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

Johnson ZI, Shapiro IM, Risbud MV - Matrix Biol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, United States.

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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.
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Figure 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.

Mentions: Importantly, aside from regulating intracellular osmolarity, TonEBP is a key regulator of the content and, therefore, osmotic status of the extracellular matrix. Tsai et al. identified a consensus TonE element in the promoter of the aggrecan gene that functionally interacted with TonEBP (Fig. 1) (Tsai et al., 2006, 2007). Suppression of TonEBP activity by dominant negative protein or siRNA dramatically decreased aggrecan promoter activity. This was the first evidence in NP cells that TonEBP transcriptionally regulates matrix genes. Later investigations by our group showed that TonEBP also regulated the expression of β1,3-glucoronosyltransferase (GlcAT-I), an enzyme required for the synthesis of chondroitin sulfate chains of aggrecan (Hiyama et al., 2009). A follow-up study revealed that BMP-2 and TGF-β-mediated induction of GlcAT-I was also dependent on TonEBP activity (Hiyama et al., 2010). Based on these findings, it is clear that by controlling the expression and synthesis of aggrecan, TonEBP permits disc cells to autoregulate and adapt to their hyperosmotic state (Fig. 2).


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

Johnson ZI, Shapiro IM, Risbud MV - Matrix Biol. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390124&req=5

Figure 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.
Mentions: Importantly, aside from regulating intracellular osmolarity, TonEBP is a key regulator of the content and, therefore, osmotic status of the extracellular matrix. Tsai et al. identified a consensus TonE element in the promoter of the aggrecan gene that functionally interacted with TonEBP (Fig. 1) (Tsai et al., 2006, 2007). Suppression of TonEBP activity by dominant negative protein or siRNA dramatically decreased aggrecan promoter activity. This was the first evidence in NP cells that TonEBP transcriptionally regulates matrix genes. Later investigations by our group showed that TonEBP also regulated the expression of β1,3-glucoronosyltransferase (GlcAT-I), an enzyme required for the synthesis of chondroitin sulfate chains of aggrecan (Hiyama et al., 2009). A follow-up study revealed that BMP-2 and TGF-β-mediated induction of GlcAT-I was also dependent on TonEBP activity (Hiyama et al., 2010). Based on these findings, it is clear that by controlling the expression and synthesis of aggrecan, TonEBP permits disc cells to autoregulate and adapt to their hyperosmotic state (Fig. 2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery and Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, PA, United States.

Show MeSH
Related in: MedlinePlus