901731

research-article2020
JDRXXX10.1177/0022034520901731Journal of Dental ResearchFunctional Analysis of PTH1R Variants

Research Reports: Biological

Journal of Dental Research
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Functional Analysis of PTH1R Variants © International & American Associations
for Dental Research 2020

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DOI: 10.1177/0022034520901731
https://doi.org/10.1177/0022034520901731
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E. Izumida1,2, T. Suzawa1, Y. Miyamoto1, A. Yamada1, M. Otsu3,4, T. Saito5,

T. Yamaguchi2, K. Nishimura6, M. Ohtaka7, M. Nakanishi8, K. Yoshimura1,
K. Sasa1, R. Takimoto1,9, R. Uyama1, T. Shirota9, K. Maki2, and R. Kamijo1

Abstract
Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of
eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R
variants reported in PFE patients—namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)—using HeLa
cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level
of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L
or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation
in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of
PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with
PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in
mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were
augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active
vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control
and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L
variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in
osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment
of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Keywords: bone biology, cell differentiation, cell signaling, osteoblasts, stem cells, receptors

Introduction 1
Department of Biochemistry, School of Dentistry, Showa University,
Tokyo, Japan
Primary failure of eruption (PFE) is a disorder characterized by 2
Department of Orthodontics, School of Dentistry, Showa University,
a posterior open bite (Ahmad et al. 2006). Variants in the Tokyo, Japan
3
PTH1R gene—which encodes parathyroid hormone 1 receptor Stem Cell Bank & Division of Stem Cell Processing, Center for Stem
(PTH1R), a receptor for parathyroid hormone (PTH) and PTH- Cell Biology and Regenerative Medicine, The Institute of Medical
Science, The University of Tokyo, Tokyo, Japan
related peptide (PTHrP)—have been identified in patients with 4
Present address: Department of Transfusion and Cell Transplantation,
PFE (Yamaguchi et al. 2011; Risom et al. 2013; Frazier-Bowers School of Medicine, Kitasato University, Sagamihara, Japan
et al. 2014; Subramanian et al. 2016). PTH and PTHrP have 5
Division of Tissue Engineering, Department of Bone and Cartilage
pivotal roles in the development and remodeling of skeletal Regenerative Medicine, University of Tokyo Hospital, The University of
tissues (Kitahara et al. 2002; Blair and Zaidi 2006; Kronenberg Tokyo, Tokyo, Japan
6
2006; Gama et al. 2015). We showed that conditional deletion Laboratory for Gene Regulation, Faculty of Medicine, University of
of PTH1R in PTHrP gene-expressing cells caused a PFE-like Tsukuba, Tsukuba, Japan
7
TOKIWA-Bio, Inc., Tsukuba, Japan
phenotype in mice (Takahashi et al. 2019). However, delayed 8
Biotechnology Research Institute for Drug Discovery, National Institute
tooth eruption was observed in mice expressing constitutive of Advanced Industrial Science and Technology, Tsukuba, Japan
active PTH1R (Tsutsui et al. 2008). Hence, it is plausible that 9
Department of Oral and Maxillofacial Surgery, School of Dentistry,
PTH1R variants found in patients with PFE cause disrupted Showa University, Tokyo, Japan
tooth eruption.
Our previous exome sequencing study indicated that 4 het- A supplemental appendix to this article is available online.
erozygous variants in PTH1R—namely, 356C>T, 395C>T, Corresponding Author:
439C>T, and 1148G>A, found in 4 patients with PFE—are Y. Miyamoto, Department of Biochemistry, School of Dentistry, Showa
closely related to PFE (Yamaguchi et al. 2011). Each is a mis- University, 1-5-8 Hatanodai, Shinagawa, Tokyo, 142-8555, Japan.
sense mutation, with P119L, P132L, R147C, and R383Q Email: yoichim@dent.showa-u.ac.jp
2 Journal of Dental Research 00(0)
substitutions in PTH1R. It was shown that a homozygous
mutation at 395C>T in the PTH1R causes Blomstrand lethal
chondrodysplasia, and functional analysis of that mutation
indicated decreased cAMP production in mutated PTH1R-
expressing cells after stimulation with PTH as compared with
those expressing wild-type PTH1R (Zhang et al. 1998).
Here, we performed functional analyses of PTH1R variants
(i.e., 356C>T, 395C>T, 439C>T, and 1148G>A) with HeLa
cells. Also, induced pluripotent stem cell (iPSC) clones were
established from hematopoietic progenitor cells from a patient
with PFE and the 395C>T variant in PTH1R to examine the
response of osteoblast-like cells derived from those clones to
PTH1-34 in comparison with osteoblast-like cells derived from
control iPSCs.
Materials and Methods
Introduction of Wild-type and Mutated PTH1R
in HeLa Cells
Wild-type and mutant PTH1R (356C>T, 395C>T, 439C>T,
1148G>A) cDNAs were prepared by polymerase chain reac-
tion (PCR) with a KOD Plus Mutagenesis Kit (Toyobo Co.,
Ltd.) with wild-type PTH1R cDNA as the template (Yano et al.
2013). Each cDNA or cDNA for green fluorescent protein
(GFP) gene (gfp) as a control was integrated into pLenti6.3
plasmid (Invitrogen); then, either each pLenti-PTH1R plasmid
or pLenti-gfp plasmid and packaging plasmids (ViraPower
Lentiviral Expression Systems; Invitrogen) were introduced into
293T cells to produce lentiviruses. HeLa cells infected with the
lentiviruses were selected in DMEM (Wako Pure Chemicals)
supplemented with 10% fetal bovine serum (FBS) and 10-µg/mL
blasticidin (Thermo Fisher Scientific).
Western Blot Analysis of PTH1R
Cells were incubated with 20-mmol/L Tris-HCl buffer (pH 7.5)
containing 0.15-mol/L NaCl, 1-mmol/L EDTA, 1-mmol/L
EGTA, 1% NP-40, 0.1% SDS, 1-mmol/L Na3VO4, and a prote-
ase inhibitor cocktail (Roche Applied Science). Following cen-
trifugation, the supernatants (7 µg of protein) were subjected to
SDS-PAGE (10% polyacrylamide) under a reducing condition,
and proteins were transferred onto a PVDF membrane. After
blocking in 1% skim milk with 10-mmol/L Tris-HCl buffer
(pH 7.6) containing 150-mmol/L NaCl and 0.05% Tween 20,
the membrane was incubated with a mouse monoclonal anti-
body against human PTH1R (1:1,000, clone 4D2; Abcam), fol-
lowed by incubation with horseradish peroxidase–conjugated
anti-mouse IgG (GE Healthcare). After enhanced chemilumi-
nescence reactions (ECL Prime Western Blot Detection
System; GE Healthcare), immunoreactive bands were observed
with a VersaDoc Imaging System (Bio-Rad).
N-glycosidase Treatment
Cell lysates (8 µg of protein) were incubated for 20 h at 37 °C
with or without 1 mU of peptide:N-glycosidase F (TaKaRa Bio
Inc.) at pH 8.6 under a denatured condition. PTH1R proteins
with or without N-glycosidase treatment were detected by
Western blotting.
Binding of Fluorescence-Labeled PTH
Lys-13(5-TAMRA)-PTH1-34, a fluorescence-labeled PTH1-34
(Castro et al. 2005), was synthesized by Peptide Institute, Inc.
Transfected HeLa cells were incubated for 1 h with Lys-13(5-
TAMRA)-PTH1-34 (100 nmol/L) in the presence or absence of
PTH1-34 (100 µmol/L) in the complete medium. After fixation
in 4% paraformaldehyde and counterstaining with 4′,6-
dimidino-2-phenylindole (DAPI), cells were observed under a
confocal fluorescence microscope (BX61WI; Olympus).
Fluorescence intensity of TAMRA was quantified with ImageJ
software (Schneider et al. 2012).
Preparation of iPSCs
Preparation and use of iPSCs for the present study were
approved by the Ethics Committee of Showa University School
of Dentistry (2014-004) and that of the Institute of Medical
Science, University of Tokyo (25-3-0701). Peripheral blood
samples were obtained from a patient with PFE (38-y-old
woman) with a nucleotide substitution of 395C>T in the
PTH1R gene and a healthy volunteer upon informed consent
following the Declaration of Helsinki standard ethics proce-
dure. We generated iPSCs according to previously reported
procedures (Lin et al. 2015; Kubara et al. 2018), with some
modifications. Yamanaka factors were introduced into CD34+
hematopoietic stem/progenitor cells isolated from a peripheral
blood sample with a Sendai virus SeVdp vector (KOSM302L;
Nishimura et al. 2017). iPSCs were cultured in Essential 8
medium (Thermo Fisher Scientific). iPSC clones fixed with
4% paraformaldehyde were incubated with primary antibodies
for SSEA-4 and Tra-1-60 (Cell Signaling Technology), which
were visualized with Alexa Fluor 488– and 594–conjugated
secondary antibodies (Invitrogen), respectively. Nuclei were
stained with DAPI, and the cells were observed under a fluo-
rescence microscope. Expression of mRNA for SOX2, OCT,
and NANOG was also examined by real-time reverse transcrip-
tion PCR (RT-PCR). The heterogeneous 395C>T mutation in
the PTH1R gene was confirmed in PFE-specific iPSCs.
Differentiation of iPSCs into Osteoblast-like Cells
Two clones of iPSCs obtained from the healthy subject and 3
from the patient with PFE were cultured for 3 wk in osteoblast-
inducing medium (α-MEM supplemented with 10% FBS,
10-mmol/L β-glycerophosphate, 0.5-mmol/L ascorbic acid,
10-nmol/L dexamethasone, 0.1-µg/mL BMP-2) or control
medium (α-MEM plus 10% FBS). Osteoblastic differentiation
was assessed by alizarin red staining of calcified nodules, as
well as real-time RT-PCR analyses of mRNA expression of
RUNX2, SP7, and BGLAP with GAPDH as a reference. We
selected osteoblast-like cells derived from a control iPSC clone
(TkPP#7) and a clone of iPSCs obtained from the patient
Functional Analysis of PTH1R Variants 3

(TkSP#11), based on the expression of RANKL mRNA after

stimulation with 10-nmol/L 1α,25-dihydroxyvitamin D3
(Sigma-Aldrich). Osteoblast-like cells derived from these
clones were used for the subsequent experiments.

Expression of RANKL in iPSC-Derived Osteoblast-like

Cells after Treatment with Active Vitamin D3
and PTH1-34
Osteoblast-like cells derived from control (TkPP#7) and PFE-
specific (TkSP#11) iPSCs were treated for 6 or 48 h with
1α,25-dihydroxyvitamin D3 (10 nmol/L) or PTH1-34 (25 nmol/L;
Asahi Kasei Pharma Co.). Real-time RT-PCR analysis was per-
formed to assess the expression of RANKL with GAPDH as a
reference.

Real-time RT-PCR
RNA was isolated with an RNeasy Plus Kit (Qiagen). First-
strand cDNA synthesis was carried out with Superscript III
reverse transcriptase (Invitrogen) primed with random hexam-
ers. With the TaqMan-based assay, quantitative PCR reactions
were performed according to the manufacturer’s instructions
(Applied Biosystems). Data were normalized by the expres-
sion of GAPDH mRNA. The TaqMan Gene Expression Assay
sets were as follows: SOX2, Hs01053049_s1; OCT4, Hs042
60367_gH; NANOG, Hs02387400_g1; RUNX2, Hs01047973_
m1; SP7, Hs01866874_s1; RANKL, Hs00243522_m1; BGLAP,
Hs01587814_g1; and GAPDH, Hs02758991_g1.

Immunocytochemistry of PTH1R
HeLa cells introduced with wild-type and mutated PTH1R
were fixed in 0.1-mol/L sodium phosphate buffer (pH 7.4) con-
taining 4% paraformaldehyde. After treatment for 1 h with
phosphate-buffered saline containing 10% goat serum, cells
were incubated overnight at 4 °C with an anti-PTH1R antibody
(clone 4D2) at a dilution ratio of 1:250, followed by incubation
for 1 h with anti-mouse goat IgG conjugated with Alexa Fluor
546 (Thermo Fisher Scientific). Osteoblast-like cells derived
from the iPSC clones (TkPP#7, TkSP#11) were fixed with
10% formalin in phosphate-buffered saline and then incubated
overnight at 4 °C with an anti-PTH1R antibody (clone 4D2) at
a dilution ratio of 1:250. The immune complex was detected
with Histofine Simple Stain MAX PO (Nichirei Biosciences
Inc.).
Measurement of Intracellular cAMP after
Treatment with PTH
Transfected HeLa cells were incubated for 15 min with
10-nmol/L PTH1-34 in the presence of 1-mmol/L IBMX (3-
isobutyl-1-methylxanthine). Osteoblast-like cells derived
Figure 1.  Expression of wild-type and mutated PTH1R in HeLa cells.
(A) Localization of PTH1R in HeLa cells transfected with wild-type and
mutant PTH1R. Immunocytochemical analysis of PTH1R protein was
performed in HeLa cells transfected with gfp (GFP), wild-type PTH1R
(Wild), or the PTH1R mutants 356C>T (P119L), 395C>T (P132L),
439C>T (R147C), and 1148G>A (R383Q). Scale bar = 50 µm.
(B) Western blot analysis of PTH1R and β-actin in lysates from HeLa
cells transfected with gfp (GFP), wild-type PTH1R (Wild), or the
PTH1R mutants 356C>T (P119L), 395C>T (P132L), 439C>T (R147C),
and 1148G>A (R383Q). Molecular weight (Mw × 10–3) values for the
major bands were deduced from their mobility and are indicated with
arrowheads. (C) Relative intensities of bands with a molecular weight
of 87 × 103 and 69 × 103 shown in panel B. (D) Western blot analysis
of PTH1R and β-actin in lysates from HeLa cells transfected with gfp
(GFP), wild-type PTH1R (Wild), or the PTH1R mutants 356C>T (P119L),
395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q) after
incubation with (+) or without (–) peptide-N-glycosylase. Molecular
weight (Mw × 10–3) values were deduced from mobility and are indicated
with bars.
4 Journal of Dental Research 00(0)

Figure 2.  Cellular response of HeLa cells transfected with wild-type and mutant PTH1R for binding to parathyroid hormone (PTH). (A) Binding
of PTH1-34 to cells. HeLa cells were transfected with gfp (GFP), wild-type PTH1R (Wild), or the PTH1R mutants 356C>T (P119L), 395C>T (P132L),
439C>T (R147C), and 1148G>A (R383Q) and then treated with TAMRA-labeled PTH1-34 in the absence (–) or presence (+) of nonlabeled PTH1-34
in a 1,000-fold excess amount. Red, TAMRA; blue, DAPI. (B) Fluorescence intensity for TAMRA was quantified with ImageJ software and expressed
as relative values to that obtained in Hela cells introduced with wild-type PTH1R (Wild). Values show the mean ± SD for 3 experiments. UD,
undetectable. (C) Intracellular accumulation of cAMP after treatment with PTH. HeLa cells were transfected with gfp (GFP), wild-type PTH1R (Wild),
or the PTH1R mutants 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q) and then treated with 10-nmol/L PTH1-34 in the
presence of IBMX (1 mmol/L) for 15 min. The amounts of cAMP are presented as ratios to that obtained in cells transfected with wild-type PTH1R.
Values are shown as the mean of 2 independent experiments.

from iPSCs were treated for 15 min with 25-nmol/L PTH1-34
or 50-µmol/L forskolin in the presence of 1-mmol/L IBMX
(Sigma-Aldrich). The amount of cAMP was determined
with a cAMP Biotrak Enzyme Immunoassay System (GE
Healthcare).
Determination of Cellular RANKL Protein
The amount of cellular RANKL was determined with the
enzyme-linked immunosorbent assay kit for human RANKL
(ab213841; Abcam).
Statistical Analysis
Mann-Whitney U test was used for comparisons of results
from 2 experimental groups. Dunnett’s test was used for mul-
tiple comparisons between the control and each treatment
group.
Results
Differential Glycosylation of Wild-type
PTH1R and Its Mutants
HeLa cells were infected with lentiviruses carrying the wild-
type PTH1R or 4 mutated PTH1R genes (356C>T, 395C>T,
439C>T, 1148G>A) that result in 4 types of amino acid substi-
tutions (i.e., P119L, P132L, R147C, and R383Q) in PTH1R
protein (Yamaguchi et al. 2011). All PTH1Rs were similarly
expressed by HeLa cells (Fig. 1A).
Western blot analysis confirmed the expression of the wild-
type and mutated PTH1R (Fig. 1B). Two major immune reac-
tive bands with higher (87 × 103) and lower (69 × 103) molecular
weights were detected in cells transfected with the wild-type
and mutated PTH1R. Wild-type PTH1R was mostly accumu-
lated in the 87 × 103 band. Approximately 20% of the P119L
and P132L proteins showed an apparent molecular weight of
87 × 103, while the remaining 80% had a molecular weight of
Functional Analysis of PTH1R Variants 5

69 × 103, and R147C PTH1R was equally divided between

proteins with those 2 molecular weights. Although the majority
of R383Q PTH1R was accumulated in the 87 × 103 band, some
showed a molecular weight of 69 × 103 (Fig. 1C).
The apparent molecular weights of the immune reactive
bands were greater than that of PTH1R (63516.18), as deduced
from its amino acid sequence (GenBank: AAI10389.1). It is
known that human PTH1R has 4 N-linked glycosylated Asp
residues in its N-terminal region: Asp-151, Asp-161, Asp-166,
and Asp-176 (Zhou et al. 1997; Gentili et al. 2003; Gan et al.
2006). Following treatment with peptide-N-glycosidase, 87 ×
103 and 69 × 103 bands were converged as a single band with
an apparent molecular weight of 62 × 103 (Fig. 1D). These
results indicated that the degree of N-glycosylation of the
P119L and P132L mutants was lower as compared with wild-
type PTH1R and the R383Q mutant, while the R147C mutant
expressed that at a level intermediate between them.

Binding of Fluorescence-Labeled PTH1-34 to HeLa

Cells Expressing Wild-type and Mutated PTH1R
HeLa cells expressing wild-type and P119L-, P132L-, R147C-,
and R383Q-substituted PTH1R were incubated with fluores-
cence-labeled PTH1-34, in the absence or presence of an excess
amount of nonlabeled PTH1-34 (Fig. 2A, B). That excessive
amount of nonlabeled PTH1-34 abolished the fluorescence of
wild-type PTH1R-expressing cells, indicating the specific
binding of TAMRA-labeled PTH1-34 to PTH1R. Fluorescence
from TAMRA-labeled-PTH1-34 bound to cells expressing
P119L-, P132L-, and R147C-mutated PTH1R was weaker than
those expressing wild-type PTH1R, indicating that the affinity
of PTH1R for PTH1-34 was lowered by these amino acid
substitutions.
Figure 3.  Expression of pluripotency markers in control and PFE-
specific iPSCs. (A) Immunofluorescence staining for SSEA-4 and Tra-1
in cultures of the control iPSC line (TkPP#7) and a PFE-specific iPSC
line (TkSP#11). Both cell lines were positive for SSEA-4 and Tra-1-60.
Scale bars = 100 µm. (B) mRNA expression levels of pluripotency genes
(SOX2, OCT, NANOG) in control (TkPP#7) and PFE-specific (TkSP#11)
iPSCs, as well as peripheral blood mononuclear cells (PBMCs) isolated
from a healthy volunteer. Values are shown as the mean ± SD of 4
experiments. iPSCs, induced pluripotent stem cells; PFE, primary failure
of eruption.

cAMP Accumulation after PTH Treatment

of HeLa Cells Introduced with Wild-type
and Mutated PTH1R Genes
Production of cAMP was not observed in control HeLa cells
expressing GFP instead of PTH1R after treatment with 10
nmol/L of PTH1-34. However, cAMP was increased in HeLa
cells expressing wild-type PTH1R by the treatment with PTH1-34.
PTH1-34 had a negligible effect on cAMP production in cells
expressing P119L- and P132L-substituted PTH1R, indicating
that these amino acid substitutions have critical effects on the
function of PTH1R. Also, the amount of PTH1-34-induced
accumulation of cAMP in cells expressing the R147C and
R383Q mutants was lower than that observed in cells express-
ing wild-type PTH1R (Fig. 2C).
Expression of Pluripotency Markers
in PFE-Specific iPSCs
The results described here (Fig. 2) are consistent with the pre-
vious report describing the impaired function of PTH1R with
P132L substitution in the PTH1R associated with Blomstrand
lethal chondrodysplasia (Zhang et al. 1998). However, there
remained a possibility that those results did not fully reproduce
the situation of PFE at the cellular level. Hence, we prepared
PFE-specific iPSCs. PFE-specific iPSCs (TkSP#11) and iPSCs
from the healthy subject (TkPP#7) expressed SSEA-4 and Tra-
1-60, cell surface markers for pluripotent stem cells (Fig. 3A).
All of the other iPSC clones originating from the patient with
PFE were also positively stained with antibodies for SSEA-4
and Tra-1-60 (not shown). Expression of mRNA for SOX2,
OCT4, and NANOG, marker genes for pluripotent stem cells,
was confirmed in both clones (TkPP#7 and TkSP#11; Fig. 3C).
Osteoblastic Differentiation of PFE-Specific iPSCs
Osteogenic differentiation of control and PFE-specific iPSCs
was examined. Mineralization by the cells derived from the
PFE-specific iPSCs (TkSP#11) after cultivation in the osteoblast-
inducing medium was comparable to that by cells derived from
the control iPSC line (TkPP#7; Fig. 4A, B). Expressions of
6 Journal of Dental Research 00(0)

PFE-specific iPSCs as in those derived

from control iPSCs during cultivation in
osteoblast-inducing medium (Fig. 4C,
D). Not only expression of osteocalcin
gene (BGLAP), a differentiation marker
of osteoblasts, but also that of PTH1R
was upregulated in cells derived from
PFE-specific (TkSP#11) and control
(TkPP#7) iPSCs (Fig. 4D, E). The
expression of PTH1R protein in both types
of cells was also confirmed (Fig. 4G).

PFE-Specific iPSCs Showed Lower

Response to PTH
The responses of osteoblast-like cells
derived from TkPP#7 and TkSP#11 iPSCs
to stimulation by PTH1-34 were examined.
Following treatment for 6 h (Fig. 5Aa,
Ab) or 48 h (Fig. 5Ac, Ad) with PTH1-34,
no augmentation of RANKL mRNA
expression in osteoblast-like cells derived
from PFE-specific iPSCs was seen (Fig.
3Ab, Ad), whereas the same stimulation
increased the expression of RANKL
mRNA cells derived from the control
iPSCs (Fig. 5Aa, Ac). However, increased
expression of RANKL mRNA was
observed in osteoblast-like cells derived
from the control and PFE-specific iPSCs
following treatment with 1α,25-
dihydroxyvitamin D3 (Fig. 5A), indicating
that both cell types possessed a capacity to
express RANKL mRNA. We also con-
firmed differential effects of PTH1-34 on
the expression of RANKL protein in cells
derived from control iPSCs and PFE-
specific iPSCs (Fig. 5B). Furthermore,
PTH1-34-induced cAMP accumulation in
cells derived from the PFE-specific iPSCs
(TkSP#11) was lower than that from the
Figure 4.  Osteoblastic differentiation of control and PFE-specific iPSCs. Control (TkPP#7)
and PFE-specific (TkSP#11) iPSCs were cultured for 3 wk in osteoblast-inducing medium (see
control iPSCs (TkPP#7; Fig. 5C). These
Materials and Methods section). (A) After cultivation for 3 wk, alizarin red staining (red) was results suggested that osteoblast-like cells
performed. Scale bars = 500 µm. (B) Alizarin red–positive areas were measured with a BZ-H3C derived from the PFE-specific iPSCs were
Hybrid Cell Count Module (Keyence). Values are expressed as the mean ± SD of 4 experiments. less responsive to PTH as compared with
Quantitative real-time reverse transcriptase polymerase chain reaction assays of control (TkPP#7,
unfilled columns) and PFE-specific (TkSP#11, filled columns) iPSCs at the baseline (day 0) and cells the control iPSCs.
derived from those iPSCs after cultivation for 1, 3, 7, and 21 d in osteoblast-inducing medium to Given that the patient with PFE has the
determine the mRNA expression of RUNX2 (C) and SP7 (D). Quantitative real-time polymerase heterozygous 395C>T (P132L) mutation
chain reaction assay of control and PFE-specific iPSCs at the baseline (0 wk) and cells derived
in the PTH1R gene, the lentivirus vector
from those iPSCs (3 wk) to determine the mRNA expression of osteocalcin gene (BGLAP) (E)
and PTH1R (F). Values were normalized by the expression of GAPDH mRNA and shown as fold of wild-type PTH1R and that of the
change relative to the mRNA expression level of each gene in the control iPSCs (TkPP#7) without 395C>T-mutated PTH1R were infected to
induction of osteoblastic differentiation. Values are shown as the mean ± SD of 4 experiments. **P HeLa cells at different ratios of the virus
< 0.01. (G) Immunocytochemical detection of PTH1R in cultures of osteoblast-like cells derived
from control (TkPP#7) and PFE-specific (TkSP#11) iPSCs. iPSCs, induced pluripotent stem cells; particles. Increasing the ratio of infection
PFE, primary failure of eruption. of the virus having the wild-type PTH1R
gene to that having the 395C>T-mutated
RUNX2 and SP7, genes for master transcription factors of PTH1R gene partially restored cAMP production in HeLa cells
osteogenesis, were upregulated in cells derived from the after stimulation by PTH1-34.
Functional Analysis of PTH1R Variants 7

Discussion
It has been reported that mutations in the PTH1R gene are
associated with the development of PFE (Yamaguchi et al.
2011; Risom et al. 2013; Frazier-Bowers et al. 2014;
Subramanian et al. 2016). In this study, we performed func-
tional analyses of PTH1R mutations found in patients with
PFE via HeLa cells expressing those mutations and PFE-
specific iPSCs.
It was suggested that the amino-terminal region of PTH1R
is N-glycosylated (Zhou et al. 1997; Gan et al. 2006). Four pos-
sible N-glycosylated Asn residues have been reported in human
as well as rat PTH1R: Asn-151, Asp-161, Asn-166, and Asn-
176 (Jüppner et al. 1991; Zhou et al. 2000). It was shown that
a PTH1R mutant with all of these Asn residues with Gln sub-
stitution was deficient in the function, though at least 1 of the 4
N-glycosylation sites was sufficient for cAMP production fol-
lowing stimulation with PTH (Zhou et al. 2000). In the present
study, we found that substitution of Pro-117 or Pro-132 with
Leu had a suppressive effect on the N-glycosylation. Since the
apparent molecular weights of the mutant PTH1R proteins
indicate that the effects of these amino acid substitutions on the
N-glycosylation were partial, the suppressed cAMP production
could not be explained by the retarded N-glycosylation.
The high-resolution crystal structure of PTH1R complexed
with a PTH1-34 analog was recently reported (Ehrenmann et al.
2018). It was indicated that Arg-20 of PTH1-34 interacts with
Trp-136 and Asp-137 of PTH1R. Interactions of Lys-27 and
Leu-28 of the ligand with Ile-115 of the receptor were also
detected. The Pro residue contributes to locking the secondary
structure of proteins due to restricted rotation of the X-Pro pep-
tide bond; thus, the substitution of Pro-117 or Pro-132 with Leu
may have an impact on conformation of their adjacent regions
containing Ile-115, Trp-136, and Asp-137, which might directly
suppress the binding of PTH1-34 and the induction of cAMP pro-
duction. While substitution of Arg-147 in the extracellular
domain of the receptor partially affected binding of PTH1-34 and
cAMP production, the substitution of Arg-383 with Gln
(R383Q) had little effect on the binding of the ligand. The par-
tial suppression of cAMP production in HeLa cells with R383Q-
PTH1R is considered to be due to other reasons, such as possible
alteration in G-protein binding (Hendricks et al. 2017).
Figure 5.  Responses of iPSC-derived osteoblast-like cells to
Also, PTH1R has been shown to exist as a dimer, and binding parathyroid hormone (PTH). Osteoblast-like cells were used that
of PTH induces dissociation of its receptors (Pioszak et al. had differentiated from control (TkPP#7) and PFE-specific (TkSP#11)
2010). Furthermore, PTH and PTHrP are found to transduce not iPSCs after cultivation for 3 wk in the osteoblast-inducing medium.
only cAMP but also Ca2+ signals (Mannstadt et al. 1999). PTH1R (A) Expression of RANKL mRNA in cells derived from control (a, c)
and PFE-specific (b, d) iPSCs after treatment with 25-nmol/L PTH1-34
has been shown to interact with other signaling molecules, (PTH) and 10-nmol/L active vitamin D3 (D3) for 6 (a, b) and 48 (c, d)
including β-catenin (Yano et al. 2013) and transforming growth h. Values were normalized by the expression of GAPDH mRNA and
factor β (TGF-β) type II receptor (Qiu et al. 2010). It would be shown as fold change relative to RANKL mRNA expression level in each
type of iPSC without induction of osteoblastic differentiation (unfilled
interesting to examine the effects of amino acid substitutions in columns). (B) Expression of RANKL protein in the control (TkPP#7)
PTH1R on their dimer formation with wild-type PTH1R, as well (a) and PFE-specific (TkSP#11) (b) iPSCs after cultivation for 3 wk
as on signals mediated by Ca2+, β-catenin, and TGF-β. in the osteoblast-inducing medium. (C) Intracellular accumulation of
PFE-specific and control iPSCs differentiated into osteoblast- cAMP after treatment with PTH1-34. Osteoblast-like cells derived from
control (TkPP#7) and PFE-specific (TkSP#11) iPSCs were treated for
like cells, each with a similar extent of mineralization and 15 min with 25-nmol/L PTH1-34 in the presence of 1 mmol/L IBMX. As
expression of osteoblast marker genes as well as the PTH1R a positive control of cAMP production, cells were treated for 15 min
gene. However, we observed a lower level of responsiveness to with 50-µmol/L forskolin in the presence of IBMX. Values are shown
as fold change relative to that obtained with control iPSC-derived
PTH in cells derived from PFE-specific iPSCs with the 395C>T
osteoblast-like cells (left column). Values are shown as the mean ± SD
mutation in the PTH1R as compared with control iPSCs, as of 4 experiments. **P < 0.01, *P < 0.05. iPSCs, induced pluripotent stem
evaluated by the accumulation of cAMP as well as the cells; PFE, primary failure of eruption.
8 Journal of Dental Research 00(0)
induction of RANKL mRNA and protein expression.
Accumulation of such differences might have effects on the
functions of osteoblasts and osteocytes, including induction of
osteoclastogenesis via expression of RANKL. Since bone
resorption by osteoclasts is required for tooth eruption, defects
in RANKL induction might be one of the causes of deficient
tooth eruption.
A previous report suggested that PTH1R with a substitution
of Gly-452 with Glu showed a dominant-negative effect on the
wild-type receptor (Subramanian et al. 2016). In our study, an
increase in the ratio of the expression of wild-type PTH1R to
that of P132L-mutated PTH1R partially restored cAMP pro-
duction after stimulation by PTH1-34. Further studies are
required to clarify whether P132L-substituted PTH1R affects
the function of wild-type PTH1R (Appendix Fig.).
To summarize, the present results show that amino acid
substitutions found in PTH1R from a patient with PFE lowered
the responsiveness of the cells to PTH. To clarify the role of the
mutations in PTH1R in PFE, the development of in vivo mod-
els of PFE with PFE-specific iPSCs is desirable. Detection of
gene variants in patients with rare diseases with techniques
such as whole-exome sequencing coupled with studies based
on disease models constructed with the disease-specific iPSCs
may be a promising approach to identify the therapeutic targets
and development of effective treatment for such diseases.
Author Contributions
E. Izumida, T. Suzawa, contributed to data acquisition, analysis,
and interpretation, drafted the manuscript; Y. Miyamoto, contrib-
uted to conception and design, drafted the manuscript; A. Yamada,
contributed to conception, drafted the manuscript; M. Otsu, T.
Saito, contributed to design, critically revised the manuscript; T.
Yamaguchi, K. Nishimura, M. Ohtaka, M. Nakanishi, K.
Yoshimura, K. Sasa, R. Takimoto, R. Uyama, contributed to data
acquisition, critically revised the manuscript; T. Shirota, K. Maki,
contributed to data analysis, critically revised the manuscript; R.
Kamijo, contributed to data interpretation, critically revised the
manuscript. All authors gave final approval and agree to be
accountable for all aspects of the work.
Acknowledgments
PTH1-34 was donated by Asahi Kasei Pharma Co. This study was
supported by grants from JSPS KAKENHI (15H05016, 16H07198,
18K09511, 18K09513, 18K19655), the MEXT-Supported Program
for the Strategic Research Foundation at Private Universities
(S1411009), and the MEXT Private University Research Branding
Project. We thank Tamaki Kurosawa for the excellent technical
assistance. The authors declare no potential conflicts of interest
with respect to the authorship and/or publication of this article.
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