic representation of the osmotic response in healthy (left panel) and degenerative (right panel) disc or articular cartilage tissues. In healthy tissue, hyperosmolarity results from high negative charge of proteoglycans resulting in influx of NaCl. Hyperosmolarity results in robust increase in TonEBP mRNA, protein, and nuclear shuttling. TonEBP binds to TonE sites in target promoters to drive expression of osmotic response genes (blue) (AR, BGT1, SMIT, TauT), protecting against cellular damage during hypertonic stress. Similarly, TonEBP induces transcription of genes involved in matrix homeostasis (green) (ACAN, GlcAT-I, AQP2, Sox9) to autoregulate the extracellular osmotic environment. In the pathological state, activation of TLR or NF-κB pathways induce TonEBP to act on a specific set of targets (red). Activity on osmoadaptation targets (blue) is decreased with this type of activation. Osmotic stress may also induce TonEBP activation of pro-inflammatory targets (green). It should be noted that in disc and cartilage relationship between pathological stimuli and TonEBP has not been studied yet. *Studies have not addressed whether TonEBP binds TonE in Sox9. AR, aldose reductase; BGT1, betaine-g-amino butyric acid transporter; SMIT, sodium myo-inositol transporter; TauT, taurine transporter (TauT); ACAN, Aggrecan; GlcAT-I, β1,3-glucuronosyl transferase 1; AQP2, Aquaporin 2; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-a; IL-6, interleukin 6;NOS2, nitric oxide synthase 2; VCAN, versican; MMP-13, matrix metallopeptidase 13. Targets in dashed border indicate results that have not been verified in NP cells or chondrocytes.