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Loss of methylation in CpG sites in the NF-?B enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes

Loss of methylation in CpG sites in the NF-?B enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes
Loss of methylation in CpG sites in the NF-?B enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes
OBJECTIVE.: To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS.: Expression of iNOS was quantified by qRT-PCR. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Co-transfections with expression vectors encoding NF-?B subunits were carried out to analyse iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS.: The 1000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both controls and OA samples; the CpG site at -289 and the sites in the starting coding region were largely un-methylated in both groups. The NF-?B enhancer region at -5.8 kb was significantly de-methylated in OA samples compared with control samples. This enhancer element was transactivated by co-transfection with the NF-?B subunits p65 alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in reporter assay. CONCLUSIONS.: These studies demonstrate the association between de-methylation of specific NF-?B-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, critically, show association with the osteoarthritic process. © 2012 American College of Rheumatology.
0004-3591
732-742
de Andrés, María C.
9b3834e7-972f-410d-a8cb-199abd035b87
Imagawa, Kei
cfdeef65-8259-4f0c-943a-0d439aab3193
Hashimoto, Ko
19b8f3be-d539-4579-a6cb-936ed0243b27
Gonzalez, Antonio
41d6e3cb-9a9a-499a-89b3-a2872864e9d3
Roach, Helmtrud I.
ca2ff1f4-1ada-4c56-9097-cd27ca4d199e
Goldring, Mary B.
67cbd000-e36a-4b82-bc50-f3c5a8d292da
Oreffo, Richard O.C.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
de Andrés, María C.
9b3834e7-972f-410d-a8cb-199abd035b87
Imagawa, Kei
cfdeef65-8259-4f0c-943a-0d439aab3193
Hashimoto, Ko
19b8f3be-d539-4579-a6cb-936ed0243b27
Gonzalez, Antonio
41d6e3cb-9a9a-499a-89b3-a2872864e9d3
Roach, Helmtrud I.
ca2ff1f4-1ada-4c56-9097-cd27ca4d199e
Goldring, Mary B.
67cbd000-e36a-4b82-bc50-f3c5a8d292da
Oreffo, Richard O.C.
ff9fff72-6855-4d0f-bfb2-311d0e8f3778

de Andrés, María C., Imagawa, Kei, Hashimoto, Ko, Gonzalez, Antonio, Roach, Helmtrud I., Goldring, Mary B. and Oreffo, Richard O.C. (2013) Loss of methylation in CpG sites in the NF-?B enhancer elements of inducible nitric oxide synthase is responsible for gene induction in human articular chondrocytes. Arthritis and Rheumatism, 65 (3), 732-742. (doi:10.1002/art.37806). (PMID:23239081)

Record type: Article

Abstract

OBJECTIVE.: To investigate whether the abnormal expression of inducible nitric oxide synthase (iNOS) by osteoarthritic (OA) human chondrocytes is associated with changes in the DNA methylation status in the promoter and/or enhancer elements of iNOS. METHODS.: Expression of iNOS was quantified by qRT-PCR. The DNA methylation status of the iNOS promoter and enhancer regions was determined by bisulfite sequencing or pyrosequencing. The effect of CpG methylation on iNOS promoter and enhancer activities was determined using a CpG-free luciferase vector and a CpG methyltransferase. Co-transfections with expression vectors encoding NF-?B subunits were carried out to analyse iNOS promoter and enhancer activities in response to changes in methylation status. RESULTS.: The 1000-bp iNOS promoter has only 7 CpG sites, 6 of which were highly methylated in both controls and OA samples; the CpG site at -289 and the sites in the starting coding region were largely un-methylated in both groups. The NF-?B enhancer region at -5.8 kb was significantly de-methylated in OA samples compared with control samples. This enhancer element was transactivated by co-transfection with the NF-?B subunits p65 alone or together with p50. Critically, methylation treatment of the iNOS enhancer element significantly decreased its activity in reporter assay. CONCLUSIONS.: These studies demonstrate the association between de-methylation of specific NF-?B-responsive enhancer elements and the activation of iNOS transactivation in human OA chondrocytes, consistent with the differences in methylation status observed in vivo in normal and human OA cartilage and, critically, show association with the osteoarthritic process. © 2012 American College of Rheumatology.

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e-pub ahead of print date: 12 December 2012
Published date: March 2013
Organisations: Human Development & Health

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Local EPrints ID: 346416
URI: http://eprints.soton.ac.uk/id/eprint/346416
ISSN: 0004-3591
PURE UUID: a541e5bd-414e-4334-aec5-aa2505b96356
ORCID for Richard O.C. Oreffo: ORCID iD orcid.org/0000-0001-5995-6726

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Date deposited: 02 Jan 2013 10:06
Last modified: 27 Oct 2023 01:41

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Contributors

Author: María C. de Andrés
Author: Kei Imagawa
Author: Ko Hashimoto
Author: Antonio Gonzalez
Author: Helmtrud I. Roach
Author: Mary B. Goldring

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