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Calcified Tissue International
https://doi.org/10.1007/s00223-018-0434-0
ORIGINAL RESEARCH
lncRNA HOTAIR Inhibits Mineralization in Osteoblastic Osteosarcoma
Cells by Epigenetically Repressing ALPL
Aya Misawa
1
· Hideo Orimo
1
Received: 31 January 2018 / Accepted: 24 May 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
HOTAIR is a lncRNA that plays critical role in gene regulation and chromatin dynamics through epigenetic mechanisms. In
this work we studied the physiological role of HOTAIR during the process of mineralization using osteoblastic osteosarcoma
cells focusing in ALPL (Tissue Non-Specific Alkaline Phosphatase), a pivotal gene that controls bone formation. HOTAIR
knockdown resulted in upregulation of ALPL, increase of alkaline phosphatase (ALP) activity, and enhanced mineralization
in osteoblastic SaOS-2 cells cultured in mineralizing medium. Luciferase assays using reporter vectors containing ALPL
promoter showed that HOTAIR repression increases ALPL promoter activity. Furthermore, HOTAIR knockdown increased
histone H3K4 methylation levels at ALPL promoter region, suggesting that ALPL repression by HOTAIR is regulated by
epigenetic mechanisms. This work supports that physiological bone formation is epigenetically regulated by a lncRNA.
Keywords ncRNA · lncRNA · Epigenetics · Alkaline phosphatase · Mineralization · Osteoblast
Introduction
Alkaline phosphatases are membrane-bound ectoenzymes
that hydrolyze monophosphate esters between pH 8 and
10. They are classified into four types: tissue non-specific,
intestinal, placental, and germ cell, of which tissue non-spe-
cific type (TNSAP) is the ubiquitously expressed in vari-
ous tissues including liver, bone, and kidney [1]. Therefore,
TNSAP is also known as liver/bone/kidney (LBK) alkaline
phosphatase, although no precise physiological roles are
known in liver and kidney. TNSAP is encoded by the ALPL
gene, which is located in the short arm of chromosome 1
(1p36.1-34) and is spread over 50 kb [2]. ALPL is ~ 1.5 kb in
length and consists of 11 coding exons (exon 2–12) and two
alternative non-coding exons (exon 1B and 1L) [3, 4]. ALPL
plays a critical role in the mineralization of bone matrix and
other tissues, by hydrolyzing inorganic pyrophosphate (PPi),
a natural inhibitor of mineralization, into phosphate (Pi).
Extracellular PPi is formed by nucleotide pyrophosphatase
phosphodiesterase 1 (ENPP1) by hydrolysis of ATP, and
is also provided by the membrane transporter ANKH, the
human homolog of the mouse progressive ankylosis gene.
Pi molecules are needed for the formation of hydroxyapatite,
and the balance between the concentrations of PPi and Pi is
critical for mineralization [5].
TNSAP deficiency causes hypophosphatasia (HPP),
which is an inherited bone disease characterized by
hypomineralization of bone and teeth [6, 7]. On the other
hand, patients with genetic disorders causing an increased
alkaline phosphatase activity present an aberrant minerali-
zation of vascular and other soft tissues [8]. To date, 349
mutations in the ALPL gene have been reported, mainly
single-nucleotide substitutions (The TNSAP Gene Muta-
tions Database http://www.sesep .uvsq.fr/03_hypo_mutat
ions.php). Although HPP is generally concordant among
affected siblings with the identical ALPL genotype, signifi-
cant discordance sometimes occurs in brothers and sisters,
and the relationship between genotype and phenotype has
not been fully elucidated [9–11]. Osteoblasts, which syn-
thesize collagen and other components of the bone matrix,
are the main source of alkaline phosphatase in bone. Dur-
ing bone formation, some osteoblasts become embedded in
the new mineralized bone matrix and experiment a series
of morphological and functional changes resulting in spe-
cific modifications in their gene expression patterns, finally
transforming into osteocytes which do not express ALPL
* Hideo Orimo
orimohd@nms.ac.jp
1
Division of Metabolism and Nutrition, Department
of Biochemistry and Molecular Biology, Nippon Medical
School, 1-1-5 Sendagi Bunkyo-ku, Tokyo 113-8602, Japan
@Phil_Robichaud
@deepthiw
@JoseServera