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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Placenta-specific Methylation of the Vitamin D 24-Hydroxylase
Gene</title><author><name sortKey="Novakovic, Boris" sort="Novakovic, Boris" uniqKey="Novakovic B" first="Boris" last="Novakovic">Boris Novakovic</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation></author><author><name sortKey="Sibson, Mandy" sort="Sibson, Mandy" uniqKey="Sibson M" first="Mandy" last="Sibson">Mandy Sibson</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation></author><author><name sortKey="Ng, Hong Kiat" sort="Ng, Hong Kiat" uniqKey="Ng H" first="Hong Kiat" last="Ng">Hong Kiat Ng</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Manuelpillai, Ursula" sort="Manuelpillai, Ursula" uniqKey="Manuelpillai U" first="Ursula" last="Manuelpillai">Ursula Manuelpillai</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Rakyan, Vardhman" sort="Rakyan, Vardhman" uniqKey="Rakyan V" first="Vardhman" last="Rakyan">Vardhman Rakyan</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Institute of Cell and Molecular Science, Barts and the London, London E1 2AT, United Kingdom, the</nlm:aff></affiliation></author><author><name sortKey="Down, Thomas" sort="Down, Thomas" uniqKey="Down T" first="Thomas" last="Down">Thomas Down</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Wellcome Trust Cancer Research UK Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom, the</nlm:aff></affiliation></author><author><name sortKey="Beck, Stephan" sort="Beck, Stephan" uniqKey="Beck S" first="Stephan" last="Beck">Stephan Beck</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">UCL Cancer Institute, University College London, London WC1E 6BT, United Kingdom,</nlm:aff></affiliation></author><author><name sortKey="Fournier, Thierry" sort="Fournier, Thierry" uniqKey="Fournier T" first="Thierry" last="Fournier">Thierry Fournier</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">INSERM U427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris 5, F-75006 Paris, France, and the</nlm:aff></affiliation></author><author><name sortKey="Evain Brion, Danielle" sort="Evain Brion, Danielle" uniqKey="Evain Brion D" first="Danielle" last="Evain-Brion">Danielle Evain-Brion</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">INSERM U427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris 5, F-75006 Paris, France, and the</nlm:aff></affiliation></author><author><name sortKey="Dimitriadis, Eva" sort="Dimitriadis, Eva" uniqKey="Dimitriadis E" first="Eva" last="Dimitriadis">Eva Dimitriadis</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia</nlm:aff></affiliation></author><author><name sortKey="Craig, Jeffrey M" sort="Craig, Jeffrey M" uniqKey="Craig J" first="Jeffrey M." last="Craig">Jeffrey M. Craig</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Morley, Ruth" sort="Morley, Ruth" uniqKey="Morley R" first="Ruth" last="Morley">Ruth Morley</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Saffery, Richard" sort="Saffery, Richard" uniqKey="Saffery R" first="Richard" last="Saffery">Richard Saffery</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19237542</idno><idno type="pmc">2685665</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685665</idno><idno type="RBID">PMC:2685665</idno><idno type="doi">10.1074/jbc.M809542200</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">001118</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001118</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Placenta-specific Methylation of the Vitamin D 24-Hydroxylase
Gene</title><author><name sortKey="Novakovic, Boris" sort="Novakovic, Boris" uniqKey="Novakovic B" first="Boris" last="Novakovic">Boris Novakovic</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation></author><author><name sortKey="Sibson, Mandy" sort="Sibson, Mandy" uniqKey="Sibson M" first="Mandy" last="Sibson">Mandy Sibson</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation></author><author><name sortKey="Ng, Hong Kiat" sort="Ng, Hong Kiat" uniqKey="Ng H" first="Hong Kiat" last="Ng">Hong Kiat Ng</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Manuelpillai, Ursula" sort="Manuelpillai, Ursula" uniqKey="Manuelpillai U" first="Ursula" last="Manuelpillai">Ursula Manuelpillai</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Rakyan, Vardhman" sort="Rakyan, Vardhman" uniqKey="Rakyan V" first="Vardhman" last="Rakyan">Vardhman Rakyan</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Institute of Cell and Molecular Science, Barts and the London, London E1 2AT, United Kingdom, the</nlm:aff></affiliation></author><author><name sortKey="Down, Thomas" sort="Down, Thomas" uniqKey="Down T" first="Thomas" last="Down">Thomas Down</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Wellcome Trust Cancer Research UK Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom, the</nlm:aff></affiliation></author><author><name sortKey="Beck, Stephan" sort="Beck, Stephan" uniqKey="Beck S" first="Stephan" last="Beck">Stephan Beck</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">UCL Cancer Institute, University College London, London WC1E 6BT, United Kingdom,</nlm:aff></affiliation></author><author><name sortKey="Fournier, Thierry" sort="Fournier, Thierry" uniqKey="Fournier T" first="Thierry" last="Fournier">Thierry Fournier</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">INSERM U427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris 5, F-75006 Paris, France, and the</nlm:aff></affiliation></author><author><name sortKey="Evain Brion, Danielle" sort="Evain Brion, Danielle" uniqKey="Evain Brion D" first="Danielle" last="Evain-Brion">Danielle Evain-Brion</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">INSERM U427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris 5, F-75006 Paris, France, and the</nlm:aff></affiliation></author><author><name sortKey="Dimitriadis, Eva" sort="Dimitriadis, Eva" uniqKey="Dimitriadis E" first="Eva" last="Dimitriadis">Eva Dimitriadis</name><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia</nlm:aff></affiliation></author><author><name sortKey="Craig, Jeffrey M" sort="Craig, Jeffrey M" uniqKey="Craig J" first="Jeffrey M." last="Craig">Jeffrey M. Craig</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Morley, Ruth" sort="Morley, Ruth" uniqKey="Morley R" first="Ruth" last="Morley">Ruth Morley</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author><author><name sortKey="Saffery, Richard" sort="Saffery, Richard" uniqKey="Saffery R" first="Richard" last="Saffery">Richard Saffery</name><affiliation><nlm:aff wicri:cut=", and" id="N0x3c0c3d0N0x3795e10">Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital</nlm:aff></affiliation><affiliation><nlm:aff id="N0x3c0c3d0N0x3795e10">Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Biological Chemistry</title><idno type="ISSN">0021-9258</idno><idno type="eISSN">1083-351X</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Plasma concentrations of biologically active vitamin D
(1,25-(OH)<sub>2</sub>D) are tightly controlled via feedback regulation of
renal 1α-hydroxylase (<italic>CYP27B1</italic>; positive) and 24-hydroxylase
(<italic>CYP24A1</italic>; catabolic) enzymes. In pregnancy, this regulation is
uncoupled, and 1,25-(OH)<sub>2</sub>D levels are significantly elevated,
suggesting a role in pregnancy progression. Epigenetic regulation of
<italic>CYP27B1</italic> and <italic>CYP24A1</italic> has previously been described in cell
and animal models, and despite emerging evidence for a critical role of
epigenetics in placentation generally, little is known about the regulation of
enzymes modulating vitamin D homeostasis at the fetomaternal interface. In
this study, we investigated the methylation status of genes regulating vitamin
D bioavailability and activity in the placenta. No methylation of the
<italic>VDR</italic> (vitamin D receptor) and <italic>CYP27B1</italic> genes was found in any
placental tissues. In contrast, the <italic>CYP24A1</italic> gene is methylated in
human placenta, purified cytotrophoblasts, and primary and cultured chorionic
villus sampling tissue. No methylation was detected in any somatic human
tissue tested. Methylation was also evident in marmoset and mouse placental
tissue. All three genes were hypermethylated in choriocarcinoma cell lines,
highlighting the role of vitamin D deregulation in this cancer. Gene
expression analysis confirmed a reduced capacity for <italic>CYP24A1</italic>induction with promoter methylation in primary cells and <italic>in vitro</italic>reporter analysis demonstrated that promoter methylation directly
down-regulates basal promoter activity and abolishes vitamin D-mediated
feedback activation. This study strongly suggests that epigenetic decoupling
of vitamin D feedback catabolism plays an important role in maximizing active
vitamin D bioavailability at the fetomaternal interface.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id><journal-id journal-id-type="publisher-id">jbc</journal-id><journal-title>The Journal of Biological Chemistry</journal-title><issn pub-type="ppub">0021-9258</issn><issn pub-type="epub">1083-351X</issn><publisher><publisher-name>American Society for Biochemistry and Molecular Biology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19237542</article-id><article-id pub-id-type="pmc">2685665</article-id><article-id pub-id-type="publisher-id">14838</article-id><article-id pub-id-type="doi">10.1074/jbc.M809542200</article-id><article-categories><subj-group subj-group-type="heading"><subject>Transcription, Chromatin, and Epigenetics</subject></subj-group></article-categories><title-group><article-title>Placenta-specific Methylation of the Vitamin D 24-Hydroxylase
Gene</article-title><subtitle><italic>IMPLICATIONS FOR FEEDBACK AUTOREGULATION OF ACTIVE VITAMIN D
LEVELS AT THE FETOMATERNAL
INTERFACE</italic><xref ref-type="fn" rid="fn1">S⃞</xref></subtitle></title-group><contrib-group><contrib contrib-type="author"><name><surname>Novakovic</surname><given-names>Boris</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref></contrib><contrib contrib-type="author"><name><surname>Sibson</surname><given-names>Mandy</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref></contrib><contrib contrib-type="author"><name><surname>Ng</surname><given-names>Hong Kiat</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§</xref></contrib><contrib contrib-type="author"><name><surname>Manuelpillai</surname><given-names>Ursula</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">¶</xref></contrib><contrib contrib-type="author"><name><surname>Rakyan</surname><given-names>Vardhman</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">∥</xref></contrib><contrib contrib-type="author"><name><surname>Down</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">**</xref></contrib><contrib contrib-type="author"><name><surname>Beck</surname><given-names>Stephan</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡‡</xref><xref ref-type="fn" rid="fn2">1</xref></contrib><contrib contrib-type="author"><name><surname>Fournier</surname><given-names>Thierry</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§§</xref></contrib><contrib contrib-type="author"><name><surname>Evain-Brion</surname><given-names>Danielle</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§§</xref></contrib><contrib contrib-type="author"><name><surname>Dimitriadis</surname><given-names>Eva</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">¶¶</xref></contrib><contrib contrib-type="author"><name><surname>Craig</surname><given-names>Jeffrey M.</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§</xref><xref ref-type="fn" rid="fn3">2</xref></contrib><contrib contrib-type="author"><name><surname>Morley</surname><given-names>Ruth</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§</xref><xref ref-type="fn" rid="fn3">2</xref></contrib><contrib contrib-type="author"><name><surname>Saffery</surname><given-names>Richard</given-names></name><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">‡</xref><xref ref-type="aff" rid="N0x3c0c3d0N0x3795e10">§</xref><xref ref-type="fn" rid="fn3">2</xref><xref ref-type="corresp" rid="cor1">3</xref></contrib></contrib-group><aff id="N0x3c0c3d0N0x3795e10"><label>‡</label>Developmental Epigenetics, Murdoch Childrens Research Institute, Royal Children's Hospital, and<label>§</label>Department of Paediatrics, University of Melbourne, Parkville, Victoria 3052, Australia, the<label>¶</label>Monash Institute of Medical Research, Monash University, Clayton, Victoria 3168, Australia, the<label>∥</label>Institute of Cell and Molecular Science, Barts and the London, London E1 2AT, United Kingdom, the<label>**</label>Wellcome Trust Cancer Research UK Gurdon Institute and Department of Genetics, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, United Kingdom, the<label>‡‡</label>UCL Cancer Institute, University College London, London WC1E 6BT, United Kingdom,<label>§§</label>INSERM U427, Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, Paris 5, F-75006 Paris, France, and the<label>¶¶</label>Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia</aff><author-notes><fn id="fn2"><label>1</label><p>Supported by <grant-sponsor>Wellcome
Trust</grant-sponsor> Grant
<grant-num>084071</grant-num>.</p></fn><fn id="fn3"><label>2</label><p>Supported by <grant-sponsor>National Health and Medical Research Council
Research Fellowships</grant-sponsor>.</p></fn><fn id="cor1"><label>3</label><p>To whom correspondence should be addressed: Murdoch Childrens Research
Institute, Flemington Road, Parkville, Victoria 3052, Australia. Tel.:
61-03-83416341; E-mail:
<email>richard.saffery@mcri.edu.au</email>.
</p></fn></author-notes><pub-date pub-type="ppub"><day>29</day><month>5</month><year>2009</year></pub-date><pub-date pub-type="pmc-release"><day>29</day><month>5</month><year>2009</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on
/art/copyright. </pmc-comment>
<volume>284</volume><issue>22</issue><fpage>14838</fpage><lpage>14848</lpage><history><date date-type="received"><day>19</day><month>12</month><year>2008</year></date><date date-type="rev-recd"><day>20</day><month>2</month><year>2009</year></date></history><permissions><copyright-statement>Copyright © 2009, The American Society for Biochemistry and
Molecular Biology, Inc.</copyright-statement><license license-type="open-access"><p><italic>Author's Choice</italic></p><p><ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">Creative
Commons Attribution Non-Commercial License</ext-link> applies to Author
Choice Articles</p></license></permissions><self-uri xlink:title="pdf" xlink:href="zbc02209014838.pdf"></self-uri><abstract><p>Plasma concentrations of biologically active vitamin D
(1,25-(OH)<sub>2</sub>D) are tightly controlled via feedback regulation of
renal 1α-hydroxylase (<italic>CYP27B1</italic>; positive) and 24-hydroxylase
(<italic>CYP24A1</italic>; catabolic) enzymes. In pregnancy, this regulation is
uncoupled, and 1,25-(OH)<sub>2</sub>D levels are significantly elevated,
suggesting a role in pregnancy progression. Epigenetic regulation of
<italic>CYP27B1</italic> and <italic>CYP24A1</italic> has previously been described in cell
and animal models, and despite emerging evidence for a critical role of
epigenetics in placentation generally, little is known about the regulation of
enzymes modulating vitamin D homeostasis at the fetomaternal interface. In
this study, we investigated the methylation status of genes regulating vitamin
D bioavailability and activity in the placenta. No methylation of the
<italic>VDR</italic> (vitamin D receptor) and <italic>CYP27B1</italic> genes was found in any
placental tissues. In contrast, the <italic>CYP24A1</italic> gene is methylated in
human placenta, purified cytotrophoblasts, and primary and cultured chorionic
villus sampling tissue. No methylation was detected in any somatic human
tissue tested. Methylation was also evident in marmoset and mouse placental
tissue. All three genes were hypermethylated in choriocarcinoma cell lines,
highlighting the role of vitamin D deregulation in this cancer. Gene
expression analysis confirmed a reduced capacity for <italic>CYP24A1</italic>induction with promoter methylation in primary cells and <italic>in vitro</italic>reporter analysis demonstrated that promoter methylation directly
down-regulates basal promoter activity and abolishes vitamin D-mediated
feedback activation. This study strongly suggests that epigenetic decoupling
of vitamin D feedback catabolism plays an important role in maximizing active
vitamin D bioavailability at the fetomaternal interface.</p></abstract></article-meta><notes><fn-group><fn><p><italic>Author's Choice</italic>—Final version full
access.</p></fn><fn id="fn1"><label>S⃞</label><p>The on-line version of this article (available at
<ext-link ext-link-type="uri" xlink:href="http://www.jbc.org">http://www.jbc.org</ext-link>)
contains supplemental Figs. 1 and 2.</p></fn></fn-group></notes></front><body><p>In eutherian mammals, proper fetal growth and development are dependent on
the formation and function of the placenta. In combination with the maternal
decidua, cells of the placenta (primarily trophoblasts) regulate the exchange
of nutrients, growth factors, oxygen, and waste between maternal and fetal
circulations. The placenta also protects the developing pregnancy from the
maternal immune system (<xref ref-type="bibr" rid="ref1">1</xref>).</p><p>Although mammalian placentas share a basic common function in modulating
exchange at the fetomaternal interface, it is clear that the human placenta is
unique in structure and function
(<xref ref-type="bibr" rid="ref2">2</xref>). Animal models may
therefore be of limited value in fully dissecting processes underlying certain
aspects of human pathologies, such as pre-eclampsia and gestational
trophoblastic disease (<xref ref-type="bibr" rid="ref3">3</xref>). An
examination of human placentation is therefore critical in understanding its
unique function and potential role in disease.</p><p>The biologically active form of vitamin D
(1,25-(OH)<sub>2</sub>D)<xref ref-type="fn" rid="fn4">4</xref>is a potent secosteroid hormone. Its wide ranging actions include regulating
calcium and phosphorus homeostasis, immune system modulation, cellular
differentiation, and apoptosis
(<xref ref-type="bibr" rid="ref4">4</xref>–<xref ref-type="bibr" rid="ref6">6</xref>).
It also has potent antiproliferative effects
(<xref ref-type="bibr" rid="ref7">7</xref>–<xref ref-type="bibr" rid="ref9">9</xref>).
Vitamin D deficiency has been linked to several adverse pregnancy outcomes,
including those associated with placental insufficiency (including
pre-eclampsia) (<xref ref-type="bibr" rid="ref10">10</xref>,
<xref ref-type="bibr" rid="ref11">11</xref>).</p><p>Although concentrations of 1,25-(OH)<sub>2</sub>D are low in the early
embryo, circulating maternal levels are elevated (∼2-fold) during
pregnancy (reviewed in Ref.
<xref ref-type="bibr" rid="ref12">12</xref>), and it is clear that a
large proportion of maternal circulating 1,25-(OH)<sub>2</sub>D is derived
from decidua and placental cells
(<xref ref-type="bibr" rid="ref13">13</xref>). There is inconsistent
evidence regarding transplacental transfer of 1,25-(OH)<sub>2</sub>D from
mother to fetus (<xref ref-type="bibr" rid="ref14">14</xref>,
<xref ref-type="bibr" rid="ref15">15</xref>), but it is clear that the
immediate precursor, 25-(OH)<sub>2</sub>D, crosses from maternal to fetal
circulations (<xref ref-type="bibr" rid="ref16">16</xref>). At birth,
fetal and maternal levels of 25-(OH)<sub>2</sub>D are comparable, and indirect
measures suggest that 1,25-(OH)<sub>2</sub>D may also be correlated
(<xref ref-type="bibr" rid="ref17">17</xref>).</p><p>Several components of the vitamin D pathway are strongly expressed in human
uterine decidua and placental tissues from early pregnancy
(<xref ref-type="bibr" rid="ref18">18</xref>,
<xref ref-type="bibr" rid="ref19">19</xref>). VDR (vitamin D
receptor)-bound 1,25-(OH)<sub>2</sub>D regulates key target genes associated
with implantation and trophoblast functioning (including specific cytokines
and homeobox genes
(<xref ref-type="bibr" rid="ref18">18</xref>–<xref ref-type="bibr" rid="ref20">20</xref>).
1,25-(OH)<sub>2</sub>D has been shown to directly modulate the production of
human chorioric gonadotrophin in human trophoblasts, and reduced expression
and activity of 1-hydroxylase has been reported in differentiating trophoblast
cells undergoing syncitialization <italic>in vitro</italic>(<xref ref-type="bibr" rid="ref18">18</xref>). A supportive role of
vitamin D in placental function is also evident by its influence on human
placental lactogen, estradiol, and progesterone gene transcription
(<xref ref-type="bibr" rid="ref21">21</xref>,
<xref ref-type="bibr" rid="ref22">22</xref>). In addition, vitamin D,
in the form of 1,25-(OH)<sub>2</sub>D can function as an intracrine regulator
of antibacterial responses in human trophoblasts that may lead to a novel mode
of activation of innate immune responses in the placenta
(<xref ref-type="bibr" rid="ref23">23</xref>). Given its potent
immunosuppressive effects, 1,25-(OH)<sub>2</sub>D may also play a role in
implantation tolerance (<xref ref-type="bibr" rid="ref19">19</xref>).
The vitamin D metabolic pathway involves multiple enzymatic reactions, each of
which is a potential target for epigenetic regulation. The <italic>CYP27B1</italic>(1α-hydroxylase) gene product, 1α-hydroxylase, is directly
responsible for the production of 1,25-(OH)<sub>2</sub>D from
25-(OH)<sub>2</sub>D. Largely expressed in the kidney, it is also expressed in
decidual cells and the placenta
(<xref ref-type="bibr" rid="ref24">24</xref>). In contrast,
1,25-(OH)<sub>2</sub>D-hydroxylase (24-hydroxylase), encoded by the
<italic>CYP24A1</italic> (vitamin D 24-hydroxylase) gene, plays a pivotal role in the
attenuation of vitamin D responsiveness by catalyzing synthesis of less active
metabolites from both 1,25-(OH)<sub>2</sub>D and 25-(OH)<sub>2</sub>D
(<xref ref-type="bibr" rid="ref25">25</xref>–<xref ref-type="bibr" rid="ref28">28</xref>).
The <italic>CYP24A1</italic> gene is up-regulated in several cancer cell types, where
it attenuates vitamin D-mediated growth inhibition
(<xref ref-type="bibr" rid="ref29">29</xref>). However, other cancers
appear to show decreased activity of this gene
(<xref ref-type="bibr" rid="ref4">4</xref>). Interestingly, a lack of
expression of <italic>CYP24A1</italic> has been directly associated with DNA
methylation of the upstream promoter and first exon regions in tumor-derived
endothelial cells in mice and rat osteoblastic ROS17/2.8 cells
(<xref ref-type="bibr" rid="ref30">30</xref>,
<xref ref-type="bibr" rid="ref31">31</xref>).</p><p>The importance of epigenetic regulation in placental development and
function is becoming increasingly apparent. General disruption of DNA
methylation using drug treatment has been shown to disrupt placental
development and also inhibit invasiveness in trophoblast cell models
(<xref ref-type="bibr" rid="ref32">32</xref>,
<xref ref-type="bibr" rid="ref33">33</xref>). In addition to the well
documented parent-of-origin specific imprinting of genes in the placenta
(reviewed in Ref. <xref ref-type="bibr" rid="ref34">34</xref>),
specific epigenetic silencing of nonimprinted genes has also recently been
reported
(<xref ref-type="bibr" rid="ref35">35</xref>–<xref ref-type="bibr" rid="ref38">38</xref>).
This is likely to play a role in many aspects of placental functioning.</p><p>Very little is known about the epigenetic regulation of enzymes modulating
vitamin D homeostasis at the fetomaternal interface. Given the likely
importance of 1,25-(OH)<sub>2</sub>Din proper placental development and
functioning, fetal development, and the modulation of maternal immune response
to the developing fetus, we investigated epigenetic regulation of vitamin D
metabolism in human placental tissue in general and in specific cell
subtypes.</p><sec sec-type="methods"><title>EXPERIMENTAL PROCEDURES</title><p><italic>Tissue Isolation</italic>—Tissues were collected for research
purposes with approval from the Human Research Ethics Committees at the Royal
Women's Hospital (03/51), Mercy Hospital for Women (R07/15), and Monash
Medical Centre (07084C). For human placental tissue sampling, a core of
full-thickness tissue was isolated from two randomly chosen sites. Tissue
sections from other species were washed briefly in saline before processing by
maceration with a scalpel blade prior to immediate DNA isolation. CVS samples
were collected and processed as previously described
(<xref ref-type="bibr" rid="ref37">37</xref>).</p><p>First trimester human cytotrophoblast cells were isolated as per Ref.
<xref ref-type="bibr" rid="ref39">39</xref>, and purity of preparations
was determined using antibodies to the trophoblast-specific cytokeratin-7
(1:100 dilution, clone OV-TL 12/30; DakoCytomation, Glostrup, Denmark) using
immunocytochemistry. Cells were fixed in ethanol, rehydrated, and treated with
0.3% hydrogen peroxide in methanol for 10 min to block endogenous peroxidase
activity. Cells were then washed with Tris-buffered saline and incubated with
nonimmune block for 30 min. Primary antibody was applied and incubated at 48
°C for 16 h followed by biotinylated horse anti-mouse IgG (1:200) for 30
min and then streptavidinbiotin-peroxidase complex ABC (DakoCytomation)
according to the manufacturer's instructions. Peroxidase activity was
visualized by application of diaminobenzidine substrate (DakoCytomation) for
3–5 min. Cells were counterstained with Harris hematoxylin (Sigma),
air-dried, and mounted. Only cell preparations in which 95% of the cells were
positive for cytokeratin-7 were used for subsequent experiments.</p><p>For CVS samples (8–10 weeks of gestation), contaminating maternal
blood and decidua were removed, and ∼5–10 mg of cleaned villi was
separated from the diagnostic sample, washed in isotonic saline, pelleted by
centrifugation, and stored at –20 °C until DNA extraction and
bisulfite modification. A cell culture from each villi sample was established
by finely macerating the villi and resuspending in 1.0 ml of 0.25% trypsin
with incubation of the suspension at 37 °C for 45 min. Cells were then
resuspended in 7 ml of RPMI 1640 medium (SAFC Biosciences), pelleted at 560
× <italic>g</italic>, and resuspended in a small volume of AmnioMAX medium
(Invitrogen) containing 0.5 μg/ml amphotericin B. The cell suspension was
then inoculated onto glass coverslips in 35-mm Petri dishes with subsequent
incubation at 37 °C in 5% CO<sub>2</sub> in humidified air. Wells were
then flooded with 2 ml of AmnioMAX medium the following day and were grown to
around 95% confluence, trypsinized, pelleted, and stored at –20 °C
until DNA extraction and bisulfite modification. Cultured cells were isolated
following 14–18 days in culture with a maximum of three passages.</p><p><italic>Cell Culturing and Transfection</italic>—JEG-3 cells (HTB-36
(<xref ref-type="bibr" rid="ref40">40</xref>)), BeWo (CCL-98
(<xref ref-type="bibr" rid="ref41">41</xref>)), and JAR cells (HTB-144
(<xref ref-type="bibr" rid="ref42">42</xref>)), were maintained in RPMI
plus 10% fetal calf serum at 37 °C in 5% CO<sub>2</sub>. The
SV40-transformed trophoblast HIPEC-65 cell line was maintained as previously
described (<xref ref-type="bibr" rid="ref43">43</xref>). Cells were
grown for a minimal number of passages (<8) and were split (1:5–1:8)
prior to confluence and harvesting of genomic DNA or transfection
analysis.</p><p><fig position="float" id="fig1"><label>FIGURE 1.</label><caption><p><bold>Methylation status of <italic>CYP24A1</italic>, <italic>CYP27B1</italic>, and
<italic>VDR</italic> in human term placenta.</bold> Schematic representation of
methylation data generated for regulatory regions of genes coding for
24-hydroxylase (<italic>A</italic>), 1α-hydroxylase (<italic>B</italic>), and the
vitamin D receptor (<italic>C</italic>). <italic>i</italic>, gene structure showing the
location of methylation assays. <italic>Gray box</italic>, CpG island; <italic>black
boxes</italic>, exon regions; <italic>arrow</italic>, translational start site.
<italic>ii</italic>, mean methylation levels seen at each CpG site for each type of
sample tested. Numbers of each type of tissue are listed in
<italic>parentheses</italic>. Between 8 and 12 individual clones were sequenced for
each sample. <italic>Circles</italic>, CpG sites denoted by <italic>vertical dashes.
Closed circles</italic>, methylation; <italic>open circles</italic>, lack of methylation.
Missing circles indicate CpG sites for which no information was obtained.</p></caption><graphic xlink:href="zbc0210973730001"></graphic></fig></p><p>For transfection experiments, a total of 8 × 10<sup>3</sup> JAR or
HIPEC65 cells were plated 24 h prior to transfection in each well of a 96-well
plate (96F Nunclon Delta white microwell). Transfection was carried out using
Lipofectamine LTX reagent (Invitrogen). Luciferase and <italic>Renilla</italic>activity was measured 24 h post-transfection using the DualGLO assay system
(Promega, Madison, WI) on a FluorSTAR Optima microplate reader (BMG Labtech,
Offenburg, Germany). For vitamin D induction experiments, cells were treated
with 10 or 100 n<sc>m</sc> 1,25-(OH)<sub>2</sub>D (Sigma) 12 h
post-transfection, with luciferase activity monitored after overnight
incubation. Endometrial stromal cells and decidua were isolated as previously
described (<xref ref-type="bibr" rid="ref44">44</xref>).</p><p><italic>Genomic DNA and Methylation Analysis</italic>—Genomic DNA isolation
and bisulfite DNA sequencing was performed as previously described
(<xref ref-type="bibr" rid="ref37">37</xref>,
<xref ref-type="bibr" rid="ref38">38</xref>). DNA samples were
processed using the Methyl Easy™ bisulfite modification kit (Human
Genetic Signatures, Sydney, Australia) kit according to the manufacturer's
instructions. Amplification was performed on converted genomic DNA using
primers directed to bisulfite modified DNA: VDRbisF1,
5′-AATATTTTTTGTTCTTTAAGTGTTAAGTA; VDRbisF2,
5′-TAAATTTTAGTGTTTTTTAGTGTTTTAGTT; VDRBisR1,
5′-CTAACCTAATCAACCCAAATAAAAATA; CYP24BisF1,
5′-GGAAAGGGGGTTTTAAGAAATAGTA; CYP24BisR1,
5′-ACTTCCAACCCCAAAAAACTCTAAC; CYP24BisF2,
5′-TGGTTTTAGTTAGATTTTAGAGGTTAGTTT; CYP24BisR2,
5′-TACTATTTCTTAAAACCCCCTTTCC; CYP24BisF3,
5′-GAGTATGTTTTGGGTGGTTAATGAG; CYP24BisR3,
5′-TCCAAACTAAAAATATCTAACTCCCC; CYP27BisF1,
5′-TTTTGGTTGTTTTATATTTGTTTTG; CYP27BisR1,
5′-AACCATAACCCAAACCCTCAAATA.</p><p>Marmoset amplification primers were marF1 (5′-GTATTTTAGGGAAGGTTTGGAG)
and marR1 (5′-CCTCCTTCCTACAACAACAAC). Mouse primers were mF1
(5′-TGTTTAGTAGTGGTTAGTTGGTGGG) and mR1
(5′-ACATCCCTAAACATCTACCTCTTCC). Amplification conditions were as
follows: 95 °C for 5 min, 56 °C for 1 min 30 s, and 72 °C for 1
min 30 s × 40 cycles, 72 °C for 7 min. Resulting amplicons were
cloned into TOPO TA Cloning Vector (Invitrogen) for automated fluorescent
sequencing as described in Ref.
<xref ref-type="bibr" rid="ref45">45</xref>. Data were analyzed using
BiQ Analyser software (<xref ref-type="bibr" rid="ref46">46</xref>),
and clones showing less than 80% conversion or that were identified as clonal
in origin were not included in subsequent analysis. Mean methylation levels
for each amplicon were determined using the formula (number of methylated CpG
sites/total CpG sites examined × 100).</p><p><italic>CYP24A1 Gene Reporter Assays</italic>—A 758-bp region of the
promoter/exon 1 of the human <italic>CYP24A1</italic> gene previously shown to confer
vitamin D responsiveness (<xref ref-type="bibr" rid="ref47">47</xref>)
was amplified using primers; Sal_CYP24_–548F
(5′-CTCTCTGTCGACTTGGAGCTCCGCAGAAAGCCAAACTTCCT) and SalI_CYP24_+209R
(5′-CTCTCTGTCGACGTACTCGAGACAGCCTCAGAGCATTGGTG) with Phusion Hi-Fidelity
DNA polymerase (Finnzymes). The SalI-digested amplicon was cloned into a
promoterless luciferase expression vector pGL3:basic (Promega, Madison, WI) to
produce the plasmid pGL3:–594/+209. Reverse orientation cloning was also
carried out to produce pGL:+209/–594. Following sequence verification,
DNA was <italic>in vitro</italic> methylated using either Sss1, HpaII, or HhaI
methylases (New England Biolabs, Ipswich, MA) according to the manufacturer's
instructions. 100 ng of pGL3:control vector (SV40 constitutive promoter),
pGL3:basic (promoterless), or unmethylated or methylated <italic>CYP24A1</italic>promoter constructs were then transfected into JAR and HIPEC65 cells.
Co-transfection with 10 ng of <italic>pRn</italic> (HSV-TK promoter linked to the
<italic>Renilla</italic> luciferase gene) was carried out in all cases to normalize
for transfection efficiency.</p><p><italic>Gene Induction Experiments</italic>—24 h prior to induction,
CVS-derived cells, skin, and placental-derived fibroblasts were seeded in a
6-well plate at a density of 250,000 cells/well. Half of the wells were then
treated with 100 n<sc>m</sc> calcitriol (Sigma), and the other half were
left untreated. Cells were then incubated for 7 h prior to total RNA isolation
using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA
synthesis was performed with the SuperScript III kit (Invitrogen), from 1
μg of total RNA. Quantitative reverse transcription-PCR was performed in a
96-well optical plate (Applied Biosystems, Foster City, CA) on a 7300 ABi
thermal cycler (Applied Biosystems). The master mix consisted of 15 μl of
Sybr Green Master Mix (Sigma), 1 μl of forward primer, 1 μl of reverse
primer (50 p<sc>m</sc> final concentration), 8 μl of water, 5 μl of
cDNA template in a total volume of 30 μl. Nondiluted cDNA was used for
<italic>CYP24A1, CYP27B1</italic>, and <italic>VDR</italic> assays, and a 1:10 dilution was
used for the <italic>GAPDH</italic> (glyceraldehyde-3-phosphate dehydrogenase) assay.
Primer sequences were as follows: CYP24A1_F, 5′-CCCAAAGGAACAGTGCTCATG;
CYP24A1_R, 5′-TCTCCTGAAGCCAACGTTCAG; CYP27B1_F,
5′-ATGAGGAACGGAGGACAGCC; CYP27B1_R, 5′-GAGCGTGTTGGACACCGTGT;
VDR_F, 5′-TTGCGCTCCAATGAGTCCTTC; VDR_R, 5′-CTGTGTCCGGCTTTGGTCAC;
GAPDH_F, 5′-TTCGACAGTCAGCCGCATCTT; GAPDH_R,
5′-CCCAATACGACCAAATCCGTT.</p></sec><sec><title>RESULTS</title><p><italic>The CYP24A1 but Not the VDR or CYP27B1 Gene Promoter Is Methylated in
Full Term Human Placental Tissue</italic>—As a first step in the
identification of potential epigenetic regulation of vitamin D levels in the
placenta, we designed specific methylation amplification assays for regulatory
regions of the <italic>VDR, CYP24A1</italic>, and <italic>CYP27B1</italic> genes. In the case
of the <italic>VDR</italic> and <italic>CYP24A1</italic> genes, assays were designed to span
the transcription start site and exon 1 within the associated CpG island,
whereas for <italic>CYP27B1</italic>, we analyzed a region of exon 1 previously shown
to be methylated in some cancers
(<xref ref-type="bibr" rid="ref48">48</xref>,
<xref ref-type="bibr" rid="ref49">49</xref>). Initial experiments on
human term placenta revealed a complete lack of methylation at the
<italic>VDR</italic> and <italic>CYP27B1</italic> gene regulatory regions, with clear (but
variable) methylation of the <italic>CYP24A1</italic> promoter region
(<xref rid="fig1" ref-type="fig">Fig. 1</xref> and
<xref ref-type="table" rid="tbl1">Table 1</xref>). This was not due to
problems with specific assays, since methylation of both <italic>VDR</italic> and
<italic>CYP27B1</italic> were detected in choriocarcinoma cells (see below). Three
independent assays spanning the <italic>CYP24A1</italic>-associated CpG island were
then examined with each showing evidence of methylation in full term placenta
(supplemental Fig. 1). A total of 10 term placentas were then examined using
the <italic>CYP24A1</italic>-2 assay spanning a region of differential methylation
recently identified in the placenta by whole genome methylation data
(<xref ref-type="bibr" rid="ref50">50</xref>). These results confirmed
methylation of the <italic>CYP24A1</italic> CpG island in all full term placental
samples, with a mean methylation level of 56.5%
(<xref ref-type="table" rid="tbl1">Table 1</xref>). Whereas some
<italic>CYP24A1</italic> alleles showed nearly complete methylation, others were
almost completely unmethylated. This is reminiscent of monoallelic
methylation, previously described for the <italic>APC</italic> tumor suppressor gene
in the placenta (<xref ref-type="bibr" rid="ref37">37</xref>), but may
equally reflect differing methylation profiles in a mixed population of
cells.</p><p><table-wrap position="float" id="tbl1"><label>TABLE 1</label><caption><p><bold>Mean promoter methylation levels of <italic>CYP24A1, CYP27B1</italic>, and
<italic>VDR</italic> genes in human placental samples and cell lines</bold></p><p>A summary of CpG island methylation status for genes regulating vitamin D
homeostasis and activity was determined by bisulfite sequencing. Placenta,
full term placental tissue; 1st trimester CT, purified cytotrophoblasts
showing >95% CK-7 positive staining; Terminated pregnancy, placental tissue
at 9–12 weeks gestation; Pre-eclampsia, diseased placental tissue at
9–12 weeks gestation; Choriocarcinoma, cell lines (BeWo, JEG-3, and/or
JAR). Placental fibroblasts were isolated following serial passaging of
Percoll gradient-separated cell fractions. Decidual cells, human endometrial
stromal cells grown in culture. HIPEC65, an SV40-transformed trophoblast cell
line. Normal tissue data consist of pooled data across a variety of different
human tissues isolated from different subjects. The number of each type of
cell/tissue is shown in parentheses.</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="top"><bold>Gene</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>Tissue</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>Mean methylation</bold></th><th colspan="1" rowspan="1" align="center" valign="top"><bold>S. D.</bold></th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="center" valign="top"><italic>%</italic></td><td colspan="1" rowspan="1" align="center" valign="top"></td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CYP24A1</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> Placenta (<italic>n</italic> = 8)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 56.53
</td><td colspan="1" rowspan="1" align="center" valign="top"> 10.49
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Uncultured CVS (<italic>n</italic> = 4)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 33.07
</td><td colspan="1" rowspan="1" align="center" valign="top"> 9.31
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Cultured CVS (<italic>n</italic> = 4)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 33.79
</td><td colspan="1" rowspan="1" align="center" valign="top"> 19.02
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> First trimester CT (<italic>n</italic> = 4)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 48.17
</td><td colspan="1" rowspan="1" align="center" valign="top"> 24.49
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Terminated pregnancy (<italic>n</italic> = 3)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 48.62
</td><td colspan="1" rowspan="1" align="center" valign="top"> 19.18
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Pre-eclampsia (<italic>n</italic> = 3)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 39.85
</td><td colspan="1" rowspan="1" align="center" valign="top"> 6.32
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Choriocarcinoma (<italic>n</italic> = 3)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 80.40
</td><td colspan="1" rowspan="1" align="center" valign="top"> 10.47
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Placenta fibroblasts (<italic>n</italic> = 3)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2.95
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2.28
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Decidual cells (<italic>n</italic> = 4)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1.57
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.57
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> HIPEC65 (<italic>n</italic> = 2)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 2.03
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.96
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Nonplacental tissue (<italic>n</italic> = 10)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1.56
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1.12
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>CYP27B1</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> Placenta (<italic>n</italic> = 4)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 5.23
</td><td colspan="1" rowspan="1" align="center" valign="top"> 4.88
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top"> Choriocarcinoma (<italic>n</italic> = 2)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 86.63
</td><td colspan="1" rowspan="1" align="center" valign="top"> 1.51
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"><italic>VDR</italic></td><td colspan="1" rowspan="1" align="left" valign="top"> Placenta (<italic>n</italic> = 2)
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.65
</td><td colspan="1" rowspan="1" align="center" valign="top"> 0.14
</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"></td><td colspan="1" rowspan="1" align="left" valign="top">Choriocarcinoma (<italic>n</italic> = 3)
</td><td colspan="1" rowspan="1" align="center" valign="top">85.84
</td><td colspan="1" rowspan="1" align="center" valign="top">11.61
</td></tr></tbody></table></table-wrap></p><p><italic>CYP24A1 Placental Methylation in Multiple First Trimester Placental
Cell Types</italic>—In order to ascertain the cell types and timing of
methylation of the <italic>CYP24A1</italic> promoter within the placenta, we examined
the methylation status of chorionic villous tissue (pre- and post-cell
culturing), first trimester termination placental tissue, purified first
trimester cytotrophoblasts (>90% cytokeratin-7-positive), and full term
placental fibroblasts. Promoter methylation comparable with that seen in full
term placental tissue was observed both in primary and cultured CVS tissue
samples (<xref rid="fig2" ref-type="fig">Fig. 2</xref> and
<xref ref-type="table" rid="tbl1">Table 1</xref>) and in first trimester
termination tissue (<xref ref-type="table" rid="tbl1">Table 1</xref>).
Since the CVS culturing conditions do not favor trophoblast growth or
division, the methylation of <italic>CYP24A1</italic> seen in the cultured samples is
probably mesodermal in origin. In addition, purified first trimester
cytotrophoblasts also show methylation of the <italic>CYP24A1</italic> gene
(<xref rid="fig2" ref-type="fig">Fig. 2</xref>), demonstrating that
this methylation is present in multiple cell types in first trimester
placenta. No methylation was detected in purified full term placental
fibroblasts or an SV40-transformed first trimester trophoblast cell line
(<xref rid="fig2" ref-type="fig">Fig. 2</xref>). A summary of mean
methylation levels obtained in this study is presented in
<xref ref-type="table" rid="tbl1">Table 1</xref>.</p><p><fig position="float" id="fig2"><label>FIGURE 2.</label><caption><p><bold>Methylation of <italic>CYP24A1</italic> in different human placental tissues and
cell types.</bold> Representative methylation data generated for the CYP24A1-2
assay in cytokeratin-7 positive cytotrophoblasts (<italic>A</italic>), uncultured CVS
(<italic>B</italic>), cultured CVS (<italic>C</italic>), full term placental fibroblasts
(<italic>D</italic>), and whole blood (<italic>E</italic>). <italic>i</italic>, between 8 and 12
individual clones were sequenced for each sample. <italic>Circles</italic>, CpG sites
denoted by <italic>vertical dashes. Closed circles</italic>, methylation; <italic>open
circles</italic>, lack of methylation. Missing circles indicate CpG sites for
which no information was obtained. <italic>Gray boxes</italic>, to GpG island
locations; <italic>arrows</italic>, start site of translation within exon 1 (<italic>black
line</italic>). <italic>ii</italic>, mean methylation levels seen at each CpG site for
each type of sample tested. Numbers of each type of tissue are listed in
<italic>parentheses</italic>.</p></caption><graphic xlink:href="zbc0210973730002"></graphic></fig></p><p><italic>CYP24A1 Methylation Is Placenta-specific</italic>—Given the
importance of the 24-hydroxylase enzyme in regulating levels of active vitamin
D, it is surprising how little is known about the epigenetic regulation of
this gene in human tissues. In order to address this, we assessed the
methylation status of the <italic>CYP24A1</italic> gene in a panel of human tissues
consisting of kidney, skeletal muscle, skin fibroblasts, brain (prefrontal
cortex), sperm, whole blood, buccal mucosa, endometrial stroma, decidualized
stroma, bone marrow, and umbilical cord tissue. The results, (summarized in
<xref rid="fig2" ref-type="fig">Fig. 2</xref> and
<xref ref-type="table" rid="tbl1">Table 1</xref>; detailed in
supplemental Fig. 2), demonstrate the specificity of placental
<italic>CYP24A1</italic> methylation in humans, with no evidence for methylation in
any human somatic tissue tested.</p><p><italic>The CYP24A1 Gene Is Differentially Methylated in Full Term Placentas
from Different Mammals</italic>—In previous studies, we have demonstrated
that some of the specific promoter methylation recently documented in human
term placental tissue is present in primates but absent in E18 (day 18 of
pregnancy) mouse placenta. We therefore examined methylation of the
<italic>CYP24A1</italic> gene promoter regions in full term placenta from marmoset and
mouse E18 and E11.5 (<xref rid="fig3" ref-type="fig">Fig. 3</xref>).
Assays were designed to span the region corresponding to the human
<italic>CYP24A1</italic>-2 assay. This revealed varying levels of <italic>CYP24A1</italic>methylation in both marmoset (mean 27 ± 18.7%) and mouse (mean 35.1
± 4.5%) placental tissue with generally lower levels relative to that
seen in human full term placentas (mean 56.5 ± 10.5%;
<xref rid="fig3" ref-type="fig">Fig. 3</xref>) Interestingly, less
methylation was seen in E18 mouse placental tissue compared with E11.5 tissue
(data not shown).</p><p><fig position="float" id="fig3"><label>FIGURE 3.</label><caption><p><bold>Variable levels of <italic>CYP24A1</italic> methylation in different mammalian
full term placentas.</bold> Representative methylation data were generated for
homologous <italic>CYP24A1</italic> promoter region in marmoset (<italic>A</italic>) and mouse
(<italic>B</italic>). <italic>i, gray boxes</italic>, GpG island locations; <italic>arrows</italic>,
orientation of transcription. <italic>Black boxes</italic>, assay location.
<italic>ii</italic>, eight individual clones were sequenced for each sample.
<italic>Circles</italic>, CpG sites denoted by <italic>black dashes. Closed circles</italic>,
methylation; <italic>open circles</italic>, lack of methylation. <italic>Shaded bars</italic>,
mean methylation levels seen at each CpG site for each type of sample
tested.</p></caption><graphic xlink:href="zbc0210973730003"></graphic></fig></p><p><italic>Methylation of the Core CYP24A1 Gene Promoter Directly Attenuates Basal
Promoter Activity in Human Trophoblast-derived Cells</italic>—Despite
circumstantial evidence of an inverse correlation of <italic>CYP24A1</italic> mRNA
levels with promoter gene methylation (above), no direct evidence for DNA
methylation-mediated silencing of this gene promoter has been reported. To
investigate this further, we PCR-amplified the previously described vitamin
D-responsive region of the human <italic>CYP24A1</italic> gene promoter
(<xref ref-type="bibr" rid="ref47">47</xref>) and generated a
luciferase reporter construct to directly test the effects of promoter
methylation on both basal and vitamin D-induced promoter activity.</p><p>The SV40-transformed human trophoblast cell line, HIPEC65, and
choriocarcinoma-derived JAR cells were transfected with two different reporter
constructs (plus and minus orientation) with or without <italic>in vitro</italic>methylation with SssI or HpaII methylases. SssI methylase methylates all CpGs;
HpaII methylase methylates only the CpG within the sequence CCGG. Results,
summarized in <xref rid="fig4" ref-type="fig">Fig. 4</xref>, reveal a
low level of promoter activity of this promoter region in the plus orientation
in both cell lines, ∼6.5-fold (HIPEC65; <italic>p</italic> = 0.028) and 12-fold
(JAR; <italic>p</italic> < 0.001) higher than that of the promoterless luciferase
vector. Promoter activity in the reverse (minus) orientation was negligible
(1.3-fold), demonstrating a directionality of the basal promoter element (data
not shown). Basal activity was completely attenuated by prior methylation with
any of SssI (<italic>p</italic> = 0.011), HpaII (<italic>p</italic> = 0.01), or HhaI
(<italic>p</italic> = 0.014) methylases in HIPEC65 cells and inhibited in the JAR cell
line (<italic>p</italic> < 0.001 for each of SssI and HpaII; supplemental Table 1).
The 768-bp region of this gene used for reporter activity assays contains a
total of 5 HhaI and 15 HpaII methylase target sites, suggesting that even low
level methylation of this promoter is sufficient to abrogate gene expression
activity.</p><p><italic>CpG Island Methylation Abrogates 1,25-(OH)<sub>2</sub>D-mediated
Up-regulation of CYP24A1 Transcription</italic>—In addition to examining the
effects of promoter methylation on the basal <italic>CYP24A1</italic> promoter
activity, we also set out to investigate the consequences of this methylation
on the previously demonstrated inducibility of this gene promoter by
1,25-(OH)<sub>2</sub>D (<xref ref-type="bibr" rid="ref47">47</xref>).
JAR and HIPEC65 cells were transfected with the 768-bp core promoter with or
without previous methylation with Sss1 methylase as above. 10 h
post-transfection, one-half of cells were supplemented with 100 n<sc>m</sc>1,25-(OH)<sub>2</sub>D, and reporter (luciferase) activity was then monitored.
Although 1,25-(OH)<sub>2</sub>D treatment resulted in a clear induction
(3.5-fold) of basal promoter activity in HIPEC65 cells in the absence of
previous promoter methylation (<italic>p</italic> = 0.008), this response was
abolished with <italic>in vitro</italic> methylation of the reporter construct prior
to transfection (<xref rid="fig4" ref-type="fig">Fig. 4</xref>).
1,25-(OH)<sub>2</sub>D treatment had no effect on promoter activity in JAR
cells (<italic>p</italic> = 0.663), most likely reflecting the methylation of the
<italic>VDR</italic> gene in this cell line (see below) that silences the gene and
inhibits exogenous 1,25-(OH)<sub>2</sub>D signaling through the VDR.</p><p><fig position="float" id="fig4"><label>FIGURE 4.</label><caption><p><bold>CYP24A1 promoter activity in human trophoblast cell lines.</bold>SV40-transformed cytotrophoblast (HIPEC-65) or choriocarcinoma (JAR) cells
were transfected (<italic>n</italic> = 8 for each cell line) with a promoterless
luciferase reporter (vector) with or without the cloned <italic>CYP24A1</italic> gene
promoter (–548/+209 relative to the start site of transcription). This
region has previously been demonstrated to contain vitamin D-responsive
elements (<xref ref-type="bibr" rid="ref47">47</xref>). Basal promoter
activity was detected in both cell lines and was abolished with <italic>in
vitro</italic> methylation of the promoter prior to transfection (Sss1, HpaII, and
HhaI methylases). 1,25-(OH)<sub>2</sub>D (active vitamin D) induction of the
promoter was observed only in HIPEC-65 cells (JAR cells are predicted to lack
the vitamin D receptor, as evidenced by complete methylation of the associated
CpG island), but this induction was attenuated with prior methylation of the
promoter region. Luminescence values were normalized to <italic>Renilla</italic>luciferase to correct for transfection efficiency, and all data are displayed
relative to the mean promoterless control vector in HIPEC-65 cells. <italic>Error
bars</italic> denote 95% confidence intervals. Statistical analysis for comparing
between transfected groups was carried out using a two-tailed Student's
<italic>t</italic> test with unequal variance.</p></caption><graphic xlink:href="zbc0210973730004"></graphic></fig></p><p>We also examined the effect of promoter methylation on the induction of
endogenous <italic>CYP24A1, CYP27B1</italic>, and <italic>VDR</italic> gene expression in
primary cells derived from CVS tissue (average methylation level of 33.4%) and
placental and skin fibroblasts (both lacking <italic>CYP24A1</italic> methylation).
Cultured skin fibroblasts showed a mean <italic>CYP24A1</italic> induction level
approaching 2000-fold (<italic>n</italic> = 2; <xref rid="fig5" ref-type="fig">Fig.
5</xref>), consistent with previous data
(<xref ref-type="bibr" rid="ref47">47</xref>,
<xref ref-type="bibr" rid="ref51">51</xref>,
<xref ref-type="bibr" rid="ref52">52</xref>). Placental fibroblasts
also showed a significant level of induction (mean 172-fold; <italic>n</italic> = 2).
In contrast, cultured CVS cells showed a reduced capacity for gene induction
(mean ∼10 fold; <italic>n</italic> = 4). In contrast to the major effects of
vitamin D induction on CYP24A1 expression in skin and placental fibroblasts,
VDR expression levels were slightly reduced (mean reduction of ∼3-fold) in
skin fibroblasts, with little change apparent in placental fibroblasts (mean
1.18-fold increase) and CVS-derived cells (mean 0.76-fold change;
<xref rid="fig5" ref-type="fig">Fig. 5</xref>). Similarly, no major
effects on CYP27B1 expression level were seen in skin fibroblasts (mean
1.19-fold increase) or CVS-derived cells (mean 1.55-fold increase) following
vitamin D induction (<xref rid="fig5" ref-type="fig">Fig.
5</xref>).</p><p><italic>Altered CYP24A1 Methylation in Placental Tissue from Pre-eclampsia
Pregnancies?</italic>—We carried out a preliminary analysis of methylation
levels of the CYP24A1-2 assay region in a small set of available preeclampsia
tissue (<xref ref-type="table" rid="tbl1">Table 1</xref>). These samples
showed a mean methylation level of only 39.5 ± 6% (<italic>n</italic> = 3),
compared with the 56.53 ± 10% level seen in nonpre-eclampsia placental
tissue (<italic>n</italic> = 8). The potential significance (<italic>p</italic> = 0.017) of
these data will require further investigation in a larger sample size in the
future.</p><p><italic>Complete Methylation of Vitamin D Regulatory Genes in Choriocarcinoma
Cell Lines</italic>—Trophoblast hyperplasia is a common feature of complete
and partial hydatidiform mole with the potential for malignant transformation
to choriocarcinoma (CCA) (<xref ref-type="bibr" rid="ref53">53</xref>).
Trophoblasts that comprise CCA resemble the primitive trophoblast of the
previllous stage during placental development, arrested in specific stages of
differentiation (<xref ref-type="bibr" rid="ref54">54</xref>).</p><p>We analyzed the methylation status of <italic>CYP24A1</italic> in three widely
studied CCA cell lines, JEG-3, BeWo, and JAR. In all three lines, methylation
at the <italic>CYP24A1</italic> promoter was increased to nearly complete methylation
(<xref rid="fig6" ref-type="fig">Fig. 6</xref>). These results
correlate with the very low levels of <italic>CYP24A1</italic> mRNA seen in each of
these cell types (data not shown). We also assessed methylation at the
<italic>VDR</italic> and <italic>CYP27B1</italic> CpG islands, which also revealed nearly
complete methylation of each of these genes in all cell lines tested. This
contrasts with the complete lack of methylation seen in full term placental
tissue and first trimester cytotrophoblast (described above).</p></sec><sec><title>DISCUSSION</title><p>Placental insufficiency, due to inadequate remodeling of the maternal
vasculature in early pregnancy, is thought to play a major role in several
adverse pregnancy outcomes, including the early stages of pre-eclampsia
(<xref ref-type="bibr" rid="ref55">55</xref>). Lowered
1,25-(OH)<sub>2</sub>D and altered calcium metabolism have been implicated in
this process (<xref ref-type="bibr" rid="ref10">10</xref>,
<xref ref-type="bibr" rid="ref11">11</xref>). For example, a recent
Cochrane review of randomized control trials demonstrated a role for calcium
supplementation in reducing pre-eclampsia
risk.<xref ref-type="fn" rid="fn5">5</xref> Altered
expression levels of 1-hydroxylase, <italic>VDR</italic>, and 24-hydroxylase genes
have all been reported in different placental cells and tissue from
preeclamptic pregnancies, possibly in response to decreasing maternal
1,25-(OH)<sub>2</sub>D (<xref ref-type="bibr" rid="ref19">19</xref>,
<xref ref-type="bibr" rid="ref57">57</xref>,
<xref ref-type="bibr" rid="ref58">58</xref>). In addition, levels of
<italic>CYP27B1</italic> mRNA are reduced in cultured syncytial trophoblasts derived
from pre-eclamptic patients compared with controls
(<xref ref-type="bibr" rid="ref57">57</xref>). These data highlight the
likely functional importance of 1,25-(OH)<sub>2</sub>D in placental function
and implicate disruption of vitamin D homeostasis in adverse pregnancy
outcomes.</p><p><fig position="float" id="fig5"><label>FIGURE 5.</label><caption><p><bold>Induction of endogenous CYP24A1 mRNA in cultured primary cells.</bold>Cells were cultured in multiple wells, and 100 n<sc>m</sc>1,25-(OH)<sub>2</sub>D<sub>3</sub> (active vitamin D) was added to half of the
cultures for 7 h, followed by total RNA isolation and quantitative real time
reverse transcription-PCR. Mean levels of induction (<italic>n</italic> = 2–4)
are listed in the corresponding <italic>table</italic>. Whereas cultured skin
fibroblasts show a nearly 2000-fold induction of CYP24A1 expression,
consistent with previous data
(<xref ref-type="bibr" rid="ref47">47</xref>,
<xref ref-type="bibr" rid="ref51">51</xref>,
<xref ref-type="bibr" rid="ref52">52</xref>), and placental fibroblasts
show a mean 172-fold induction, cultured CVS biopsies (containing cells with
CYP24A1 methylation) show a greatly reduced level of gene induction
(∼10-fold). No major effects on VDR or CYP27B1 expression were observed.
<italic>Error bars</italic> denote 95% confidence interval. <italic>ND</italic>, no data
available.</p></caption><graphic xlink:href="zbc0210973730005"></graphic></fig></p><p>Epigenetic regulation of steroid signaling is an emerging field of research
and hints at a complex interplay between environmental or dietary influences
with underlying genetic variation, epigenetic variation, steroid metabolism,
and subsequent downstream effects
(<xref ref-type="bibr" rid="ref59">59</xref>). However, at present,
very little is known about the consequences of altered environmental, genetic,
or epigenetic regulation of vitamin D homeostasis at the fetomaternal
interface. In this study, we found no evidence for methylation of the
<italic>VDR</italic> or <italic>CYP27B1</italic> genes in human full term or first trimester
placental tissue. Since methylation is generally associated with gene
silencing, this supports previous data demonstrating high levels of expression
of 1α-hydroxylase and VDR in the placenta
(<xref ref-type="bibr" rid="ref18">18</xref>,
<xref ref-type="bibr" rid="ref19">19</xref>,
<xref ref-type="bibr" rid="ref60">60</xref>). In contrast, we have
identified methylation of the <italic>CYP24A1</italic> gene, encoding
24α-hydroxylase, the major catabolic enzyme of 1,25-(OH)<sub>2</sub>D
and 25-(OH)<sub>2</sub>D in placental tissue and cell subtypes. We have
demonstrated that this methylation is present in both the trophoblast
compartment and cells of mesodermal origin and is present (albeit at variable
levels) in primate and rodent placentas. This contrasts with the recently
described placenta-specific methylation of the <italic>APC</italic> tumor suppressor
gene that is primate-specific and localizes solely to the trophoblast
compartment (<xref ref-type="bibr" rid="ref37">37</xref>) and the
<italic>SFRP2</italic> tumor suppressor methylation that is primate-specific but
localizes to multiple placental cell subtypes
(<xref ref-type="bibr" rid="ref38">38</xref>).</p><p>Importantly, we have demonstrated (by <italic>in vitro</italic> reporter studies)
that methylation of the <italic>CYP24A1</italic> gene promoter directly down-regulates
basal gene activity and abolishes vitamin D induction of the proximal promoter
region. This is further supported by data demonstrating a reduced inducibility
(by 2–3 orders of magnitude) of the methylated endogenous
<italic>CYP24A1</italic> gene in cultured primary human CVS-derived cells relative to
unmethylated fibroblast cells. We observed much higher levels of induction of
the endogenous gene in cultured primary fibroblast cells in comparison with
that seen with a transfected reporter construct (containing only the proximal
promoter region) in transformed trophoblast cells. This is consistent with
previous data demonstrating a requirement for distal upstream sequence
elements, in addition to the two previously described vitamin D-responsive
elements, contained within the proximal promoter region, for maximal induction
of the <italic>CYP24A1</italic> gene
(<xref ref-type="bibr" rid="ref47">47</xref>). Interestingly, we did
not find any major effects of exogenous 1,25-(OH)<sub>2</sub>D on the levels
of VDR or CYP27B1 expression in skin or placental fibroblast cells. This
contrasts with previous studies showing down-regulation of each of these genes
in some cancer cell lines (<xref ref-type="bibr" rid="ref61">61</xref>)
in response to 1,25-(OH)<sub>2</sub>D treatment. In addition, the CYP27B1 gene
was recently reported to be down-regulated in response to
1,25-(OH)<sub>2</sub>D in cultured trophoblasts undergoing syncitialization
(<xref ref-type="bibr" rid="ref62">62</xref>). The proximal human
CYP27B1 gene promoter contains putative vitamin D-responsive elements and has
been shown to mediate 1,25-(OH)<sub>2</sub><sc>d</sc>-mediated
down-regulation of this gene in <italic>in vitro</italic> reporter experiments in
mouse proximal tubule cells
(<xref ref-type="bibr" rid="ref63">63</xref>). However, in primary
human keratinocytes, 1,25-(OH)<sub>2</sub>D does not regulate
1α-hydroxylase mRNA or protein expression
(<xref ref-type="bibr" rid="ref64">64</xref>), supporting our findings
in this study.</p><p>Methylation of the proximal promoter region of the mouse <italic>Cyp24a1</italic>gene has been previously reported in tumor-derived endothelial cells, where it
contributes to selective growth inhibition by 1,25-(OH)<sub>2</sub>D
(<xref ref-type="bibr" rid="ref30">30</xref>). Conversely, a lack of
methylation in corresponding non-tumor-derived mouse endothelial cells is
associated with a lack of 1,25-(OH)<sub>2</sub>D-mediated growth inhibition
(<xref ref-type="bibr" rid="ref30">30</xref>). No equivalent data have
previously been reported for the human <italic>CYP24A1</italic> gene.</p><p>It is well documented that maternal circulating 1,25-(OH)<sub>2</sub>D
levels show a dramatic increase during pregnancy (reviewed in Ref.
<xref ref-type="bibr" rid="ref12">12</xref>), with ∼50% of maternal
circulating 1,25-(OH)<sub>2</sub>D derived from maternal decidua and placenta
(<xref ref-type="bibr" rid="ref13">13</xref>). This observation
demonstrates that the tight regulation of 1,25-(OH)<sub>2</sub>D levels is
uncoupled during pregnancy. There are two potential mechanisms by which this
can be achieved. First, this can be achieved through an increase in expression
of the <italic>CYP27B1</italic> gene, which results in increasing 1α-hydroxylase
and elevated production of 1,25-(OH)<sub>2</sub>D from less active precursors.
The second potential mechanism involves a decrease in the catabolism of
1,25-(OH)<sub>2</sub>D by the 24-hydroxylase enzyme encoded by
<italic>CYP24A1</italic>. Increasing levels of maternal circulating
1,25-(OH)<sub>2</sub>D during pregnancy have been linked to increased
synthesis rather than decreasing catabolic activity in animal studies
(<xref ref-type="bibr" rid="ref65">65</xref>–<xref ref-type="bibr" rid="ref67">67</xref>).
In addition, <italic>CYP27B1</italic> expression is increased in early pregnancy
decidua in comparison with nonpregnant endometrium
(<xref ref-type="bibr" rid="ref19">19</xref>,
<xref ref-type="bibr" rid="ref68">68</xref>). In this study, we have
demonstrated that the increasing synthesis of 1,25-(OH)<sub>2</sub>D occurs in
concert with a placenta-specific epigenetic down-regulation of the
<italic>CYP24A1</italic> (24-hydroxylase) gene. Thus, both mechanisms for elevating
1,25-(OH)<sub>2</sub>D levels are present during pregnancy.</p><p><fig position="float" id="fig6"><label>FIGURE 6.</label><caption><p><bold>Methylation analysis of vitamin D-associated genes in CCA-derived
trophoblast cell lines.</bold> High levels of methylation were detected for both
<italic>VDR</italic> (<italic>A</italic>) and <italic>CYP27B1</italic> (<italic>C</italic>) in choriocarcinoma
cell lines (<italic>n</italic> = 2). All choriocarcinoma cell lines (<italic>n</italic> = 3;
BeWo not shown) examined showed hypermethylation of the <italic>CYP24A1</italic> gene
relative to full term placental tissue or purified first trimester
trophoblasts. Numbers of each type of tissue are listed in
<italic>parentheses</italic>. Between 8 and 12 individual clones were sequenced for
each sample. <italic>Circles</italic>, CpG sites within the assayed region. <italic>Closed
circles</italic>, methylation; <italic>open circles</italic>, lack of methylation. Missing
circles indicate CpG sites for which no information was obtained. <italic>Gray
boxes</italic> correspond to GpG island locations, and <italic>arrows</italic> denote
start site of translation within exon 1 (<italic>black line</italic>). <italic>iii</italic>,
mean methylation levels seen at each CpG site for each type of sample
tested.</p></caption><graphic xlink:href="zbc0210973730006"></graphic></fig></p><p>We are currently investigating the specific role of 1,25-(OH)<sub>2</sub>D
in mediating various aspects of placental functioning. 1,25-(OH)<sub>2</sub>D
has widely diverse roles in regulating cell division, apoptosis, and immune
modulation. It also shows antiproliferative effects in many different cell
types, is proapoptotic, and is inhibitory to cell migration and invasion
(<xref ref-type="bibr" rid="ref5">5</xref>,
<xref ref-type="bibr" rid="ref6">6</xref>,
<xref ref-type="bibr" rid="ref69">69</xref>–<xref ref-type="bibr" rid="ref71">71</xref>).
Each of these processes plays an important role in the development and
function of the mammalian placenta.</p><p>The interesting finding of simultaneous methylation of the <italic>VDR,
CYP24A1</italic>, and <italic>CYP27B1</italic> genes in choriocarcinoma-derived
trophoblast cell lines is unprecedented in cancer. Methylation of the
<italic>VDR</italic> gene is consistent with recent data demonstrating a lack of
expression in BeWo and JEG-3 choriocarcinoma cell lines
(<xref ref-type="bibr" rid="ref72">72</xref>), and dysregulation of all
three genes has been reported in cancers of different origin
(<xref ref-type="bibr" rid="ref73">73</xref>). Although abolition of
<italic>VDR</italic> and <italic>CYP27B1</italic> expression is anticipated to attenuate
vitamin D-mediated growth inhibition in tumor cells, silencing of
<italic>CYP24A1</italic> would have the effect of maximizing active vitamin D
bioavailability. It is possible that the precursor cells from which CCA
developed already had the <italic>CYP24A1</italic> methylation described in this study
and that acquisition of epigenetic silencing of the <italic>VDR</italic> and
<italic>CYP27B1</italic> genes was associated with the development of this tumor type.
It is interesting to speculate that a sequential series of methylation events
may occur in tumor development and progression. Future studies examining the
epigenetic regulation of this and other pathways in hydatidiform mole,
recurring mole, and choriocarcinoma, may shed valuable light on the
acquisition of methylation changes in gestational tumors.</p><p>Confirmation of epigenetic regulation of active vitamin D levels at the
fetomaternal interface provides compelling circumstantial evidence for the
intersection of two metabolic pathways in regulating pregnancy outcome, namely
vitamin D and 1-carbon (folate or methyl donor) metabolism. One-carbon
metabolism is a process by which methyl groups are passed from one donor
molecule to the next (<xref ref-type="bibr" rid="ref74">74</xref>,
<xref ref-type="bibr" rid="ref75">75</xref>). This cycle produces
<italic>S</italic>-adenosylmethionine, which donates its methyl group to cytosine to
produce methylated CpG. Each of these pathways relies on external inputs for
the production of precursor molecules, and enzymes involved in each pathway
(including VDR and CYP27B1) have known functional polymorphic variants
implicated in moderating disease risk
(<xref ref-type="bibr" rid="ref76">76</xref>–<xref ref-type="bibr" rid="ref83">83</xref>),
including adverse pregnancy outcomes
(<xref ref-type="bibr" rid="ref84">84</xref>–<xref ref-type="bibr" rid="ref89">89</xref>).
There are numerous polymorphisms throughout the <italic>CYP24A1</italic> exonic,
intronic, and regulatory gene regions
(<xref ref-type="bibr" rid="ref56">56</xref>), but as yet there is
little information regarding the functionality or clinical relevance of such
variants. The convergence/intersection of 1-carbon metabolism with other
metabolic and signaling pathways is potentially very important in modulating
disease risk and warrants further investigation.</p><p>The placenta-specific epigenetic regulation of several genes and metabolic
pathways, independent of well characterized imprinted regions, is now a firmly
established phenomenon. Uncovering the mechanisms leading to the establishment
of such markings, their timing, and sensitivity to environmental and genetic
disruption has the potential to reveal valuable insights into the evolution of
different placental functions and may reveal novel risk pathways associated
with adverse pregnancy outcomes and tumor development.</p></sec></body><back><ack><p>We thank Dr. Nicole Brooks and Tina Vaino (Mercy Hospital for Women,
Australia) and Sarah Healy (Royal Women's Hospital) for help with collection
of placental tissue, Professor Lois Salamonson (Prince Henry's Institute of
Medical Research, Australia) and Professor Samuel Breit (University of NSW,
Australia) for CCA cancer cell lines, Drs. Patrick Western and Craig Smith
(Murdoch Childrens Research Institute) for mouse placental tissue, and Dr.
Mark Pertile for human CVS samples. We especially thank Lavinia Gordon for
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