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CDKN2A: a predictive marker for bone mineral density or direct role in osteoblast function

CDKN2A: a predictive marker for bone mineral density or direct role in osteoblast function
CDKN2A: a predictive marker for bone mineral density or direct role in osteoblast function
As the population is becoming more aged, osteoporosis is becoming more prevalent and the number of fragility fractures that are occurring is increasing. One of the main predictors of developing osteoporosis in later life is a bone mineral density that is greater than 2.5 standard deviations below the young adult sex-matched mean, though studies have only been able to explain 5% of the variance seen in bone mineral densities between individuals. There is now increasing evidence that the development of osteoporosis can begin in utero and that epigenetic processes, such as DNA methylation, may be central to the mechanism by which early development influences bone mineral density and later bone health.

Previous work within the group has identified associations of a 300bp differentially methylated region within the CDKN2A locus with bone mineral content, bone area and areal bone mineral density in offspring from the Southampton Women’s Survey(SWS) cohort. As methylation of CDKN2A increases, bone mineral content, bone area and areal bone mineral density all decrease at both 4 and 6 years of age. The two cell cycle regulators p16INK4A and p14ARF are transcribed from this gene as well as the lncRNA ANRIL.

Various techniques were used to determine whether this differentially methylated region has a functional role in osteoblasts or whether it is just a potential marker for predicting bone mineral density. It was shown that there was specific binding in the osteoblast-like SaOS-2 cell line to this region and that methylation of these CpGs led to a decrease in transcription factor binding. Treatment of both SaOS-2 cells and primary osteoblasts with siRNAs against ANRIL caused a decrease in cell number, a decrease in cell proliferation, an increase in cells in G0/G1 phase of the cell cycle and, in SaOS-2 cells, caused an increase in the expression of bone differentiation markers RUNX2 and ALP. Knock down of ANRIL expression also caused a change in genes that have roles in osteoblast specific pathways as well as genes that were involved in molecular functions such as nuclear regulation and acetylation. Associations were found between CpG methylation in RXRA and offspring bone outcomes at birth in both the SWS and MAVIDOS cohorts. Daily maternal cholecalciferol supplementation was also shown to decrease the methylation of CpG loci within RXRA in offspring umbilical cord and further investigation showed that this effect of maternal supplementation was only seen in children born during the winter months.

Together, these findings suggest that the association between CDKN2A methylation at birth and later bone health could indicate a potential role for CDKN2A in bone development and function and raises the possibility that differential methylation of CDKN2A may allow the identification of individuals at increased risk of osteoporosis in later life. Due to the effect of ANRIL knock down in primary osteoblasts, which could not be explained through normal cell cycle pathways, results suggest that ANRIL has potential trans-regulatory functions in these cells. These findings suggest that ANRIL could potentially play a role in bone differentiation, cell proliferation and metabolism and that dysregulation of the expression of this lncRNA could have adverse effects on bone health, potentially increasing the risk of individuals developing osteoporosis in later life.
Cook, Eloise
5ace6df3-08fc-4a52-b663-13e0b90af2de
Cook, Eloise
5ace6df3-08fc-4a52-b663-13e0b90af2de
Lillycrop, Karen
eeaaa78d-0c4d-4033-a178-60ce7345a2cc
Harvey, Nicholas
ce487fb4-d360-4aac-9d17-9466d6cba145
Lanham, Stuart
28fdbbef-e3b6-4fdf-bd0f-4968eeb614d6

(2016) CDKN2A: a predictive marker for bone mineral density or direct role in osteoblast function. University of Southampton, Faculty of Medicine, Doctoral Thesis, 283pp.

Record type: Thesis (Doctoral)

Abstract

As the population is becoming more aged, osteoporosis is becoming more prevalent and the number of fragility fractures that are occurring is increasing. One of the main predictors of developing osteoporosis in later life is a bone mineral density that is greater than 2.5 standard deviations below the young adult sex-matched mean, though studies have only been able to explain 5% of the variance seen in bone mineral densities between individuals. There is now increasing evidence that the development of osteoporosis can begin in utero and that epigenetic processes, such as DNA methylation, may be central to the mechanism by which early development influences bone mineral density and later bone health.

Previous work within the group has identified associations of a 300bp differentially methylated region within the CDKN2A locus with bone mineral content, bone area and areal bone mineral density in offspring from the Southampton Women’s Survey(SWS) cohort. As methylation of CDKN2A increases, bone mineral content, bone area and areal bone mineral density all decrease at both 4 and 6 years of age. The two cell cycle regulators p16INK4A and p14ARF are transcribed from this gene as well as the lncRNA ANRIL.

Various techniques were used to determine whether this differentially methylated region has a functional role in osteoblasts or whether it is just a potential marker for predicting bone mineral density. It was shown that there was specific binding in the osteoblast-like SaOS-2 cell line to this region and that methylation of these CpGs led to a decrease in transcription factor binding. Treatment of both SaOS-2 cells and primary osteoblasts with siRNAs against ANRIL caused a decrease in cell number, a decrease in cell proliferation, an increase in cells in G0/G1 phase of the cell cycle and, in SaOS-2 cells, caused an increase in the expression of bone differentiation markers RUNX2 and ALP. Knock down of ANRIL expression also caused a change in genes that have roles in osteoblast specific pathways as well as genes that were involved in molecular functions such as nuclear regulation and acetylation. Associations were found between CpG methylation in RXRA and offspring bone outcomes at birth in both the SWS and MAVIDOS cohorts. Daily maternal cholecalciferol supplementation was also shown to decrease the methylation of CpG loci within RXRA in offspring umbilical cord and further investigation showed that this effect of maternal supplementation was only seen in children born during the winter months.

Together, these findings suggest that the association between CDKN2A methylation at birth and later bone health could indicate a potential role for CDKN2A in bone development and function and raises the possibility that differential methylation of CDKN2A may allow the identification of individuals at increased risk of osteoporosis in later life. Due to the effect of ANRIL knock down in primary osteoblasts, which could not be explained through normal cell cycle pathways, results suggest that ANRIL has potential trans-regulatory functions in these cells. These findings suggest that ANRIL could potentially play a role in bone differentiation, cell proliferation and metabolism and that dysregulation of the expression of this lncRNA could have adverse effects on bone health, potentially increasing the risk of individuals developing osteoporosis in later life.

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Published date: July 2016
Organisations: University of Southampton, Human Development & Health

Identifiers

Local EPrints ID: 400683
URI: http://eprints.soton.ac.uk/id/eprint/400683
PURE UUID: 146c0407-a9a6-4314-a39a-471ddf76b56e
ORCID for Karen Lillycrop: ORCID iD orcid.org/0000-0001-7350-5489
ORCID for Nicholas Harvey: ORCID iD orcid.org/0000-0002-8194-2512
ORCID for Stuart Lanham: ORCID iD orcid.org/0000-0002-4516-264X

Catalogue record

Date deposited: 08 Nov 2016 14:29
Last modified: 01 Feb 2019 01:36

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Contributors

Author: Eloise Cook
Thesis advisor: Karen Lillycrop ORCID iD
Thesis advisor: Nicholas Harvey ORCID iD
Thesis advisor: Stuart Lanham ORCID iD

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